ABCMdb

ABCC6 p.Arg1141*

ClinVar:	c.3421C>T , p.Arg1141* D , Pathogenic
LOVD-ABCC6:	p.Arg1141* D

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[hide] Multidrug resistance associated proteins as determ... Curr Drug Metab. 2007 Dec;8(8):787-802. Yu XQ, Xue CC, Wang G, Zhou SF
Multidrug resistance associated proteins as determining factors of pharmacokinetics and pharmacodynamics of drugs.
Curr Drug Metab. 2007 Dec;8(8):787-802., [PMID:18220559]

Abstract [show]
The multidrug resistance associated proteins (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, MRP8 and MRP9) belong to the ATP-binding cassette superfamily (ABCC family) of transporters. They are expressed differentially in the liver, kidney, intestine, brain and other tissues. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. Several MRPs (mainly MRP1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. MRPs transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. Most MRPs are subject to induction and inhibition by a variety of compounds. Several nuclear receptors, including pregnane X receptor (PXR), liver X receptor (LXR), and farnesoid receptor (FXR) participate in the regulation of MRPs. MRPs play an important role in the absorption, distribution and elimination of various drugs in the body and thus may affect their efficacy and toxicity and cause drug-drug interactions. MRPs located in the blood-brain barrier can restrict the penetration of compounds into the central nervous system. Mutation of MRP2 causes Dubin-Johnson syndrome, while mutations in MRP6 are responsible for pseudoxanthoma elasticum. More recently, mutations in mouse Mrp6/Abcc6 gene is associated with dystrophic cardiac calcification (DCC), a disease characterized by hydroxyapatite deposition in necrotic myocytes. A single nucleotide polymorphism, 538G>A in the MRP8/ABCC11 gene, is responsible for determination of earwax type. A better understanding of the function and regulating mechanism of MRPs can help minimize and avoid drug toxicity, unfavourable drug-drug interactions, and to overcome drug resistance.

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406 MRP Chromosomal location Amino acid variation Nucleotide variation Location References Lys13Asn G39GC Exon1 His68Tyr C202T Exon2 Ser346Phe C1037T Exon9 Gln513Lys C1537A Exon12 Arg1297His G3890A Exon27 MRP3 17q21.3 Gly1423Arg G4267A Exon29 [241] MRP4 13q32.1 Unknown MRP5 3q27 Unknown L63L W64R 189G>C 190T>C Exon2 Exon2 [250] T364R Q378X 1091C>G 1132C>T Exon9 Exon9 [260, 261] R518X R518Q 1552 C>T 1553G>A Exon12 Exon12 [247, 262] R1141X R1138Q T1130M R1114C M1127T 3421C>T 3413G>A 3389C>T 3340C>T 3380C>T Exon24 Exon24 Exon24 Exon24 Exon24 [246, 247] R1275X 3823C>T Exon27 [246] P1346S 4036C>T Exon28 [246] MRP6 16p13.1 E1400K 4198G>A Exon29 [247] MRP7 6p12-21 Unknown MRP8 16q12.1 Unknown MRP9 16q12.1 Unknown CONCLUSIONS AND FUTURE DIRECTIONS MRPs which belong to the ABC transporter family are able to transport a remarkable array of diverse endo- and xenobiotics and their metabolites. Login to comment

[hide] Pseudoxanthoma elasticum: mutations in the MRP6 ge... Proc Natl Acad Sci U S A. 2000 May 23;97(11):6001-6. Ringpfeil F, Lebwohl MG, Christiano AM, Uitto J
Pseudoxanthoma elasticum: mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter.
Proc Natl Acad Sci U S A. 2000 May 23;97(11):6001-6., 2000-05-23 [PMID:10811882]

Abstract [show]
Pseudoxanthoma elasticum (PXE), the prototypic heritable connective tissue disorder affecting the elastic structures in the body, manifests with cutaneous, ophthalmologic, and cardiovascular findings, with considerable morbidity and mortality. The molecular basis of PXE has remained unknown, but the disease locus has recently been mapped to an approximately 500-kb interval on chromosome 16p13.1, without evidence for locus heterogeneity. In this study, we report pathogenetic mutations in MRP6, a member of the ABC transporter gene family, in eight kindreds with PXE. The mutation detection strategy consisted of heteroduplex scanning of coding sequences in the MRP6 gene, which were amplified by PCR by using genomic DNA as template, followed by direct nucleotide sequencing. A total of 13 mutant MRP6 alleles were disclosed in the eight probands with PXE. These genetic lesions consisted of either single base pair substitutions resulting in missense, nonsense, or splice site mutations, or large deletions resulting in allelic loss of the MRP6 locus. Examination of clinically unaffected family members in four multiplex families identified heterozygous carriers, consistent with an autosomal recessive inheritance pattern. Collectively, identification of mutations in the MRP6 gene provides the basis to examine the pathomechanisms of PXE and allows development of DNA-based carrier detection, prenatal testing, and preimplantation genetic diagnosis in families with a history of this disease.

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74 The mutation in exon 24 resulted in substitution of a codon for arginine by a stop codon, a mutation designated R1141X. Login to comment
77 MRP6 mutations in families with PXE Family Age and sex of proband Mutation Exon Consequence Verification* 1 53 F 3421C 3 T 24 R1141X BsiYI 3803G 3 A 27 R1268Q BstXI 2 29 F 3412C 3 T 24 R1138W MspI 3 40 F 3421C 3 T 24 R1141X BsiYI Partial deletion 24† Allelic loss D16S2720 MRP6 D16B9622 4 53 F 3736-1G 3 A 27 Altered splicing of exon 27 AciI Partial deletion 27† Allelic loss D16S2720 MRP6 D16B9622 5 60 M 3413G 3 A 24 R1138Q MspI 3803G 3 A 27 R1268Q BstXI 6 28 F 3421C 3 T 24 R1141X BsiYI 7 41 M 3803G 3 A 27 R1268Q BstXI 8 25 F 3421C 3 T 24 R1141X BsiYI *Mutations were verified in the proband and his/her family members by digestion with restriction enzyme, or in case of deletion, by microsatellite markers indicated. Login to comment
98 Examination of the proband`s DNA by CSGE and nucleotide sequencing (Fig. 2 A and B) revealed a homozygous mutation in exon 24, 3421C3T, which resulted in a premature termination codon R1141X. Login to comment
101 Examination of the parents revealed that the father (I-2) was heterozygous for the mutation R1141X. Login to comment
111 These findings are consistent with the interpretation that the proband and her sister had inherited a nonsense mutation from the father and a deletion mutation affecting exon 24 of MRP6 from the mother, thus reducing the paternal mutation R1141X to hemizygosity. Login to comment
113 A search for other mutations in the MRP6 gene was unyielding, and he may therefore manifest with mild features of PXE, reflecting his carrier status for the mutation R1141X. Login to comment
142 Evaluation of their DNA revealed that the patient in family 5 was compound heterozygous for nucleotide substitutions 3413G3A and 3803G3A in exons 24 and 27, which resulted in the amino acid substitutions R1138Q and R1268Q, respectively. In families 6-8, heterozygous nucleotide substitutions were discovered, resulting in the mutations R1141X (families 6 and 8), and R1268Q (family 7). Login to comment
159 Although only one mutation in each of the latter three individuals was observed, it is likely that PXE in these cases was also autosomal recessive, because each of the two distinct mutations (R1141X and R1268Q) discovered in these three probands were also present in the multiplex families, and the heterozygous carriers did not show definitive signs of the disease. Login to comment

[hide] Homozygosity for the R1268Q mutation in MRP6, the ... Biochem Biophys Res Commun. 2000 Aug 2;274(2):297-301. Germain DP, Perdu J, Remones V, Jeunemaitre X
Homozygosity for the R1268Q mutation in MRP6, the pseudoxanthoma elasticum gene, is not disease-causing.
Biochem Biophys Res Commun. 2000 Aug 2;274(2):297-301., 2000-08-02 [PMID:10913334]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an inherited systemic disorder of connective tissue, characterized by progressive calcification of the elastic fibers in the eye, the skin, and the cardiovascular system, resulting in decreased vision, skin lesions, and life-threatening vascular disease, with highly variable phenotypic expression. The PXE locus has been mapped to chromosome 16p13.1, and was recently further refined to a 500 kb-region, containing two pseudogenes and four candidate genes. In a comprehensive mutational screening, we were able to exclude the responsibility of pM5, UNK, and MRP1 genes, candidate on the basis of their genetic localization. Conversely, we have found pathogenetic mutations in the MRP6 gene, in patients affected with PXE, indicating that human MRP6, which encodes a 1503 amino-acids membrane protein, member of the human ATP binding cassette (ABC) transporters superfamily, is the gene responsible for PXE. In one large PXE pedigree for which we had identified a nonsense mutation (R1141X), we came across a G to A transition at position 3803 of the MRP6 cDNA sequence (R1268Q). Astonishingly, this latter variant was found at the homozygous state in the proband's unaffected husband. We investigated the R1268Q mutation, and found the Q1268 allele at a relatively high frequency (0.19) in a Caucasian control population (n = 62 subjects). Genotype frequencies were in Hardy-Weinberg equilibrium, and three healthy volunteers were homozygous for the Q1268 allele. These data indicate that the R1268Q variant in the MRP6 gene does not cause PXE per se. Further studies will elucidate if it may play a role when found in compound heterozygotes.

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4 In one large PXE pedigree for which we had identified a nonsense mutation (R1141X), we came across a G to A transition at position 3803 of the MRP6 cDNA sequence (R1268Q). Login to comment
18 During our molecular analysis of the MRP6 gene in PXE patients, we came across a G to A transition at position 3803 of the cDNA sequence, altering the codon (CGG) for arginine to the codon (CAG) for glutamine (R1268Q) in a pedigree in which we had previously identified a nonsense mutation (R1141X). Login to comment
49 During our mutational analysis, we found a heterozygous C to T transition at cDNA position 3421 of the MRP6 gene, predicting termination of translation at codon 1141 (R1141X) (Fig. 2). Login to comment
75 Detection of the R1141X nonsense mutation by direct sequencing of the MRP6 gene in the proband. Login to comment

[hide] Abstracts: mutations in the MRP6 gene cause pseudo... J Invest Dermatol. 2000 Aug;115(2):332. Ringpfeil F, Lebwohl MG, Uitto J
Abstracts: mutations in the MRP6 gene cause pseudoxanthoma elasticum
J Invest Dermatol. 2000 Aug;115(2):332., [PMID:10951257]

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8 A particular stop codon mutation, R1141X, occurred independently in six unrelated kindreds. Login to comment

[hide] Compound heterozygosity for a recurrent 16.5-kb Al... Am J Hum Genet. 2001 Mar;68(3):642-52. Epub 2001 Feb 9. Ringpfeil F, Nakano A, Uitto J, Pulkkinen L
Compound heterozygosity for a recurrent 16.5-kb Alu-mediated deletion mutation and single-base-pair substitutions in the ABCC6 gene results in pseudoxanthoma elasticum.
Am J Hum Genet. 2001 Mar;68(3):642-52. Epub 2001 Feb 9., [PMID:11179012]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality. Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed. Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations. In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern. In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans. The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide. The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently. Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.

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24 OF AFFECTED FAMILY MEMBERS COMPLEMENTARY MUTATION PHENOTYPE a When Studied At Disease Onset 1 (German) 61 9 2 R1164X Skin-cobblestoning on neck, antecubital fossae, and wrists, sagging skin in axillae and bilateral groin; eyes-angioid streaks, central vision loss; CVS-claudication, ischemic attack; other-ovarian cancer 2 (British) 60 Unknown 3 R1164X Skin-moderate to severe involvement; eyes-loss of vision 3 (British) 41 Unknown 3 R1141X Skin-cobblestoning on neck, axillae, and bilateral groin; eyes-angioid streaks; CVS-coronary artery disease, GI bleeding 4 (Greek) 60 51 2 3736-1GrA Skin-cobblestoning in axillae, bilateral groin, and antecubital fossae; eyes-angioid streaks, central vision loss, macular degeneration; CVS-angina, abdominal pain, loss of peripheral pulses; other-depression, chronic fatigue syndrome a CVS p cardiovascular system; GI p gastrointestinal. Login to comment
56 Clinical Assessment of Families with PXE Members of families 1, 3, and 4 were personally examined at least by one of the authors; information on Table 2 Haplotypes of Affected Individuals in Four Unrelated Families with PXE MARKER a HAPLOTYPE FOR b del/R1164X del/R1141X; Family 3 del/3736-1GrA; Family 4Family 1 Family 2 D16S3114 4 4 10 5 9 6 9 4 D16S500 6 3 4 10 4 8 6 10 D16S2619 2 1 3 2 2 3 2 2 D16S3079 2 3 2 3 1 9 8 7 D16S3060 4 2 8 2 7 4 5 8 D16S405 8 3 4 3 4 8 3 4 D16B9622 2 2 2 2 1 2 3 1 D16S764 4 2 3 2 2 2 3 2 D16S79 8 0 3 3 3 3 3 2 D16S3103 7 1 3 1 9 3 7 4 D16S3017 3 2 4 1 5 4 2 4 D16S499 1 5 5 1 8 7 5 8 D16S3036 8 8 7 7 8 11 4 6 a The distances between the listed markers are as follows: telomere, D16S3114 (1.9 cM) D16S500 (0.5 cM) D16S2619 (0.7 cM) D16S3079 (0.5 cM) D16S3060 (22 kb) D16S405 (430 kb) D16B9622 (0.7 kb) ABCC6 (317 kb) D16S764 (8 kb) D16S79 (1.5 cM) D16S3103 (0.4 cM) D16S3017 (0.9 cM) D16S499 (1.5 cM) D16S3036, centromere. Login to comment
99 In these families, designated here as "family 3" (family 3 in Ringpfeil et al. 2000) and "family 4" (family 4 in Ringpfeil et al. 2000), a nonsense mutation (R1141X) and a splicing mutation (3736-1GrA) are located in exon 24 and in the 3 acceptor splice site of intron 26, respectively. Login to comment
154 In each family, the proband was initially shown to be apparently homozygous for a nucleotide substitution, resulting in either a nonsense mutation (R1164X, R1164X, and R1141X in families 1, 2, and 3, respectively) or a splice-site mutation (3736-1GrA in family 4). Login to comment
180 Exon 24 encodes a segment of MRP6 residing within TMSD3, and the mutations R1141X and R1164X are predicted to result in the synthesis of a truncated polypeptide entirely devoid of NBF2 (fig. 4A). Login to comment

[hide] Molecular genetics of pseudoxanthoma elasticum: a ... Trends Mol Med. 2001 Jan;7(1):13-7. Uitto J, Pulkkinen L, Ringpfeil F
Molecular genetics of pseudoxanthoma elasticum: a metabolic disorder at the environment-genome interface?
Trends Mol Med. 2001 Jan;7(1):13-7., [PMID:11427982]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a relatively rare heritable disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality. The disease affects the elastic fibers of affected organs, which become progressively calcified. Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system. Recently, PXE has been linked to mutations in the MRP6/ABCC6 gene, a member of the ABC transporter family, expressed primarily in the liver and the kidneys. This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment-genome interface.

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68 A particularly common mutation appears to be R1141X, which has been independently described in families of various ethnic backgrounds10-13,17. Login to comment
75 Thisraisesthequestionoftherelationshipbetweenthe MRP6mutationsandthemanifestationsinPXE affectingtheelasticfibersinvariousorgans.Itmaywell Opinion CR1339C G1345R TRENDS in Molecular Medicine 10 kb NBF2NBF1 0.5 kb Extracellular Intracellular GS 5' 3' R1138Q R1164X R1141X R1138W 2787+1G T A455P R518Q R1114P R1314W (a) (b) (c) EcoRI SmaI SmaI SmaI SacI SacI SmaI N GS 2542delG 1944del22 4220insAGAA 3775delT 3736-1G A Fig. 3. Login to comment

[hide] Molecular genetics of pseudoxanthoma elasticum. Exp Dermatol. 2001 Aug;10(4):221-8. Ringpfeil F, Pulkkinen L, Uitto J
Molecular genetics of pseudoxanthoma elasticum.
Exp Dermatol. 2001 Aug;10(4):221-8., [PMID:11493310]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a systemic heritable connective tissue disorder, is characterized by progressive calcification of elastic structures in the skin, the eyes and the cardiovascular system, with considerable intra- and interfamilial phenotypic variability. Recently, underlying genetic defects have been identified in the ABCC6 gene, which resides on the chromosomal locus 16p13.1 and encodes the MRP6 protein, a member of the ATP-binding cassette (ABC) family of proteins. The affected individuals are homozygous or compound heterozygous for a spectrum of genetic lesions, including nonsense and missense mutations, or deletions and splice-site alterations, confirming the autosomal recessive nature of this condition. Analysis of the deduced primary sequence suggests that MRP6 is a transmembrane transporter, but its function has not been delineated yet. Surprisingly, however, MRP6 is expressed primarily, if not exclusively, in the liver and the kidneys, suggesting that PXE may be a primary metabolic disorder with secondary involvement of elastic fibers. Identification of mutations in the ABCC6 gene in PXE provides a means for prenatal and presymptomatic testing in families at risk for recurrence. DNA-based analyses will also identify heterozygous carriers who may be at risk for development of limited manifestations of the disease as a result of compounding genetic factors and/or environmental modifiers.

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74 Three recurrent mutations, R1141X, R1164X, and Del exon 23-29, are boxed (see text). Login to comment
87 Among the PTC causing mutations, two recurrent nonsense mutations, R1141X and R1164X, have been identified Figure . Login to comment
93 In particular, the R1141X mutation, which results from C»T transition in nucleotide position 3421, has been encountered in approximately 15% of the mutated alleles disclosed thus far. Login to comment

[hide] A spectrum of ABCC6 mutations is responsible for p... Am J Hum Genet. 2001 Oct;69(4):749-64. Epub 2001 Aug 31. Le Saux O, Beck K, Sachsinger C, Silvestri C, Treiber C, Goring HH, Johnson EW, De Paepe A, Pope FM, Pasquali-Ronchetti I, Bercovitch L, Marais AS, Viljoen DL, Terry SF, Boyd CD
A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum.
Am J Hum Genet. 2001 Oct;69(4):749-64. Epub 2001 Aug 31., [PMID:11536079]

Abstract [show]
To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

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8 R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. Login to comment
9 ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Login to comment
85 PXE Mutations The most-prevalent mutations detected in the ABCC6 gene were missense substitutions (21 [58.3%] mutations, Table 1 ABCC6 Mutations in a Cohort of Patients with PXE CHANGE IN STATUS a ORIGIN(S)b EXON(S)c REFERENCE(S)Amino Acid Nucleotide … 179-195del ht Belgium 2 Present study … 938-939insT ch, ht SA, UK 8 Present study N411K 1233TrG ht US 10 Present study A455P 1363GrC Nd Nd 11 Uitto et al. (2001) R518Q 1553GrA ch, ht Belgium 12 Present study, Uitto et al. (2001) F568S 1703TrC ch US 13 Present study … ABCC6del15 hm SA 15 Present study … 1944del22 ht Holland 16 Bergen et al. (2000) … 1995delG ht Germany 16 Present study L673P 2018TrC ch SA 16 Present study R765Q 2294GrA ht Germany 18 Present study Y768X 2304CrA ch, ht SA 18 Present study … 2322delC ht US 18 Present study … 2542delG Nd Nd 19 Uitto et al. (2001) … IVS21ϩ1GrT ch US, Germany i-21 Present study, Uitto et al. (2001) R1030X 3088CrT ht SA, UK 23 Present study R1114P 3341GrC hm UK 24 Present study S1121W 3362CrG ch Germany 24 Present study R1138W 3412CrT hm Nd 24 Ringpfeil et al. (2000) R1138P 3413GrC ch Germany 24 Present study R1138Q 3413GrA ch UK, US 24 Present study, Ringpfeil et al. (2000) R1141X 3421CrT All All 24 Present study and othersd R1164X 3490CrT ch Germany, UK 24 Ringpfeil et al. (2001) G1203D 3608GrA ch Germany 25 Present study … IVS26-1GrA ch Belgium i-26 Present study, Ringpfeil et al. (2000, 2001) Q1237X 3709CrT ch Belgium 26 Present study … 3775delT ht, hm SA, US, Holland 27 Present study, Bergen et al. (2000) V1298F 3892GrT ht US 28 Present study T1301I 3902CrT ch Belgium 28 Present study G1302R 3904GrA hm US 28 Present study A1303P 3907GrC ch Belgium 28 Present study R1314W 3940CrT hm US 28 Present study R1314Q 3941GrA ch Germany 28 Present study G1321S 3961GrA ht US 28 Present study R1339C 4015CrT All SA, US 28 Present study, Struk et al. (2000) Q1347H 4041GrC hm US 28 Present study D1361N 4081GrA ch Germany 29 Present study … 4104delC ch Belgium 29 Present study R1398X 4192CrT ch Belgium 29 Present study … ABCC6del23-29 ch US 23-29 Present study, Ringpfeil et al. (2001) … 4220insAGAA ht Holland 30 Bergen et al. (2000) I1424T 4271TrC ht US 30 Present study … ABCC6del ht Holland all Bergen et al. (2000) a Nd p not determined; hm p homozygote; ht p heterozygote; ch p compound heterozygote. Login to comment
94 Although most of the mutations reported here appear to be unique, a few disease-causing variants have been found to occur frequently in apparently unrelated individuals; R1141X was found at Table 2 Frequencies of Mutant Alleles Found in a Cohort of 101 Unrelated Patients with PXE MUTATION a OVERALL EUROPE UNITED STATES No. of Alleles Frequency (%) No. of Alleles Frequency (%) No. of Alleles Frequency (%) R1141X 38 18.8 33 28.4 3 4.1 ABCC6del23-29 26 12.9 5 4.3 21 28.4 IVS21ϩ1GrT 7 3.5 4 3.4 3 4.1 G1302R 4 2.0 0 .0 4 5.4 A1303P 4 2.0 3 2.6 1 1.4 R1314W 3 1.5 0 .0 3 4.1 R518Q* 3 1.5 1 .9 1 1.4 3775delT* 3 1.5 2 1.7 0 .0 R1138Q 2 1.0 1 .9 1 1.4 V1298F 2 1.0 0 .0 2 2.7 R1339C 2 1.0 0 .0 2 2.7 Q1347H 2 1.0 0 .0 2 2.7 4104delC* 2 1.0 1 .9 0 .0 179-195del 1 .5 1 .9 0 .0 938-939insT* 1 .5 0 .0 0 .0 N411K 1 .5 0 .0 1 1.4 F568S 1 .5 0 .0 1 1.4 1995delG 1 .5 1 .9 0 .0 R765Q 1 .5 1 .9 0 .0 2322delC 1 .5 0 .0 1 1.4 R1030X* 1 .5 0 .0 0 .0 R1114P 1 .5 1 .9 0 .0 S1121W 1 .5 1 .9 0 .0 R1138P 1 .5 1 .9 0 .0 G1203D 1 .5 1 .9 0 .0 IVS26-1GrA 1 .5 1 .9 0 .0 Q1237X 1 .5 1 .9 0 .0 W1241C 1 .5 1 .9 0 .0 T1301I 1 .5 1 .9 0 .0 R1314Q 1 .5 1 .9 0 .0 D1361N 1 .5 1 .9 0 .0 R1398X 1 .5 1 .9 0 .0 G1321S 1 .5 0 .0 1 1.4 I1424T 1 .5 0 .0 1 1.4 ? Login to comment
102 Interestingly, alleles R1141X and ABCC6del23-29 were found to occur at different frequencies when patient origin was considered. Login to comment
103 R1141X was far more frequently represented in the European cohort (33 [28.4%] of 116 alleles) than in the U.S. cohort of patients with PXE (3 [4.1%] of 74). Login to comment
105 Indeed, 15 of 26 Belgians, 8 of 15 Germans, 3 of 12 British, 2 of 6 Dutch, and 1 of 1 Italian carried an R1141X allele in a homozygous, compound heterozygous, or heterozygous state. Login to comment
106 In the U.S. cohorts, three patients carried a single R1141X allele. Login to comment
107 Among the South African patients, 2 of the 5 who were of British ancestry had the nonsense mutation in the heterozygous or compound heterozygous state, and 2 of the 17 of the Afrikaner patients carried a heterozygous or compound heterozygous R1141X. Login to comment
121 The frequency of individuals homozygous for these two frequently occurring disease-causing alleles (R1141X and ABCC6del23-29) was observed to be in Hardy-Weinberg equilibrium in the European and United States cohorts, respectively. Login to comment
139 One nonsense mutation (R1141X) that we have previously characterized in a homozygous state was indeed associated with no detectable ABCC6 messenger in RNA samples obtained from skin fibroblasts (Le Saux et al. 2000), and the absence of any ABCC6 mRNA is probably caused by NMD and would result in a null allele. Login to comment
157 Characterization of Two Alu-Mediated Deletions ABCC6del23-29.-A loss of heterozygosity of an R1141X allele was detected in three affected children of a two-generation family from the United States. These affected individuals seemed homozygous for R1141X; whereas the mother appeared to be heterozygous for this allele, and the father appeared to have two normal alleles. Login to comment
159 These results suggested that the affected offspring in this family had inherited a single maternal R1141X mutation and a paternally derived deletion involving exon 24, thereby reducing the nonsense mutation to hemizygosity in all three patients. Login to comment
213 Among the four Belgian patients with homozygous R1141X alleles, three had typical skin lesions, and all patients displayed moderate-to- severe ocular involvement. Login to comment
228 Although most mutations observed in ABCC6 were unique, two variants (R1141X and ABCC6del23-29) occurred at a high frequency (table 2). Login to comment
230 However, when patient ancestries were considered, R1141X was found to occur at a greater frequency in Europe, whereas ABCC6del23-29 was preponderant in the United States (table 2). Login to comment
236 A similar hypothesis could explain the high frequency of R1141X alleles in the European sample. Login to comment
237 However, to demonstrate that the various R1141X and ABCC6del23-29 alleles are identical by descent, the analysis of additional microsatellite markers and single-nucleotide polymorphisms within and in close proximity to ABCC6 will be required. Login to comment

[hide] Identification of ABCC6 pseudogenes on human chrom... Hum Genet. 2001 Sep;109(3):356-65. Pulkkinen L, Nakano A, Ringpfeil F, Uitto J
Identification of ABCC6 pseudogenes on human chromosome 16p: implications for mutation detection in pseudoxanthoma elasticum.
Hum Genet. 2001 Sep;109(3):356-65., [PMID:11702217]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a heritable disorder affecting the skin, eyes, and the cardiovascular system, has recently been linked to mutations in the ABCC6 gene on chromosome 16p13.1. The original mutation detection strategy employed by us consisted of the amplification of each exon of the ABCC6 gene with primer pairs placed on the flanking introns, followed by heteroduplex scanning and direct nucleotide sequencing. However, this approach suggested the presence of multiple copies of the 5'-region of the gene when total genomic DNA was used as a template. In this study, we have identified two pseudogenes containing sequences highly homologous to the 5'-end of ABCC6. First, by the use of allele-specific polymerase chain reaction (PCR), two bacterial artificial chromosome (BAC) clones containing a putative pseudogene of ABCC6, designated as ABCC6-psi 1, were isolated from the human BAC library. Sequence analysis of ABCC6-psi 1 revealed it to be a truncated copy of ABCC6, which contains the upstream region and exon 1 through intron 9 of the gene. Secondly, a homology search of a high-throughput sequence database revealed the presence of another truncated copy of ABCC6, which was designated as ABCC6-psi 2, and which was shown to harbor upstream sequences and a segment spanning exon 1 through intron 4 of ABCC6. In addition to several nucleotide differences in the flanking introns and the upstream region, both pseudogenes contain several nucleotide changes in the exonic sequences, including stop codon mutations, which complicate mutation analysis in patients with PXE. Nucleotide differences in flanking introns between these two pseudogenes and ABCC6 allowed us to design allele-specific primers that eliminated the amplification of both pseudogene sequences by PCR and provided reliable amplification of ABCC6-specific sequences only. The use of allele-specific PCR has revealed, thus far, two novel 5'-end PXE mutations, 179del9 and T364R in exons 2 and 9, respectively, and several polymorphisms within the upstream region and exons 1-9 of ABCC6. These strategies facilitate comprehensive analysis of ABCC6 for mutations in PXE.

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31 Specifically, NciI restriction enzyme was used for the detection of mutation 179del9, BstNI for the detection of mutation G1354R, and BsiYI for mutation R1141X. Login to comment
112 In addition to the missense mutation T364R, this patient was shown to have a recurrent nonsense mutation R1141X in exon 24 in the other ABCC6 allele, as shown by sequence analysis (Fig.4C). Login to comment
129 Screening of other parts of the gene revealed a recurrent nonsense mutation R1141X in exon 24 in the other allele of patient P2 (C). Login to comment
130 Thus, the patient is a compound heterozygote for mutations T364R/R1141X Table 3 Polymorphisms characterized in the region spanning 5`-UTR and exon 1 to intron 9 of ABCC6, as identified by the use of allele-specific primers aThe nucleotide positions refer to numbers in ABCC6 cDNA and are counted from the first nucleotide of the translation initiation codon ATG (Belinsky and Kruh 1999; GenBank accession nos. XM_007798, NM_001171) Location Nucleotide positiona Major allele Minor allele Allele frequences 5`-flanking 1-219 A C 0.950/0.050 5`-flanking 1-132 C T 0.953/0.047 5`-flanking 1-127 C T 0.857/0.143 Exon 3 232 G (Ala) T (Thr) - Intron 3 346-6 G A - Exon 6 645 G (Thr) A (Thr) - Intron 6 662+12 C T - Intron 6 662+114 T C 0.937/0.063 Intron 6 662+298 C G - Intron 6 662+305 T G - Intron 6 662+403 T A - Exon 7 793 A (Arg) G (Gly) 0.914/0.086 a number of duplicated regions (Doggett et al. 1995; Loftus et al. 1999; Cai et al. 2000). Login to comment
149 In this work, we have found three novel mutations, 179del9, T364R, and G1354R, and a recurrent nonsense mutation R1141X in two families with PXE (Fig.5). Login to comment
153 The proband in Family 2 is a compound heterozygote for R1141X and T364R mutations. Login to comment
154 The recurrent R1141X nonsense mutation predicts the elimination of the entire C-terminus from the protein, including part of the third transmembrane domain and the second nucleotide-binding fold (Fig.5). Login to comment

[hide] Frequent mutation in the ABCC6 gene (R1141X) is as... Circulation. 2002 Aug 13;106(7):773-5. Trip MD, Smulders YM, Wegman JJ, Hu X, Boer JM, ten Brink JB, Zwinderman AH, Kastelein JJ, Feskens EJ, Bergen AA
Frequent mutation in the ABCC6 gene (R1141X) is associated with a strong increase in the prevalence of coronary artery disease.
Circulation. 2002 Aug 13;106(7):773-5., 2002-08-13 [PMID:12176944]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is an inborn disorder of the connective tissue with specific skin, ocular, and cardiovascular disease (CVD) manifestations. Recently, we and others have identified mutations in the gene coding for the ABCC6 transporter in PXE patients with ocular and skin involvement. In the Netherlands, as in the rest of Europe, a particular premature truncation variant ABCC6 (R1141X) was found in a large cohort of PXE patients. Given the association between CVD and PXE, we hypothesized that heterozygosity of this ABCC6 mutation could also confer an increased risk for CVD. METHODS AND RESULTS: To assess the relationship between the frequent R1141X mutation in the ABCC6 gene and the prevalence of premature coronary artery disease (CAD), we conducted a case-control study of 441 patients under the age of 50 years who had definite CAD and 1057 age- and sex-matched population-based controls who were free of coronary disease. Strikingly, the prevalence of the R1141X mutation was 4.2 times higher among patients than among controls (3.2% versus 0.8%; P<0.001). Consequently, among subjects with the R1141X mutation, the odds ratio for a coronary event was 4.23 (95% CI: 1.76 to 10.20, P= 0.001). CONCLUSION: The presence of the R1141X mutation in the ABCC6 gene is associated with a sharply increased risk of premature CAD.

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0 Feskens and Arthur A.B. Bergen Boer, Jacoline B. ten Brink, Aeilko H. Zwinderman, John J.P. Kastelein, Edith J.M. Mieke D. Trip, Yvo M. Smulders, Jurgen J. Wegman, Xiaofeng Hu, Jolanda M.A. Increase in the Prevalence of Coronary Artery Disease Frequent Mutation in the ABCC6 Gene (R1141X) Is Associated With a Strong ISSN: 1524-4539 Copyright (c) 2002 American Heart Association. Login to comment
7 Fax: Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters http://circ.ahajournals.org//subscriptions/ Subscriptions: Information about subscribing to Circulation is online at at UNIV OF N CAROLINA CHAPEL HILL on August 8, 2011http://circ.ahajournals.org/Downloaded from Frequent Mutation in the ABCC6 Gene (R1141X) Is Associated With a Strong Increase in the Prevalence of Coronary Artery Disease Mieke D. Trip, MD; Yvo M. Smulders, MD, PhD; Jurgen J. Wegman, MD; Xiaofeng Hu, MD; Jolanda M.A. Boer, PhD; Jacoline B. ten Brink, Ing; Aeilko H. Zwinderman, PhD; John J.P. Kastelein, MD, PhD; Edith J.M. Feskens, PhD; Arthur A.B. Bergen, PhD Background-Pseudoxanthoma elasticum (PXE) is an inborn disorder of the connective tissue with specific skin, ocular, and cardiovascular disease (CVD) manifestations. Login to comment
9 In the Netherlands, as in the rest of Europe, a particular premature truncation variant ABCC6 (R1141X) was found in a large cohort of PXE patients. Login to comment
11 Methods and Results-To assess the relationship between the frequent R1141X mutation in the ABCC6 gene and the prevalence of premature coronary artery disease (CAD), we conducted a case-control study of 441 patients under the age of 50 years who had definite CAD and 1057 age-and sex-matched population-based controls who were free of coronary disease. Login to comment
12 Strikingly, the prevalence of the R1141X mutation was 4.2 times higher among patients than among controls (3.2% versus 0.8%; PϽ0.001). Login to comment
13 Consequently, among subjects with the R1141X mutation, the odds ratio for a coronary event was 4.23 (95% CI: 1.76 to 10.20, Pϭ0.001). Login to comment
14 Conclusion-The presence of the R1141X mutation in the ABCC6 gene is associated with a sharply increased risk of premature CAD. Login to comment
22 In parallel studies (Xiaofeng Hu, unpublished data), we found that the R1141X mutation is the most common mutation in Dutch PXE patients and families, and it seems as though this is the case for the rest of Europe as well. Login to comment
23 We therefore studied the prevalence of the R1141X mutation in the ABCC6 gene in patients with premature CAD and in a large population-based group of healthy controls to further delineate the role of this genetic variation as a risk factor for CAD. Login to comment
51 In cases, 14 of 441 (3.2%, 95% CI: 1.9 to 5.6) were carriers of the R1141X truncation variant, whereas 8 of 1057 controls (0.8%, 95% CI: 0.3 to 1.5) carried this ABCC6 mutation, yielding a statistically significant difference at a probability value Ͻ0.001 with an odds ratio corrected for age and sex of 4.23 (95% CI: 1.76 to 10.20). Login to comment
52 We subsequently categorized the premature CAD patients in carriers (nϭ14) and noncarriers (nϭ427) of the R1141X variant of the ABCC6 gene (Table 2). Login to comment
54 Discussion We demonstrate in a large case-control study that a strong association exists between a frequent mutation in the ABCC6 gene (R1141X) and the presence of premature CAD. Login to comment
64 Baseline Characteristics of Patients According to R1141X Genotype Variable Heterozygous nϭ14 Wild Type nϭ427 P Age, y 48.1Ϯ6.0 47.3Ϯ6.0 0.64 Male sex 10 (71) 322 (80) 0.43 Age at diagnosis 39.6Ϯ6.9 40.6Ϯ6.1 0.56 History of MI 11 (79) 312 (78) 0.93 Smoking 4 (29) 113 (28) 0.84 BMI, kg/m2 26.4Ϯ3.6 26.8Ϯ4.0 0.71 Hypertension 8 (57) 145 (36) 0.13 DM II 6 (43) 111 (27) 0.13 Family history CAD 9 (64) 229 (57) 0.33 Total cholesterol, mmol/L 5.96Ϯ1.36 5.81Ϯ1.60 0.73 HDL cholesterol, mmol/L 1.01Ϯ0.17 1.10Ϯ0.31 0.29 LDL cholesterol, mmol/L 3.96Ϯ1.27 3.82Ϯ1.53 0.74 Triglycerides, mmol/L 2.19Ϯ1.43 2.09Ϯ2.12 0.43 Values are meanϮSD or n (%). Login to comment

[hide] Mutation affecting arterial elasticity associated ... Lancet. 2002 Aug 10;360(9331):468. Bradbury J
Mutation affecting arterial elasticity associated with premature heart disease.
Lancet. 2002 Aug 10;360(9331):468., 2002-08-10 [PMID:12241726]

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8 To find out, Trip and co-workers examined DNA samples from 441 patients younger than 50 years referred to an outpatient atherosclerosis clinic and from 1057 healthy controls for the R1141X mutation, "the most common ABCC6 mutation associated with PXE in the Netherlands", explains Trip. Login to comment
9 3·2% of patients had this mutation compared with only 0·8% of healthy controls, giving an odds ratio for coronary events among people with the R1141X mutation of 4·23 (95% CI 1·76-10·20; Circulation; published online Aug 5, 2002; DOI: 10.1161/01.CIR.0000028420.27813 .C0). Login to comment
10 "We are now looking at other mutations associated with PXE in the same population", says Trip, "and plan to extend our work on the R1141X mutation to other populations". Login to comment

[hide] Evidence for a founder effect for pseudoxanthoma e... Hum Genet. 2002 Oct;111(4-5):331-8. Epub 2002 Sep 7. Le Saux O, Beck K, Sachsinger C, Treiber C, Goring HH, Curry K, Johnson EW, Bercovitch L, Marais AS, Terry SF, Viljoen DL, Boyd CD
Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa.
Hum Genet. 2002 Oct;111(4-5):331-8. Epub 2002 Sep 7., [PMID:12384774]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable elastic tissue disorder recently shown to be attributable to mutations in the ABCC6 ( MRP6) gene. Whereas PXE has been identified in all ethnic groups studied to date, the prevalence of this disease in various populations is uncertain, although often assumed to be similar. A notable exception however is the prevalence of PXE among South African Afrikaners. A previous report has suggested that a founder effect may explain the higher prevalence of PXE in Afrikaners, a European-derived population that first settled in South Africa in the 17th century. To investigate this hypothesis, we performed haplotype and mutational analysis of DNA from 24 South African families of Afrikaner, British and Indian descent. Among the 17 Afrikaner families studied, three common haplotypes and six different disease-causing variants were identified. Three of these mutant alleles were missense variants, two were nonsense mutations and one was a single base-pair insertion. The most common variant accounted for 53% of the PXE alleles, whereas other mutant alleles appeared at lower frequencies ranging from 3% to 12%. Haplotype analysis of the Afrikaner families showed that the three most frequent mutations were identical-by-descent, indicating a founder origin of PXE in this population.

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53 Two nonsense variants (Y768X and R1141X) and a single frameshift mutation (939insT) were also identified (Table 1). Login to comment
55 Two of the mutations found in the Afrikaner group, R1141X and 939insT, were also found in PXE individuals of British ancestries (Tables 1, 2). Login to comment
56 One of the latter individuals (from Family 212) carried the R1141X allele in association with 939insT. Login to comment
58 Moreover, two of the four R1141X alleles found in Afrikaner and British individuals occurred in similar haplotypes, also suggesting a possible consanguineous relationship between Family 206 (Afrikaner) and Family 216 (British ancestry). Login to comment
59 The remaining R1141X mutations, characterized in Family 224 (Afrikaner) and Family 212 (British ancestry) were found in different haplotypes indicating that 333 Table 1 ABCC6 mutations identified in a cohort of South African PXE patients of Afrikaner and other ancestries (nt nucleotide, aa amino acid, ni not identified, UK United Kingdom, Black black South Africans) Family Allele 1 Allele 2 Ancestry nt change aa change Exon Haplotype nt change aa change Exon Haplotype 201 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner 203 4015C→T R1339C 28 I 3413G→A R1138Q 24 III Afrikaner 205 3413G→A R1138Q 24 III 2304C→A Y768X 18 II Afrikaner 206 4015C→T R1339C 28 I 3421C→T R1141X 24 Other Afrikaner 208 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner 209 4015C→T R1339C 28 I 2018T→C L673P 16 Other Afrikaner 211 4015C→T R1339C 28 I 3413G→A R1138Q 24 III Afrikaner 222 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner 223 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner 225 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner 226 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner 228 4015C→T R1339C 28 I 2304C→A Y768X 18 II Afrikaner 229 4015C→T R1339C 28 I 4015C→T R1339C 28 I Afrikaner 213 939insT Frameshift 8 Other ni ni ni Other Afrikaner 214 4015C→T R1339C 28 I ni ni ni Other Afrikaner 224 3421C→T R1141X 24 Other ni ni ni Other Afrikaner 204 ni ni Other ni ni ni Other Afrikaner 212 3421C→T R1141X 24 Other 939insT Frameshift 8 Other UK 215 3775delT Frameshift 27 Other 4104delC Frameshift 29 Other UK 217 ABCC6del15 Frameshift 15 Other ABCC6del15 Frameshift 15 Other Indian 207 3088C→T R1030X 23 Other ni ni ni Other UK 216 3421C→T R1141X 24 Other ni ni ni Other UK 219 1553G→A R518Q 12 Other ni ni ni Other UK 221 ni ni Other ni ni ni Other Black Table 2 Frequencies of mutant ABCC6 alleles found in a cohort of PXE patients of Afrikaner and other ancestries (? Login to comment
60 unidentified alleles, MDR mutation detection rate) Mutation Overall Afrikaner ancestries Others ancestries Allele Allele Allele Allele Allele Allele count frequency (%) count frequency (%) count frequency (%) R1339C 18 37.5 18 52.9 0 0 Y768X 4 8.3 4 11.8 0 0 R1141X 4 8.3 2 5.9 2 14.3 R1138Q 3 6.3 3 8.8 0 0 939insT 2 4.2 1 2.9 1 7.1 ABCC6del15 2 4.2 0 0.0 2 14.3 L673P 1 2.1 1 2.9 0 0.0 R1030X 1 2.1 0 0.0 1 7.1 R518Q 1 2.1 0 0.0 1 7.1 3775delT 1 2.1 0 0.0 1 7.1 4104delC 1 2.1 0 0.0 1 7.1 ? Login to comment
83 All other mutated alleles, R1141X, L673P, 939insT and particularly the five unidentified mutations, were found in divergent haplotypes. Login to comment
84 The latter three alleles (R1141X, L673P and 939insT) could have been introduced by founder individuals or could represent de novo mutation events in the Afrikaner population. Login to comment

[hide] Anomalous structure of urinary glycosaminoglycans ... Clin Chem. 2003 Mar;49(3):380-8. Maccari F, Gheduzzi D, Volpi N
Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum.
Clin Chem. 2003 Mar;49(3):380-8., [PMID:12600949]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides. METHODS: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography. RESULTS: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio. CONCLUSIONS: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.

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154 Four of them were heterozygous for a stop codon mutation on one allele and a missense mutation on the other allele, one was heterozygous for a deletion on one allele and a nonsense mutation on the second allele, and five are still under investigation. The great majority of patients had the R1141X stop codon mutation in homozygosity or heterozygosity with another mutation. Login to comment

[hide] Arterial remodeling and stiffness in patients with... Arterioscler Thromb Vasc Biol. 2003 May 1;23(5):836-41. Epub 2003 Mar 20. Germain DP, Boutouyrie P, Laloux B, Laurent S
Arterial remodeling and stiffness in patients with pseudoxanthoma elasticum.
Arterioscler Thromb Vasc Biol. 2003 May 1;23(5):836-41. Epub 2003 Mar 20., 2003-05-01 [PMID:12649085]

Abstract [show]
OBJECTIVE: Proteoglycans organize the extracellular matrix, act as signaling molecules, and are involved in cell migration and proliferation. They may play an important role in arterial geometric and elastic properties. The aim of the present study was to determine large artery phenotype in patients with pseudoxanthoma elasticum (PXE), a genetic disease characterized by proteoglycan accumulation and fragmented elastic fibers in connective tissues. METHODS AND RESULTS: In 27 patients with PXE (25 females and 2 male) and 27 control subjects matched by age, sex, and blood pressure, we noninvasively determined the common carotid and radial artery diameter, intima-media thickness (IMT), and distensibility with high-definition echo-tracking systems and applanation tonometry. Patients with PXE had a significantly higher carotid IMT (611+/-106 versus 520+/-76 microm, P<0.001) independently of body surface area, age, and mean blood pressure. The increase in carotid IMT predominated in older patients with PXE at the time of examination. No significant difference in carotid elastic properties was observed between patients with PXE and control subjects. At the site of the radial artery, distensibility was significantly higher in patients with PXE than in control subjects (11.6+/-11.4 versus 5.9+/-3.4 kPa(-1) x 0.10(-3); P=0.02) and internal diameter was lower (1.66+/-0.51 versus 2.07+/-0.36 mm; P<0.01) without change in intima-media thickness and Young's elastic modulus. CONCLUSIONS: Phenotypic changes of superficial arteries in patients with PXE were represented by a thickening of the carotid artery and a reduced stiffness of the radial artery and predominated in older female patients.

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13 Vascular involvement is common, and patients with PXE typically present with arteriosclerosis, hypertension, transient ischemic attacks, and occlusive vascular changes at young ages.2 Cardiovascular complications, mainly coronary artery disease, are rare but can be life threatening.2 The estimated prevalence of PXE is 1 in 70 000 to 100 000.3 Although autosomal recessive inheritance (OMIM 264800) is most frequently found in PXE, autosomal dominant segregation (OMIM 177850) has also been proposed.4 The PXE locus has been mapped to chromosome 16p13.1, and mutations in the ABCC6 gene (previously known as MRP6 or eMOAT), encoding a 1503-amino acid putative membrane transporter of unknown function, have recently been simultaneously disclosed by 5 research groups as the genetic defect responsible for PXE.5-9 The existence of pseudogenes has been subsequently documented.10-13 The R1141X mutation in the ABCC6 gene is associated with a sharply increased risk of premature coronary artery disease.14 Histopathology of biopsies from clinically affected skin shows morphologically altered elastic fibers, which are fragmented and swollen and appear calcified when examined by special stains (eg, von Kossa), whereas PXE has also been characterized by proteoglycans accumulation, including heparan-sulfate and chondroitin-6-sulfate proteoglycans, glycosaminoglycan hyaluronic acid, decorin, and biglycan.15-17 Despite identification of the genetic defect, little is known about the pathogenesis of vascular lesions in PXE and the means for preventing arterial complications in young adults. Login to comment
29 Various mutations were identified, among which the nonsense R1141X was the most prevalent. Login to comment

[hide] ABCC6/MRP6 mutations: further insight into the mol... Eur J Hum Genet. 2003 Mar;11(3):215-24. Hu X, Plomp A, Wijnholds J, Ten Brink J, van Soest S, van den Born LI, Leys A, Peek R, de Jong PT, Bergen AA
ABCC6/MRP6 mutations: further insight into the molecular pathology of pseudoxanthoma elasticum.
Eur J Hum Genet. 2003 Mar;11(3):215-24., [PMID:12673275]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a hereditary disease characterized by progressive dystrophic mineralization of the elastic fibres. PXE patients frequently present with skin lesions and visual acuity loss. Recently, we and others showed that PXE is caused by mutations in the ABCC6/MRP6 gene. However, the molecular pathology of PXE is complicated by yet unknown factors causing the variable clinical expression of the disease. In addition, the presence of ABCC6/MRP6 pseudogenes and multiple ABCC6/MRP6-associated deletions complicate interpretation of molecular genetic studies. In this study, we present the mutation spectrum of ABCC6/MRP6 in 59 PXE patients from the Netherlands. We detected 17 different mutations in 65 alleles. The majority of mutations occurred in the NBF1 (nucleotide binding fold) domain, in the eighth cytoplasmatic loop between the 15th and 16th transmembrane regions, and in NBF2 of the predicted ABCC6/MRP6 protein. The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. The second most frequent mutation, an intragenic deletion from exon 23 to exon 29 in ABCC6/MRP6, was detected in 11 (18.6%) of the patients. Our data include 11 novel ABCC6/MRP6 mutations, as well as additional segregation data relevant to the molecular pathology of PXE in a limited number of patients and families. The consequences of our data for the molecular pathology of PXE are discussed.

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8 The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. Login to comment
30 of patients Allele 1 Consequence Exon Allele 2 Consequence Exon Mode of inheritance in family 1 2247C>T Q749X 17 s 1 3421C>T R1141X 24 2247C>T Q749X 17 ar 9 3421C>T R1141X 24 ar,s, n 1 3421C>T R1141X 24 1944del22 Frameshift 16 n 3 3421C>T R1141X 24 Deletion A995del405 23-29 ar 1 3421C>T R1141X 24 4182delG Frameshift 29 ar 1 3421C>T R1141X 24 3775delT Frameshift 27 s 3 3421C>T R1141X 24 3421C>T R1141X 24 ar, s 1 2294G>A R765Q 18 3775delT Frameshift 27 ar 1 3341G>A R1114H 24 n 1 3390C>T T1130M 24 3390C>T T1130M 24 ar 1 3663C>T R1221C 26 3775delT Frameshift 27 n 1 3904G>C G1302R 28 s 1 3907G>A A1303P 28 Deletion A995del405 23-29 ar 1 4182G>T K1394N 29 Deletion A995del405 23-29 ar 1 4182delG Frameshift 29 n 1 4182delG Frameshift 29 4182delG Frameshift 29 ar 1 4377C>T R1459C 30 ad?, s,n 2 3775delT Frameshift 27 s,n 1 3775delT Frameshift 27 Deletion all? Login to comment
38 Table 2 Summary of ABCC6/MRP6 mutations associated with PXE known today: our data combined with those of the literature Mutation Protein alteration Nucleotide substitution Location Reference Nonsense Q378X 1132C > T Exon 9 19,20 R518X 1552C > T Exon 2 41 Q749X 2247C > T Exon 17 This study Y768X 2304C > A Exon 18 22 R1030X 3088C > T Exon 23 22 R1141X 3421C > T Exon 24 12,20,22,38,39, this study R1164X 3490C > T Exon 24 12,41 Q1237X 3709C > T Exon 26 22 R1398X 4192C >T Exon 29 22 T364R Missense N411K 1091C > G Exon 9 20 A455P 1233T > G Exon 10 22 R518Q 1363G > C Exon 11 38 F568S 1553G > A Exon 12 22,38 L673P 1703T > C Exon 13 22 R765Q 2018T > C Exon 16 22 R1114P 2294G > A Exon 18 22, this study R1114H 3341G > C Exon 24 22 S1121W 3341G > A Exon 24 This study T1130M 3362C > G Exon 24 22 R1138W 3390C > T Exon 24 This study R1138Q 3412C > T Exon 24 12 R1138P 3413G > A Exon 24 12,22 G1203D 3413G > C Exon 24 22 R1221C 3608G > A Exon 25 22 V1298F 3663C > T Exon 26 This study T1301I 3892G > T Exon 28 22 G1302R 3902C > T Exon 28 22 A1303P 3904G > A Exon 28 22, this study R1314W 3907G > C Exon 28 22, this study R1314Q 3940C > T Exon 28 22 G1321S 3941G > A Exon 28 22 R1339C 3961G > A Exon 28 22 Q1347H 4015C > T Exon 28 22,39 G1354R 4041G > C Exon 28 22 D1361N 4060G > C Exon 29 20,38 K1394N 4081G > A Exon 29 22 I1424T 4182G > T Exon 29 This study R1459C 4271T > C Exon 30 22 4377C > T Exon 30 This study Frameshift IVS17-12delT T Intron 17 This study IVS21+1G>T Intron 21 22,38 IVS26-1G>A Intron 26 12,21,22 179del 9 Exon 2 20 179-195del Exon 2 22 960del C Exon 8 41 1944del22 Exon 16 This study 1995delG Exon 16 22 2322delC Exon 18 22 2542delG Exon 19 41 3775delT Exon 27 This study 4104delC Exon 29 22 4182delG Exon 29 This study 938-939insT Exon 8 22 4220insAGAA Exon 30 This study Large deletion Exons 23-29 21, This study Exon 15 22 ABCC1, ABCC6 41, this study Mutation types The mutation types found in this study are summarized in Table 1. Login to comment
39 We observed two distinct nonsense mutations, R1141X and Q749X in 24 out of 117 alleles (20.5%). Login to comment
40 R1141X occurred in 22/117 alleles (18.8%) and was found in a homozygous, heterozygous, or compound heterozygous form in 19 patients (32.2%). Login to comment
52 The latter deletion was found on 13 alleles (11.1%) of 11 patients (18.6%) and was the second most frequent mutation in this study after R1141X. Login to comment
69 The R1141X mutation, the most common PXE mutation found in our cohort, is located in this domain. Login to comment
76 Molecular analysis revealed that she was homozygous for the ABCC6/MRP6 R1141X mutation. Login to comment
95 The family consisted of an affected mother, a healthy father, three severely affected, two mildly affected, Table 3 Clinical characteristics of patients from the pedigrees described in Figure 2 Genotype Pedigree Family member Age Age of onset Skin Biopsy Eyes Cardiovascular Allele 1 Allele 2 26101 II-1 44 12 + d AS ht,TIA R1141X R1141X I-1 69 n d n MI R1141X WT I-2 69 n d n Chest pain R1141X WT 26098 II-1 46 22 + d AS,MD GI hemorrhage delABCC6 del exon 23-29 II-2 40 n d AS n delABCC6 del exon 23-29 I-1 71 n 7 Drusen Multiple CI delABCC6 WT I-2 73 d d d MI del exon 23-29 WT 26095 I-3 83 52 + + AS,MD ht d II-1 66 n d n n WT WT II-2 63 48 + d MD n R1459C WT II-3 61 61 7 7 AS n R1459C WT II-4 59 n n AS,PdO n R1459C WT II-5 57 55 + d AS,neo,PdO n R1459C WT II-6 56 n d n n WT WT II-7 54 49 + d AS,neo n R1459C WT II-8 52 n d n n WT WT AS = angioid streaks; CI = cerebral infarct; GI = gastrointestinal; ht = hypertension; MD = macula degeneration; MI = myocardial infarct; n = normal; neo = neovascularization; PdO = peau d`orange; RD = retinal detachment; TIA = transient ischaemic attack; WT = wild type; + = affected; 7 = possibly affected; d = not tested. Login to comment
99 The R1141X mutant allele leads to loss of a BsiYI restriction site; the mutated allele is therefore not cut and presents with a 500 bp band. Login to comment
128 Further alignment showed that the R765Q mutation in ABCC6/MRP6 is the positional equivalent of both the R560T mutation in ABCC7,28 and the R842G mutation in ABCC8.29 Similarly, additional possible positional equivalent clusters of conserved and mutated residues were found between ABCC6/ MRP6 and ABCC2 (R1114H and R1150H),30 ABCC6/MRP6 and ABCC7 (3775 del T and W1204X),31 ABCC6/MRP6 and ABCR (R1459C and H2128R, 4220InsAGAA and R2077W, R1141X and L1631P).32,33 Interestingly, for both ABCC7 and ABCR, models were postulated in which the severity of the disease shows an inverse correlation with the predicted transport activity of the ABC protein. Login to comment
165 The ABCC6/MRP6-specific RT-PCR primers used to analyse the mutations indicated were as follows: (R1141X): ABCC6/MRP6F, 50 -CTGTCTCCAAGCCATTGGGC- 30 (cDNA position 3008-3027) and ABCC6/MRP6R, 50 - AGCCACCAGTCGCGGGAAAC-30 (cDNA position 3524- 3505); (deletion exon 23-29): ABCC6/MRP6F3, 50 - ATACGGCAGGGTGAAGGCCA-30 (cDNA position 2801- 2820) and ABCC6/MRP6R3, 50 -CAGTGCACTGTGCAAAC CAGC-30 (cDNA position 4380-4360); (R1459C): ABCC6/ MRP6F4, 50 -CTGGCTCTCTGCGGATGAAC-30 (cDNA position 4081-4100); ABCC6/MRP6R4,50 -AGAACCCGGGCA CAGTCCAT-30 (cDNA position 4432-4413). Login to comment

[hide] Analysis of the frequent R1141X mutation in the AB... Invest Ophthalmol Vis Sci. 2003 May;44(5):1824-9. Hu X, Peek R, Plomp A, ten Brink J, Scheffer G, van Soest S, Leys A, de Jong PT, Bergen AA
Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum.
Invest Ophthalmol Vis Sci. 2003 May;44(5):1824-9., [PMID:12714611]

Abstract [show]
PURPOSE: To characterize the ABCC6 R1141X nonsense mutation, which is implicated in more than 25% of a cohort of patients from The Netherlands with pseudoxanthoma elasticum (PXE). METHODS: A combination of single-strand conformational polymorphism (SSCP), PCR, sequencing, and Southern blot analysis was used to identify mutations in the ABCC6 gene in 62 patients. Haplotypes of 16 patients with the R1141X mutation were determined with eight polymorphic markers spanning the ABCC6 locus. The effect of the R1141X mutation on the expression of ABCC6 was studied in leukocytes and cultured dermal fibroblasts from affected skin in patients heterozygous or homozygous for the R1141X mutation. ABCC6 expression was analyzed by RT-PCR and immunocytochemistry with ABCC6-specific monoclonal antibodies. RESULTS: The ABCC6 R1141X mutation was found on 19 alleles in 16 patients with PXE and occurred in heterozygous, homozygous, or compound heterozygous form. All R1141X alleles were associated with a common haplotype, covering at least three intragenic ABCC6 markers. None of the patients or healthy control subjects had a similar ABCC6 haplotype. Furthermore, the results showed that the expression of the normal allele in R1141X heterozygotes was predominant, whereas no detectable, or very low, ABCC6 mRNA levels were found in R1141X homozygotes. Immunocytochemical staining of cultured dermal fibroblasts with ABCC6-specific monoclonal antibodies showed no evidence of the presence of a truncated protein in patients with PXE who were homozygous for R1141X. CONCLUSIONS: A specific founder effect for the R1141X mutation exists in Dutch patients with PXE. The R1141X mutation induces instability of the aberrant mRNA. Functional haploinsufficiency or loss of function of ABCC6 caused by mechanisms, such as nonsense-mediated decay (NMD), may be involved in the PXE phenotype.

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0 Analysis of the Frequent R1141X Mutation in the ABCC6 Gene in Pseudoxanthoma Elasticum Xiaofeng Hu,1 Ron Peek,1 Astrid Plomp,1,2 Jacoline ten Brink,1 George Scheffer,3 Simone van Soest,1 Anita Leys,4 Paulus T. V. M. de Jong,1,5,6 and Arthur A. B. Bergen1,2 PURPOSE. Login to comment
1 To characterize the ABCC6 R1141X nonsense mutation, which is implicated in more than 25% of a cohort of patients from The Netherlands with pseudoxanthoma elasticum (PXE). Login to comment
4 Haplotypes of 16 patients with the R1141X mutation were determined with eight polymorphic markers spanning the ABCC6 locus. Login to comment
5 The effect of the R1141X mutation on the expression of ABCC6 was studied in leukocytes and cultured dermal fibroblasts from affected skin in patients heterozygous or homozygous for the R1141X mutation. Login to comment
8 The ABCC6 R1141X mutation was found on 19 alleles in 16 patients with PXE and occurred in heterozygous, homozygous, or compound heterozygous form. Login to comment
9 All R1141X alleles were associated with a common haplotype, covering at least three intragenic ABCC6 markers. Login to comment
11 Furthermore, the results showed that the expression of the normal allele in R1141X heterozygotes was predominant, whereas no detectable, or very low, ABCC6 mRNA levels were found in R1141X homozygotes. Login to comment
12 Immunocytochemical staining of cultured dermal fibroblasts with ABCC6-specific monoclonal antibodies showed no evidence of the presence of a truncated protein in patients with PXE who were homozygous for R1141X. Login to comment
14 A specific founder effect for the R1141X mutation exists in Dutch patients with PXE. Login to comment
15 The R1141X mutation induces instability of the aberrant mRNA. Login to comment
42 In this study, the recurrence of the R1141X mutation in 16 patients with PXE and the level of expression of ABCC6 mRNA with the R1141X mutation in (PXE) leukocytes and (PXE) fibroblasts were analyzed. Login to comment
43 In addition, the ABCC6 protein in cultured dermal fibroblasts from a patient homozygous for R1141X was studied with ABCC6-specific monoclonal antibodies. Login to comment
46 Sixteen Dutch families and patients with the R1141X mutation were included in the study. Login to comment
65 Restriction Analysis of the R1141X Mutation and Polymorphisms After identification of the R1141X mutation and detection of other intragenic polymorphisms, optimized protocols were designed by using PCR and restriction analyses. Login to comment
66 The R1141X mutation leads to the loss of a BsiYI restriction site, which was confirmed by a restriction fragment length polymorphism assay. Login to comment
74 We constructed (phase-known) haplotypes of all informative alleles from patients with the R1141X mutation. Login to comment
83 The presence or absence of the R1141X mutation in individual clones was checked with digestion with BsiYI, and, accordingly, the inserts of individual clones were assigned to be expression products from the mutant or wild-type allele. Login to comment
87 The monoclonal antibod- ies for immunocytochemical staining were M6II-7, MRPr1, and M5I-1 reactive to ABCC6, ABCC1, and ABCC5, respectively.11,12,17 RESULTS R1141X Mutation Analysis Mutation analysis of the ABCC6 gene resulted in the identification of the R1141X mutation, which accounted for up to 30% of all mutations detected in the ABCC6 gene in our cohort of patients with PXE. Login to comment
88 The R1141X mutation was caused by a C3T substitution at nucleotide 3421 in the putative eighth intracellular loop. Login to comment
90 Previously, we found that the R1141X mutation was associated with a strong increase in the prevalence of coronary artery disease.18 In our PXE cohort, R1141X was found in 19 of 29 nonde- letion alleles in DNA of 16 patients with PXE. Login to comment
92 In three patients, the R1141X mutation was observed in a homozygous form; in seven, it was in heterozygous form; and in six, it occurred in combination with other mutations in the second allele in compound heterozygous form. Login to comment
96 In the three other compound heterozygotes, the combination R1141X with a del22 bp in exon 16, a 3375delT, or Q749X was identified. Login to comment
99 The last patient, P26240, inherited the R1141X mutation and another nonsense mutation, Q749X. Login to comment
101 Founder Effect for the R1141X Mutation To determine whether the R1141X mutation originates from recurrent de novo mutational events or from founder effects, ABCC6-associated haplotypes of all patients carrying the R1141X mutation and of control subjects were constructed. Login to comment
112 The minimum common haplotype shared by all 19 alleles with the R1141X mutation was represented by the haplotype G(3803G3A)-G(2490C3G)-A(1896C3A) (Table 2). Login to comment
117 Genotype and Clinical Features of 16 Patients with the R1141X Mutation Pedigree Allele 1 Allele 2 Skin Eyes Cardiovascular 25494 R1141X Delex23-29 ϩ AS, MD HT 26026 R1141X Delex23-29 ϩ, bϩ D D 26241 R1141X Delex23-29 ϩ, bϩ PdO, AS N 26007 R1141X 1944del22 ϩ RPE changes N 26240 R1141X Q749X ϩ, bϩ AS MVP 26273 R1141X 3775delT D AS, neo D 26091 R1141X R1141X ϩ, bϩ AS GI hemorrhage 26101 R1141X R1141X ϩ AS TVI 26107 R1141X R1141X D Neo D 26093 R1141X WT ϩ AS N 26123 R1141X WT ϩ, bϩ PdO, comet N 24694 R1141X WT ϩ AS, MD CI 25908 R1141X WT ϩ AS D 26109 R1141X WT ϩ AS, neo D 26215 R1141X WT ϩ ϩ D 26242 R1141X WT ϩ, bϩ PdO MVP WT, wild type; ϩ, affected; bϩ, biopsy specimen obtained and histologic PXE changes determined after von Kossa staining; D, declined and/or no data available; AS, angioid streaks, MD, macula degeneration, PdO, peau d`orange, RPE involvement; neo, neovascularisation; comet, presence of comets; HT, hypertension; N, normal, MVP, mitral valve prolaps; GI, gastrointestinal; TVI, tricuspid valve insufficiency; CI, cerebral infarct. Login to comment
118 Effect of R1141X on ABCC6 Expression in Dermal Fibroblasts The ABCC6 mRNA with the R1141X mutation encodes a C-terminally truncated ABCC6 protein that lacks part of one of the transmembrane domains and one of the ATP-binding cassette domains. Login to comment
119 To determine the effect of this mutation on the expression of the ABCC6 gene, we analyzed dermal fibroblasts from individuals homozygous or heterozygous for R1141X and healthy control subjects. Login to comment
120 The presence of the mutations was confirmed by PCR amplification of exon 24 containing the R1141X mutation followed by restriction fragment length polymorphism analysis (Fig. 2) and direct sequencing (not shown). Login to comment
121 Next, we analyzed the amount of mRNA from alleles with the R1141X mutation. Login to comment
122 RT-PCR analyses of cultured PXE fibroblasts with the R1141X in homozygous form did not contain detectable ABCC6 mRNA. Login to comment
123 Fibroblasts from those heterozygous for the R1141X mutation appeared to have a reduced level of ABCC6 mRNA compared with the healthy control subjects (Fig. 3). Login to comment
124 The latter result indicates that the R1141X mutation affects the abundance of the mutant mRNA. Login to comment
125 To examine this in more detail, we determined the ratio of steady state transcript levels between wild-type and mutated mRNA in mononuclear blood cells and fibroblasts of patients heterozygous for the R1141X mutation. Login to comment
127 In fibroblasts of patients heterozygous for the R1141X mutation, no mRNA containing the mutation was found, whereas in blood cells, only 5% of the ABCC6 mRNA was from the mutated allele (Table 3). Login to comment
129 In parallel experiments, we determined the expression of the R1141X truncated protein in cells of patients with PXE. Login to comment
130 Cultured dermal fibroblasts of a patient with PXE homozygous for the R1141X mutation and that of a healthy control subject were analyzed by immunocytochemistry. Login to comment
134 Haplotype of the R1141X Mutation Alleles Pedigree Marker D16S 3060 972AA AG1 962 CA2 3803 G3A *3421* C3T 2490 C3G 1896 C3A CA (18) D16S 764 25908 7 3 4 G T G A 1 3 26273 5 3 4 G T G A 1 3 25494 2 3 4 G T G A 1 3 26240 3 2 4 G T G A 1 3 24694 1 2 4 G T G A 1 3 26109 2 4 G T G A 1 3 26241 4 2 4 G T G A 1 3 26101 2 3 4 G T G A 1 2 26101 5 3 4 G T G A 5 1 26026 4 3 4 G T G A 1 1 26091 4 3 4 G T G A 1 1 26091 4 12 4 G T G A 1 2 26093 2 2 4 G T G A 1 1 26007 4 12 4 G T G A 1 2 26107 2 13 4 G T G A 3 2 26107 2 13 4 G T G A 3 3 26123 2 2 4 G T G A 5 4 26215 1 2 3 G T G A 4 3 26242 2 3 2 G T G A 4 2 Distinct alleles are indicated by number. Login to comment
135 Identical haplotypes for the R1141X mutation are shaded. Login to comment
137 Underline shows homozygosity for the R1141X mutation in the proband(s). Login to comment
148 The presence of the R1141X mutation was confirmed by digestion with BsiYI of PCR products containing exon 24. Login to comment
151 The R1141X mutant allele leads to the loss of the BsiYI restriction site and produces a single fragment of 100 bp. Login to comment
153 These results suggest that the R1141X mutation in ABCC6 does not lead to detectable amounts of truncated protein. Login to comment
154 Phenotypes of Patients with the R1141X Mutation A summary of clinical data available from the 16 patients with PXE with R1141X mutations is shown in Table 1. Login to comment
159 In summary, in all patients homozygous or compound heterozygous for the R1141X mutation, we observed ocular and skin abnormalities and, less frequently, cardiovascular problems. Login to comment
160 However, because the expression of the disease in these tissues is highly variable among our patients, we could not correlate a distinct phenotype with the R1141X mutation. Login to comment
161 DISCUSSION R1141X Mutation Analysis We detected the R1141X mutation in homozygous, heterozygous, and compound heterozygous forms. Login to comment
162 In nine patients the R1141X mutation was present in a homozygous form or a compound heterozygous form. Login to comment
164 In seven patients, we detected R1141X in heterozygous form. Login to comment
166 However, despite extensive screening, we have not yet found another mutation or deletion in the second, non-R1141X, ABCC6 allele. Login to comment
168 Consequently, the pathologic molecular aspect of the R1141X mutation is most likely compatible with the frequently occurring autosomal recessive inheritance in PXE. Login to comment
169 Nonetheless, potential mild expression of the disease in carriers of the R1141X mutation warrants further investigation. Login to comment
170 Founder Effect for the R1141X Mutation Mutation analysis of the ABCC6 gene in patients with PXE has yielded 57 different ABCC6 mutations to date.15 The R1141X mutation was reported to be the most common mutation by us and others, especially in European patients.14,15 Recently, we also found that R1141X may be associated with a strong increase in the prevalence of coronary artery disease.18 The association between its high frequency and the geographical distribution could reflect a founder effect from a common ancestor. Login to comment
171 To test this hypothesis, we analyzed the R1141X mutation in more detail in this study. Login to comment
172 The majority of our R1141X mutant alleles (17/19) shared a common haplotype spanning at least one ABCC6 flanking marker. Login to comment
179 Top: RT-PCR analysis of ABCC6 expression in cultured dermal fibroblasts from patients heterozygous or homozygous for the R1141X mutation. Login to comment
183 Staining with MRP1 and MRP6 monoclonal antibodies of a monolayer of dermal fibroblasts from a healthy individual and a patient with PXE heterozygous for the R1141X mutation. Login to comment
185 The Expression Ratio of ABCC6 Wild-Type and Mutated mRNA Genotype Tissue WT Allele (%) Mutant Allele (%) WT/WT Blood 20/20 (100) No R1141X/WT Blood 38/40 (95) 2/40 (5) R1459C/WT Blood 52/100 (52) 48/100 (48) The number of PXE heterozygotes carrying an ABCC6 R1141X (R1141/X) or R1459C (R1459C/WT) mutation and a healthy control subject with wild-type ABCC6 (WT/WT). Login to comment
187 Predominant Expression of the Normal ABCC6 Allele in Patients with PXE Heterozygous for the R1141X Mutation For several mammalian mRNAs, it has been shown that a nonsense mutation or a frameshift mutation that generates a nonsense codon may greatly influence the abundance of these transcripts. Login to comment
188 A specific mechanism called nonsense-mediated mRNA decay (NMD) accelerates decay of transcripts coding for truncated proteins and thus minimizes potential metabolic damage.19,20 We found no detectable ABCC6 mRNA in patients with PXE who were homozygous for the R1141X mutation. Login to comment
189 Consistent with this observation, no ABCC6 protein was detected in cultured dermal fibroblasts of a patient homozygous for R1141X. Login to comment
190 Using a more quantitative approach, we found that in cultured dermal fibroblasts of a R1141X heterozygote, only transcripts from the wild-type allele were detected. Login to comment
191 In mononuclear blood cells of a R1141X heterozygote the mutated transcript was detected, but the abundance was reduced to 5% of total ABCC6 mRNA. Login to comment
192 Our results suggest that the R1141X mutation induces instability of the aberrant ABCC6 mRNA, which leads to a reduced abundance of the corresponding transcript due to alterations in RNA processing by NMD. Login to comment
197 Features of the Phenotype The clinical variability in PXE was demonstrated previously2,24 and also occurred in our R1141X patient cohort. Login to comment
203 CONCLUSION In summary, this study presents evidence that the frequent occurrence of the ABCC6 R1141X mutation in Dutch patients with PXE was due to a founder effect. Login to comment
204 The PXE phenotype of the R1141X mutation is most likely due to a complete loss of function or functional haploinsufficiency of the ABCC6 gene. Login to comment
205 No clear correlation between the R1141X genotype and phenotype could be established in the cohort studied. Login to comment

[hide] Pseudoxanthoma elasticum: a clinical, histopatholo... Surv Ophthalmol. 2003 Jul-Aug;48(4):424-38. Hu X, Plomp AS, van Soest S, Wijnholds J, de Jong PT, Bergen AA
Pseudoxanthoma elasticum: a clinical, histopathological, and molecular update.
Surv Ophthalmol. 2003 Jul-Aug;48(4):424-38., [PMID:12850230]

Abstract [show]
Pseudoxanthoma elasticum is an autosomally inherited disorder that is associated with the accumulation of mineralized and fragmented elastic fibers in the skin, Bruch's membrane in the retina, and vessel walls. The ophthalmic and dermatologic expression of pseudoxanthoma elasticum and its vascular complications are heterogeneous, with considerable variation in phenotype, progression, and mode of inheritance. Using linkage analysis and mutation detection techniques, mutations in the ABCC6 gene were recently implicated in the etiology of pseudoxanthoma elasticum. ABCC6 encodes the sixth member of the ATP-binding cassette transporter and multidrug resistance protein family (MRP6). In humans, this transmembrane protein is highly expressed in the liver and kidney. Lower expression was found in tissues affected by pseudoxanthoma elasticum, including skin, retina, and vessel walls. So far, the substrates transported by the ABCC6 protein and its physiological role in the etiology of pseudoxanthoma elasticum are not known. A functional transport study of rat MRP6 suggests that small peptides such as the endothelin receptor antagonist BQ123 are transported by MRP6. Similar molecules transported by ABCC6 in humans may be essential for extracellular matrix deposition or turnover of connective tissue at specific sites in the body. One of these sites is Bruch's membrane. This review is an update on etiology of pseudoxanthoma elasticum, including its clinical and genetic features, pathogenesis, and biomolecular basis.

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179 The R1141X mutation in exon 24 was reported by four research groups and appears to be a common mutation underlying PXE. Login to comment
180 R1141X generates a stop codon at cDNA position 3421 and presumably results in a reduction in mutant mRNA levels by nonsense-mediated RNA decay.25,69 Mutations appear to be also frequent in exons 28-30, corresponding to the NBF2 region, where the function of the protein might be abolished by the change in a single amino acid. Login to comment
193 TABLE 3 Summary of ABCC6 Mutations in PXE Patients Mutation Protein Alteration Nucleotide Substitution Location Reference Nonsense Q378X 1132C Ͼ T Exon 9 16,107 R518X 1552C Ͼ T Exon 12 88 Y768X 2304C Ͼ A Exon 18 67 R1030X 3088C Ͼ T Exon 23 67 R1141X 3421C Ͼ T Exon 24 12,45,67,107,111,112,133 R1164X 3490C Ͼ T Exon 24 88,112 Q1237X 3709C Ͼ T Exon 26 67 R1398X 4192C Ͼ T Exon 29 67 Missense T364R 1091C Ͼ G Exon 9 107 N411K 1233T Ͼ G Exon 10 67 A455P 1363G Ͼ C Exon 11 142 R518Q 1553G Ͼ A Exon 12 67,142 F568S 1703T Ͼ C Exon 13 67 L673P 2018T Ͼ C Exon 16 67 R765Q 2294G Ͼ A Exon 18 67 R1114P 3341G Ͼ C Exon 24 67 S1121W 3362C Ͼ G Exon 24 67 R1138W 3412C Ͼ T Exon 24 111 R1138Q 3413G Ͼ A Exon 24 67,111 R1138P 3413G Ͼ C Exon 24 67 G1203D 3608G Ͼ A Exon 25 67 V1298F 3892G Ͼ T Exon 28 67 T13011 3902C Ͼ T Exon 28 67 G1302R 3904G Ͼ A Exon 28 67 A1303P 3907G Ͼ C Exon 28 67 R1314W 3940C Ͼ T Exon 28 67 R1314Q 3941G Ͼ A Exon 28 67 G1321S 3961G Ͼ A Exon 28 67 R1339C 4015C Ͼ T Exon 28 67,133 Q1347H 4041G Ͼ C Exon 28 67 G1354R 4060G Ͼ C Exon 29 107,142 D1361N 4081G Ͼ A Exon 29 67 11424T 4271T Ͼ C Exon 30 67 Frameshift Splicing IVS21 ϩ 1G ϾT Intron 21 67,142 IVS26-1G ϾA Intron 26 67,111,112 Deletion 179del9 Exon 2 107 179-195del Exon 2 67 960delC Exon 8 88 1944del22 Exon 16 12 1995delG Exon 16 67 2322delC Exon 18 67 2542delG Exon 19 67 3775delT Exon 27 12,67 4101delC Exon 29 67 Insertion 938-939insT Exon 8 67 4220insAGAA Exon 30 12 Intragenic deletion Exon 23-29 67,112 Exon 15 67 Intergenic deletion ABCC6 12,88 LOCAL RETINAL TRANSPORT FUNCTION OF ABCC6 ABCC6 Expression in the Retina Bergen et al detected ABCC6 expression in various tissues in man.12 Low expression levels of ABCC6 were observed in the retina as well as other tissues usually affected by PXE, including skin and vessel wall. Login to comment

[hide] Subcellular localization and N-glycosylation of hu... Biochem Biophys Res Commun. 2003 Aug 22;308(2):263-9. Sinko E, Ilias A, Ujhelly O, Homolya L, Scheffer GL, Bergen AA, Sarkadi B, Varadi A
Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells.
Biochem Biophys Res Commun. 2003 Aug 22;308(2):263-9., 2003-08-22 [PMID:12901863]

Abstract [show]
Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the basolateral membrane. The human ABCC6 in MDCKII cells was found to be glycosylated, in contrast to the underglycosylated form of the protein, as expressed in Sf9 cells. In order to localize the major glycosylation site(s) in ABCC6, we applied limited proteolysis on the fully glycosylated and underglycosylated forms, followed by immunodetection with region-specific antibodies for ABCC6. Our results indicate that Asn15, which is located in the extracellular N-terminal region of human ABCC6, is the only N-glycosylation site in this protein. The polarized mammalian expression system characterized here provides a useful tool for further examination of routing, glycosylation, and function of the normal and pathological variants of human ABCC6.

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11 Furthermore, it was recently found that the presence of the most common mutation (R1141X) in the ABCC6 gene is associated with an increased risk of premature coronary artery disease [10]. Login to comment

[hide] Heterozygous carriers of Pseudoxanthoma elasticum ... J Neurol. 2003 Aug;250(8):983-6. Morcher M, Hausser I, Brandt T, Grond-Ginsbach C
Heterozygous carriers of Pseudoxanthoma elasticum were not found among patients with cervical artery dissections.
J Neurol. 2003 Aug;250(8):983-6., [PMID:12928920]

Abstract [show]
In this study of patients with spontaneous cervical artery dissections (sCAD) we searched for mutations in ABCC6, the candidate gene for Pseudoxanthoma elasticum (PXE). Genomic DNA samples from 12 sCAD patients with pronounced electron microscopic alterations in their dermal connective tissue and from 2 patients with PXE were analysed. One patient with PXE was compound heterozygous for two missense point mutations, in the second patient with PXE we did not find changes in the ABCC6 gene. We observed several missense mutations (H623Q, R3190W and R1268Q) in the patients with sCAD, but these mutations were not disease specific,since they were also detected in a series of 25 healthy control subjects.The finding of several sequence variants in sCAD patients and of disease causing mutations in one of the PXE patients suggests that our strategy of mutation search is reliable. Since we did not find disease causing mutations in our series of patients with sCAD we suggest that ABCC6 is not a candidate gene for sCAD.

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45 However, the R1141X muTable ABCC6sequencevariationin12patientswithsCADand2patientswithPXE exon nucleotide Amino-acid Occurrence Occurrence position change among patients among controls 10 1233T¡C synonymous sCAD not tested 10 1245G¡A synonymous sCAD not tested 15 1890C¡G synonymous sCAD not tested 15 1896C¡A H623Q sCAD + 19 2490C¡T synonymous sCAD not tested 20 2631C¡A synonymous sCAD not tested 22 2835C¡T synonymous sCAD not tested 22 2836C¡A L946I PXE - 23 3190C¡T R1064W sCAD + 24 3389C¡T T1130M PXE - 27 3803G¡A R1268Q sCAD + tation is common in European patients, whereas a large deletion of exons 23-26 is repeatedly found in patients from the United States [22]. Login to comment

[hide] Assessment of a rapid-cycle PCR assay for the iden... Lab Invest. 2004 Jan;84(1):122-30. Gotting C, Schulz V, Hendig D, Grundt A, Dreier J, Szliska C, Brinkmann T, Kleesiek K
Assessment of a rapid-cycle PCR assay for the identification of the recurrent c.3421C>T mutation in the ABCC6 gene in pseudoxanthoma elasticum patients.
Lab Invest. 2004 Jan;84(1):122-30., [PMID:14631379]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. Mutations in the ABCC6 gene could be linked to this disease and, just recently, the c.3421C>T mutation was also associated with a high risk of coronary artery disease. We have now developed new real-time PCR assays for the accurate and rapid determination of the c.3421C>T genotype. Using our new assay, we analyzed the presence of the c.3421C>T mutation in the largest collection of DNA samples from unrelated German PXE patients (n=64) and in a control cohort (n=910). For assay setup, two sets of samples with known genotype for the c.3421C>T mutation were analyzed over a period of 14 days. Results were confirmed by restriction endonuclease mapping, sequence-specific PCR and DNA sequencing. In order to ensure that no further mutations or deletions interfered with the c.3421C>T genotyping, we scanned the exon 24 of the ABCC6 gene by DHPLC and investigated the presence of the ABCC6del23-29 deletion in all patients. The assay has been set up on a group of patients with known genotype and validated on 64 PXE patients. In this group four PXE patients (6.3%) were found to be homozygous and 25 (39.0%) to be heterozygous carriers of the c.3421C>T mutation. The common ABCC6del23-29 deletion, possibly interfering with genotype determination, was searched and excluded. Furthermore, two novel mutations in the ABCC6 gene could be identified in two patients. The novel mutations c.3389C>T and c.3341G>A did not interfere with our new assay. Our new c.3421C>T genotyping assays can be used for the rapid identification of this frequent mutation in PXE patients and of the recently newly proposed cardiac risk factor in young patients with myocardial infarcts of unknown origin.

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23 The first mutations in the ABCC6 gene were identified in the genomic DNA isolated from PXE patients, and comparative family analysis could confirm most of these mutations to be associated with the PXE phenotype.7-9,14-18 A frequent mutation detected in the ABCC6 gene in PXE patients is the nonsense mutation c.3421C4T (p.R1141X), resulting in a truncated protein that lacks the second ATP-binding domain of the native MRP6.7-9,19,20 Another mutation, which is frequently found in American PXE patients, is a large deletion including exons 23-29,17 which also leads to a loss of the second ATP-binding site of MRP6. Login to comment
122 (a) Schematic structure of the MRP6 protein and localization of the mutations c.3421C4T (p.R1141X), c.3389C4T (p.T1130 M) and c.3341G4A (p.R1114 H). Login to comment

[hide] Multidrug resistance protein-6 (MRP6) in human der... Matrix Biol. 2003 Nov;22(6):491-500. Boraldi F, Quaglino D, Croce MA, Garcia Fernandez MI, Tiozzo R, Gheduzzi D, Bacchelli B, Pasquali Ronchetti I
Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients.
Matrix Biol. 2003 Nov;22(6):491-500., [PMID:14667841]

Abstract [show]
Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE.

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3 In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). Login to comment
33 We compared the efficiency of the MDRyMRP system of normal fibroblasts with that of fibroblasts from PXE patients homozygous for the nucleotide change 3421 C™T in exon 24 of the ABCC6 gene resulting in the creation of a premature stop codon at R1141X (Le Saux et al., 2000, 2001). Login to comment
92 Only fibroblasts from patients homozygous for the R1141X mutation were used in the present study. Login to comment
112 Therefore, MRP6 deficiency, due to the R1141X mutation, induces fibroblasts to accumulate calcein in higher amount and to release it in a time 25% longer than normal cells. Login to comment
149 Patients were homozygous for the mutation R1141X (see further for details) showing no detectable mRNA for MRP6 (Le Saux et al., 2000). Login to comment
160 R1141X mutation identification Genomic DNA was isolated from blood leukocytes of PXE patients and purified with a QIAamp blood kit (Qiagen), according to standard procedures. Login to comment

[hide] High levels of desmosines in urine and plasma of p... Eur J Clin Invest. 2004 Feb;34(2):156-64. Annovazzi L, Viglio S, Gheduzzi D, Pasquali-Ronchetti I, Zanone C, Cetta G, Iadarola P
High levels of desmosines in urine and plasma of patients with pseudoxanthoma elasticum.
Eur J Clin Invest. 2004 Feb;34(2):156-64., [PMID:14764080]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE), a rare heritable disorder caused by mutations of the ABCC6 gene, is characterized by fragmentation and mineralization of elastic fibres. We determined the extent of degradation of elastin by measuring and comparing the amount of desmosines in plasma and urine of PXE patients, healthy carriers and normal subjects. METHODS: Using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) we measured the amount of desmosines in the urine of 46 individuals (14 PXE patients, 17 healthy carriers and 15 controls) and in the plasma of 56 subjects (18 PXE patients, 23 healthy carriers and 15 controls). Pseudoxanthoma elasticum patients and carriers were identified by clinical, structural and molecular biology analyses. RESULTS: The urinary excretion of desmosines was two-fold higher in PXE patients than in controls (P < 0.01); the values for healthy carriers were intermediate between those of PXE patients and controls. A very similar trend between patients and their relatives was observed for plasma desmosines. There was a significant correlation between the amount of the desmosines in plasma and urine. Moreover, a positive correlation was observed between urinary desmosine content and age of the patients as well as between urinary desmosine content and severity of clinical manifestations. CONCLUSIONS: Both the urinary and plasma desmosine concentrations indicate that elastin degradation is higher in PXE patients and, to a lesser extent, in healthy carriers than in normal subjects. Data seem to indicate that the amount of elastin breakdown products correlates with the age of patients as well as with the severity of the disease.

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62 Four patients were homozygous for the R1141X mutation on both alleles, two patients were homozygous for a mutation on exons 9 and 30, respectively.All the others were compound heterozygous with mutations on exons 9, 10, 12, 23, 24 and 28. Login to comment

[hide] Novel ABCC6 mutations in pseudoxanthoma elasticum. J Invest Dermatol. 2004 Mar;122(3):608-13. Chassaing N, Martin L, Mazereeuw J, Barrie L, Nizard S, Bonafe JL, Calvas P, Hovnanian A
Novel ABCC6 mutations in pseudoxanthoma elasticum.
J Invest Dermatol. 2004 Mar;122(3):608-13., [PMID:15086542]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in an ABC (ATP-Binding Cassette) transporter gene (ABCC6), which manifests with cutaneous, ophthalmologic, and cardiovascular findings. We studied a cohort of 19 families with PXE, and identified 16 different mutations, nine of which were novel variants. The mutation detection rate was about 77%. We found that arginine codon 518 was, with the previously described R1141X and EX23_29del, a recurrently mutated amino acid (11.5% of the mutations detected for each variant R518Q and R518X). No clear delineation of genotype/phenotype correlation was identified, and marked intra-familial variability of the disease was seen in one family. One family with pseudodominant inheritance displayed three distinct ABCC6 mutations, providing further evidence for the probable exclusive recessive transmission of PXE. These data contribute to the expanding database of ABCC6 mutations, to the description of phenotypic variability, and inheritance in PXE, and should be helpful for genetic counselling.

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3 We found that arginine codon 518 was, with the previously described R1141X and EX23_29del, a recurrently mutated amino acid (11.5% of the mutations detected for each variant R518Q and R518X). Login to comment
28 Phenotype and genotype of the patients studied Patient Origin Sex Age (y) Amino acid variation Nucleotide variation Exon Phenotype C O CV 1 France/Greece F 26 R1141X 3421C4T 24 1 0 0 E1400K 4198G4A 29 2 France F 31 V74del 220-222del 3 1 1 0 V74del 220-222del 3 3-1 Morocco F 20 E1400K 4198G4A 29 1 0 0 E1400K 4198G4A 29 3-2 M 18 E1400K 4198G4A 29 1 0 2 E1400K 4198G4A 29 4 France F 36 A999_S1403del EX23_29del 23-29 1 1 0 Splicing alteration IVS25-3C4A 5 France F 44 ?/? Login to comment
29 2 0 0 6 France M 15 R1141X 3421C4T 24 2 0 1 R1141X 3421C4T 24 7 Morocco F 26 R518Q 1553G4A 12 1 1 1 R518Q 1553G4A 12 8 Turkey F 21 A766D 2297C4A 18 1 0 0 A766D 2297C4A 18 9 France F 41 R518Q 1553G4A 12 1 0 0 T1130M 3389C4T 24 10 France F 30 R518X 1552C4T 12 1 0 0 R518X 1552C4T 12 11-1 Algeria F 75 NA 1 0 0 T1130M 3389C4T 24 11-2 M 39 D1238H 3712G4C 26 1 0 0 11-3 F 36 Q363_R373del 1088-1120del 9 2 0 0 D1238H 3712G4C 26 12 France M 58 ?/? Login to comment
33 1552C4T 12 1 0 0 18 France F 31 R1141X 3421C4T 24 1 0 0 W1223X 3668G4A 26 19-1 France M 18 R1164X/? Login to comment
46 The two known recurrent mutations (R1141X and EX23_29del) account respectively for 13% and 7% of the mutations detected in the French families included in our study, and were absent from the families originating from Algeria, Morocco, and Turkey. Login to comment
58 The results are summarized in Table II. The recurrent mutated allele R1141X shares a common haplotype identical-by-descent in the French families (families 1, 6, and 18), which is consistent with the same geographic origin of these patients. Login to comment
59 R1141X is the most frequent mutation found in the European population, and haplotype analysis of additional European patients would be helpful to establish whether this mutant allele is inherited from a common ancestor. Login to comment
90 In two families, the recurrent recessive mutation R1141X was identified in affected patients (Bergen et al, 2000). Login to comment

[hide] Does autosomal dominant pseudoxanthoma elasticum e... Am J Med Genet A. 2004 May 1;126A(4):403-12. Plomp AS, Hu X, de Jong PT, Bergen AA
Does autosomal dominant pseudoxanthoma elasticum exist?
Am J Med Genet A. 2004 May 1;126A(4):403-12., 2004-05-01 [PMID:15098239]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a progressive disorder of elastic fibers in skin, eyes, and arterial walls. It is caused by mutations in the ABCC6 gene. Most patients are sporadic cases. The majority of familial cases show autosomal recessive (AR) inheritance, but autosomal dominant (AD) inheritance has also been reported. We reviewed the literature on AD PXE and we studied in detail, both clinically and by DNA studies, a selection of potentially AD pedigrees from our patient population consisting of 59 probands and their family members. Individuals were considered to have definite PXE if they had two of the following three criteria: characteristic ophthalmologic signs, characteristic dermatologic signs, and a positive skin biopsy. In the literature we found only three families with definite PXE in two successive generations and no families with definite PXE in three or more generations. Our own data set comprised three putative AD families. Extensive DNA studies revealed a mutation in only one ABCC6 allele in the patients of these families. Only one of our families showed definite PXE in two generations. Linkage studies revealed that pseudodominance was unlikely in this family. In the other two families AD PXE could not be confirmed after extensive clinical examinations and application of our criteria, since definite PXE was not present in two or more generations. Conclusion: the inheritance pattern in PXE usually is AR. Part of the phenotype in family members of PXE patients might be due to expression in heterozygous carriers of an AR disease. AD inheritance in PXE may exist, but is both after careful literature study and in our patient material much rarer than previously thought.

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130 Mother35Peaud`orangeIncreasedechogenicityofkidneys andpancreas AppelmansandLebas[1953]a Femaleproband50AS,bleeding,MDCharacteristicofPXEHypertension Son28AS,yellowishretinallesionsManypapuleshighonback Daughter25AS,yellowishretinallesionsNormal Extensive screening of the ABCC6 gene revealed a R1141X mutation in only one allele. Login to comment
133 However, he did have the R1141X mutation. Login to comment
138 She also had the R1141X mutation in a single allele. An aunt (II-3) had some yellowish papules on the neck and the cubital fossae. Login to comment
141 She also was heterozygous for the R1141X mutation. Login to comment
144 One of the uncles had the R1141X mutation. Login to comment
203 ABCC6 mutation analysis of a control population of 1,057 persons in our lab yielded 8 carriers of the R1141X mutation [Trip et al., 2002]. Login to comment
223 The R1141X mutation was found in one allele of these three individuals and in a healthy uncle Fig. 2. Pedigree of family 1. Login to comment
234 Expression studies in our lab suggested that the R1141X mutation leads to absence of protein by nonsense-mediated RNA decay [Hu et al., 2003c]. Login to comment
236 The patients with definite PXE, in whom one R1141X mutation was found, could have a second, as yet unknown, mutation. Login to comment
260 Recently, heterozygosity for the R1141X mutation was found to be associated with increased risk of cardiovascular disease [Trip et al., 2002]. Login to comment

[hide] ABCC6 mutations in Italian families affected by ps... Hum Mutat. 2004 Nov;24(5):438-9. Gheduzzi D, Guidetti R, Anzivino C, Tarugi P, Di Leo E, Quaglino D, Ronchetti IP
ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE).
Hum Mutat. 2004 Nov;24(5):438-9., [PMID:15459974]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter. The ABCC6 gene was sequenced in 38 unrelated PXE Italian families. The mutation detection rate was 82.9%. Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes. Twenty-three different mutations were identified, among which 11 were new. Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%). The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected. Among the others, exons 9 and 12 were particularly involved. Almost all mutations were located in the intracellular site of MRP6. A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations. The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder.

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64 PXE-causative Mutations Recognized (on one and both alleles) in Italian Patients Family/ Proband Affected subjects Age / gender Clinical score Tot Mutations* Allele 1 Allele 2 Mutation type 001 32 F S2,E2 4 p.R518Q p.T1130MI-3097 002 36 M S3,E2,V2,C2 9 p.R518Q p.T1130M I-3013 001 46 F S1,E3 4 p.R1339C None found I-3094 001 57 F S2,E2 4 p.C440G p.P1346S I-3103 001 57 M E2 2 p.V810M p.R1114C I-3076 001 57 F S2,E4,V3 9 p.R1339C p.R1339C I-3016 001 69 F S3,E2,V2 7 p.N411K p.R1138Q missense I-3082 001 23 M S1,E2 3 p.R518Q p.R1141X I-3074 001 27 F S2,E2 4 p.T364R p.R518X I-3015 001 27 F S2,E3 5 p.Q378X p.R600G I-3062 001 45 M S2,E4,V2 8 p.R1141X p.E1400K 001 50 F S1 1 p.R1275X p.E1400K 002 60 F S3,E3 6 p.R1275X p.E1400K I-3067 003 66 F S2,E2 4 p.R1275X p.E1400K 001 61 F S3,E2 4 p.R518Q p.R1141XI-3027 002 63 F S3,E4,V3 10 p.R518Q p.R1141X missense + nonsense 001 23 F S3,E2 5 p.R1141X p.R1141XI-3056** 002 32 M S2,E2 4 p.R1141X p.R1141X 001 27 F S1,E2 3 p.R1141X p.R1141XI-3057** 002 31 M S3,E2 5 p.R1141X p.R1141X 001 28 M S1,E2 3 p.R1141X None foundI-3045 002 32 F S3,E2,V1 6 p.R1141X None found I-3107 001 29 M S2,E1 3 p.R1030X p.R1141X I-3073 001 31 F S3,E2 5 p.R1141X p.R1141X I-3111 001 32 F S1,E2 3 p.R1141X p.R1141X I-3090 001 34 F S2,E1 3 p.R1141X p.R1141X I-3001 001 37 F S3,E2,V2 7 p.R1030X None found 001 40 F S2,E2 4 p.R1141X p.R1141XI-3007** 002 48 F S1,E2 3 p.R1141X p.R1141X I-3114 001 40 M S2,E2,V3,C1,G2 10 p.R518X p.R518X I-3054 001 44 F S2,E3 5 p.R518X p.R518X 001 47 F S3,E4,C2,G1 10 p.R1141X p.R1141XI-3055** 002 50 F S3,E3 6 p.R1141X p.R1141X 001 50 F S3,E4,V3,C2 12 p.R518X p.R1030X 002 52 F S3,E4,V3 10 p.R518X p.R1030X I-3017 003 55 F S3,E2 5 p.R518X p.R1030X I-3100 001 52 M S3,E3 6 p.Q378X p.Q378X I-3051 001 53 F S3,E4,V2 9 p.R1141X p.R1141X I-3034 001 53 M S3,E4,V3 10 p.R1141X p.R1141X I-3093 001 57 F S3,E3,V2,C3 11 p.R518X None found I-3087 001 57 F S3,E4,V2,C2 11 p.Q378X p.Q378X I-3040 001 60 F S3,E4,V2 9 p.R1141X None found I-3033 001 62 F S3,E4 7 p.R1141X p.R1141X nonsense I-3026 001 36 F S3,E2,G1 6 p.R518X c.2248-2_2248- 1del I-3024 001 40 F S1,E2,V3 6 p.R518X p.L1182PfsX96 I-3072 001 41 F S2,E2,C2 6 p.M1127T c.3736-1G>A I-3002 001 50 F S3,E2 5 p.A820P c.3736-1G>A others Family/ Proband Affected subjects Age / gender Clinical score Tot Mutations* Allele 1 Allele 2 Mutation type 002 57 F S2,E4 6 p.A820P c.3736-1G>A I-3008 001 53 F S2,E2,C1 5 p.M1440CfsX24 p.M1440CfsX24 Patients are identified by an international code: I = Italian, 3001 = family number (European patients are numerated from 3000), 001 = subject number. Login to comment
72 ABCC6/MRP6 Mutations Found in Italian PXE Patients Number of families INTRON EXON cDNA* PROTEIN* References 1 9 c.1091C>G p.T364R Pulkkinen et al., 2001 3 9 c.1132C>T p.Q378X Pulkkinen et al., 2001; Cai et al., 2001 1 10 c.1318T>G p.C440G Present study 1 10 c.1233T>G p.N411K Le Saux et al., 2001 7 12 c.1552C>T p.R518X Meloni et al., 2001; Chassaing et al., 2004 3 12 c.1553G>A p.R518Q Le Saux et al., 2001; Chassaing et al., 2004 1 14 c.1798C>T p.R600G Present study 1 17 c.2248-2_2248- 1del - Present study 1 19 c.2428G>A p.V810M Present study 1 19 c.2458G>C p.A820P Present study 3 23 c.3088C>T p.R1030X Le Saux et al., 2001 1 24 c.3340C>T p.R1114C Present study 1 24 c.3380C>T p.M1127T Present study 1 24 c.3389C>T p.T1130M Chassaing et al., 2004; Gotting et al., 2004 1 24 c.3413G>A p.R1138Q Le Saux et al., 2000; Ringpfeil et al., 2000; Le Saux et al., 2001 13 24 c.3421C>T p.R1141X Bergen et al., 2000; Germain et al., 2000; Ringpfeil et al., 2000; Le Saux et al., 2001; Pulkkinen et al., 2001; Uitto et al., 2001; Hu et al., 2003 ; Gotting et al., 2004 1 25 c.3544_3544dupC p.L1182PfsX96 Present study 2 26 c.3736-1G>A - Ringpfeil et al., 2000; Le Saux et al., 2001 1 27 c.3823C>T p.R1275X Present study 2 28 c.4015C>T p.R1339C Le Saux et al., 2001 1 28 c.4036C>T p.P1346S Present study 2 29 c.4198G>A p.E1400K Chassaing et al., 2004 1 30 c.4318_4318delA p.M1440CfsX24 Present study The number of families in which a specific mutation was found (in heterozygous and in homozygous state) is reported. Login to comment
75 In the PXE patients examined, exon 24 of the ABCC6 gene was the most affected one, and c.3421C>T (p.R1141X) was the most frequent mutation, being homozygous in 7 of 38 families (18.4%), and heterozygous in another 6 unrelated families (15.8%), including the 2 families (3 patients) in whom it was the only detected mutation. Login to comment
76 Therefore, the p.R1141X mutation was present in 26.3% of all the alleles examined (20/76). Login to comment
81 In order to identify a possible founder origin of recurrent disease-associated alleles, a limited haplotype analysis was performed in 11 unrelated families carrying the same mutation (p.Q378X, p.R518X, p.R1141X) in homozygous state (data not shown). Login to comment
84 The analysis of the seven families with the p.R1141X mutated allele showed common haplotypes, suggesting the presence of a common ancestor. Login to comment
85 Comparison of the analysis of 9 intragenic single-nucleotide polymorphisms, evaluated in Italian as well as in French patients (Chassaing et al., 2004), showed identical alleles, suggestive for a founder origin of p.R1141X mutation in European PXE patients. Login to comment
111 In particular, by considering patients homozygous for the most recurrent p.R1141X mutation, that is associated with the almost complete absence of MRP6 (Le Saux et al., 2001; Hu et al., 2003), the correlation between total clinical score and age was highly consistent (p<0.01). Login to comment
118 Some were recurrent mutations (p.Q378X, p.R518X, p.R518Q, p.R1141X), however the majority of mutations were sporadic variants. Login to comment
119 The most frequent p.R1141X PXE-related allele shared a common haplotype identical-by-descent in seven Italian families homozygous for this mutation. Login to comment
128 As a further example, patient I- 3055-001, who was also a strong smoker, was the only PXE patient homozygous for the p.R1141X mutation with heart and gastro-intestinal alterations, whereas her sister had only eye and skin lesions, underlining the importance of exogenous factors that may interfere with elastin deposition and degradation. Login to comment

[hide] Identification of two novel missense mutations (p.... Intern Med. 2004 Dec;43(12):1171-6. Noji Y, Inazu A, Higashikata T, Nohara A, Kawashiri MA, Yu W, Todo Y, Nozue T, Uno Y, Hifumi S, Mabuchi H
Identification of two novel missense mutations (p.R1221C and p.R1357W) in the ABCC6 (MRP6) gene in a Japanese patient with pseudoxanthoma elasticum (PXE).
Intern Med. 2004 Dec;43(12):1171-6., [PMID:15645653]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a rare, inherited, systemic disease of elastic tissue that in particular affects the skin, eyes, and cardiovascular system. Recently, the ABCC6 (MRP6) gene was found to cause PXE. A defective type of ABCC6 gene (16pl3.1) was determined in two Japanese patients with PXE. In order to determine whether these patients have a defect in ABCC6 gene, we examined each of 31 exons and flanking intron sequences by PCR methods (SSCP screening and direct sequencing). We found two novel missense variants in exon 26 and 29 in a compound heterozygous state in the first patient. One is a missense mutation (c.3661C>T; p.R1221C) in exon 26 and the other is a missense mutation (c.4069C>T; p.R1357W) in exon 29. These mutations have not been detected in our control panel of 200 alleles. To our knowledge, this is the first report of mutation identification in the ABCC6 gene in Japanese PXE patients. The second patient was homozygous for 2542_2543delG in ABCC6 gene and heterozygous for 6 kb deletion of LDL-R gene. This case is the first report of a genetically confirmed case of double mutations both in PXE and FH loci.

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This is erroneously identified as a reported sequence variant. In the cited article E18F is the name of a PCR primer.
aranyi on 2012-05-05 13:19:37

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16 Le Saux et al demonstrated that the nonsense mutation p.R1141X and a large deletion spanning from exon 23 to exon 29 were relatively common mutations in a cohort of 122 patients from the United Kingdom, the United States, South Africa, Italy, Germany, and Belgium (5). Login to comment
56 We have found neither the nonsense variant p.R1141X nor the large deletion ABCC6del23-29 in our patients. Login to comment
85 In two families, the recurrent recessive mutation p.R1141X was identified in affected patients (3). Login to comment

[hide] New ABCC6 gene mutations in German pseudoxanthoma ... J Mol Med (Berl). 2005 Feb;83(2):140-7. Epub 2004 Nov 10. Hendig D, Schulz V, Eichgrun J, Szliska C, Gotting C, Kleesiek K
New ABCC6 gene mutations in German pseudoxanthoma elasticum patients.
J Mol Med (Berl). 2005 Feb;83(2):140-7. Epub 2004 Nov 10., [PMID:15723264]

Abstract [show]
Pseudoxanthoma elasticum (PXE; OMIM 177850 and 264800) is a rare heritable disorder of the connective tissue affecting the extracellular matrix of the skin, eyes, gastrointestinal system, and cardiovascular system. It has recently been found that mutations in the ABCC6 gene encoding the multidrug resistance-associated protein (MRP) 6 cause PXE. This study examined novel mutations in the ABCC6 gene in our cohort of 76 German PXE patients and 54 unaffected or not yet affected relatives with a view to expanding the known mutational spectrum of the gene. Mutational analysis was performed using denaturing high-performance liquid chromatography and direct sequencing. The mutational screening revealed a total of 22 different ABCC6 sequence variations. We identified seven novel and four previously described PXE-associated mutations as well as eight novel neutral ABCC6 sequence variants. The new PXE-associated mutations included five missense mutations, one single base pair deletion, and one larger out-of-frame deletion. We suspect that the novel missense mutations lead to an impaired function of MRP6. Both deletions are predicted to result in a dysfunctional MRP6 protein. The seven new ABCC6 mutations were not present in 200 alleles from healthy blood donors which served as a control cohort. Most of the PXE patients who were found to carry PXE-causing ABCC6 mutations were assumed to manifest the PXE phenotype because of a compound heterozygous genotype. However, a genotype-phenotype correlation could not be established for the detected ABCC6 mutations. In summary, our data give a further insight into the spectrum of ABCC6 mutations in PXE patients.

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35 The most frequent mutation in PXE patients of European descent is the nonsense mutation p.R1141X [15, 18, 19]. Login to comment
74 In addition, the presence of the frequent c.3421C>T (p.R1141X) mutation was analyzed in all patients and relatives as previously reported by us [18]. Login to comment
75 The nonsense mutation p.R1141X was the most frequent mutation found in the panel of PXE patients investigated in the present study. Login to comment
77 Heterozygous carriers but no homozygous carriers of the p.R1141X mutation were identified among the 54 unaffected relatives (data not shown). Login to comment
78 One heterozygous carrier of the nonsense mutation p.R1141X was detected among 910 healthy Westphalian blood donors. Login to comment
104 The nonsense mutation p.R1141X, which was identified in 34 (44.7%) PXE patients, was the most frequent mutation in our cohort of German PXE patients. Login to comment
107 In contrast to the p.R1141X mutation, most of these variations were unique in the cohort studied. Login to comment
134 All but two of the PXE patients who were identified to be heterozygous carriers of the six novel mutations (p.M751K, p.R760W, p.L851P, p.S1403R, and c.2835_ 2850del16) were also found to carry one other PXE-causing ABCC6 mutation (c.2787+1G>T, p.R1141X, c.3736-1G>A, p.R1268Q). Login to comment

[hide] Efficient molecular diagnostic strategy for ABCC6 ... Genet Test. 2004 Fall;8(3):292-300. Hu X, Plomp A, Gorgels T, Brink JT, Loves W, Mannens M, de Jong PT, Bergen AA
Efficient molecular diagnostic strategy for ABCC6 in pseudoxanthoma elasticum.
Genet Test. 2004 Fall;8(3):292-300., [PMID:15727254]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a hereditary disorder of connective tissue with skin, cardiovascular, and visual involvement. In familial cases, PXE usually segregates in an autosomal recessive fashion. The aim of this manuscript is to describe an efficient strategy for DNA diagnosis of PXE. The two most frequent mutations, R1141X and an ABCC6 del exons 23-29, as well as a core set of mutations, were identified by restriction enzyme digestion and size separation on agarose gels. Next, in the remaining patient group in which only one or no mutant allele was found, the complete coding sequence was analyzed using denaturing high-performance liquid chromatography (dHPLC). All variations found were confirmed by direct DNA sequencing. Finally, Southern blot was used to investigate the potential presence of small or large deletions. Twenty different mutations, including two novel mutations in the ABCC6 gene, were identified in 80.3% of the 76 patients, and 58.6% of the 152 ABCC6 alleles analyzed. With this strategy, 70 (78.7%) out of 89 mutant alleles could be detected within a week. We conclude that this strategy leads to both reliable and time-saving screening for mutations in the ABCC6 gene in sporadic cases and in families with PXE.

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4 The two most frequent mutations, R1141X and an ABCC6 del exons 23-29, as well as a core set of mutations, were identified by restriction enzyme digestion and size separation on agarose gels. Login to comment
57 Six nucleotide changes, including 2,294g Ǟ a (R765Q in exon 18), 3,421c Ǟ t (R1141X in exon 24), 3904g Ǟ a (G1302R in exon 28), 3,907g Ǟ c (A1303P in exon 28), 3,775del T (W1259 frameshift in exon 27), and 4,377c Ǟ t (R1459C in exon 30) were determined by the digestion of PCR fragments with restriction enzymes Sma I, Bsi YI, Nci I, Hea III, Bst NI, and Aci I, respectively. Login to comment
95 RESULTS OF THE MUTATION ANALYSIS IN THE ABBC6 GENE IN 76 PATIENTS Sequence Type AA change variation Location Alleles Statusa Phaseb Method Nonsense Q749X 2247 C → T Exon 17 2 ht 3 DHPLC R1141X 3421 C → T Exon 24 35 hm.ht.ch 1 Bsi YI Missense R765Q 2294G → A Exon 18 1 ht 2 Sma I R1114H 3341G → A Exon 24 1 ht 3 dHPLC T1130M 3390C → T Exon 24 2 ch 3 dHPLC R1221C 3663C → T Exon 26 1 ch 3 dHPLC G1302R 3904G → A Exon 28 1 ht 2 Nci I A1303P 3907G → C Exon 28 1 ch 2 Hae III D1326N 3999G → A Exon 28 1 ht 3 dHPLC K1394N 4182G → T Exon 29 1 ch 3 dHPLC R1459C 4377C → T Exon 30 1 ht 2 Aci I Frameshift Splicing IVS17-12delTT Intron 17 1 ht 3 dHPLC IVS26-1G → A Intron 26 1 ht 3 dHPLC Deletion 1944del22 Exon 16 2 ht,ch 2 PCR 4182delG Exon 29 6 hm,ht 3 dHPLC 3775delT Exon 27 11 hm,ht 2 Bst NI 3821del48 Exon 27 1 ht 2 PCR Insertion 4220insAGAA Exon 30 1 ht 3 dHPLC Deletion A995del405 del exon 23-29 Exon 23-29 17 hm,ht,ch,hem 1 PCR ABCC6 2 ht 4 FISH ahm, homozygote; ht, heterozygote; ch, compound heterozygote; hem, hemizygote. Login to comment
98 ABCC6 mutation analysis. Phase I: Detection of the R1141X mutation in exon 24 and deletion of exons 23-29. Login to comment
100 Lane 1, the R1141X mutation disrupts the Bsi YI site in the wild-type sequence and results in a single PCR fragment of 100 bp; lane 2, the wild-type allele was cleaved into two Bsi YI fragments of 88 bp and 12 bp (latter band not visible). Login to comment
115 Phase 1: Rapid detection of the founder mutation R114X and deletion of exons 23-29 The founder mutation, R1141X (Hu et al., 2003b), was found in 35 out of 89 mutant alleles (39.3%). Login to comment
116 R1141X was the most common PXE mutation in our cohort. Login to comment
154 Phase 2: Rapid detection of a core set of mutations After screening of R1141X and del exons 23-29, a core set of seven additional mutations was rapidly identified by PCR and PCR-RFLP. Login to comment
172 The current strategy is especially efficient for the European population, in which a number of PXE mutations occur very frequent, i.e., R1141X (39.3%) and del exon 33-29 (19%). Login to comment

[hide] Patients with premature coronary artery disease wh... Int J Cardiol. 2005 Apr 28;100(3):389-93. Wegman JJ, Hu X, Tan H, Bergen AA, Trip MD, Kastelein JJ, Smulders YM
Patients with premature coronary artery disease who carry the ABCC6 R1141X mutation have no Pseudoxanthoma Elasticum phenotype.
Int J Cardiol. 2005 Apr 28;100(3):389-93., 2005-04-28 [PMID:15837081]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is an inherited disorder of elastic tissue. We recently found that heterozygosity for the frequent (0.8% prevalence in Dutch population) R1141X mutation in the PXE gene coding for the ABCC6 transporter, is associated with a fourfold risk of premature coronary artery disease. Yet, it is not clear whether or not heterozygosity for this mutation results in a mild PXE phenotype. The objective of our study was to determine if skin and/or eye abnormalities related to a PXE phenotype could be found in patients with premature coronary artery disease, with and without the R1141X mutation. METHODS: R1141X mutation carriers with premature coronary artery disease (cases) and patients with premature coronary artery disease with no-or not known-mutation (controls) were studied. Cases and controls were examined for PXE-like skin changes and retinal angioid streaks, peau d'orange or pigment epithelium changes. RESULTS: 7 cases and 31 controls were analysed. In both the mutation-positive and the control group, skin inspection and eye fundus examination did not reveal any dermatological or ocular signs of PXE. CONCLUSIONS: Carriers for the ABCC6 R1141X mutation, which is frequent and confers a high risk of premature coronary artery disease, do not commonly have skin or eye abnormalities consistent with a mild PXE phenotype.

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0 Patients with premature coronary artery disease who carry the ABCC6 R1141X mutation have no Pseudoxanthoma Elasticum phenotype Jurgen J. Wegmana , Xiaofeng Hub , Hendra Tanc , Arthur A.B. Bergenb , Mieke D. Tripd , John J.P. Kastelein, Yvo M. Smulderse,* a Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands b The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands c Department of Ophthalmology, Academic Medical Center, University of Amsterdam, The Netherlands d Department of Cardiology, Academic Medical Center, University of Amsterdam, The Netherlands e Department of Internal Medicine, VU University Medical Center, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands Received 26 February 2004; received in revised form 28 June 2004; accepted 5 July 2004 Available online 2 February 2005 Abstract Background: Pseudoxanthoma elasticum (PXE) is an inherited disorder of elastic tissue. Login to comment
1 We recently found that heterozygosity for the frequent (0.8% prevalence in Dutch population) R1141X mutation in the PXE gene coding for the ABCC6 transporter, is associated with a fourfold risk of premature coronary artery disease. Login to comment
3 The objective of our study was to determine if skin and/or eye abnormalities related to a PXE phenotype could be found in patients with premature coronary artery disease, with and without the R1141X mutation. Login to comment
4 Methods: R1141X mutation carriers with premature coronary artery disease (cases) and patients with premature coronary artery disease with no-or not known-mutation (controls) were studied. Login to comment
8 Conclusions: Carriers for the ABCC6 R1141X mutation, which is frequent and confers a high risk of premature coronary artery disease, do not commonly have skin or eye abnormalities consistent with a mild PXE phenotype. Login to comment
32 We subsequently observed a relatively high prevalence of 3.2% heterozygosity for the R1141X mutation in the ABCC6 gene, in a population with premature coronary artery disease, as opposed to 0.8% prevalence in a large population-based sample [17]. Login to comment
33 Heterozygosity for the R1141X mutation certainly does not commonly lead to the typical PXE phenotype, but how often a mild PXE phenotype characterises such patients is unknown. Login to comment
34 In the present study, we specifically looked for typical skin changes and ocular abnormalities in patients with premature coronary artery disease and heterozygosity for the R1141X mutation, in order to assess if these patients have mild PXE-like characteristics. Login to comment
36 This was done for two reasons: First, R1141X is not the only mutation causing PXE in Dutch patients [7] and, second, the phenotypical presentations, particularly the angioid streaks in fundo, are not entirely specific for PXE [11]. Login to comment
42 Seven patients who were under the age of 50 years at the time of their coronary artery disease event (occurring between 1995 and 2001), who carried the R1141X mutation in the ABCC6 gene, and who we were able to contact and obtain consent from, were selected as cases from the database of the atherosclerosis outpatient clinic [17]. Login to comment
45 In 21 patients in the control group, the absence of the R1141X mutation in the ABCC6 gene was confirmed by DNA analysis. Login to comment
64 Results The summary demographic and clinical data of the 7 R1141X mutation-positive patients and the 31 controls are listed in Table 1. Login to comment
74 Discussion Our study examined discrete PXE-like characteristics in 38 patients with premature coronary artery disease, 7 of whom were identified as carriers of the R1141X mutation in the ABCC6 gene. Login to comment
78 Second, more importantly, our results suggest that, although heterozygosity for the R1141X PXE mutation is frequent and confers a sharply increased risk of coronary artery disease [17], these patients, or at least the majority of them, cannot easily be identified by a discrete PXE phenotype. Login to comment
82 The absence of a macroscopically detectable dermatological and retinal PXE phenotype in coronary artery disease patients who are carriers of the R1141X mutation is important. Login to comment
83 The prevalence of this mutation in a large (n=1057) sample of the Dutch population was 0.8%, and the odds ratio for coronary artery disease associated with the Table 1 Summary of demographic and clinical data on ABCC6/MRP6 R1141X mutation-positive patients and controls R1141X positive Control Number of patients 7 31 Male gender 5 (71%) 24 (77%) Age (years) 42.9 (4.8) 40.6 (5.7) History of smoking 4 (57%) 24 (77%) First-degree family history of CAD 4 (57%) 7 (23%) Systolic blood pressure (mmHg) 127.1 (14.1) 129.5 (14.7) Diastolic blood pressure (mmHg) 78.6 (6.3) 79.3 (8.3) Total cholesterol (mmol/l) 5.7 (1.0) 5.4 (1.1) HDL cholesterol (mmol/l) 1.1 (0.2) 1.1 (0.3) LDL cholesterol (mmol/l) 3.8 (1.1) 3.5 (0.9) Triglycerides (mmol/l) 1.9 (0.9-3.1) 1.4 (0.4-4.6) Skin/fundus abnormalities none none Data are number (%), mean (standard deviation), or median (range). Login to comment
84 Table 2 Individual patient data on the ABCC6/MRP6 R1141X mutation-positive patients, who all had a coronary event before the age of 50 years Patient #1 Patient #2 Patient #3 Patient #4 Patient #5 Patient #6 Patient #7 Gender male male male male female female male Age at time of first coronary event 37 50 43 48 42 38 42 Family history parental parental parental none sibs none none Smoking history (pack years) 25 30 none 60 none none 12 Blood pressure (mmHg) 115/80 120/80 130/80 110/70 125/75 150/90 140/75 Total cholesterol (mmol/l) 5.4 5.2 5.2 6.7 6.7 4.1 6.6 LDL cholesterol (mmol/l) 3.1 3.4 3.2 4.4 5.1 2.3 5.0 HDL cholesterol (mmol/l) 0.9 1.0 1.3 1.0 0.9 1.2 1.2 Triglycerides (mmol/l) 3.1 1.7 1.6 2.8 1.6 1.4 0.9 Skin abnormalities absent absent absent absent absent absent absent Fundus examination normal normal normal normal normal normal normal J.J. Wegman et al. / International Journal of Cardiology 100 (2005) 389-393 391 mutation was 4.2 [17]. Login to comment
86 However, given our present findings in coronary artery disease patients, and the rarity of the PXE phenotype in the population, it appears that the majority of individuals who are heterozygous for R1141X do not have the typical PXE phenotype. Login to comment
101 Secondly, the R1141X mutation was not determined in all controls. Login to comment
102 Statistically, however, the chance of finding an appreciable number of R1141X mutations in these 10 patients is small. Login to comment
103 Even if 1 or 2 of these patients would be R1141X-positive, this would in no way affect our conclusion, but only strengthen our deduction that such patients do not commonly have a PXE phenotype. Login to comment
109 In a recent study, in which we presented the mutation spectrum of ABCC6 in 59 PXE patients from The Netherlands, we identified the R1141X to be the most common mutation [7]. Login to comment
110 The R1141X mutation was identified in 32.2 % of Dutch patients [24]. Login to comment

[hide] Disruption of Abcc6 in the mouse: novel insight in... Hum Mol Genet. 2005 Jul 1;14(13):1763-73. Epub 2005 May 11. Gorgels TG, Hu X, Scheffer GL, van der Wal AC, Toonstra J, de Jong PT, van Kuppevelt TH, Levelt CN, de Wolf A, Loves WJ, Scheper RJ, Peek R, Bergen AA
Disruption of Abcc6 in the mouse: novel insight in the pathogenesis of pseudoxanthoma elasticum.
Hum Mol Genet. 2005 Jul 1;14(13):1763-73. Epub 2005 May 11., 2005-07-01 [PMID:15888484]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue, affecting mainly skin, eye and the cardiovascular system. PXE is characterized by dystrophic mineralization of elastic fibres. The condition is caused by loss of function mutations in ABCC6. We generated Abcc6 deficient mice (Abcc6-/-) by conventional gene targeting. As shown by light and electron microscopy Abcc6-/- mice spontaneously developed calcification of elastic fibres in blood vessel walls and in Bruch's membrane in the eye. No clear abnormalities were seen in the dermal extracellular matrix. Calcification of blood vessels was most prominent in small arteries in the cortex of the kidney, but in old mice, it occurred also in other organs and in the aorta and vena cava. Newly developed monoclonal antibodies against mouse Abcc6 localized the protein to the basolateral membranes of hepatocytes and the basal membrane in renal proximal tubules, but failed to show the protein at the pathogenic sites. Abcc6-/- mice developed a 25% reduction in plasma HDL cholesterol and an increase in plasma creatinine levels, which may be due to impaired kidney function. No changes in serum mineral balance were found. We conclude that the phenotype of the Abcc6-/- mouse shares calcification of elastic fibres with human PXE pathology, which makes this model a useful tool to further investigate the aetiology of PXE. Our data support the hypothesis that PXE is in fact a systemic disease.

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31 Furthermore, we found that a frequent (founder) mutation in ABCC6, R1141X (10), may be associated with a strong increase in the prevalence of premature coronary artery disease (5). Login to comment

[hide] Pseudoxanthoma elasticum: a clinical, pathophysiol... J Med Genet. 2005 Dec;42(12):881-92. Epub 2005 May 13. Chassaing N, Martin L, Calvas P, Le Bert M, Hovnanian A
Pseudoxanthoma elasticum: a clinical, pathophysiological and genetic update including 11 novel ABCC6 mutations.
J Med Genet. 2005 Dec;42(12):881-92. Epub 2005 May 13., [PMID:15894595]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an inherited systemic disease of connective tissue primarily affecting the skin, retina, and cardiovascular system. It is characterised pathologically by elastic fibre mineralisation and fragmentation (so called "elastorrhexia"), and clinically by high heterogeneity in age of onset and the extent and severity of organ system involvement. PXE was recently associated with mutations in the ABCC6 (ATP binding cassette subtype C number 6) gene. At least one ABCC6 mutation is found in about 80% of patients. These mutations are identifiable in most of the 31 ABCC6 exons and consist of missense, nonsense, frameshift mutations, or large deletions. No correlation between the nature or location of the mutations and phenotype severity has yet been established. Recent findings support exclusive recessive inheritance. The proposed prevalence of PXE is 1/25,000, but this is probably an underestimate. ABCC6 encodes the protein ABCC6 (also known as MRP6), a member of the large ATP dependent transmembrane transporter family that is expressed predominantly in the liver and kidneys, and only to a lesser extent in tissues affected by PXE. The physiological substrates of ABCC6 remain to be determined, but the current hypothesis is that PXE should be considered to be a metabolic disease with undetermined circulating molecules interacting with the synthesis, turnover, or maintenance of elastic fibres.

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378 Interestingly, among the 49 different missense mutations in ABCC6 (42 previously published and seven new ones in the present study), the majority (43) replace critical amino acids in intracellular domains (seven and 19 mutations are located in I1424T R1459C 4220insAGAA 4318delA G1354R D1361N K1394N E1400K R1298X 410delC 418delG 3775delT R1275X R1221C D1238H W1223X Q1237X IVS26-1G→A R1114C R1114H R1114P S1121W M1127T T1130M R1138P R1138Q R1138W R1141X R1164X R765Q A766D Y768X A781V 2322delC IVS19-2delAG T364R R391G Q378X Q363_R373del 938_939insT 960delC IVS8+2delTG G199X Y227X 179_195del 179_187del G226R V74del Q749X IVS17-12delTT IVS14-5T→G IVS13-29T→A R600G V1298F G1299S T1301I G1302R A1303P S1307P R1314Q R1314W G1321S L1335P R1339C P1346S Q1347H R1030X F1048del R807Q V810M A820P 254delG L673P 1944_1966del 1995delG R518Q R518X K502M A455P G992R IVS21+1G→T G1203DF568SN411K C440G IVS25-3C→A 3544dupC Ex23_29del 30 Ex15del ABCC6del 252015105 Figure 10 Position of the mutations in the ABCC6 gene. Login to comment
379 Table 2 ABCC6 mutations Nucleotide variation Protein alteration Location (gene ) Location (protein) Reference Missense 676 GRA G226R Exon 7 CL 3 This study 1091 CRG T364R Exon 9 TS 7 63, 78 1171 ARG R391G Exon 9 CL 4 88 1233 TRG N411K Exon 10 CL 4 63, 90 1318 TRG C440G Exon 10 TS 8 63 1363 GRC A455P Exon 11 TS 9 86 1505 ART K502M Exon 12 CL 5 This study 1553 GRA R518Q Exon 12 CL 5 63, 86, 88, 90 1703 TRC F568S Exon 13 ECL 5 90 1798 CRT R600G Exon 14 CL 6 63 2018 TRC L673P Exon 16 NBF 1 90 2294 GRA R765Q Exon 18 NBF 1 87, 90 2297 CRA A766D Exon 18 NBF 1 88 2342 CRT A781V Exon 18 NBF 1 This study 2420 GRA R807Q Exon 19 NBF 1 This study 2428 GRA V810M Exon 19 NBF1 63 2458 GRC A820P Exon 19 NBF1 63 2965 GRC G992R Exon 22 ECL 6 This study 3340 CRT R1114C Exon 24 CL 8 63 3341 GRA R1114H Exon 24 CL 8 87 3341 GRC R1114P Exon 24 CL 8 90 3362 CRG S1121W Exon 24 CL 8 90 3380 CRT M1127T Exon 24 CL 8 63 3389 CRT T1130M Exon 24 CL 8 63, 87, 88 3412 CRT R1138W Exon 24 CL 8 17 3413 GRC R1138P Exon 24 CL 8 90 3413 GRA R1138Q Exon 24 CL 8 17, 63, 88, 90 3608 GRA G1203D Exon 25 TS17 90 3663 CRT R1221C Exon 26 COOH 87 3712 GRC D1238H Exon 26 COOH 88 3892 GRT V1298F Exon 28 NBF 2 90 3895 GRA G1299S Exon 28 NBF 2 This study 3902 CRT T1301I Exon 28 NBF 2 90 3904 GRA G1302R Exon 28 NBF 2 87, 90 3907 GRC A1303P Exon 28 NBF 2 87, 90 3919 TRC S1307P Exon 28 NBF 2 This study 3940 CRT R1314W Exon 28 NBF 2 90 3941 GRA R1314Q Exon 28 NBF 2 90 3961 GRA G1321S Exon 28 NBF 2 90 4004 TRC L1335P Exon 28 NBF 2 88 4015 CRT R1339C Exon 28 NBF 2 18, 63, 90 4036 CRT P1346S Exon 28 NBF 2 63 4041 GRC Q1347H Exon 28 NBF 2 90 4060 GRC G1354R Exon 29 NBF 2 78, 86 4081 GRA D1361N Exon 29 NBF 2 90 4182 GRT K1394N Exon 29 NBF 2 87 4198 GRA E1400K Exon 29 NBF 2 63, 88 4271 TRC I1424T Exon 30 NBF 2 90 4377 CRT R1459C Exon 30 NBF 2 87 Nonsense 595 CRT G199X Exon 5 89 681 CRG Y227X Exon 7 84 1132 CRT Q378X Exon 9 63, 78, 83 1552 CRT R518X Exon 12 63, 84, 88 2245 CRT Q749X Exon 17 87 2304 CRA Y768X Exon 18 90 3088 CRT R1030X Exon 23 63, 90 3421 CRT R1141X Exon 24 15, 17, 18, 63, 78, 85, 87, 88, 90 3490 CRT R1164X Exon 24 84, 85, 88 3668 GRA W1223X Exon 26 88 3709 CRT Q1237X Exon 26 90 3823 CRT R1275X Exon 27 63 4192 CRT R1398X Exon 29 90 Splicing alteration IVS8+2delTG Intron 8 This study IVS13-29 TRA Intron 13 This study IVS14-5 TRG Intron 14 This study IVS17-12delTT Intron 17 87 IVS18-2delAG Intron 17 63 IVS21+1 GRT Intron 21 86, 90 IVS25-3 CRA Intron 25 88 IVS26-1 GRA Intron 26 17, 63, 90 Insertion 938_939insT Frameshift Exon 8 90 3544dupC Frameshift Exon 25 63 4220insAGAA Frameshift Exon 30 15, 87 Small deletion 179_187del Frameshift Exon 2 78 179_195del Frameshift Exon 2 90 Pseudoxanthoma elasticum www.jmedgenet.com NBF1 and NBF2, respectively), four are located in transmembrane domains, and only two mutations have been identified in extracellular domains. Login to comment
383 Although most of the 90 pathogenic mutations have been identified in one or a limited number of families, two variants (Ex23_29del and R1141X) are recurrent mutations. Login to comment
384 The frequency of these two recurrent mutations differs according to the population studied: mutation Ex23_29del represents ,28% of the detected mutations in the US population and ,4% in the European population, whereas mutation R1141X represents ,4% of the detected mutations in the US population and ,28% in the European population.90 Furthermore, frequency of the R1141X differs between European countries (for example, 30% in the Dutch population,91 92 26% in Italian patients,63 and 13% in the French population88 ). Login to comment
385 A common founder effect was identified for mutation R1141X in French and Italian populations.63 88 We found that arginine codon 518 was a recurrently mutated amino acid in a cohort of 19 French families with PXE (11.5% of the detected mutations for each variant R518Q and R518X).88 These two mutations represent 19% of the mutations detected in the Italian population.63 In Japanese patients, neither R1141X nor Ex23_29del mutations were identified, whereas mutations 2542delG and Q378X account for 53% and 25%, respectively.93 In South African families of Afrikaaners, mutation R1339C represents more than half the mutations detected,28 with a common haplotype indicating a founder effect.27 28 These mutations are rarely identified in American or European populations.90 The detection rate in different studies varies from 0.55 to 0.83.63 87 88 90 Lack of mutation detection in some patients could reflect exonic deletions (for example, deletion of exon 15), splice site mutations distant from the coding sequence, mutations in the gene regulatory sequences, or investigation of patients with acquired PXE-like syndrome not related to ABCC6 mutations, such as seen in b thalassaemia and sickling syndromes (see below).94 95 Locus heterogeneity of PXE is unlikely, but cannot currently be ruled out. Login to comment
399 * In 2002, Trip et al reported that the prevalence of the sole R1141X mutation was significantly increased in young individuals with coronary artery disease (3.2%), but was also frequent in the general Dutch population.100 They identified eight heterozygous carriers for mutation R1141X among 1057 controls (0.76%). Login to comment
409 A few reports have emphasised the carriage of a sole ABCC6 mutation as a cardiovascular risk factor.70 100 102 In the study by Trip et al, mutation R1141X in ABCC6 appeared to be an independent risk factor for coronary heart disease in young people.100 This observation, if confirmed in other similar cohorts, could be of course of considerable concern for public health. Login to comment

[hide] Molecular genetics of pseudoxanthoma elasticum: ty... Hum Mutat. 2005 Sep;26(3):235-48. Miksch S, Lumsden A, Guenther UP, Foernzler D, Christen-Zach S, Daugherty C, Ramesar RK, Lebwohl M, Hohl D, Neldner KH, Lindpaintner K, Richards RI, Struk B
Molecular genetics of pseudoxanthoma elasticum: type and frequency of mutations in ABCC6.
Hum Mutat. 2005 Sep;26(3):235-48., [PMID:16086317]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder that affects the elastic tissue in the skin, eye, and cardiovascular system. Mutations in the ABCC6 gene cause PXE. We performed a mutation screen in ABCC6 using haplotype analysis in conjunction with direct sequencing to achieve a mutation detection rate of 97%. This screen consisted of 170 PXE chromosomes in 81 families, and detected 59 distinct mutations (32 missense, eight nonsense, and six likely splice-site point mutations; one small insertion; and seven small and five large deletions). Forty-three of these mutations are novel variants, which increases the total number of PXE mutations to 121. While most mutations are rare, three nonsense mutations, a splice donor site mutation, and the large deletion comprising exons 23-29 (c.2996_4208del) were identified as relatively frequent PXE mutations at 26%, 5%, 3.5%, 3%, and 11%, respectively. Chromosomal haplotyping with two proximal and two distal polymorphic markers flanking ABCC6 demonstrated that most chromosomes that carry these relatively frequent PXE mutations have related haplotypes specific for these mutations, which suggests that these chromosomes originate from single founder mutations. The types of mutations found support loss-of-function as the molecular mechanism for the PXE phenotype. In 76 of the 81 families, the affected individuals were either homozygous for the same mutation or compound heterozygous for two mutations. In the remaining five families with one uncovered mutation, affected showed allelic compound heterozygosity for the cosegregating PXE haplotype. This demonstrates pseudo-dominance as the relevant inheritance mechanism, since disease transmission to the next generation always requires one mutant allelic variant from each parent. In contrast to other previous clinical and molecular claims, our results show evidence only for recessive PXE. This has profound consequences for the genetic counseling of families with PXE.

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122 Initial Haplotype-Based Mutation Screening Strategy for Common Mutations and Deletions Initially all of the DNA samples were tested for the nonsense mutation c.3421C4T (p.R1141X) by restriction analysis of PCR products, since the c.3421C4T mutation causes loss of a Bsl I restriction site. Login to comment
246 PXE Mutations While most mutations are unique variants that represent the typical allelic heterogeneity of a recessive disease, we observed five mutations (p.R1141X [26%], p.I1000_S1403delW1404fsX1463 [11%], p.R1164X [5.3%], p.Q378X [3.5%], and c.278711G4T [3%]) at higher frequencies that accounted for almost 50% (exactly 48.8%) of the PXE mutations. Login to comment
251 The most frequently observed PXE mutation was p.R1141X. Login to comment
258 Hu et al. [2003a] reported p.R1141X in allelic linkage disequilibrium with the c.2490C4G polymorphism in his family cohort. Login to comment
260 This clearly demonstrates that while the p.R1141X mutation exhibits a founder effect, it has arisen independently in different population cohorts at least four times. Login to comment

[hide] Analysis of sequence variations in the ABCC6 gene ... J Vasc Res. 2005 Sep-Oct;42(5):424-32. Epub 2005 Aug 26. Schulz V, Hendig D, Schillinger M, Exner M, Domanovits H, Raith M, Szliska C, Kleesiek K, Gotting C
Analysis of sequence variations in the ABCC6 gene among patients with abdominal aortic aneurysm and pseudoxanthoma elasticum.
J Vasc Res. 2005 Sep-Oct;42(5):424-32. Epub 2005 Aug 26., [PMID:16127278]

Abstract [show]
Abdominal aortic aneurysm (AAA) is characterized by dilatation of arterial walls, which is accompanied by degradation of elastin and collagen molecules. Biochemical and environmental factors are known to be relevant for AAA development, and familial predisposition is well recognized. A connective tissue disorder that is also associated with fragmentation of elastic fibers is Pseudoxanthoma elasticum (PXE). PXE is caused by mutations in the ABCC6 gene and mainly affects dermal, ocular and all vascular tissues. To investigate whether variations in ABCC6 are found in AAA patients and to determine mutations in PXE patients, we analyzed seven selected ABCC6 exons of 133 AAA and 54 PXE patients subjected to mutational analysis. In our cohort of AAA patients, we found five ABCC6 alterations, which result in missense or silent amino acid variants. The allelic frequencies of these sequence variations were not significantly different between AAA patients and healthy controls. Therefore, we suggest that alterations in ABCC6 are not a genetic risk factor for AAA. Mutational screening of the PXE patients revealed 19 different ABCC6 variations, including two novel PXE-causing mutations. These results expand the ABCC6 mutation database in PXE.

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This is erroneously identified as a reported sequence variant. In the cited article E18L is the name of a PCR primer.
aranyi on 2012-05-05 13:15:49

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105 The most common PXE mutation was the nonsense mutation p.R1141X which was observed in a homozygous form in 4 (7.4%) and in a heterozygous state in 20 (37.0%) PXE patients. Login to comment
116 The variations p.R1114C and p.G1311E occurred in a heterozygous form in 2 PXE patients, and RFLP or DHPLC analysis revealed that they were not present in our groups of healthy controls Exona Sequence variation Allele frequency AAA patients PXE patients PXE relatives blood donors 16 c.1964A>G (p.Q655R) 1 0 0 0/286 16 c.1990C>T (p.P664S) 0 0 0 1/286 16 c.1995delG (frameshift) 0 3 0 0/286 17 c.2171G>A (p.R724K) 3 1 1 2/254 17 c.2175A>T (p.V725V) 3 1 1 2/254 17 c.2224A>G (p.I742V) 3 1 1 2/254 i-17 IVS17+22T>G 1 0 0 0/254 18 c.2252T>A (p.M751K) 0 2 0 0/204 18 c.2278C>T (p.R760W) 0 1 0 0/204 18 c.2294G>A (p.R765Q) 0 3 0 0/204 24 c.3340C>T (p.R1114C) 0 1 0 0/400 24 c.3341G>A (p.R1114H) 0 1 0 0/400 24 c.3389C>T (p.T1130M) 0 2 0 0/400 24 c.3413G>A (p.R1138Q) 0 2 0 ND 24 c.3421C>T (p.R1141X) 0 28 9 1/1,820b i-24 IVS24+15G>A 1 0 0 ND 28 c.3902C>T (p.T1301I) 0 1 0 ND 28 c.3932G>A (p.G1311E) 0 1 0 0/400 28 c.3940C>T (p.R1314W) 0 1 0 ND 28 c.3941G>A (p.R1314Q) 0 1 1 ND i-28 IVS28+49C>T 59 ND ND ND i-28 IVS28-30C>T 48 ND ND ND 29 c.4182delG (frameshift) 0 3 0 0/400 i-29 IVS29+9G>A 5 ND ND ND 30 c.4209C>A (p.S1403R) 0 1 0 0/244 30 c.4254G>A (p.R1418R) 6 0 0 2/244 i-30 IVS30+11C>G 0 2 0 0/244 23-29 Ex23_Ex29del 0 5 3 ND i = intron; ND = not determined. Login to comment
123 DNA sequencing of all 31 ABCC6 exons showed that the first PXE patient was compound heterozygous for p.R1114C and the known PXE mutation c.1995delG in exon 16, whereas the second carried the new variant p.G1311E and the PXE-causing exon 24 mutation p.R1141X in a compound heterozygous state. Login to comment

[hide] Elevated xylosyltransferase I activities in pseudo... J Mol Med (Berl). 2005 Dec;83(12):984-92. Epub 2005 Aug 24. Gotting C, Hendig D, Adam A, Schon S, Schulz V, Szliska C, Kuhn J, Kleesiek K
Elevated xylosyltransferase I activities in pseudoxanthoma elasticum (PXE) patients as a marker of stimulated proteoglycan biosynthesis.
J Mol Med (Berl). 2005 Dec;83(12):984-92. Epub 2005 Aug 24., [PMID:16133423]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue characterized by extracellular matrix alterations with elastin fragmentation and excessive proteoglycan deposition. Xylosyltransferase I (XT-I, E.C. 2.4.2.26) is the initial enzyme in the biosynthesis of the glycosaminoglycan chains in proteoglycans and has been shown to be a marker of tissue remodeling processes. Here, we investigated for the first time serum XT-I activities in a large cohort of German PXE patients and their unaffected relatives. XT-I activities were measured in serum samples from 113 Caucasian patients with PXE and 103 unaffected first-degree family members. The occurrence of the frequent ABCC6 gene mutation c.3421C>T (R1141X) and the hypertension-associated genetic variants T174M and M235T in the angiotensinogen (AGT) gene were determined. Serum XT-I activities in male and female PXE patients were significantly increased compared to unaffected family members (male patients, mean value 0.96 mU/l, SD 0.37; male relatives, 0.78 mU/l, SD 0.29; female patients, 0.91 mU/l, SD 0.31; female relatives, 0.76 mU/l, SD 0.34; p<0.05). The mean XT-I activities in PXE patients with hypertension were 24% higher than in patients without increased blood pressure (p<0.05). The AGT T174M and M235T frequencies were not different in hypertensive PXE patients, normotensive PXE patients, family members or blood donors. Our data show that the altered proteoglycan biosynthesis in PXE patients is closely related to an increased XT-I activity in blood. Serum XT-I, the novel fibrosis marker, may be useful for the assessment of extracellular matrix alterations and disease activity in PXE.

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11 The occurrence of the frequent ABCC6 gene mutation c.3421C>T (R1141X) and the hypertension-associated genetic variants T174M and M235T in the angiotensinogen (AGT) gene were determined. Login to comment

[hide] Targeted ablation of the abcc6 gene results in ect... Mol Cell Biol. 2005 Sep;25(18):8299-310. Klement JF, Matsuzaki Y, Jiang QJ, Terlizzi J, Choi HY, Fujimoto N, Li K, Pulkkinen L, Birk DE, Sundberg JP, Uitto J
Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.
Mol Cell Biol. 2005 Sep;25(18):8299-310., [PMID:16135817]

Abstract [show]
Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

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45 In addition, it was recently suggested that a recurrent mutation in the ABCC6 gene, R1141X, which is frequently encountered in Caucasian populations, is associated with a significantly increased risk for cardiovascular disease (59). Login to comment

[hide] Identification of a novel deletion in the ABCC6 ge... J Dermatol Sci. 2005 Nov;40(2):115-21. Epub 2005 Sep 23. Katona E, Aslanidis C, Remenyik E, Csikos M, Karpati S, Paragh G, Schmitz G
Identification of a novel deletion in the ABCC6 gene leading to Pseudoxanthoma elasticum.
J Dermatol Sci. 2005 Nov;40(2):115-21. Epub 2005 Sep 23., [PMID:16183260]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is an inherited systemic disorder, characterized by dermal, ocular and cardiovascular lesions. Genetic defects of the ABCC6 (MRP6) transporter are known to cause PXE. OBJECTIVES: The purpose of this study was to identify the genetic background of a PXE patient with a very early onset of the disease and severe systemic involvement. METHODS: Direct sequencing of genomic DNA obtained from peripheral whole blood. RESULTS: Our patient was found to be compound heterozygous with both ABCC6 alleles having genomic deletions. A novel exon 24-25 deletion was identified on one allele, while the frequently observed exon 23-29 deletion was found on the other allele. The novel deletion is 4.68 kb long and was shown to extend from intron 23 to 25. DNA-sequencing of a 2.03 kb fusion fragment revealed the deletion breakpoints within introns 23 and 25 originating in the middle of two Alu-repeats. CONCLUSION: In a patient with severe clinical symptoms, we found two genomic deletions in regions that might be important for function of the ABCC6 transporter. Genomic deletions in ABCC6 may occur more frequently in PXE patients than previously expected and future genetic analysis should focus on these mutations as well.

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24 The most common mutations are R1141X and a deletion from exon 23 to 29, with the latter being present in 13% of PXE cases [5,9,11]. Login to comment

[hide] Pseudoxanthoma elasticum is a recessive disease ch... J Invest Dermatol. 2006 Apr;126(4):782-6. Ringpfeil F, McGuigan K, Fuchsel L, Kozic H, Larralde M, Lebwohl M, Uitto J
Pseudoxanthoma elasticum is a recessive disease characterized by compound heterozygosity.
J Invest Dermatol. 2006 Apr;126(4):782-6., [PMID:16410789]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene. Historically, PXE has been suggested to be inherited either in an autosomal dominant or autosomal recessive manner. To determine the exact mode of inheritance of PXE and to address the question of phenotypic expression in mutation carriers, we identified seven pedigrees with affected individuals in two different generations and sequenced the entire coding region of ABCC6 in affected individuals, presumed carriers with a limited phenotype and unaffected family members. Two allelic mutations were identified in each individual with unambiguous diagnosis of PXE, as well as in those with only minimal clinical signs suggestive of PXE but with positive skin biopsy. Missense mutations were frequently detected in the latter cases. In conclusion, PXE is inherited in an autosomal recessive manner and presence of disease in two generations is due to pseudodominance.

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28 Five of the alleles harbored the recurrent R1141X mutation, which is prevalent in Caucasian populations, six of the alleles contained the R1138W mutation, and seven alleles harbored the deletion mutation del23-29, all of which have been previously reported in a number of families with PXE (Le Saux et al., 2001; Ringpfeil et al., 2001a; Uitto et al., 2001; Pulkkinen et al., 2002; Chassaing et al., 2004). Login to comment
30 In addition, a previously unpublished nonsense mutation W1324X was F568S/R1141X W1324X/R1141X Family 4 R1138W/R1138W R1138W/R1138W -/R1138W R1138W/- R1138W/- R1138W/- Family 6 R391G/R1138W R391G/R1138W Family 7 Family 2 Del23-29/W218C R391G/W218C Del23-29/W218C Del23-29/R391G W218C/- Del23-29/- Del23-29/- ? Login to comment
31 R1164Q/R518X R1164Q/R1164Q R1164Q/- -/- Family 5 Del23-29/R391G Del23-29/Del23-29 Family 3 R1141X/del23-29 R1141X/del23-29 Del23-29/- R1141X/T811M Family 1 Figure 1. Login to comment
40 In Family 1, the two affected daughters of a clinically unaffected mother and an affected father were compound heterozygotes (R1141X/del23-29), and the parents were carriers of the corresponding mutations. Login to comment
41 In addition, the father with asymptomatic angioid streaks, evidence of coronary artery disease, history of gastrointestinal bleeding, and positive skin biopsy, but no clinically obvious skin involvement, was compound heterozygous for R1141X and a novel missense mutation T811M. Login to comment
48 In Family 4, the two affected individuals, a father and a son, were compound heterozygotes, both for R1141X mutation in one allele, in combination with either F568S (father) or W1324X (son). Login to comment
53 However, probing the two most common mutations in ABCC6, namely R1141X and deletion of exons 23-29 in 254 control alleles of Caucasian origin, revealed no carriers (data not shown). Login to comment

[hide] Serum factors from pseudoxanthoma elasticum patien... J Invest Dermatol. 2006 Jul;126(7):1497-505. Epub 2006 Mar 16. Le Saux O, Bunda S, VanWart CM, Douet V, Got L, Martin L, Hinek A
Serum factors from pseudoxanthoma elasticum patients alter elastic fiber formation in vitro.
J Invest Dermatol. 2006 Jul;126(7):1497-505. Epub 2006 Mar 16., [PMID:16543900]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable disorder mainly characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, a gene encoding an ABC transporter predominantly expressed in liver and kidneys. The functional relationship between ABCC6 and elastic fiber calcification is unknown. We speculated that ABCC6 deficiency in PXE patients induces a persistent imbalance in circulating metabolite(s), which may impair the synthetic abilities of normal elastoblasts or specifically alter elastic fiber assembly. Therefore, we compared the deposition of elastic fiber proteins in cultures of fibroblasts derived from PXE and unaffected individuals. PXE fibroblasts cultured with normal human serum expressed and deposited increased amounts of proteins, but structurally normal elastic fibers. Interestingly, normal and PXE fibroblasts as well as normal smooth muscle cells deposited abnormal aggregates of elastic fibers when maintained in the presence of serum from PXE patients. The expression of tropoelastin and other elastic fiber-associated genes was not significantly modulated by the presence of PXE serum. These results indicated that certain metabolites present in PXE sera interfered with the normal assembly of elastic fibers in vitro and suggested that PXE is a primary metabolic disorder with secondary connective tissue manifestations.

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177 21 Fibroblast U21 (M) PXE R1141X/R1141X ? Login to comment
178 Serum samples Control #1 (M) Normal wt/wt 45 Control #2 (M) Normal wt/wt 34 Control #3 (pool)1 Normal wt/wt Av. 68 Control #4 (pool)1 Normal wt/wt Av. 18 PXE 1 (F) PXE R391G/del23-29 50 PXE 2 (F) PXE IVS21+1 G4T/4104delC 51 PXE 3 (F) PXE IVS13-29 T4A/R1141X 41 PXE 4 (M) PXE ?/? Login to comment

[hide] ABCC6 and pseudoxanthoma elasticum. Pflugers Arch. 2007 Feb;453(5):685-91. Epub 2006 Apr 8. Bergen AA, Plomp AS, Hu X, de Jong PT, Gorgels TG
ABCC6 and pseudoxanthoma elasticum.
Pflugers Arch. 2007 Feb;453(5):685-91. Epub 2006 Apr 8., [PMID:16604369]

Abstract [show]
ABCC6 belongs to the adenosine triphosphate-binding cassette (ABC) gene subfamily C. This protein family is involved in a large variety of physiological processes, such as signal transduction, protein secretion, drug and antibiotic resistance, and antigen presentation [Kool et al. (1999) 59:175-182; Borst and Elferink (2002) 71:537-592]. ABCC6 is primarily and highly expressed in the liver and kidney [Kool et al. (1999) 59:175-182; Bergen et al. (2000) 25:228-2231]. The precise physiological function and natural substrate(s) transported by ABCC6 are unknown, but the protein may be involved in active transport of intracellular compounds to the extracellular environment [Kool et al. (1999) 59:175-182] [Scheffer et al. (2002) 82:515-518]. Recently, it was shown that loss of function mutations in ABCC6 cause pseudoxanthoma elasticum (PXE) [Bergen et al. (2000) 25:228-2231; Le Saux et al. (2000) 25:223-227]. PXE is an autosomal recessively inherited multi-organ disorder [Goodman et al. (1963) 42:297-334; Lebwohl et al. (1994) 30:103-107]. PXE is primarily associated with the accumulation of mineralized and fragmented elastic fibers of the connective tissue in the skin [Neldner (1988) 6:1-159], Bruch's membrane in the retina [Hu et al. (2003) 48:424-438], and vessel walls [Kornet et al. (2004) 30:1041-1048]. PXE patients usually have skin lesions and breaks in Bruch's membrane of the retina (angioid streaks). Also, a variety of cardiovascular complications has been observed [Hu et al. (2003) 48:424-438]. Recently, a mouse model for PXE was created by targeted disruption of Abcc6 [Gorgels et al. (2005) 14:1763-1773; Klement et al. (2005) 25:8299-8310], which may be useful to elucidate the precise function of Abcc6 and to develop experimental therapies.

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82 Two ABCC6 mutations, R1141X and del(ex23_29), occur very frequently, probably due to founder effects and genetic drift. Login to comment
83 R1141X accounts for up to 30% of mutations found in European populations [9, 18], but accounts for only 4% of the US PXE population. Login to comment
85 R1141X probably produces an instable ABCC6 mRNA which is rapidly degraded by nonsense mediated RNA decay [18, 29]. Login to comment
86 Finally, R1141X may be associated with premature coronary artery disease [48]. Login to comment

[hide] Early and severe amyloidosis in a patient with con... Br J Dermatol. 2006 Jun;154(6):1190-3. Cattan D, Bouali B, Chassaing N, Martinez F, Dupont JM, Dode C, Martin L
Early and severe amyloidosis in a patient with concurrent familial Mediterranean fever and pseudoxanthoma elasticum.
Br J Dermatol. 2006 Jun;154(6):1190-3., [PMID:16704654]

Abstract [show]
A young woman patient had early and extensive familial Mediterranean fever (FMF)-related amyloidosis and pseudoxanthoma elasticum (PXE). She had the novel G1042S mutation in the ATP-binding cassette subfamily C member 6 (ABCC6) gene, responsible for PXE, and the mutation M694I in MEFV, the FMF gene. Both mutations were homozygous, in agreement with consanguinity in the parents. ABCC6 deficiency may have increased the severity of amyloidosis by increasing the deposition in target tissues of heparan sulphate, which colocalizes spatially and temporally with amyloid proteins, and/or by decreasing the therapeutic activity of colchicine.

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75 The fact that ABCC1 transports colchicine and decreases the activity of this drug could be one explanation for the poor efficacy of colchicine in preventing the extension of amyloidosis in our patient.26 A Dutch epidemiological study has demonstrated that R1141X, the most prevalent ABCC6 mutation in Europe, was not rare in the general population (0Æ8%).27 Accordingly, it can be speculated that ABCC6 Fig 4. Login to comment

[hide] Acute myocardial infarction and a new ABCC6 mutati... Int J Cardiol. 2007 Mar 20;116(2):261-2. Epub 2006 Jul 18. Kiec-Wilk B, Surdacki A, Dembinska-Kiec A, Michalowska J, Stachura-Deren M, Dubiel JS, Dudek D, Rakowski T, Szastak G, Bodzioch M, Aslanidis C, Schmitz G
Acute myocardial infarction and a new ABCC6 mutation in a 16-year-old boy with pseudoxanthoma elasticum.
Int J Cardiol. 2007 Mar 20;116(2):261-2. Epub 2006 Jul 18., 2007-03-20 [PMID:16854481]

Abstract [show]
Pseudoxanthoma elasticum is caused by mutations of the ABCC6 gene. We hereby report a case of pseudoxanthoma elasticum with clinical features dominated by early coronary involvement in addition to typical skin and ocular abnormalities. A 16-year-old survivor of acute myocardial infarction with 3-vessel coronary artery disease exhibited compound heterozygosity for the well-known nonsense mutation (c.3421C>T; R1141X) in exon 24 and a novel missense mutation (c.3662G>A; R1221H) in exon 26 of the ABCC6 gene.

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2 A 16-year-old survivor of acute myocardial infarction with 3-vessel coronary artery disease exhibited compound heterozygosity for the well-known nonsense mutation (c.3421C>T; R1141X) in exon 24 and a novel missense mutation (c.3662G>A; R1221H) in exon 26 of the ABCC6 gene. Login to comment
8 Interestingly, the most common disease-causing mutation in the European population (c.3421C>T; R1141X) predisposes to premature coronary artery disease in the absence of even mild ocular or dermatological PXE signs [4], whereas in PXE subjects abnormalities within the skin and eyes usually predominate and coronary symptoms are relatively rare generally occurring after the age of 35 [5]. Login to comment
22 The PXE boy exhibited compound heterozygosity for the well-recognized mutation (c.3421C>T; R1141X) in exon 24, and a novel one (c.3662G>A; R1221H) in exon 26 (Fig. 2). Login to comment
25 That a heterozygous form of the common R1141X nonsense mutation in exon 24 appears insufficient for the appearance of PXE, confirms earlier observations [5]. Login to comment
26 We suggest that the coincidence of the heterozygous R1141X mutation and a novel one (c.3662G>A; R1221H) in exon 26 on the other allele might have been responsible for PXE in the presented case. Login to comment

[hide] Fundus autofluorescence. Ophthalmology. 2007 Jun;114(6):1233; author reply 1233. Querques G, delle Noci N
Fundus autofluorescence.
Ophthalmology. 2007 Jun;114(6):1233; author reply 1233., [PMID:17544787]

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26 Patients with premature coronary artery disease who carry the ABCC6 R1141X mutation have no pseudoxanthoma elasticum phenotype. Login to comment

[hide] Mutation detection in the ABCC6 gene and genotype-... J Med Genet. 2007 Oct;44(10):621-8. Epub 2007 Jul 6. Pfendner EG, Vanakker OM, Terry SF, Vourthis S, McAndrew PE, McClain MR, Fratta S, Marais AS, Hariri S, Coucke PJ, Ramsay M, Viljoen D, Terry PF, De Paepe A, Uitto J, Bercovitch LG
Mutation detection in the ABCC6 gene and genotype-phenotype analysis in a large international case series affected by pseudoxanthoma elasticum.
J Med Genet. 2007 Oct;44(10):621-8. Epub 2007 Jul 6., [PMID:17617515]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder with considerable phenotypic variability, mainly affects the eyes, skin and cardiovascular system, characterised by dystrophic mineralization of connective tissues. It is caused by mutations in the ABCC6 (ATP binding cassette family C member 6) gene, which encodes MRP6 (multidrug resistance-associated protein 6). OBJECTIVE: To investigate the mutation spectrum of ABCC6 and possible genotype-phenotype correlations. METHODS: Mutation data were collected on an international case series of 270 patients with PXE (239 probands, 31 affected family members). A denaturing high-performance liquid chromatography-based assay was developed to screen for mutations in all 31 exons, eliminating pseudogene coamplification. In 134 patients with a known phenotype and both mutations identified, genotype-phenotype correlations were assessed. RESULTS: In total, 316 mutant alleles in ABCC6, including 39 novel mutations, were identified in 239 probands. Mutations were found to cluster in exons 24 and 28, corresponding to the second nucleotide-binding fold and the last intracellular domain of the protein. Together with the recurrent R1141X and del23-29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Genotype-phenotype analysis failed to reveal a significant correlation between the types of mutations identified or their predicted effect on the expression of the protein and the age of onset and severity of the disease. CONCLUSIONS: This study emphasises the principal role of ABCC6 mutations in the pathogenesis of PXE, but the reasons for phenotypic variability remain to be explored.

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187 Together with the recurrent R1141X and del23-29 mutations, these mutations accounted for 71.5% of the total individual mutations identified. Login to comment
200 Two recurrent mutations, R1141X and a large deletion of exons 23-29 (del23-29, p.A999_S1403del) have been described in a significant proportion of patients, leading to a mutation-detection strategy that first identifies the recurrent mutations by restriction-enzyme digestion, followed by sequencing of the remaining exons.39 In this study, we collected genotype data on 270 patients with PXE from 239 families and were able to characterise the phenotype in 198 of these patients to improve diagnosis, identify genotype-phenotype associations and facilitate genetic counselling of people at risk for PXE. Login to comment
211 Our mutation-detection strategy was based on: (1) identification of the recurrent mutations R1141X and del23-29 by restriction-enzyme digestion; (2) optimised denaturing high-performance liquid chromatography (dHPLC) scanning of PCR products corresponding to all exons in subjects in whom the two recurrent mutations were not identified on both alleles, followed by (3) sequencing of exons with altered dHPLC patterns; and (4) confirmation of novel mutations by restriction-enzyme digestion or resequencing. Login to comment
212 Screening for the recurrent mutations R1141X and del23-29 was performed as previously described.30 31 Conditions and primers for generating PCR products spanning all exons of the coding regions and flanking intronic sequences of the ABCC6 gene were identified for optimum dHPLC screening (supplementary table 1; available at http://jmg.bmj.com/supplemental). Login to comment
241 The two most common mutations were R1141X (29.3%, 92/316) and del23-29 (18%, 57/316). Login to comment
242 In all, 23 subjects (9.6%) were homozygous for either the R1141X (n = 11) or del23-29 (n = 2) or compound heterozygous (n = 10) for these two mutations. Login to comment
254 Collectively, the mutations in exons 24 and 28, including the common mutations R1141X and del 23-29, accounted for 71.5% of all the 316 mutations identified in this study (table 2), and the 11 most prevalent mutations (R1141X, del23-29, R1339C, R1164X, 2787+1GRT, G1302R, R1138Q, R1138W, Q378X, R1314W, R518Q) accounted for 70% (223 of 316) of the mutant alleles identified (table 2). Login to comment
288 The recurrent mutations R1141X and del23-29 were the most prevalent, consistent with previous reports. Login to comment
262 Genotype-phenotype correlations The comparison of subjects whose mutations would probably have resulted in no functional protein with those whose mutations would probably have resulted in some functional Table 2 Distinct mutations identified in the international case series of 271 patients with PXE Nucleotide change*À Predicted consequenceÀ Frequency (alleles) Exon-intron location Domain affected` Mutant alleles (%) References1 c.105delA p.S37fsX80 2 2 0.6 28 c.177-185del9 p.R60_Y62del 1 2 0.3 9, 28 c.179del12ins3 p. R60_W64del L60_R61ins 1 2 0.3 c.220-1gRc SJ 1 IVS 2 0.3 c.724gRt p.E242X 1 7 0.3 c.938insT FS 1 8 0.3 25 c.998+2delT SJ 1 IVS 8 0.3 2, 21 c.998+2del2 SJ 1 IVS 8 0.3 18 c.951cRg p.S317R 2 9 TM6 0.6 28 c.1087cRt p.Q363X 1 9 0.3 c.1091gRa p.T364R 1 9 TM7 0.3 9, 19, 21, 28 c.1132cRt p.Q378X 4 9 1.2 9, 17-19, 28, 37 c.1144cRt p.R382W 2 9 IC4 0.6 c.1171aRg p.R391G 3 9 IC4 0.9 9, 18, 28, 37 c.1176gRc p.K392N 1 9 IC4 0.3 c.1388tRa p.L463H 1 11 TM9 0.3 c.1484tRa p.L495H 1 12 IC5 0.3 28 c.1552cRt p.R518X 2 12 0.6 18, 19, 27, 28, 37 c.1553gRa p.R518Q 4 12 IC5 1.2 18, 19, 24, 28, 31 c.1603tRc p.S535P 1 12 TM10 0.3 c.1703tRc p.F568S 1 13 TM11 0.3 24 c.1798cRt p.R600C 1 14 TM11 0.3 c.1857insC FS 1 14 0.3 c.1987gRt p.G663C 1 16 NBF1 0.3 c.1999delG FS 1 16 0.3 c.2070+5GRA SJ 2 IVS 16 0.6 c.2093aRc p.Q698P 2 17 NBF1 0.6 c.2097gRt p.E699D 1 17 NBF1 0.3 c.2177tRc p.L726P 1 17 NBF1 0.3 c.2237ins10 FS 2 17 0.6 c.2252tRa p.M751K 1 18 NBF1 0.3 20, 37 c.2263gRa p.G755R 2 18 NBF1 0.6 c.2278cRt p.R760W 3 18 NBF1 0.9 20, 28, 32, 37 c.2294gRa p.R765Q 2 18 NBF1 0.6 20-22, 25, 28, 32, 37 c.2329gRa p.D777N 1 18 NBF1 0.3 c.2359gRt p.V787I 1 18 NBF1 0.3 c.2432cRt p.T811M 1 19 IC6 0.3 6 c.2643gRt p.R881S 1 20 IC6 0.3 c.2787+1GRT SJ 9 IVS 21 2.8 17, 20, 24, 28, 31, 37 c.2814cRg p.Y938X 1 22 0.3 c.2820insC FS 1 22 0.3 c.2831cRt p.T944I 1 22 TM12 0.3 c.2848gRa p.A950T 1 22 TM12 0.3 c.2974gRc p.G992R 1 22 TM13 0.3 2, 42 c.3340cRt p.R1114C 2 24 IC8 0.6 19, 28, 32, 37, 41 c.3389cRt p.T1130M 3 24 IC8 0.9 18, 19, 21, 22, 28, 30, 32, 37, 41 c.3398gRc p.G1133A 1 24 IC8 0.3 c.3412gRa p.R1138W 7 24 IC8 2.2 28, 30, 37 c.3413cRt p.R1138Q 7 24 IC8 2.2 18, 19, 24, 25, 28, 30, 32, 37, 41 c.3415gRa p.A1139T 2 24 IC8 0.6 c.3415gRa & c.2070+5GRA* p.A1139T & SJ 1 24, IVS 16 IC8 0.3 c.3415gRa & c.4335delG* p.A1139T & FS 1 24, 30 IC8 0.3 c.3421cRt p.R1141X 92 24 29.3 5, 9, 15,18, 19, 21, 22, 24, 28, 30-32, 33, 37, 41 c.3427cRt p.Q1143X 1 24 0.3 c.3490cRt p.R1164X 15 24 4.7 18, 27, 28, 31, 33 c.3491gRa p.R1164Q 1 24 IC8 0.3 28 c.3661cRt p.R1221C 1 26 IC9 0.3 21, 22, 28, 29 c.3662gRa p.R1221H 2 26 IC9 0.6 40 c.3676cRa p.L1226I 1 26 IC9 0.3 c.3722gRa p.W1241X 2 26 0.6 c.3774insC FS 2 27 0.6 c.3775delT p.G1259fsX1272 3 27 0.9 15, 25, 28, 41 c.3880-3882del p.K1294del 1 27 0.3 c.3883-5GRA SJ 1 IVS 27 0.3 c.3892gRt p.V1298F 1 28 NBF2 0.3 25 c.3904gRa p.G1302R 7 28 NBF2 2.2 21, 22, 25, 28 c.3907gRc p.A1303P 1 28 NBF2 0.3 21, 22, 25, 28 c.3912delG FS 1 28 0.3 28 c.3940cRt p.R1314W 4 28 NBF2 1.2 24, 25, 32, 36 c.3941gRa p.R1314Q 1 28 NBF2 0.3 25, 28, 32, 36, 41 c.4004tRa p.L1335Q 1 28 NBF2 0.3 c.4015cRt p.R1339C 16 28 NBF2 5.0 19, 25, 28, 33 c.4016gRa p.R1339H 2 28 NBF2 0.6 c.4025tRc p.I1342T 1 28 NBF2 0.3 protein did not yield significant differences. Login to comment

[hide] Pseudoxanthoma elasticum-like fibers in the inflam... J Cutan Pathol. 2007 Oct;34(10):777-81. Bowen AR, Gotting C, LeBoit PE, McCalmont TH
Pseudoxanthoma elasticum-like fibers in the inflamed skin of patients without pseudoxanthoma elasticum.
J Cutan Pathol. 2007 Oct;34(10):777-81., [PMID:17880583]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is an inherited disorder leading to characteristic calcified elastic fibers in skin, eyes and vasculature. PXE-like fibers have not been described in inflammatory skin disease in the absence of other signs of PXE. METHODS: The histopathology of inflamed skin from 13 patients that contained PXE-like fibers but lacked clinical evidence of PXE were studied. Six of these and six comparison specimens from known patients with PXE were subjected to polymerase chain reaction amplification and sequencing of exons 24 and 28 of the PXE-associated gene ABCC6. This genetic analysis employed a novel assay utilizing paraffin-embedded tissue. RESULTS: Incidental PXE-like fibers were found in patients without clinical suspicion of PXE in lesional tissue showing lipodermatosclerosis, granuloma annulare, lichen sclerosus, morphea profunda, erythema nodosum, septal panniculitis, basal cell carcinoma and fibrosing dermatitis. Two patients with PXE-like fibers but without clinical findings of PXE were heterozygous for a PXE-associated ABCC6 sequence alteration. CONCLUSIONS: This pilot study shows elastic fibers similar to those of PXE in the lesional skin of patients with a variety of inflammatory skin diseases in the absence of clinical evidence of PXE; and some of these patients harbor changes in ABCC6.

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42 The analysis of the c.3421C.T (R1141X) mutation through rapid-cycle PCR and hybridization analysis with the LightCycler (Roche, Penzberg, Germany) was performed as described previously,7 with the following modifications: (1) an improved primer mixture containing the oligonucleotides E24LCU (5#-CTCCCATCCATCCTTCT-3#), E24LCL (5#-CCTCGCTACCATACAATATGA-3#) and Ex24LCL_T (5#-CCTCTCTACCATACAA- TATGA-3#) in equal amounts was used in the assay to improve genotyping reliability in patients carrying the intronic polymorphism IVS24 1 83C.A in the ABCC6 gene, (2) 3 ml of DNA isolated from paraffin-embedded tissues was used in the reaction mixture (20 ml) and (3) amplification was performed using 50 cycles of 95°C for 1 s, 56°C for 10 s and 72°C for 20 s to increase signal intensity. Login to comment

[hide] Novel clinico-molecular insights in pseudoxanthoma... Hum Mutat. 2008 Jan;29(1):205. Vanakker OM, Leroy BP, Coucke P, Bercovitch LG, Uitto J, Viljoen D, Terry SF, Van Acker P, Matthys D, Loeys B, De Paepe A
Novel clinico-molecular insights in pseudoxanthoma elasticum provide an efficient molecular screening method and a comprehensive diagnostic flowchart.
Hum Mutat. 2008 Jan;29(1):205., [PMID:18157818]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder characterized by ocular, cutaneous and cardiovascular manifestations. It is caused by mutations in the ABCC6 gene (chr. 16p13.1), encoding a transmembrane transporter protein, the substrate and biological function of which are currently unknown. A comprehensive clinical and molecular study of 38 Belgian PXE probands and 21 relatives (4 affected and 17 carriers) was performed. An extensive clinical evaluation protocol was implemented with serial fundus, skin and cardiovascular evaluation. We report on 14 novel mutations in the ABCC6 gene. We observed extensive variability in severity of both cutaneous and ocular lesions. The type of skin lesion however usually remained identical throughout the evolution of the disorder, while ophthalmological progression was mainly due to functional decline. Peripheral artery disease (53%) and stroke (15%) were significantly more prevalent than in the general population (10-30% and 0.3-0.5% respectively). Interestingly, we also observed a relatively high incidence of subclinical peripheral artery disease (41%) in our carrier population. We highlight the significance of peripheral artery disease and stroke in PXE patients as well as the subclinical manifestations in carriers. Through follow-up data we gained insight into the natural history of PXE. We propose a cost- and time-efficient two-step method of ABCC6 analysis which can be used in different populations. Additionally, we created a diagnostic flowchart and attempted to define the role of molecular analysis of ABCC6 in the work-up of a PXE patient.

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127 12-001(II-1) C F I:1 I:2 II:1 II:2 II:3II:4 III:1 III:2 II:5 III:3 III:4 R1141X/ WT R1141X/ WT R1141X/ WT R1141X/ WT R1141X/ WT R1141X/ R1141X D E A B R1141X/ R1141X R1141X/ WT 12-002(II-3) Table 2. Prevalence of the Cutaneous (A) and Ocular (B) Manifestations of PXE in This Study at Initial Presentation and at Last Consultation Initial presentation Last consultation A. Login to comment
28 One is the nonsense mutation c.3421C>T (p.R1141X), which, in a homozygous state, leads to complete loss of ABCC6 function and is more frequent among the European population [Chassaing et al., 2005]. Login to comment
78 Mutations were most frequently located in exons 24 (47%), mainly due to the p.R1141X mutation, 18 (8%), 28 (7%) and 29 (5%). Login to comment
80 The nonsense mutation c.3421C>T (p.R1141X) in exon 24 was most frequently found (31/76 alleles or 41%), both in homozygous (52%) and compound heterozygous (43%) state. Login to comment
83 Genotype and Phenotype of 42 Belgian PXE Patients Patient S e x Age/Clinical score at initial presentation Age/Clinical score at most recent follow-up Mutations* Allele 1 Allele 2 01-001 F 52 - S0, E2 65 - S0, E3, HT p.R1141X c.3421C>T p.R760Q c.2279G>A 02-001 M 18 - S1, E2, VR-I 18 - S1, E2, VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 03-001 F 59 - S1, E4 75 - S1, E4, HT, IC, VR-I p.R1141X c.3421C>T p.N793L c.2379C>G 04-001 F 36 - S3, E2 36 - S3, E2 p.N466Y c.1396A>T p.R1339H c.4016G>A 05-001 F 26 - S1, E4 43 - S3, E4, VR-I p.R1141X c.3421C>T p.T364M c.1091C>T 06-001 F 36 - S2, E4 44 - S2, E4, P p.A1303P c.3907G>C None found - 07-001 M 48 - S1, E2, HT 58 - S1, E4, HT p.R1141X c.3421C>T p.R1141X c.3421C>T 08-001 F 26 - S1, E0 44 - S2, E2 p.R1141X c.3421C>T p.R760Q c.2279G>A 09-001 M 49 - S0, E3, P, GIB 65 - S2, E4, P, HT, VR-I, GIB p.A1303P c.3907G>C None found - 10-001 F 46 - S1, E2 63 - S3, E4, HT, AP,VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 11-001 M 25 - S1, E2, GIB 37 - S1, E3, GIB p.R1141X c.3421C>T None found - 12-001 F 52 - S1, E4, CI, HT, VR-I 52 - S1, E4, IC, HT, VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 12-002 F 40 - S1, E2, HT, MVP, VR-I 40 - S1, E2, HT, MVP, VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 13-001 F 65 - S0, E2 80 - S0, E2, P, VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 13-002 F 57 - S3, E4 73 - S3, E4, HT, CI, VR-I p.R1141X c.3421C>T p.R1141X c.3421C>T 14-001 F 23 - S1, E2 27 - S1, E2 p.S398R c.1194C>G - c.3364delT 15-001 F 27 - S1, E2 27 - S1, E2 p.R1138W c.3412C>T p.R1221H c.3662G>A 16-001 M 51 - S2, E2 54 - S2, E2 p.R1141X c.3421C>T p.R1141X c.3421C>T 17-001 M 42 - S1, E3, IC 58 - S1, E3, IC Del23-29 - p.R518Q c.1553G>A 18-001 M 63 - S1, E4 63 - S1, E4 p.E1400K c.4198G>A None found - 19-001 F 34 - S2, E2 50 - S2, E2 p.A1303P c.3907G>C p.R1398X c.4192C>T 20-001 F 52 - S2, E2, HT, IC, GIB 68 - S2, E4, HT, IC, GIB p.R1141X c.3421C>T None found - 21-001 M 20 - S1, E2 26 - S1, E2 p.R1141X c.3421C>T p.R1141X c.3421C>T 22-001 M 53 - S2, E2, IC, AP 69 - S2, E2, HT, IC, AP p.M751K c.2252T>A p.R1164Q c.3491G>A 23-001 F 20 - S1, E2 27 - S1, E2, P, VR-I p.G666V c.1996G>T - c.1868-5T>G 24-001 M 54 - S1, E2 57 - S1, E2 p.T500P c.1498A>C p.E521D c.1563G>C 25-001 F 50 - S1, E3, HT, MI 57 - S2, E3, HT, MI p.R1141X c.3421C>T p.R1141X c.3421C>T 26-001 M 52 - S2, E4, HT 68 - S2, E4, HT, CI p.M751K c.2252T>A Del23-29 - 27-001 F 61 - S3, E4 68 - S3, E4, P, CI, AP p.R1141X c.3421C>T - c.4104delC Allele 2 28-001 F 31 - S1, E2 32 - S1, E2 - c.1674DelC p.R765W c.2293C>T Patient S e x Age/Clinical score at initial presentation Age/Clinical score at most recent follow-up Mutations* Allele 1 Allele 2 29-001 M 30 - S1, E3 32 - S1, E3 p.E125K c.373G>A p.L1025P c.3074T>C 30-001 M 65 - S0, E2, HT, CI, MI 66 - S0, E2, HT, CI, MI p.G1405S c.4213G>A None found - 31-001 F 38 - S1, E4 39 - S1, E4 p.R1141X c.3421C>T Del23-29 - 32-001 M 22 - S1, E2 36 - S1, E2 p.R1141X c.3421C>T p.R518Q c.1553G>A 33-001 F 45 - S2, E3, P 61 - S2, E3, P, VR-II p.R1141X c.3421C>T p.R1141X c.3421C>T 34-001 F 65 - S1, E4, HT 81 - S1, E4, HT, AP p.R1141X c.3421C>T p.T1301I c.3902C>T 35-001 F 62 - S2, E2 78 - S2, E2, HT - c.175_179del p.G1354R c.4060G>C 35-002 F 58 - S2, E2 74 - S2, E4 - c.175_179del p.G1354R c.4060G>C 35-003 M 67 - S2, E2 79 - S2, E3, HT, VR-I - c.175_179del p.G1354R c.4060G>C 36-001 M 53 - S1, E4 59 - S1, E4, HT, AP p.R1114H c.3341G>A p.Q1237X c.3709C>T 37-001 M 18 - S3, E2 18 - S3, E2 p.Q981H c.2943G>T - c.3507-3C>A 38-001 F 27 - S1, E2 27 - S1, E2 p.G1263R c.3787G>A - c.4182delG Table 1 represents the sex of all patients (M = male; F= female) and the age (in years - italics), respectively at initial presentation and last follow-up. Login to comment
141 Especially in patients homozygous for the p.R1141X mutation - previously associated with a more severe phenotype [Hu et al., 2003]-, no significant difference with patients with either one or no nonsense mutation was observed. Login to comment
145 An initial analysis can be limited to the latter four exons (including p.R1141X) and the Del23-29 with a direct sequencing based approach. Login to comment

[hide] ABCC6 mutations in pseudoxanthoma elasticum: an up... Mol Vis. 2008 Jan 24;14:118-24. Plomp AS, Florijn RJ, Ten Brink J, Castle B, Kingston H, Martin-Santiago A, Gorgels TG, de Jong PT, Bergen AA
ABCC6 mutations in pseudoxanthoma elasticum: an update including eight novel ones.
Mol Vis. 2008 Jan 24;14:118-24., [PMID:18253096]

Abstract [show]
PURPOSE: Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder of connective tissue, affecting the retina, the skin, and the cardiovascular system. PXE is caused by mutations in ABCC6. Up to now, the literature reports that there are 180 different ABCC6 mutations in PXE. The purpose of this paper is to report eight novel mutations in ABCC6 and to update the spectrum and frequency of ABCC6 mutations in PXE patients. METHODS: Eye, skin, and DNA examinations were performed using standard methodologies. We newly investigated the gene in 90 probands by denaturing high-performance liquid chromatography (dHPLC) and direct sequencing. We examined a total of 166 probands. RESULTS: Eight novel ABCC6 mutations (c.1685T>C, p.Met562Thr; c.2477T>C, p.Leu826Pro; c.2891G>C, p.Arg964Pro; c.3207C>A, p.Tyr1069X; c.3364delT, p.Ser1122fs; c.3717T>G, p.Tyr1293X; c.3871G>A, p.Ala1291Thr; c.4306_4312del, p.Thr1436fs) were found in seven unrelated patients. Currently, our mutation detection score is at least one ABCC6 mutation in 87% of patients with a clinical diagnosis of PXE. CONCLUSIONS: Our results support that ABCC6 is the most important, and probably the only, causative gene of PXE. In total, 188 different ABCC6 mutations have now been reported in PXE in the literature.

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265 Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum. Login to comment

[hide] Mutations in the GGCX and ABCC6 genes in a family ... J Invest Dermatol. 2009 Mar;129(3):553-63. Epub 2008 Sep 18. Li Q, Grange DK, Armstrong NL, Whelan AJ, Hurley MY, Rishavy MA, Hallgren KW, Berkner KL, Schurgers LJ, Jiang Q, Uitto J
Mutations in the GGCX and ABCC6 genes in a family with pseudoxanthoma elasticum-like phenotypes.
J Invest Dermatol. 2009 Mar;129(3):553-63. Epub 2008 Sep 18., [PMID:18800149]

Abstract [show]
A characteristic feature of classic pseudoxanthoma elasticum (PXE), an autosomal recessive disorder caused by mutations in the ABCC6 gene, is aberrant mineralization of connective tissues, particularly the elastic fibers. Here, we report a family with PXE-like cutaneous features in association with multiple coagulation factor deficiency, an autosomal recessive disorder associated with GGCX mutations. The proband and her sister, both with severe skin findings with extensive mineralization, were compound heterozygotes for missense mutations in the GGCX gene, which were shown to result in reduced gamma-glutamyl carboxylase activity and in undercarboxylation of matrix gla protein. The proband's mother and aunt, also manifesting with PXE-like skin changes, were heterozygous carriers of a missense mutation (p.V255M) in GGCX and a null mutation (p.R1141X) in the ABCC6 gene, suggesting digenic nature of their skin findings. Thus, reduced gamma-glutamyl carboxylase activity in individuals either compound heterozygous for a missense mutation in GGCX or with haploinsufficiency in GGCX in combination with heterozygosity for ABCC6 gene expression results in aberrant mineralization of skin leading to PXE-like phenotype. These findings expand the molecular basis of PXE-like phenotypes, and suggest a role for multiple genetic factors in pathologic tissue mineralization in general.

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31 Patient (III-3) Patient (III-3) Patient (III-3) Patient (II-2) Patient (II-3) Patient (II-3) a b c d e f g Skin Coagulation GGCX ABCC6 GGCX ABCC6 V255M S300F R1141X V255M S300F R1141X 1 1 2 3 4 3 2 21 5 II I III - -+ + + - - - - + + + + + + + + + Figure 1. Login to comment
3 The proband`s mother and aunt, also manifesting with PXE-like skin changes, were heterozygous carriers of a missense mutation (p.V255M) in GGCX and a null mutation (p.R1141X) in the ABCC6 gene, suggesting digenic nature of their skin findings. Login to comment
41 The recurrent mutation, p.R1141X, which is particularly prevalent (about 30% of all detected mutations in PXE patients of the European extraction; Pfendner et al., 2007) was found in the DNA from the father, mother, the maternal aunt and the younger brother, but not in the proband herself or her sister (Figures 1g and 3a). Login to comment
64 The latter individuals were also carriers of the ABCC6 nonsense mutation p.R1141X. Login to comment
65 Specifically, the mother and her twin sister were heterozygous for the GGCX missense mutation p.V255M and the ABCC6 nonsense mutation p.R1141X, suggesting digenic inheritance of their cutaneous findings. Login to comment
66 However, the proband`s younger brother and father were heterozygous carriers of the p.S300F mutation in the GGCX gene although they also carried the p.R1141X mutation in the ABCC6 gene; they did not display any signs of cutaneous findings or hematologic disorder. Login to comment
67 Assay of c-glutamyl carboxylase activity Previous studies have clearly demonstrated that the p.R1141X mutation in the ABCC6 gene in heterozygous carriers does not cause PXE (Wegman et al., 2005; Pfendner et al., 2007). Login to comment
77 (a) Identification of the recurrent nonsense mutation p.R1141X in the ABCC6 gene. Login to comment
131 The recurrent mutation p.R1141X was found in the DNA from the father, mother, the maternal aunt and the younger brother, but not in the proband herself or her sister. Login to comment
140 The latter individuals were also carriers of the ABCC6 nonsense mutation p.R1141X. Login to comment
141 It should be noted that the mother and her twin sister were heterozygous for one of the GGCX missense mutation p.V255M and one ABCC6 nonsense mutation p.R1141X, suggesting digenic inheritance of their cutaneous findings. Login to comment
144 It should be noted, however, that in a cohort of 4300 families with PXE analyzed in our laboratory for mutations in the ABCC6 gene, five individuals were heterozygous carriers of a mutation (p.R1141X) in one ABCC6 allele only, yet none of them carried a mutation in the GGCX gene (unpublished). Login to comment
163 Mutations in the ABCC6 gene have been shown to cause PXE, an autosomal recessive disorder (Ringpfeil et al., 2006), but heterozygous carriers of mutations, such as p.R1141X detected in the proband`s mother and aunt, are asymptomatic (Wegman et al., 2005; Pfendner et al., 2007). Login to comment
170 These two individuals were heterozygous carriers of p.R1141X mutation in ABCC6 and p.V255M in GGCX. Login to comment
171 As heterozygous carriers of p.R1141X in ABCC6 alone do not manifest PXE and GGCX mutations with respect to coagulation disorder are recessive, these findings suggest that the skin phenotype in these two individuals may be because of digenic inheritance. Login to comment
173 The reasons for the fact that the proband`s father and her brother were heterozygous carriers of mutations in the ABCC6 gene (p.R1141X) and the GGCX gene (p.S300F) yet did not display any cutaneous findings are not clear. Login to comment

[hide] Gene expression profiling of ABC transporters in d... Lab Invest. 2008 Dec;88(12):1303-15. Epub 2008 Oct 20. Hendig D, Langmann T, Kocken S, Zarbock R, Szliska C, Schmitz G, Kleesiek K, Gotting C
Gene expression profiling of ABC transporters in dermal fibroblasts of pseudoxanthoma elasticum patients identifies new candidates involved in PXE pathogenesis.
Lab Invest. 2008 Dec;88(12):1303-15. Epub 2008 Oct 20., [PMID:18936737]

Abstract [show]
Mutations in the ABCC6 gene, encoding the multidrug resistance-associated protein 6 (MRP6), cause pseudoxanthoma elasticum (PXE). This heritable disorder leads to pathological alterations in connective tissues. The implication of MRP6 deficiency in PXE is still unknown. Moreover, nothing is known about a possible compensatory expression of other ATP binding-cassette (ABC) transporter proteins in MRP6-deficient cells. We investigated the gene expression profile of 47 ABC transporters in human dermal fibroblasts of healthy controls (n=2) and PXE patients (n=4) by TaqMan low-density array. The analysis revealed the expression of 37 ABC transporter genes in dermal fibroblasts. ABCC6 gene expression was not quantifiable in fibroblasts derived from PXE patients. Seven genes (ABCA6, ABCA9, ABCA10, ABCB5, ABCC2, ABCC9 and ABCD2) were induced, whereas the gene expression of one gene (ABCA3) was decreased, comparing controls and PXE patients (with at least twofold changes). We reanalyzed the gene expression of selected ABC transporters in a larger set of dermal fibroblasts from controls and PXE patients (n=6, each). Reanalysis showed high interindividual variability between samples, but confirmed the results obtained in the array analysis. The gene expression of ABC transporter genes, as well as lineage markers of PXE, was further examined after inhibition of ABCC6 gene expression by using specific small-interfering RNA. These experiments corroborated the observed gene expression alterations, most notably in the ABCA subclass (up to fourfold, P<0.05). We therefore conclude that MRP6-deficient dermal fibroblasts exhibit a distinct gene expression profile of ABCA transporters, potentially to compensate for MRP6 deficiency. Moreover, our results point to a function for ABCC6/MRP6 in sterol transport, as sterols are preferential regulators of ABCA transporter activity and expression. Further studies are now required to uncover the role of ABCA transporters in PXE.

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305 Hu X, Peek R, Plomp A, et al. Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum. Login to comment
62 Table 1 Main characteristics of dermal fibroblasts derived from PXE patients and healthy controls used in the present study Sample ID Gender Agea Biopsy source ABCC6 genotypeb Statusc Age at disease onseta Number of involved organs PXE patients P60F Female 58 Axilla c.37-1G4A (SSM) c.37-1G4A (SSM) hm 56 3 P229F Female 50 NA c.1171A4G (p.R391G) c.1208C4A (p.A413N) c.2252T4A (p.M751K) cht NA NA P265F Female 62 Cervix c.1132C4T (p.Q378X) c.3421C4T (p.R1141X) cht 16 3 P3M Male 57 Cervix c.3421C4T (p.R1141X) c.3883-6G4A (SSM) cht 46 5 P128M Male 51 Cervix c.3769_3770insC (p.L1259fsX1277) c.3769_3770insC (p.L1259fsX1277) hm 48 3 P308M Male 42 NA c.3421C4T (p.R1141X) c.-90ins14 (c)ht NA NA P341M Male 41 NA c.1552C4T (p.R518X) ND ht NA NA Healthy controls F37A Female 37 Abdomen - - - wt - - F42A Female 42 Abdomen - - - wt - - F52C Female 52 Cheek - - - wt - - M2FS Male 2 Foreskin - - - wt - - M45D Male 45 Face - - - wt - - M56D Male 56 Face - - - wt - - hm, homozygote; cht, compound heterozygote; ht, heterozygote; wt, wild type; SSM, splice site mutation; NA, not applicable; ND, nondetected. Login to comment

[hide] Pseudoxanthoma elasticum: genetics, clinical manif... Surv Ophthalmol. 2009 Mar-Apr;54(2):272-85. Finger RP, Charbel Issa P, Ladewig MS, Gotting C, Szliska C, Scholl HP, Holz FG
Pseudoxanthoma elasticum: genetics, clinical manifestations and therapeutic approaches.
Surv Ophthalmol. 2009 Mar-Apr;54(2):272-85., [PMID:19298904]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an inherited disorder that is associated with accumulation of mineralized and fragmented elastic fibers in the skin, vascular walls, and Bruch's membrane in the eye. Clinically, patients exhibit characteristic lesions of the posterior segment of the eye including peau d'orange, angioid streaks, and choroidal neovascularisations, of the skin including soft, ivory-colored papules in a reticular pattern that predominantly affect the neck and large flexor surfaces, and of the cardiovascular system with peripheral and coronary arterial occlusive disease as well as gastrointestinal bleedings. There is yet no definitive therapy. Recent studies suggest that PXE is inherited almost exclusively as an autosomal recessive trait. Its prevalence has been estimated to be 1:25,000-100,000. Very recently, the ABCC6 gene on chromosome 16p13.1 was found to be associated with the disease. Mutations within ABCC6 cause reduced or absent transmembraneous transport that leads to accumulation of extracellular material. Presumably, this mechanism causes calcification of elastic fibers. Despite the characteristic clinical features, the variability in phenotypic expressions, and the low prevalence may be responsible for the disease being underdiagnosed. This review compiles and summarizes current knowledge of PXE pathogenesis and clinical findings. Furthermore, different therapeutic strategies to treat retinal manifestations are discussed, including thermal laser coagulation, photodynamic therapy, and intravitreal injections of drugs inhibiting vascular endothelial growth factor.

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21 The exact prevalence is unknown, with a recent estimated range between 1:25,000 and 1:100,000.21 Prevalence seems to be higher in South Africa compared to other regions, possibly due to a founder effect.28 Applying the Hardy-Weinberg equilibrium, this leads to a frequency of 1.25% heterozygotes (1:80).21 In 2002, a prevalence of 3% of heterozygous carriers of the R1141X mutation of the ABCC6 gene was reported in a Dutch population of young patients affected by coronary heart disease. Login to comment

[hide] Spectrum of genetic variation at the ABCC6 locus i... J Dermatol Sci. 2009 Jun;54(3):198-204. Epub 2009 Mar 31. Ramsay M, Greenberg T, Lombard Z, Labrum R, Lubbe S, Aron S, Marais AS, Terry S, Bercovitch L, Viljoen D
Spectrum of genetic variation at the ABCC6 locus in South Africans: Pseudoxanthoma elasticum patients and healthy individuals.
J Dermatol Sci. 2009 Jun;54(3):198-204. Epub 2009 Mar 31., [PMID:19339160]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is an autosomal recessive metabolic disorder with ectopic mineralization in the skin, eyes and cardiovascular system. PXE is caused by mutations in ABCC6. OBJECTIVE: To examine 54 unrelated South African PXE patients for ABCC6 PXE causing mutations. METHODS: Patients were screened for mutations in ABCC6 using two strategies. The first involved a comprehensive screening of all the ABCC6 exons and flanking regions by dHPLC or sequencing whereas the second involved screening patients only for the common PXE mutations. The ABCC6 gene was screened in ten white and ten black healthy unrelated South Africans in order to examine the level of common non-PXE associated variation. RESULTS: The Afrikaner founder mutation, R1339C, was present in 0.41 of white ABCC6 PXE alleles, confirming the founder effect and its presence in both Afrikaans- (34/63 PXE alleles) and English-speakers (4/28). Eleven mutations were detected in the white patients (of European origin), including two nonsense mutations, 6 missense mutations, two frameshift mutations and a large deletion mutation. The five "Coloured" patients (of mixed Khoisan, Malay, European and African origin) included three compound heterozygotes with R1339C as one of the mutations. The three black patients (sub-Saharan African origin) were all apparent homozygotes for the R1314W mutation. Blacks showed a trend towards a higher degree of neurtral variation (18 variants) when compared to whites (12 variants). CONCLUSION: Delineation of the ABCC6 mutation profile in South African PXE patients will be used as a guide for molecular genetic testing in a clinical setting and for genetic counselling.

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80 Exons 12, 18 and 24 were PCR amplified and sequenced to detect the R518Q, Y768X, R1141X and R1138Q mutations and the common del23-29 mutation was detected as previously described [16]. Login to comment
98 The next three more common mutations are R1141X (0.09), Y768X (0.08) and R1138Q (0.06). Login to comment
121 of alleles with mutation Frequency R1339C c.4015C>T 28 NBF2 18 0.375 21 0.447 0.411 (39) [30-32] R1138Q c.3413G>A 24 CL8 5 0.104 1 0.021 0.063 (6) [3,7,32] Y768X c.2304C>A 18 NBF1 5 0.104 3 0.064 0.084 (8) [31] R1141X c.3421C>T 24 CL8 4 0.083 5 0.106 0.095 (9) [3,6,13] R518Q c.1553G>A 12 CL5 2 0.042 1 0.021 0.032 (3) [31-33] Deletion ABCC6del23-29 23-29 - 1 0.021 2 0.043 0.032 (3) [6,16,31,32] L1335P c.4004T>C 28 NBF2 2 0.042 - - Other = 0.063 (6) [3] G1302R c.3904G>A 28 NBF2 1 0.021 - - [31] L726P c.2177T>C 17 NBF1 1 0.021 - - This Study Frameshift c.3775delT 27 CL9 1 0.021 - - [6] Frameshift c.4104delC 29 NBF2 1 0.021 - - [31] Unknown - - - 7 0.146 14 0.298 0.221 (21) - Total 48 47 Mutation detection rate 0.855 0.702 0.717 a ''Coloured``, black and Indian patients are excluded from the table because of the small sample size. Login to comment
145 Mutation Afrikaans-speakersa English-speakersa No. Frequency No. Frequency R1339C 34 0.54 4 0.14 R1138Q 5 0.08 1 0.04 Y768X 5 0.08 3 0.11 R1141X 5 0.08 4 0.14 R518Q 1 0.02 2 0.07 del23-29 0 - 3 0.11 Other 0 - 4 0.14 Unknown 13 0.21 7 0.25 Total no. alleles 63 28 a Only 2 families were bilingual Afrikaans/English speaking and not included in this table. Login to comment
160 Three further mutations have frequencies above 0.06, namely R1138Q, Y768X and R1141X. Login to comment
195 It detects 79.2% of the PXE mutations in white South African patients including: R1339C, R1138Q, Y768X, R1141X, R518Q, del23-29, L1335P and G1302R (calculated according to detection Strategy 1, Table 1). Login to comment

[hide] The R1141X loss-of-function mutation of the ABCC6 ... Genet Test Mol Biomarkers. 2010 Feb;14(1):75-8. Koblos G, Andrikovics H, Prohaszka Z, Tordai A, Varadi A, Aranyi T
The R1141X loss-of-function mutation of the ABCC6 gene is a strong genetic risk factor for coronary artery disease.
Genet Test Mol Biomarkers. 2010 Feb;14(1):75-8., [PMID:19929409]

Abstract [show]
Loss-of-function mutations of ABCC6 cause pseudoxanthoma elasticum (PXE). This Mendelian disorder is characterized by elastic calcification leading to dermal, ocular, and cardiovascular symptoms like coronary artery disease (CAD) and stroke. Although PXE is a recessive disease, microscopic dermal lesions, serum alterations, and higher anecdotal incidence of stroke or CAD among carriers were reported. Here we investigated the association of the c.3421C>T loss-of-function mutation of ABCC6 and CAD and stroke. A previous study demonstrated the association of the c.3421C>T mutation with CAD; however, the frequency found in the control population was unexpectedly high, contradicting, thus, the prevalence of PXE. In the present study, genomic DNA from 749 healthy blood donors was used as control, while 363 and 361 patients suffering from stroke and CAD were investigated, respectively. One carrier was found in our control group, which is in accordance with the reported prevalence of this mutation. No significant association was found between carrier status and stroke in our cohort. In contrast, a significant association of carrier status and CAD was observed (5/361 carriers: p = 0.016, odds ratio [OR] = 10.5). We propose that carriers of ABCC6 loss-of-function mutations benefit from CAD prevention therapy.

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0 The R1141X Loss-of-Function Mutation of the ABCC6 Gene Is a Strong Genetic Risk Factor for Coronary Artery Disease Gabriella Ko¨blo¨s,1 Hajnalka Andrikovics,2 Zolta´n Proha´szka,3 Attila Tordai,2 Andra´s Va´radi,1 and Tama´s Ara´nyi1 Loss-of-function mutations of ABCC6 cause pseudoxanthoma elasticum (PXE). Login to comment
13 The most frequent mutation, accounting for 20-30% of all mutations, is the c.3421C>T nucleotide change leading to the R1141X nonsense mutation (Pfendner et al., 2008). Login to comment
32 Indeed, a strong correlation between a rare sequence variant of the ABCC6 gene (c.3421C>T leading to the R1141X nonsense mutation) and CAD has been demonstrated in a Dutch cohort (Trip et al., 2002). Login to comment
37 We analyzed the allele frequency (AF) of the c.3421C > T (R1141X) mutation in healthy blood donors, patients with CAD, and patients suffering from ischemic stroke. Login to comment
61 The following equation was used to calculate the prevalence of PXE (P) in the control population: P ¼ 1=(AF · 4 · 0:01)2 (1) The maximal and minimal prevalence (Pm) were calculated by taking into account the 95% CI of AF determined in the various studies: Pm ¼ 1=[(AF Æ 95% CI · 0:01) · 4]2 (2) Results and Discussion Determination of the frequency of R1141X in the control population Before investigating the eventual association of the R1141X loss-of-function mutation of ABCC6 with CAD or stroke, we determined the frequency of this allele in our control population consisting of 749 healthy blood donors. Login to comment
62 This seemed to be important, since the two existing studies on the AF of the R1141X mutation in healthy Caucasians reported significantly different results ( p ¼ 0.028; Table 1) (Trip et al., 2002; Gotting et al., 2004). Login to comment
65 The corresponding frequency of the R1141X allele is almost identical to the German cohort (Gotting et al., 2004) ( p ¼ 0.495). Login to comment
68 Determination of the frequency of R1141X in patients with ischemic stroke Various anecdotal reports exist about a higher incidence of stroke in PXE patients than in unaffected individuals. Login to comment
73 This frequency of heterozygotes among stroke patients is not significantly different from controls ( p ¼ 0.44); thus, the carrier status of R1141X mutation of the ABCC6 gene is not a risk factor for stroke. Login to comment
77 Determination of the frequency of R1141X in patients with CAD Trip et al. found that the carrier status for the R1141X mutation of the ABCC6 gene is a risk factor for the development of CAD. Login to comment
79 We determined the number of heterozygotes among the CAD patients and found five individuals carrying the R1141X mutation (Table 2). Login to comment
88 Accordingly, Trip et al. found a significantly higher R1141X AF in the CAD group than we did. Login to comment
90 Nevertheless, both this and the previous study demonstrated a very strong correlation between CAD and the haploinsufficiency of ABCC6 due to the R1141X mutation. Login to comment
95 Based on the frequency of ABCC6 R1141X allele, approximately 0.5% of the control Caucasian population is a carrier of a loss-of-function mutation concerning several hundred thousands of individuals. Login to comment
97 Frequency of R1141X Heterozygotes in the Control Groups of the Three Different Studies Population Dutch German Hungarian n 1057 910 749 Carriers 8 1 1 AF 95% CI 0.38 Æ 0.27% 0.06 Æ 0.11% 0.07 Æ 0.13% n, cohort size; AF, allele frequency; CI, confidence interval. Login to comment
99 Frequency of R1141X Heterozygotes in the Stroke, Control, and Coronary Artery Disease Groups Cohort Stroke Blood donors Coronary artery disease n 363 749 361 Carriers 1 1 5 AF 95% CI 0.14 Æ 0.28% 0.07 Æ 0.13% 0.69 Æ 0.62% ABCC6 AND CORONARY ARTERY DISEASE screening would not be appropriate, but as most of them are relatives of PXE patients they can easily be identified. Login to comment

[hide] Novel deletions causing pseudoxanthoma elasticum u... J Hum Genet. 2010 Feb;55(2):112-7. Epub 2010 Jan 15. Costrop LM, Vanakker OO, Van Laer L, Le Saux O, Martin L, Chassaing N, Guerra D, Pasquali-Ronchetti I, Coucke PJ, De Paepe A
Novel deletions causing pseudoxanthoma elasticum underscore the genomic instability of the ABCC6 region.
J Hum Genet. 2010 Feb;55(2):112-7. Epub 2010 Jan 15., [PMID:20075945]

Abstract [show]
Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a heritable disease that affects elastic fibers. Thus far, >200 mutations have been characterized by various PCR-based techniques (primarily direct sequencing), identifying up to 90% of PXE-causing alleles. This study wanted to assess the importance of deletions and insertions in the ABCC6 genomic region, which is known to have a high recombinational potential. To detect ABCC6 deletions/insertions, which can be missed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA) was applied in PXE patients with an incomplete genotype. MLPA was performed in 35 PXE patients with at least one unidentified mutant allele after exonic sequencing and exclusion of the recurrent exon 23-29 deletion. Six multi-exon deletions and four single-exon deletions were detected. Using MLPA in addition to sequencing, we expanded the ABCC6 mutation spectrum with 9 novel deletions and characterized 25% of unidentified disease alleles. Our results further illustrate the instability of the ABCC6 genomic region and stress the importance of screening for deletions in the molecular diagnosis of PXE.

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97 Surprisingly, we identified deletions removing the region of exon 24 in two of these patients, indicating that a non-negligible proportion of patients with R1141X were in fact compound heterozygous. Login to comment
13 Two of these mutations are particularly prevalent, a deletion of exon 23-29 (del23-29) and p.R1141X (c.3421C4T).6-8 To date, only 16 different large ABCC6 deletions (entire exons, whole gene deletions) have been identified in PXE patients.7,9-14 Nevertheless, ABCC6 is extremely prone to genomic rearrangements because of the high content of repetitive elements in all introns and in the genomic sequences surrounding the gene.14 We hypothesized that because of the documented instability of the ABCC6 genomic region, the unidentified mutant alleles remaining after direct sequencing and screening for the recurrent exon 23-29 deletion, may consist of deletions and/or insertions. In this study, we aimed to screen for the presence of such deletions and/or insertions using the multiplex ligation-dependent probe amplification (MLPA) technique. Login to comment
83 Finally, we performed MLPA analysis in eight patients with a previously identified apparently homozygous p.R1141X mutation (exon 24) to verify whether the subjects were indeed homozygotes Patient 3 1 0.5 0 -0.5 -1 -1.5 Patient 13 1 0.5 0 -0.5 -1 -1.5 0Mb Patient 26 1 0.5 0 -0.5 -1 -1.5 Patient 34 1 0.5 0 -0.5 -1 -1.5 EXON27 EXON26 EXON25 EXON24 EXON23 16.16Mb 16.16Mb 16.16Mb 16.16Mb 16.16Mb 16.17Mb 16.17Mb 16.17Mb 9.87Mb 19.74Mb 29.61Mb 0Mb 9.87Mb 19.74Mb 29.61Mb 0Mb Chr.16 9.87Mb 19.74Mb 29.61Mb p.13.3 p.13.2 p.13.12 p.12.3 p.12.1 p.11.1 p.13.3 p.13.2 p.13.12 p.12.3 p.12.1 p.11.1p.13.3 p.13.2 p.13.12 p.12.3 p.12.1 p.11.1 Chr.16 Chr.16 Chr.16 Patient 32 0.5 0 -0.5 1 16146Kb EXON31 EXON30Chr.16 16148Kb 16150Kb 16151Kb 16153Kb 16156Kb -1 a b Figure 2 Array CGH based results. Login to comment

[hide] Mutation analysis (ABCC6) in a family with pseudox... Br J Dermatol. 2010 Sep;163(3):641-3. doi: 10.1111/j.1365-2133.2010.09856.x. Epub 2010 May 11. Li Q, Torok L, Kocsis L, Uitto J
Mutation analysis (ABCC6) in a family with pseudoxanthoma elasticum: presymptomatic testing with prognostic implications.
Br J Dermatol. 2010 Sep;163(3):641-3. doi: 10.1111/j.1365-2133.2010.09856.x. Epub 2010 May 11., [PMID:20491760]

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55 9 Trip MD, Smulders YM, Wegman JJ et al. Frequent mutation in the ABCC6 gene (R1141X) is associated with a strong increase in the prevalence of coronary artery disease. Login to comment
57 10 Ko¨blo¨s G, Andrikovics H, Proha´szka Z et al. The R1141X loss-of- function mutation of the ABCC6 gene is a strong genetic risk factor for coronary artery disease. Login to comment

[hide] Pseudoxanthoma elasticum: a streamlined, ethnicity... Clin Transl Sci. 2010 Dec;3(6):295-8. doi: 10.1111/j.1752-8062.2010.00243.x. Larusso J, Ringpfeil F, Uitto J
Pseudoxanthoma elasticum: a streamlined, ethnicity-based mutation detection strategy.
Clin Transl Sci. 2010 Dec;3(6):295-8. doi: 10.1111/j.1752-8062.2010.00243.x., [PMID:21167005]

Abstract [show]
Pseudoxanthoma elasticum (PXE), an autosomal recessive multisystem disorder, is caused by mutations in the ABCC6 gene, and approximately 300 distinct mutations representing >1000 mutant alleles have been disclosed thus far. Few population-based studies have reported mutational hotspots in some geographic areas. In this study, we attempted to correlate recurring mutations with the individuals' ethnic origin. Specifically, we plotted our international database of 70 families from distinct or mixed ethnic backgrounds against their mutations. The frequent p.R1141X mutation was distributed widely across Europe, while deletion of exons 23-29 (del23-29) was encountered in Northern Europe and in Northern Mediterranean countries. p.R1138W may be a marker for French descent, evidenced by its presence also in French Canadians. The splice site transition mutation 3736-1G-->A was seen in the neighboring countries Greece and Turkey, whereas 2542 delG occurs only in the Japanese. Two mutations seem to be present worldwide without evidence of a founder effect, p.Q378X and p.R1339C, suggesting the presence of mutational hotspots. Knowledge of this distribution will allow us to streamline mutation screening through a targeted, stepwise approach when the ethnicity of a patient is known. This will facilitate the identification of individuals at risk, improving their care to prevent ophthalmological and vascular disease.

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43 In our PXE cohort, the recurring nonsense mutation p.R1141X was present in all five ethnic groups, with the exception of Asian, representing 15.4% of French Canadian, 19.2% of Mediterranean, 20.5% of Northern European, and 36.4% of Scandinavian mutations; overall it occurred in 22.9% (32/140) of all the alleles examined. The high frequency of this mutation in the Mediterranean and Northern European population may be explained by a founder effect.18 The allele p.R1141X was found to share a common haplotype identical by descent in French19 and Italian patients.20 In three different studies examining German,21 Italian,20 and French PXE patients,19 R1141X was found to be the most frequent mutation. Login to comment
20 The frequent p.R1141X mutation was distributed widely across Europe, while deletion of exons 23-29 (del23-29) was encountered in Northern Europe and in Northern Mediterranean countries. Login to comment
42 A total of eight recurrent mutations were distinguished in the cohort, including nonsense (p.Q378X and p.R1141X), missense (p.R1138W and p.R1339C), splice site (3736-1G/A, 2787 + 1G/T), frameshift (2542 delG), and multiexon deletion (del exon 23-29). Login to comment
44 p.R1141X segregated in a compound heterozygous mode in 50% of French Canadians, 80% in Mediterranean, 75% in Northern Europeans, and in 33.3% of Scandinavians. Login to comment
46 The p.R1141X mutation can be detected by BsiYI restriction enzyme digestion and agarose gel electrophoresis, and confirmation can be attained by sequencing. Login to comment
64 Analysis of mutations found in Japanese patients by Noji et al.,22 combined with our study, suggested that neither the nonsense mutation p.R1141X nor the large deletion ABCC6 del 23-29 are frequent in this ethnicity group. Login to comment
83 17, 23, and24), suggest future screening strategies that incorporate the patient`s ethnic background.Suchdiagnosticstrategyshouldbeespeciallyefficient for the European and Mediterranean populations in which a number of PXE mutations occur frequently (p.R1141X and del exons 23-29). Login to comment

[hide] Role of serum fetuin-A, a major inhibitor of syste... Clin Chem. 2006 Feb;52(2):227-34. Epub 2005 Dec 29. Hendig D, Schulz V, Arndt M, Szliska C, Kleesiek K, Gotting C
Role of serum fetuin-A, a major inhibitor of systemic calcification, in pseudoxanthoma elasticum.
Clin Chem. 2006 Feb;52(2):227-34. Epub 2005 Dec 29., [PMID:16384891]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue affecting the skin, retina, and cardiovascular system and characterized by progressive calcification of abnormal and fragmented elastic fibers in the extracellular matrix. The aim of the present study was to investigate the association of fetuin-A, a major systemic inhibitor of calcification, with PXE. METHODS: Fetuin-A was measured by quantitative sandwich enzyme immunoassay in sera from 110 German patients with PXE, 53 unaffected first-degree family members, and 80 healthy blood donors. We determined the distribution of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in these same 3 groups. The occurrences of the frequent ABCC6 gene mutations c.3421C>T (p.R1141X) and c.EX23_EX29del were also assessed. RESULTS: Serum fetuin-A concentrations in male and female PXE patients were lower than in unaffected first-degree relatives and controls [mean (SD) concentrations, 0.55 (0.11) g/L in patients; 0.70 (0.23) g/L in relatives; and 0.80 (0.23) g/L in controls (P <0.0001)]. Serum fetuin-A was higher in female PXE patients with cardiovascular involvement than in the corresponding male group (P <0.05). The fetuin-A polymorphism frequencies did not differ among PXE patients, family members, and blood donors. CONCLUSION: A deficiency of multidrug resistance-associated protein 6 leads to alteration of circulating substrates, e.g., inhibitors of calcification as fetuin-A, leading to progressive mineralization of elastic fibers in PXE.

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4 The occurrences of the frequent ABCC6 gene mutations c.3421C>T (p.R1141X) and c.EX23_ EX29del were also assessed. Login to comment

[hide] [Pseudoxanthoma elasticum]. Ophthalmologe. 2006 Jun;103(6):537-51; quiz 552-3. Ladewig MS, Gotting C, Szliska C, Issa PC, Helb HM, Bedenicki I, Scholl HP, Holz FG
[Pseudoxanthoma elasticum].
Ophthalmologe. 2006 Jun;103(6):537-51; quiz 552-3., [PMID:16763870]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an inherited disorder that is associated with accumulation of mineralized and fragmented elastic fibers in the skin, vessel walls, and Bruch's membrane. Clinically, patients exhibit characteristic lesions of the skin (soft, ivory-colored papules in a reticular pattern that predominantly affect the neck), the posterior segment of the eye (peau d'orange, angioid streaks, choroidal neovascularizations), and the cardiovascular system (peripheral arterial occlusive disease, coronary occlusion, gastrointestinal bleeding). There is no causal therapy. Recent studies suggest that PXE is inherited almost exclusively as an autosomal recessive trait. Its prevalence has been estimated to be 1:25,000-100,000. The ABCC6 gene on chromosome 16p13.1 is associated with the disease. Mutations within the ABCC6 gene cause reduced or absent transmembraneous transport that leads to accumulation of substrate and calcification of elastic fibers. Although based on clinical features the diagnosis appears readily possible, variability in phenotypic expressions and the low prevalence may be responsible that the disease is underdiagnosed. This review covers current knowledge of PXE and presents therapeutic approaches.

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272 Internetadressen PXE-Selbsthilfegruppe Deutschland : http://www.pxe-groenblad.de PXE International: http://www.pxe.org Tabelle 5 PXE verursachende Mutationen imabcc6-Gen Klassifikation Lokalisation Gen Protein Missense Exon 9 Exon 9 Exon 10 Exon 10 Exon 11 Exon 12 Exon 13 Exon 14 Exon 16 Exon 18 Exon 18 Exon 18 Exon 18 Exon 19 Exon 19 Exon 19 Exon 22 Exon 24 Exon 24 Exon 24 Exon 24 Exon 24 Exon 24 Exon 24 Exon 24 Exon 24 Exon 25 Exon 26 Exon 26 Exon 26 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 28 Exon 29 Exon 29 Exon 29 Exon 29 Exon 29 Exon 30 Exon 30 Exon 30 c.1091CaG c.1171AaG c.1233TaG c.1318TaG c.1363GaC c.1553GaA c.1703TaC c.1798CaT c.2018TaC c.2252TaA c.2278CaT c.2294GaA c.2297CaA c.2428GaA c.2458GaC c.2552TaC c.2855TaG c.3340CaT c.3341GaA c.3341GaC c.3362CaG c.3380CaT c.3389CaT c.3412CaT c.3413GaA c.3413GaC c.3608GaA c.3661CaT c.3712GaC c.3715TaC c.3892GaT c.3902CaT c.3904GaA c.3907GaC c.3932GaA c.3940CaT c.3941GaA c.3961GaA c.3976GaA c.4004TaC c.4015CaT c.4036CaT c.4041GaC c.4060GaC c.4069CaT c.4081GaA c.4182GaT c.4198GaA c.4209CaA c.4271TaC c.4377CaT p.T364R p.R391G p.N411K p.C440G p.A455P p.R518Q p.F568S p.R600G p.L673P p.M751K p.R760W p.R765Q p.A766D p.V810M p.A820P p.L851P p.F952C p.R1114C p.R1114H p.R1114P p.S1121W p.M1127T p.T1130M p.R1138W p.R1138Q p.R1138P p.G1203D p.R1221C p.D1238H p.Y1239H p.V1298F p.T1301I p.G1302R p.A1303P p.G1311E p.R1314W p.R1314Q p.G1321S p.D1326N p.L1335P p.R1339C p.P1346S p.Q1347H p.G1354R p.R1357W p.D1361N p.K1394N p.E1400K p.S1403R p.I1424T p.R1459C Klassifikation Lokalisation Gen Protein Nonsense Exon 9 Exon 12 Exon 17 Exon 18 Exon 23 Exon 24 Exon 24 Exon 26 Exon 26 Exon 27 Exon 29 c.1132CaT c.1552CaT c.2247CaT c.2304CaA c.3088CaT c.3421CaT c.3490CaT c.3668GaA c.3709CaT c.3823CaT c.4192CaT p.Q378X p.R518X p.Q749X p.Y768X p.R1030X p.R1141X p.R1164X p.W1223X p.Q1237X p.R1275X p.R1398X Spleißstellen Intron 21 Intron 25 Intron 26 c.2787+1GaT c.3634-3CaA c.3736-1GaA Insertion Exon 8 Exon 25 Exon 30 c.938-939insT c.3544dupC c.4220insAGAA Deletion Exon 2 Exon 2 Exon 3 Exon 8 Exon 9 Exon 16 Exon 16 Exon 18 Exon 19 Exon 22 Exon 27 Exon 29 Exon 29 Exon 30 Exon 31 c.179del9 c.179-195del c.220-222del c.960delC c.1088-1120del c.1944del22 c.1995delG c.2322delC c.2542delG c.2835-2850del16 c.3775delT c.4101delC c.4182delG c.4318delA c.4434delA Intragenische Deletion Exon 15 Exon 18 Exon 23-29 delEx15 delEx18 delEx23-29 Intergenische Deletion ABCC6 delABCC6 Fazit für die Praxis Eine spezifische Behandlung der Grunderkrankung ist nicht bekannt. Login to comment

[hide] Mutational analysis of the ABCC6 gene and the prox... Hum Mutat. 2006 Aug;27(8):831. Schulz V, Hendig D, Henjakovic M, Szliska C, Kleesiek K, Gotting C
Mutational analysis of the ABCC6 gene and the proximal ABCC6 gene promoter in German patients with pseudoxanthoma elasticum (PXE).
Hum Mutat. 2006 Aug;27(8):831., [PMID:16835894]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by calcification of elastic fibers in dermal, ocular, and cardiovascular tissues. Recently, ABCC6 mutations were identified as causing PXE. In this follow-up study we report the investigation of 61 German PXE patients from 53 families, hitherto the largest cohort of German PXE patients screened for the complete ABCC6 gene. In addition, we characterized the proximal ABCC6 promoter of PXE patients according to mutation. In this study we identified 32 disease-causing ABCC6 variants, which had been described previously by us and others, and 10 novel mutations (eight missense mutations and two splice site alterations). The mutation detection rate among index patients was 87.7%. Frequent alterations were the PXE-mutations p.R1141X, Ex23,_Ex29del, and c.2787+1G > T. In the ABCC6 promoter we found the polymorphisms c.-127C > T, c.-132C > T, and c.-219A > C. The difference in the c.-219A > C frequencies between PXE patients and controls were determined as statistically significant. Interestingly, c.-219A > C is located in a transcriptional activator sequence of the ABCC6 promoter and occurred in a binding site for a transcriptional repressor, predominantly found in genes that participate in lipid metabolism. Obtaining these genetic data signifies our contribution to elucidating the pathogenetics of PXE.

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8 Frequent alterations were the PXE-mutations p.R1141X, Ex23,_Ex29del, and c.2787+1G>T. Login to comment
72 The most common PXE mutations were the nonsense mutation p.R1141X, the splice site mutation c.2787+1G>T and the large deletion Ex23_Ex29del. Login to comment
82 Summary of ABCC6/MRP6 mutations identified in German PXE patients Change in Number of Allelic frequency Exona nucleotideb Amino acid Statusc families in blood donorsd Referenceg i-1e c.37-1G>Af altered splicing hm 1 0 / 200 This study 2 c.113G>C p.W38S ht 1 0 / 200 This study i-3 c.346-6G>A altered splicing ht 2 Nd A, B 7 c.754C>T p.L252F ht 1 0 / 200 This study 9 c.1132C>T p.Q378X ht 4 Nd B, C 9 c.1171A>G p.R391G ht 1 Nd B, D 10 c.1244T>C p.V415A ht 1 0 / 200 This study 12 c.1460G>A p.R487Q ht 1 0 / 200 This study 12 c.1491C>A p.N497K ht 1 0 / 200 This study 12 c.1552C>T p.R518X ht 1 Nd B, E i-12 c.1574_1575insG p.L525fsX73 ht 1 0 / 200 This study 16 c.1995delG p.A667fsX20 ht 3 Nd A, F, G 18 c.2252T>A p.M751K ht 3 Nd F, G 18 c.2278C>T p.R760W ht 2 Nd B, F, G Change in Number of Allelic frequency Exona nucleotideb Amino acid Statusc families in blood donorsd Referenceg 18 c.2294G>A p.R765Q ht 2 Nd A, F, G, H 19 c.2552T>C p.L851P ht 1 Nd F i-21 c.2787+1G>T altered splicing ht 7 Nd B, C, F, I, J 22 c.2835_2850del16 p.P946fsX17 ht 1 Nd F 22 c.2855T>G p.F952C ht 1 Nd F 23 c.3145T>G p.S1049A ht 1 0 / 200 This study 23 c.3188T>G p.L1063R ht 1 0 / 200 This study 24 c.3340C>T p.R1114C ht 1 Nd B, K, G, L 24 c.3341G>A p.R1114H ht 1 Nd G, H, L, M 24 c.3389C>T p.T1130M ht 1 Nd B, D, G, H, K, L, M, N 24 c.3413G>A p.R1138Q ht 1 Nd A, B, D, J, K, L, N 24 c.3412C>T p.R1138W ht 1 Nd N 24 c.3421C>T p.R1141X hm, ht 26 Nd B, G, J, K, L, M, N, O, P, Q, R, S i-24 c.3505_3506+2delA GGT altered splicing ht 1 0 / 200 This study i-24 c.3507-3C>T altered splicing ht 2 Nd B 26 c.3715T>C p.Y1239H ht 1 Nd L 26 c.3723G>C p.W1241C ht 1 Nd A, L i-26 c.3736-1G>A altered splicing ht 1 Nd B, L, N 27 c.3775delT p.W1259fsX13 ht 1 Nd B, J, L, O i-27 c.3883-6G>A altered splicing ht 1 Nd B 28 c.3902C>T p.T1301I ht 1 Nd A, G, L 28 c.3932G>A p.G1311E ht 1 Nd L 28 c.3940C>T p.R1314W ht 1 Nd A, G, L 28 c.3941G>A p.R1314Q ht 1 Nd A, B, G, L 29 c.4182delG p.N1394fsX8 ht 2 Nd G, H, L 30 c.4209C>A p.S1403R ht 1 Nd F 31 c.4434delA p.R1479fsX25 hm 1 Nd F 23-29 Ex23_Ex29del p.A999_S1403del ht 5 Nd A, B, D, E, G, H, O, R a The exon that contains the ABCC6 sequence variation. Login to comment
89 Genotypes and phenotypes of the PXE patients analyzed in this study Phenotype Genotypeb No.a Sex, Age Age on diagnosis Organ involvement Mutations 1 M 36 11 E, S, G p.R1141X p.R1141X 2 F 44 39 E, S, G, A p.R1141X Ex23_Ex29del 3 F 41 7 E, S p.R1141X p.R1141X 4 F 46 19 E, S, A p.R1141X p.R1141X 5 F 59 55 E, S, A c.37-1G>A c.37-1G>A 6c F 63 16 E, S, H, V, A Ex23_Ex29del c.4182delG 7 F 24 15 E, S c.4434delA c.4434delA 8 M 60 23 E, S p.Q378X p.R1141X 9 F 79 65 E, S, A c.2787+1G>T p.R1141X 10 F 55 35 E, S, G, H, V, A p.Q378X c.2787+1G>T 11 F 47 14 S c.1995delG c.2787+1G>T 12c F 36 24 E, S c.2787+1G>T c.4182delG 13 F 56 8 E, S p.R1141X c.3507-3C>T 14 M 72 55 E, S, H, V p.R1141X 15 F 69 51 E, S c.1995delG p.R765Q 16 F 19 11 S p.R760W p.R1141X 17c F 59 50 E, S, H, V, A p.R1141X p.G1311E 18c M 54 32 E, S p.R1141X p.Y1239H 19-1 M 63 53 E, H p.L252F p.V415A p.R765Q 19-2 F 58 48 E, S p.L252F p.V415A p.R765Q 20 M 54 44 E, S, V, A c.3775delT c.346-6G>A 21 M 52 43 E, S, A p.R1141X c.3883-6G>A 22-1 M 47 36 E, S, G, H, V p.R518X 22-2 M 45 34 E, S, H p.R518X 23 F 35 22 E, S, A p.W38S 24 F 40 30 E c.346-6G>A 25-1 M 58 46 E, S, A p.R1141X c.3883-6G>A 25-2 M 19 10 S p.R1141X c.3883-6G>A 26-1 F 46 18 E, S, V p.R487Q c.3883-6G>A 27c F 62 30 E, S, A p.Q378X p.R1114H 28 F 59 49 E, A p.R1314Q c.3507-3C>T 29c F 30 10 E, S c.1995delG p.R1114C 30 M 67 52 E p.L1063R p.R1141X 31 F 50 46 E, S, V p.M751K p.R1141X 32 F 27 24 S Ex23_Ex29del 33c F 34 19 E, S Ex23_Ex29del p.T1130M 34 F 33 19 E, S c.2787+1G>T p.W1241C 35 M 47 15 E, S, G, H, V, A Ex23_Ex29del 36 M 72 63 E, S p.S1049A c.3736-1G>A p.S1403R 37 F 34 16 E, S c.2787+1G>T 38 F 42 8 E, S, V p.R1141X p.R1314W 39 F 37 20 E, S p.N497K 40 F 54 33 E, S, V, A p.M751K p.R1141X 41 M 53 49 E, S, G, H, V p.R1141X 42-1 F 52 38 E, S p.R391G p.R1141X 42-2 F 43 28 E, S p.R391G p.R1141X 43 F 64 58 S, A 44-1 F 51 27 E, S, A p.R1141X 44-2 F 18 9 E, S 44-3 F 54 26 E, S, V, A p.R1141X 45-1 F 64 49 E, S, G, V p.R1138Q 45-2 F 62 48 E, S, A p.R1138Q 46 M 56 25 E, S, V p.R1141X p.T1301I 47 F 34 23 E, S p.R760W c.2787+1G>T 48 M 47 24 E, S, V, A c.2835_2850del16 p.F952C p.R1141X 49 F 28 11 E, S, G, V p.M751K p.R1141X 50 F 39 25 E, S, V p.L851P p.R1141X c.3505_3506+2 delAGGT 51 F 61 16 E, S, H, A p.Q378X p.R1141X 52-1 F 40 20 E, S p.R1138W p.R1141X 52-2 F 43 23 E, S p.R1138W p.R1141X 53 M 68 66 E, H, V, G, A c.1574_1575insG p.R1141X F = female, M = male, wt = wild-type, hm = homozygote, ht = heterozygote, cht = compound heterozygote, nd = not determined, MSM = microsatellite marker, E = eyes, S = skin, G = gastrointestinum, H = heart, V = vascular tissue and A = arterial hypertension. Login to comment
97 The other 7 PXE patients were compound heterozygous for the novel DNA variations p.L252F, p.V415A, p.R487Q, p.S1049A, p.L1063R, c.1574_1575insG and c.3505_3506+2delAGGT, as well as for the known PXE-causing mutations p.R765Q, p.R1141X, p.L851P, c.3736-1G>A and p.S1403R (Bergen et al., 2000; Germain et al., 2000; Ringpfeil et al., 2000; Struk et al., 2000; Le Saux et al., 2001; Uitto et al., 2001; Hendig et al., 2005). Login to comment
123 The frequencies of these mutations were similar to those reported in previous European studies, in which the nonsense mutation p.R1141X and the large deletion Ex23_Ex29del were described as frequent ABCC6 mutations in French, Italian and Dutch PXE patients and most of the further ABCC6 alterations were determined as unique mutations (Chassaing et al., 2004; Gheduzzi et al., 2004; Hu et al., 2004). Login to comment
152 For the p.L1063R variant that was found in a compound heterozygous state with the nonsense mutation p.R1141X, segregation with the PXE phenotype within the patient's family was observed (Table 2). Login to comment
162 The mutations p.R1141X, which is found in 2 apparently non-consanguineous parts of the family, p.R487Q and c.3883-6G>A could be located. Login to comment
165 The index patient and her sister were heterozygous for p.R1141X, but no mutation could be detected in the ABCC6 gene of the daughter. Login to comment

[hide] Development of a rapid, reliable genetic test for ... J Mol Diagn. 2007 Feb;9(1):105-12. Shi Y, Terry SF, Terry PF, Bercovitch LG, Gerard GF
Development of a rapid, reliable genetic test for pseudoxanthoma elasticum.
J Mol Diagn. 2007 Feb;9(1):105-12., [PMID:17251343]

Abstract [show]
Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), a hereditary disorder that impacts the skin, eyes, and cardiovascular system. Currently, the diagnosis of PXE is based on physical findings and histological examination of a biopsy of affected skin. We have combined two simple, polymerase chain reaction (PCR)-based methods to develop a rapid, reliable genetic assay for the majority of known PXE mutations. After PCR amplification and heteroduplex formation, mutations in exon 24 and exon 28 of the ABCC6 gene were detected with Surveyor nuclease, which cleaves double-stranded DNA at any mismatch site. Mutations originating from deletion of a segment of the ABCC6 gene between exon 23 and exon 29 (ex23_ex29del) were detected by long-range PCR. Size analysis of digestion fragments and long-range PCR products was performed by agarose gel electrophoresis. The methods accurately identified mutations or the absence thereof in 16 affected individuals as confirmed by DNA sequencing. Fifteen patients had one or two point mutations, and two of these individuals carried the ex23_ex29del in their second allele. This mutation detection and mapping strategy provides a simple and reliable genetic assay to assist in diagnosis of PXE, differential diagnosis of PXE-like conditions, and study of PXE genetics.

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12 The ABCC6 gene (Online Mendelian Inheritance of Man no. 603234) consists of 31 exons on human chromosome 16p13.1.36 The gene encodes a protein (ABCC6/MRP6) belonging to the ATP-binding cassette membrane transporter family with 1503 amino acid residues, three transmembrane segments consisting of 17 hydrophobic helices, and two conserved nucleotide binding domains (NBD1 and NBD2).7-9 ABCC6 gene mutations have been associated with autosomal recessive and sporadic forms of PXE.5,10 -13 At present, some 150 causative mutations in this gene have been observed in different populations, with most mutations being missense, nonsense, deletion/insertion, or splice site alterations clustered toward the large carboxyl-terminal end of ABCC6/MRP6 in NBD1 and NBD2.5,10 -30 The most frequent mutations in North American, European, and South African populations are c.3421CϾT (p.R1141X) in exon 24 and Alu-mediated deletion of sequences between exon 23 and 29 (ex23_ex29del).14,16,18,19,21,23 Mutations in the ABCC6 gene that cause PXE allow development of genetic tests for accurate clinical diagnosis, differential diagnosis from PXE-like phenotypes (eg, PXE-like papillary dermal elastolysis and fibroelastolytic papulosis, periumbilical perforating PXE, PXE-like presentation of beta-thalassemia, and acquired PXE syndromes), and predictive preclinical diagnosis to allow for possible intervention and for timely genetic counseling. Login to comment
31 The c.3421CϾT (p.R1141X) mutation in exon 24 and the deletion between exon 23 and exon 29 (ex23_ex29del) are those most commonly found in phenotypically positive samples.14,16,18,19,21,23 This published study and unpublished research sponsored by PXE International, in collaboration with Transgenomic, Inc.; Jefferson Medical College, Philadelphia, PA; Ghent University, Ghent, Belgium; and the University of Witwatersrand, Johannesburg, South Africa, determined that mutations in exons 24 and 28 and the deletion of exons 23 to 29 account for ϳ70% of the ABCC6 gene mutations in individuals affected by PXE. Login to comment
76 Because of the one base position difference between mutation c.3412CϾT (p.R1138W) and c.3413GϾA (p.R1138Q) in exon 24 and the 8- and 9-base position differences between the c.3413GϾA and c.3412CϾT mutations and the c.3421CϾT (p.R1141X) mutation in exon 24, the cleavage fragments from these mutations could not be distinguished by agarose gel electrophoresis. Login to comment
82 Nuclease Digestion Fragment Sizes of Common Mutations in PXE Exon 24 and 28 Amino acid change Base change Fragment lengths (bp) p.T1130M c.3389CϾT 251,257/508 p.R1138W c.3412CϾT 274,234/508 p.R1138Q c.3413GϾA 275,233/508 p.R1141X c.3421CϾT 281,227/508 p.R1164X c.3490CϾT 352,156/508 p.G1302R c.3904GϾA 116,289/405 p.R1314Q c.3941GϾA 153,252/405 p.R1339C c.4015CϾT 227,178/405 The total lengths of the amplicons are listed after the slash. Login to comment
107 The two most prevalent mutations were the nonsense mutations c.3421CϾT (p.R1141X) and c.3490CϾT (p.R1164X) in exon 24. c.3421CϾT is the most common mutation found in PXE patients of European origin.14,19,20,23,29 c.3490CϾT is a common mutation in individuals of British descent.16,21 Two DNA samples carried the c.3490CϾT (p.R1339C) missense mutation in one exon 28 allele, and in both cases, IVS28 ϩ 49CϾT was also present. Login to comment
130 Recently, a multiphase strategy was described to screen for PXE mutations in the total coding sequence of the ABCC6 gene.25 PCR-RFLP and long-range PCR were used initially to detect the most common PXE mutations, c.3421CϾT (p.R1141X) and ex23_ex29del, respectively. Login to comment

[hide] Pseudoxanthoma elasticum: genetic variations in an... Clin Chem. 2007 Oct;53(10):1734-40. Epub 2007 Aug 10. Zarbock R, Hendig D, Szliska C, Kleesiek K, Gotting C
Pseudoxanthoma elasticum: genetic variations in antioxidant genes are risk factors for early disease onset.
Clin Chem. 2007 Oct;53(10):1734-40. Epub 2007 Aug 10., [PMID:17693525]

Abstract [show]
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers in connective tissues. PXE is caused by mutations in the ABCC6 gene, which encodes the membrane transporter multidrug resistance-associated protein 6. Chronic oxidative stress was recently suggested to play a crucial role in the pathogenesis of the disease. Our aim was to investigate the association of PXE with genetic variation in genes coding for antioxidant enzymes. METHODS: We used restriction fragment length polymorphism and allele-specific PCR analyses to evaluate the distribution of single-nucleotide polymorphisms in the genes encoding catalase (CAT), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 (GPX1) in DNA samples from 117 German PXE patients and 117 healthy age- and sex-matched control individuals. RESULTS: The investigated genetic variants had previously been shown to affect the activities of these antioxidant enzymes. We found a correlation between genotype and age of disease onset for polymorphisms in CAT (c.-262C>T), SOD2 (c.47C>T), and GPX1 (c.593C>T). Furthermore, the age of disease onset was inversely correlated with the number of mutated alleles, indicating a cumulative effect on the time of disease onset [mean (SD) age of 40.9 (13.6) years, 32.4 (16.3) years, and 25.7 (15.9) years for carriers of 0, 1-2, and >2 mutated alleles, respectively; P = 0.03]. CONCLUSION: Our findings demonstrate that increased oxidative stress due to activity-affecting polymorphisms in genes encoding antioxidant enzymes leads to earlier PXE onset.

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53 The most common causative PXE mutations among these patients were the nonsense mutation c.3421CϾT (p.R1141X), the splice site mutation c.2787 ϩ 1GϾT, and the large deletion Ex23_Ex29del. Login to comment
142 b Patients who are (a) homozygous for PXE mutation p.R1141X, c.2787 ϩ 1GϾT, or c.4434delA; and (b) compound heterozygotes for 2 of the PXE mutations p.R1141X, c.2787 ϩ 1GϾT, Ex23_Ex29del, c.4182delG, p.Q378X, c.1995delG, p.E507X, and c.2835_2850del16. Login to comment

[hide] Pseudoxanthoma elasticum with generalized retinal ... Invest Ophthalmol Vis Sci. 2007 Sep;48(9):4250-6. Audo I, Vanakker OM, Smith A, Leroy BP, Robson AG, Jenkins SA, Coucke PJ, Bird AC, De Paepe A, Holder GE, Webster AR
Pseudoxanthoma elasticum with generalized retinal dysfunction, a common finding?
Invest Ophthalmol Vis Sci. 2007 Sep;48(9):4250-6., [PMID:17724214]

Abstract [show]
PURPOSE: Pseudoxanthoma elasticum (PXE; [MIM 264800]) is an autosomal recessive systemic disorder characterized by progressive degeneration and calcification of elastic fibers in connective tissue. The phenotype is variable, with cutaneous, vascular, and ophthalmic abnormalities. The disorder is a consequence of mutations in the ABCC6 gene. Visual impairment is mainly due to neovascular complications, and retinal function is usually assumed to be normal. The purpose of this study was the objective assessment of macular and generalized retinal function in unrelated patients with clinical and/or genetic features of PXE. METHODS: Four unrelated patients carrying a clinical diagnosis of PXE presented with unexplained visual loss. After ophthalmic examination, retinal and macular function was assessed by full-field electroretinogram (ERG) and pattern ERG, respectively, according to ISCEV (International Society for Clinical Electrophysiology of Vision) recommendations. Molecular analysis of the ABCC6 gene was performed in three patients by dHPLC (denaturing high-performance liquid chromatography) and direct sequencing. RESULTS: Full-field ERG revealed significant reduction of cone and rod responses in all four patients. Funduscopic appearances varied. Three patients were found to carry ABCC6 mutations. In case 1, a novel nonsense mutation (p.L1474X) was detected in exon 31 paired with a splice-site mutation. Mutation analyses in cases 3 and 4 revealed previously reported ABCC6 mutations. CONCLUSIONS: These findings suggest that retinal dysfunction in PXE may not be uncommon. The mechanism underlying retinal dysfunction is unknown but may result from metabolic disturbance leading to retinal toxicity with a possible role of modifying genetic or environmental factors rather than specific ABCC6 mutations.

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112 On mutation analysis, this patient was homozygous for the p.R1141X nonsense mutation (c.3421C3T) in exon 24 (Fig. 5c). Login to comment

[hide] Compound heterozygosity for a novel and a recurren... J Dermatol Sci. 2008 Mar;49(3):252-5. Epub 2007 Oct 29. Drera B, Brezzi A, Zoppi N, Venturini M, Barlati S, Pinton PG, Colombi M
Compound heterozygosity for a novel and a recurrent ABCC6 gene mutation in an Italian family with Pseudoxanthoma elasticum.
J Dermatol Sci. 2008 Mar;49(3):252-5. Epub 2007 Oct 29., [PMID:18029147]

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11 In particular, the p.R1141X mutation represents about 28% of the mutations detected in the European patients and about 26% in Italian ones [1,6]. Login to comment
22 Sequencing analysis of proband`s genomic DNA, disclosed in exon 24 of ABCC6 gene the c.3397G>T transversion and the c.3421C>T transition, leading to p.G1133C missense and p.R1141X nonsense mutations, respectively (Fig. 2A). Login to comment
28 The specific amplification of both alleles, using a wild type and a mutated forward primer at nucleotide c.3397, and the sequence analysis of the PCR products showed that the p.G1133C substitution was in trans with the p.R1141X nonsense mutation (Fig. 2B). Login to comment
37 The recurrent p.R1141X nonsense mutation has been shown to result in a null allele due to the nonsense-mediated decay of the aberrant mRNA [9]. Login to comment
41 Previously, the p.R1141X mutation was reported to be associated with an increased risk of premature, i.e. under the 50 years, coronary artery disease in patients without PXE signs [10]. Login to comment
43 In the members of our family carrying the p.R1141X, indeed, cardiovascular involvement, i.e. hypertension, premature coronary artery disease, ischemic stroke or mucosal bleeding, was absent. Login to comment
46 In conclusion, we reported a new Italian PXE family showing intra-familiar variability in the onset and in the systems involvement, in which the disease was due to compound heterozygosity for the novel p.G1133C and the recurrent p.R1141X mutations. Login to comment

[hide] Parameters of oxidative stress are present in the ... Biochim Biophys Acta. 2008 Jul-Aug;1782(7-8):474-81. Epub 2008 May 10. Garcia-Fernandez MI, Gheduzzi D, Boraldi F, Paolinelli CD, Sanchez P, Valdivielso P, Morilla MJ, Quaglino D, Guerra D, Casolari S, Bercovitch L, Pasquali-Ronchetti I
Parameters of oxidative stress are present in the circulation of PXE patients.
Biochim Biophys Acta. 2008 Jul-Aug;1782(7-8):474-81. Epub 2008 May 10., [PMID:18513494]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an inherited disorder characterized by calcification of elastic fibres leading to dermatological and vascular alterations associated to premature aged features and to life threatening clinical manifestations. The severity of the disease is independent from the type of mutation in the ABCC6 gene, and it has been suggested that local and/or systemic factors may contribute to the occurrence of clinical phenotype. The redox balance in the circulation of 27 PXE patients and of 50 healthy subjects of comparable age was evaluated by measuring the advanced oxidation protein products (AOPP), the lipid peroxidation derivatives (LOOH), the circulating total antioxidant status (TAS), the thiol content and the extracellular superoxide dismutase activity (EC-SOD). Patients were diagnosed by clinical, ultrastructural and molecular findings. Compared to control subjects, PXE patients exhibited significantly lower antioxidant potential, namely circulating TAS and free thiol groups, and higher levels of parameters of oxidative damage, as LOOH and of AOPP, and of circulating EC-SOD activity. Interestingly, the ratio between oxidant and antioxidant parameters was significantly altered in PXE patients and related to various score indices. This study demonstrates, for the first time, that several parameters of oxidative stress are modified in the blood of PXE patients and that the redox balance is significantly altered compared to control subjects of comparable age. Therefore, in PXE patients the circulating impaired redox balance may contribute to the occurrence of several clinical manifestations in PXE patients, and/or to the severity of disease, thus opening new perspectives for their management.

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74 Table 1 Clinical data of patients Patients' gender/age Clinical scores Mutations Allele 1 Allele 2 M/10 S2E2 c.3413GNA (p.R1138Q) c.3413GNA (p.R1138Q) F/16 S1 c.1171ANG (p.R391G) c.1552CNT (p.R518X) F/18 S3E2V2 c.1484TNA (p.L495H) c.1484TNA (p.L495H) F/21 S2E2 c.2420GNA (p.R807Q) ND F/21 S2E2 c.184TNC (p.Y62H) c.2996_4208del (p.A999_S1403del) F/24 S2E2 c.1799GNA (p.R600H) c.2420GNA (p.R807Q) F/27 S3E2 c.184TNC (p.Y62H) c.2996_4208del (p.A999_S1403del) F/30 S2E2G1 c.2996_4208del (p.A999_S1403del) c.4198GNA (p.E1400K) F/30 S2E3 c.2996_4208del (p.A999_S1403del) c.4198GNA (p.E1400K) M/30 S2E1 c.3421CNT (p.R1141X) c.3735GNA F/32 S2 c.3421CNT (p.R1141X) c.3735GNA F/33 S3E2 c.1987GNA (p.G663S) ND F/33 S3E3 c.1609_1609delG (p.V537fsX26) c.1763_1769del ins56 F/36 S3E2V3 c.3421CNT (p.R1141X) ND F/36 S3E3V2G1 c.3421CNT (p.R1141X) c.3421CNT (p.R1141X) M/39 S1E2V2 c.1552CNT (p.R518X) c.2996_4208del (p.A999_S1403del) M/42 S1E3V2G1 c.1552CNT (p.R518X) c.2996_4208del (p.A999_S1403del) F/43 S3E3 c.1552CNT (p.R518X) c.1552CNT (p.R518X) F/44 S3E2 c.3341GNA (p.R1114H) c.3542GNA (p.G1181D) F/45 S3E3V2C1G1 c.3421CNT (p.R1141X) c.3421CNT (p.R1141X) F/48 S2E2V2 c.1553GNA (p.R518Q) ND M/51 S1E3 c.3662GNA (p.R1221H) ND F/52 S3E3V2 c.3088CNT (p.R1030X) c.3088CNT (p.R1030X) M/54 S1E2G1 c.1799GNA (p.R600H) c.3941GNA (p.R1314Q) F/56 S3E3V2 c.3662GNA (p.R1221H) ND F/60 S2E3V2C1G1 c.951CNA (p.S317R) c.3421CNT (p.R1141X) F/62 S2E3 c.1552CNT (p.R518X) c.3421CNT (p.R1141X) Scores describe the severity of clinical manifestations. Login to comment

[hide] Pseudoxanthoma elasticum: clinical phenotypes, mol... Exp Dermatol. 2009 Jan;18(1):1-11. Epub 2008 Oct 22. Li Q, Jiang Q, Pfendner E, Varadi A, Uitto J
Pseudoxanthoma elasticum: clinical phenotypes, molecular genetics and putative pathomechanisms.
Exp Dermatol. 2009 Jan;18(1):1-11. Epub 2008 Oct 22., [PMID:19054062]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a prototype of heritable multisystem disorders, is characterised by pathologic mineralisation of connective tissues, with primary clinical manifestations in the skin, eyes and the cardiovascular system. The causative gene was initially identified as ABCC6 which encodes an ABC transporter protein (ABCC6) expressed primarily in the liver and the kidneys. The critical role of ABCC6 in ectopic mineralisation has been confirmed by the development of Abcc6(-/-) knock-out mice which recapitulate the features of connective tissue mineralisation characteristic of PXE. Over 300 distinct loss-of-function mutations representative of over 1000 mutant alleles in ABCC6 have been identified by streamlined mutation detection strategies in this autosomal recessive disease. More recently, missense mutations in the GGCX gene, either in compound heterozygous state or digenic with a recurrent ABCC6 nonsense mutation (p.R1141X), have been identified in patients with PXE-like cutaneous findings and vitamin K-dependent coagulation factor deficiency. GGCX encodes a carboxylase which catalyses gamma-glutamyl carboxylation of coagulation factors as well as of matrix gla protein (MGP) which in fully carboxylated form serves as a systemic inhibitor of pathologic mineralisation. Collectively, these observations suggest the hypothesis that a consequence of loss-of-function mutations in the ABCC6 gene is the reduced vitamin K-dependent gamma-glutamyl carboxylation of MGP, with subsequent connective tissue mineralisation. Further progress in understanding the detailed pathomechanisms of PXE should provide novel strategies to counteract, and perhaps cure, this complex heritable disorder at the genome-environment interface.

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4 More recently, missense mutations in the GGCX gene, either in compound heterozygous state or digenic with a recurrent ABCC6 nonsense mutation (p.R1141X), have been identified in patients with PXE-like cutaneous findings and vitamin K-dependent coagulation factor deficiency. Login to comment
64 Two mutations are recurrent and of high frequency: First, the most common recurring mutation, particularly in Caucasian individuals, is p.R1141X in exon 24, with a prevalence of approximately 30% of all PXE mutations. Login to comment
67 Nevertheless, only approximately 10% of all patients in a recent large US and European study (21) were homozygous or compound heterozygous for p.R1141X and del23-29 mutations, indicating that approximately 90% of all individuals in these PXE populations will require further analysis to identify the second mutation or both mutations in the ABCC6 gene causing their disease. Login to comment
173 The analysis revealed the presence of a recurrent mutation, p.R1141X, in the peripheral blood DNA in several family members but not in the proband herself or in her sister (Fig. 5). Login to comment
174 These individuals were heterozygous for the p.R1141X mutation, and no other ABCC6 mutation could be disclosed in this family. Login to comment
178 In contrast, individuals who were heterozygous carriers of the ABCC6 nonsense mutation p.R1141X were also heterozygous carriers of one of the GGCX missense mutations. Login to comment
179 Specifically, the finding of the GGCX missense mutation p.V255M in combination of the ABCC6 nonsense mutation p.R1141X suggests the possibility of digenic inheritance of their cutaneous findings (59). Login to comment
184 Mutation analysis in the nuclear pedigree identified the presence of the p.R1141X nonsense mutation in the ABCC6 gene and two missense mutations, p.V255M and p.S300F, in the GGCX gene, as indicated on the left. Login to comment

[hide] Co-existent pseudoxanthoma elasticum and vitamin K... Am J Pathol. 2009 Feb;174(2):534-40. Epub 2008 Dec 30. Li Q, Schurgers LJ, Smith AC, Tsokos M, Uitto J, Cowen EW
Co-existent pseudoxanthoma elasticum and vitamin K-dependent coagulation factor deficiency: compound heterozygosity for mutations in the GGCX gene.
Am J Pathol. 2009 Feb;174(2):534-40. Epub 2008 Dec 30., [PMID:19116367]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a multisystem disorder characterized by ectopic mineralization of connective tissues with primary manifestations in the skin, eyes, and cardiovascular system. The classic forms of PXE are due to mutations in the ABCC6 gene that encodes the ABCC6 protein, a putative transmembrane transporter expressed primarily in the liver and the kidneys. PXE-like clinical findings have been encountered in association with vitamin K-dependent coagulation factor deficiency, an autosomal recessive disorder that is due to mutations in either the GGCX or VKORC1 genes. In this study, we investigated a family with two siblings with characteristic features of PXE and vitamin K-dependent coagulation factor deficiency. Mutation analysis identified two GGCX mutations in the affected individuals (p. R83W and p.Q374X); however, no mutations in either ABCC6 or VKORC1 could be found. GGCX encodes a gamma-glutamyl carboxylase necessary for activation of both coagulation factors in the liver and matrix gla protein, which, in fully carboxylated form, is able to prevent ectopic mineralization. Analysis of skin by specific antibodies demonstrated that matrix gla protein was found predominantly in undercarboxylated form and was associated with the mineralized areas in the patients' lesional skin. These observations pathomechanistically suggest that, in our patients, reduced carboxylase activity results in a reduction of matrix gla protein carboxylation, thus allowing peripheral mineralization to occur. Our findings also confirm GGCX as the second gene locus causing PXE.

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148 Genetic Heterogeneity of PXE The complementary nature of the ABCC6 and GGCX gene defects has been demonstrated by previous phenotypic observations of a family with features of PXE in association with vitamin K-dependent coagulation factor deficiency.20 While some of the members of that family were compound heterozygotes for missense mutations in the GGCX gene, two patients with characteristic PXE-like cutaneous findings were found to be heterozygous for a missense mutation in the GGCX gene (p.V255M) and a recurrent nonsense mutation (p.R1141X) in the ABCC6 gene in trans. Login to comment

[hide] Added value of infrared, red-free and autofluoresc... Br J Ophthalmol. 2010 Apr;94(4):479-86. Epub 2009 Sep 1. De Zaeytijd J, Vanakker OM, Coucke PJ, De Paepe A, De Laey JJ, Leroy BP
Added value of infrared, red-free and autofluorescence fundus imaging in pseudoxanthoma elasticum.
Br J Ophthalmol. 2010 Apr;94(4):479-86. Epub 2009 Sep 1., [PMID:19726431]

Abstract [show]
PURPOSE: Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder caused by mutations in the ABCC6 gene and primarily affects the oculocutaneous and cardiovascular systems. However, the phenotype, including the ophthalmological manifestations, varies in severity. The present study aims to evaluate the added value of novel funduscopic imaging techniques, such as near-infrared reflectance, red-free and autofluorescence imaging in PXE. METHODS: In 22 molecularly proven PXE patients and 25 obligate carriers, PXE retinopathy was evaluated using funduscopy, white light, red-free, infrared and autofluorescence imaging. RESULTS: At least one characteristic of PXE retinopathy was evident on funduscopy of all eyes. Angioid streaks could be subdivided in those with (brick red) or without (feathered) adjacent RPE alterations. Infrared imaging showed the brick-red-coloured streaks as well-demarcated dark fissures, even when these passed unnoticed on funduscopy. Feathered types were detected as triangular areas of hypoautofluorescence. The peau d'orange was much more visible and much more widespread on infrared imaging, with extension from the posterior pole towards the whole midperiphery. Comets and comet tails were best seen with red-free imaging. CONCLUSIONS: Infrared, red-free and autofluorescence imaging are more sensitive than white light funduscopy and imaging in visualising early retinal signs of PXE. In addition, this specialised imaging allows a better appreciation of the extent of lesions. Hence, such imaging increases the chances of making a correct diagnosis early, and aids in the accurate evaluation of evolution of disease in the ophthalmic follow-up of PXE patients.

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78 In 55% of fundi (24/44), the Table 1 Ophthalmological characteristics of patients Case Age, sex Eye BCVA AS Pd`O C(T) ODD NV PDT Anti-VEGF ABCC6 mutations 1 61, F OD 2/10 + + + À + + À p.R1459C/e OS 9/10 + + + À + + À 2 43, F OD 12/10 + + + À À À À p.R1141X/p.R1141X OS 12/10 + + + À À À À 3 35, M OD 11/10 + + + À À À À p.E125K/p.L1025P OS 1/300 + + + À + + + 4 21, M OD 10/10 + + + À À À À p.R1141X/p.R1141X OS 10/10 + + + + À À À 5 11, M OD 10/10 À + + À À À À c.3506+2T/C/e 3506+2T/COS 10/10 À + + À À À À 6 55, F OD 1/20 + + + À + À À p.R1141X/p.R1141X OS 1.5/10 + + + À + À À 7 40, M OD 4/10 + + + À + + À p.R265G/p.R1141X OS 1/10 + + + À + + À 8 22, F OD 10/10 + + + À À À À p.R1141X/p.R1141X OS 10/10 + + + À À À À 9 29, F OD 10/10 + + + À À À À p.G1263R/c.4182delGG OS 10/10 + + + À À À À 10 20, M OD 10/10 + + + + À À À c.3507-3C/A/p.T944I OS 10/10 + + + + À À À 11 37, F OD 10/10 + + + + À À À p.Q154R/e OS 9/10 + + + + À À À 12 66, F OD 1/20 + À À À + À À p.R1141X/p.R1141X OS 1/10 + À À À + À À 13 68, F OD 10/10 + À À À À À À p.R760Q/p.R1141X OS 10/10 + À À À À À À 14 68, M OD 2/10 + À + À + À À p.A1303P/p.L946I OS CF + À + À + À À 15 58, F OD 7/10 + + + À + À + p.R1141X/c.4103delC OS CF + + + À + + À 16 62,M OD 1/20 + + + À + À + p.R1141X/p.Q1237X OS CF + + + À + À À 17 47, F OD 10/10 + À À À À À À c.3506+2T/C/e OS 10/10 + À À À À À À 18 54, F OD 10/10 + + + À + À + p.R1141X/p.A1303P OS 10/10 + + + À À À À 19 30, F OD 10/10 + + + À À À À p.S398R/c.3364delT OS 10/10 + + + À À À À 20 39, F OD 12/10 + + + À À À À p.R1141X/e OS 10/10 + + + À À À À 21 30, F OD 10/10 + + + À À À À p.G666R/c.1868-5T/G OS 10/10 + + + + À À À 22 34, M OD 12/10 + + + À À À À c.3364delT/p.R518X OS 12/10 + + + À À À À anti-VEGF, anti-vascular endothelial growth factor antibodies; AS, angioid streaks; BCVA, best-corrected visual acuity (Snellen); CF, counting fingers; C(T), Comet (tails); F, female; M, male; MD, macular degeneration; NV, neovascularisation; ODD, optic disc drusen; Pd`O, Peau d`Orange; PDT, photodynamic therapy. Login to comment

[hide] Fundus autofluorescence in Pseudoxanthoma elasticu... Retina. 2009 Nov-Dec;29(10):1496-505. Finger RP, Charbel Issa P, Ladewig M, Gotting C, Holz FG, Scholl HP
Fundus autofluorescence in Pseudoxanthoma elasticum.
Retina. 2009 Nov-Dec;29(10):1496-505., [PMID:19823106]

Abstract [show]
PURPOSE: Pseudoxanthoma elasticum (PXE) is an inherited multisystem disorder of the elastic tissue. Typical ocular manifestations include angioid streaks, peau d'orange, salmon spots, and choroidal neovascularization (CNV). Changes in Bruch membrane lead to progressive atrophy of the retinal pigment epithelium (RPE), secondary CNVs, and visual loss. The RPE-photoreceptor complex was studied in vivo using fundus autofluorescence (FAF) imaging. METHODS: Forty-six patients (92 eyes) with PXE were investigated using digital fundus photography, fluorescein angiography (FA), and FAF imaging. The diagnosis was confirmed by multisystem clinical examination, mutation analysis of the ABCC6 gene, and skin biopsy. RESULTS: The mean age of the patient cohort was 50 years (range, 13-74 years), and mean visual acuity was 20/125. Fundus changes typical for PXE were observed in all eyes. Angioid streaks were detected in all but six eyes. Peau d'orange was hardly detectable on FAF, whereas comet tail lesions were apparent. Retinal pigment epithelium atrophy typically was widespread and heterogeneous, located mostly adjacent to angioid streaks or CNVs. Pattern dystrophy-like changes were only found in patients with previous CNV formation in the same or the contralateral eye. CONCLUSION: Abnormalities of the RPE-photoreceptor complex detected by FAF imaging were more diverse and widespread than expected from conventional fundus imaging. The exhibition of pattern dystrophy-like changes may be a transitional state toward a neovascular event in a subgroup of patients. The extensive alteration of the RPE suggests an important role of pathologic RPE changes in the evolution of visual loss in PXE.

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31 The analysis of genetic variations in the ABCC6 gene was performed as described elsewhere.18 Briefly, genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA)-whole blood, and the presence of the frequent ABCC6 gene mutation c.3421CϾT (p.R1141X) and the deletion Ex23-29del were analyzed by real-time polymerase chain reaction assay19 and a polymerase chain reaction method according to Hu et al.20 The exons 1 to 31 of the ABCC6 gene were polymerase chain reactions amplified with ABCC6-specific primers, excluding the amplification of the two ABCC6-pseudogenes ␺1 and ␺2, and analyzed by denaturating high-performance liquid chromatography and direct sequencing as described previously.18 Skin biopsies were taken from areas of visible skin lesions using a punch under local anesthesia. Login to comment
47 of Mutations 1 46 F Positive CA c.3421CϾT (p.R1141X) c.3412CϾT (p.R1138W) 2 2 44 F Positive PA 0 3 39 M NA CA c.3421CϾT (p.R1141X) c.3421CϾT (p.R1141X) 2 4 47 F Positive CA c.3421CϾT (p.R1141X) Ex23-29del 2 5 49 F Positive PA c.3421CϾT (p.R1141X) 1 6 39 M Positive NA 7 57 F NA PA c.3421CϾT (p.R1141X) 1 8 56 F Positive NA 9 51 F Positive PA 0 10 47 M Positive NA 11 39 F NA PA 0 12 58 M NA CA c.3421CϾT (p.R1141X) c.3715TϾC (p.Y1239H) 2 13 24 M Positive CA c.3421CϾT (p.R1141X) Deletion of unknown size 2 14 59 M Positive PA c.3421CϾT (p.R1141X) 1 15 47 F Positive PA c.3421CϾT (p.R1141X) 1 16 41 M Positive CA c.3421CϾT (p.R1141X) IVS27-6 GϾA 2 17 35 F Positive PA 0 18 74 M NA CA c.3421CϾT (p.R1141X) c.3421CϾT (p.R1141X) 2 19 67 F Positive CA c.4182delG Ex23-29del 2 20 70 M Positive CA c.3421CϾT (p.R1141X) c.3188TϾG (p.L1063R) 2 21 46 M Positive CA c.3421CϾT (p.R1141X) c.3421CϾT (p.R1141X) 2 22 61 M Positive NA 23 61 F NA CA c.754CϾT (p.L252F) c.2294GϾA (p.R765Q) 2 24 58 F NA PA 0 25 54 F NA CA c.3421CϾT (p.R1141X) 1 26 50 M Positive NA 27 38 F Positive CA c.113GϾC (p.W38S) 1 28 54 M Positive PA 0 29 52 F Positive NA 30 45 F Positive PA 0 31 45 F NA CA c.3421CϾT (p.R1141X) c.3940CϾT (p.R1314W) 2 32 27 M NA PA c.3421CϾT (p.R1141X) 1 33 59 F Positive NA 34 65 F Positive NA 35 50 M Positive CA c.3421CϾT (p.R1141X) c.2835_2850del16, c.2855TϾG (p.F952C) 3 36 62 F Positive NA 37 48 M Positive NA 38 20 F Positive PA c.3421CϾT (p.R1141X) 1 39 65 F Positive PA c.3421CϾT (p.R1141X) 1 40 13 F Positive CA c.3421CϾT (p.R1141X) c.3421CϾT (p.R1141X) 2 41 65 F Positive PA c.3412CϾT (p.R1138W) 1 42 72 M NA CA c.3421CϾT (p.R1141X) c.1574_1575insG 2 43 39 F NA PA 0 44 67 F NA CA c.3413GϾA (p.R1138Q) 1 45 43 F Positive NA 46 66 F Positive NA *GenBank accession no. Login to comment
50 CA, complete analysis of the coding regions of all exons and adjacent intron sequences, including analysis of the most frequent ABCC6 gene deletion Ex23-29del; PA, partial ABCC6 gene analysis with at least detection of the frequent mutation c.3421CϾT (p.R1141X) and the deletion Ex23-29del, plus potential DHPLC analysis and direct sequencing of selected exons; NA, not available. Login to comment

[hide] Pseudoxanthoma elasticum and familial hypercholest... Atherosclerosis. 2010 May;210(1):173-6. Epub 2009 Nov 24. Pisciotta L, Tarugi P, Borrini C, Bellocchio A, Fresa R, Guerra D, Quaglino D, Ronchetti I, Calandra S, Bertolini S
Pseudoxanthoma elasticum and familial hypercholesterolemia: a deleterious combination of cardiovascular risk factors.
Atherosclerosis. 2010 May;210(1):173-6. Epub 2009 Nov 24., [PMID:20018285]

Abstract [show]
BACKGROUND AND OBJECTIVE: Pseudoxanthoma Elasticum (PXE), an autosomal recessive disease due to mutations in ABCC6 gene, is characterised by fragmentation of elastic fibres with involvement of the cardiovascular system. We investigated a 60-year-old female with angina pectoris found to have PXE, associated with elevated plasma LDL-C suspected to be due to autosomal-co-dominant hypercholesterolemia. METHODS: ABCC6, LDLR, PCSK9 and exon 26 of APOB genes were re-sequenced. Cardiovascular involvement was assessed by coronary angiography, single-photon emission computed tomography (SPECT) and ultrasound examination. RESULTS AND CONCLUSIONS: The patient was a compound heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and heterozygous for a novel LDLR mutation (p.R574H). She had severe coronary stenosis and calcification of the arteries of the lower limbs. Treatment with ezetimibe/simvastatin 10/60mg/day, maintained over a 4.5-year period, reduced of LDL-C and the myocardial ischemic area. In PXE patients LDL-lowering treatment might contribute to delay macrovascular complications.

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4 Results and conclusions: The patient was a compound heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and heterozygous for a novel LDLR mutation (p.R574H). Login to comment
28 The proband, indicated by an arrow, was a compound heterozygous for the following ABCC6 gene mutations: (i) c.951 C > A in exon 8 (p.S317R); (ii) c.3421 C > T in exon 24 (p.R1141X). Login to comment
45 Subject (gender) I.1 (F) I.2 (F) I.3 (F) II.1 (F) II.2 (F) II.3 (M) III.1 (F) III.2 (F) III.3 (M) ABCC6 genotype M1/M2 W/M2 W/W W/M1 W/M1 W/M1 W/M1 W/W W/M1 LDLR genotype W/M3 W/W W/W W/M3 W/M3 W/M3 W/W W/M3 W/M3 Age (years) 60 56 34 40 39 36 7 15 8 BMI (kg/m2 ) 24.9 20.5 24.1 20.8 23.4 28.3 17.7 19.9 13.6 TC (mmol/L) 11.50 ± 0.59 7.88 5.53 8.22 7.03 7.19 4.55 5.74 5.71 LDL-C (mmol/L) 9.08 ± 0.54 5.44 3.29 4.96 4.78 5.22 2.75 3.75 3.77 HDL-C (mmol/L) 1.70 ± 0.16 2.01 1.83 2.66 1.89 0.90 1.55 1.52 1.68 TG (mmol/L) 1.93 ± 0.30 0.92 0.87 1.32 0.77 2.31 0.53 1.03 0.57 ApoA-I (mg/dL) 173 ± 10 209 202 226 182 112 192 183 194 ApoB (mg/dL) 246 ± 13 144 97 132 122 135 68 92 95 APOE genotype ␧3␧3 ␧3␧3 ␧3␧3 ␧2␧3 ␧3␧4 ␧2␧3 ␧2␧3 ␧3␧3 ␧3␧4 Values are mean ± SD; all values are before pharmacological treatment; ABCC6 genotype: W = wild type, M1 = c.951 C > A (p.S317R), M2 = c.3421 C > T (p.R1141X); LDLR genotype: W = wild type, M3 = c.1721 G > A (p.R574H). Login to comment
50 Sequence of ABCC6 gene The proband was compound heterozygous for mutations in ABCC6 gene: (i) a C > A transversion in exon 8 (c.951 C > A), which results in arginine for serine conversion at position 317 (p.S317R); (ii) a C > T transition in exon 24 (c.3421 C > T), which converts arginine codon at position 1141 into a termination codon (p.R1141X). Login to comment
51 One of the proband`s sisters (subject I.2) was a carrier of the p.R1141X mutation. Login to comment
64 Screening for the ABCC6 mutations Both mutations, p.S317R and p.R1141X, were screened in 106 healthy controls. Login to comment
66 The p.R1141X was also screened in 173 patients with premature CAD (before 55 years in males and 65 years in females) (Supplementary Table 1) Three carriers (two males and one female) were found among subjects of this group (1.7%). Login to comment
84 In this context it is noteworthy the observation that the frequency of the p.R1141X mutation of ABCC6 gene (the most common mutation in PXE) was found to be increased (3.3% vs. 0.8%) in a large group of patients with premature coronary heart disease [8]. Login to comment

[hide] Pseudoxanthoma elasticum: molecular genetics and p... J Invest Dermatol. 2010 Mar;130(3):661-70. Epub 2009 Dec 24. Uitto J, Li Q, Jiang Q
Pseudoxanthoma elasticum: molecular genetics and putative pathomechanisms.
J Invest Dermatol. 2010 Mar;130(3):661-70. Epub 2009 Dec 24., [PMID:20032990]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a prototypic heritable disorder with ectopic mineralization, manifests with characteristic skin findings, ocular involvement and cardiovascular problems, with considerable morbidity and mortality. The classic forms of PXE are due to loss-of-function mutations in the ABCC6 gene, which encodes ABCC6, a transmembrane efflux transporter expressed primarily in the liver. Several lines of evidence suggest that PXE is a primary metabolic disorder, which in the absence of ABCC6 transporter activity, displays reduced plasma anti-mineralization capacity due to reduced fetuin-A and matrix gla-protein (MGP) levels. MGP requires to be activated by gamma-glutamyl carboxylation, a vitamin K-dependent reaction, to serve in an anti-mineralization role in the peripheral connective tissue cells. Although the molecules transported from the hepatocytes to circulation by ABCC6 in vivo remain unidentified, it has been hypothesized that a critical vitamin K derivative, such as reduced vitamin K conjugated with glutathione, is secreted to circulation physiologically, but not in the absence of ABCC6 transporter activity. As a result, activation of MGP by gamma-glutamyl carboxylase is diminished, allowing slow yet progressive mineralization of connective tissues characteristic of PXE. Understanding of the pathomechanistic details of PXE provides a basis for the development of targeted molecular therapies for this currently intractable disease.

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71 Two recurrent mutations of high frequency have been identified: p.R1141X in exon 24 is the most common, particularly in Caucasian individuals, with a prevalence of approximately 30% of all pathologic PXE mutations (Pfendner et al., 2007). Login to comment
118 In a particularly illustrative family, individuals were shown to be heterozygous for a nonsense mutation (p.R1141X) in ABCC6 and a missense mutation (p.V255M) in GGCX. Login to comment

[hide] Pseudoxanthoma elasticum: progress in diagnostics ... Am J Med Genet A. 2011 Jul;155A(7):1517-26. doi: 10.1002/ajmg.a.34067. Epub 2011 Jun 10. Uitto J, Bercovitch L, Terry SF, Terry PF
Pseudoxanthoma elasticum: progress in diagnostics and research towards treatment : Summary of the 2010 PXE International Research Meeting.
Am J Med Genet A. 2011 Jul;155A(7):1517-26. doi: 10.1002/ajmg.a.34067. Epub 2011 Jun 10., [PMID:21671388]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a prototypic heritable disorder with ectopic mineralization, manifests with characteristic skin findings, ocular involvement, and cardiovascular problems. The classic forms of PXE are due to loss-of-function mutations in the ABCC6 gene, which encodes ABCC6, a putative transmembrane efflux transporter expressed primarily in the liver. While considerable progress has recently been made in understanding the molecular genetics and pathomechanisms of PXE, no effective or specific treatment is currently available for this disorder. PXE International, the premiere patient advocacy organization, organized a workshop in November 2010 to assess the current state of diagnostics and research to develop an agenda towards treatment of PXE. This overview summarizes the progress in PXE research, with emphasis on molecular therapies for this, currently intractable, disorder.

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25 There are recent suggestions, however, that certain mutations in ABCC6 may bestatistically associatedwithinvolvementofspecifictargetorgans, such as the p.R1268Q mutation being associated with early onset of angioidstreaks[Satoetal.,2009;Lietal.,2011a],andthestopcodon mutation p.R1141X possibly predisposing the individuals to car- diovascularinvolvementindependentofhyperlipidemiainpatients with PXE [K€obl€os et al., 2010; Pisciotta et al., 2010]. Login to comment

[hide] ABCC6 expression is regulated by CCAAT/enhancer-bi... J Invest Dermatol. 2012 Dec;132(12):2709-17. doi: 10.1038/jid.2012.218. Epub 2012 Jul 5. Ratajewski M, de Boussac H, Sachrajda I, Bacquet C, Kovacs T, Varadi A, Pulaski L, Aranyi T
ABCC6 expression is regulated by CCAAT/enhancer-binding protein activating a primate-specific sequence located in the first intron of the gene.
J Invest Dermatol. 2012 Dec;132(12):2709-17. doi: 10.1038/jid.2012.218. Epub 2012 Jul 5., [PMID:22763786]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a rare recessive genetic disease causing skin, eye, and cardiovascular lesions, is characterized by the calcification of elastic fibers. The disorder is due to loss-of-function mutations of the ABCC6 gene, but the pathophysiology of the disease is still not understood. Here we investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assay. We identified three DNase I hypersensitive sites (HSs) specific to cell lines expressing ABCC6. These HSs are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HSs by luciferase assay and demonstrated the transcriptional activity of the intronic HS. We identified the CCAAT/enhancer-binding protein beta (C/EBPbeta) as a factor binding the second intronic HS by chromatin immunoprecipitation and corroborated this finding by luciferase assays. We also showed that C/EBPbeta interacts with the proximal promoter of the gene. We propose that C/EBPbeta forms a complex with other regulatory proteins including the previously identified regulatory factor hepatocyte nuclear factor 4alpha (HNF4alpha). This complex would account for the tissue-specific expression of the gene and might serve as a metabolic sensor. Our results point toward a better understanding of the physiological role of ABCC6.

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20 Stop codon mutation p.R1141X represents 25% of all observed mutations. Login to comment

[hide] The molecular and physiological roles of ABCC6: mo... Front Genet. 2012 Dec 12;3:289. doi: 10.3389/fgene.2012.00289. eCollection 2012. Le Saux O, Martin L, Aherrahrou Z, Leftheriotis G, Varadi A, Brampton CN
The molecular and physiological roles of ABCC6: more than meets the eye.
Front Genet. 2012 Dec 12;3:289. doi: 10.3389/fgene.2012.00289. eCollection 2012., [PMID:23248644]

Abstract [show]
Abnormal mineralization occurs in the context of several common conditions, including advanced age, diabetes, hypercholesterolemia, chronic renal failure, and certain genetic conditions. Metabolic, mechanical, infectious, and inflammatory injuries promote ectopic mineralization through overlapping yet distinct molecular mechanisms of initiation and progression. The ABCC6 protein is an ATP-dependent transporter primarily found in the plasma membrane of hepatocytes. ABCC6 exports unknown substrates from the liver presumably for systemic circulation. ABCC6 deficiency is the primary cause for chronic and acute forms of ectopic mineralization described in diseases such as pseudoxanthoma elasticum (PXE), beta-thalassemia, and generalized arterial calcification of infancy (GACI) in humans and dystrophic cardiac calcification (DCC) in mice. These pathologies are characterized by mineralization of cardiovascular, ocular, and dermal tissues. PXE and to an extent GACI are caused by inactivating ABCC6 mutations, whereas the mineralization associated with beta-thalassemia patients derives from a liver-specific change in ABCC6 expression. DCC is an acquired phenotype resulting from cardiovascular insults (ischemic injury or hyperlipidemia) and secondary to ABCC6 insufficiency. Abcc6-deficient mice develop ectopic calcifications similar to both the human PXE and mouse DCC phenotypes. The precise molecular and cellular mechanism linking deficient hepatic ABCC6 function to distal ectopic mineral deposition is not understood and has captured the attention of many research groups. Our previously published work along with that of others show that ABCC6 influences other modulators of calcification and that it plays a much greater physiological role than originally thought.

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167 However, this is not without controversy as a much larger study based on 66,831 individuals has found no risk for ischemic heart diseases associated with the ABCC6 p.R1141X mutation (Hornstrup et al., 2011). Login to comment
168 Stroke is also a vascular-related condition frequently reported in PXE patients (Aessopos et al., 1997; van den Berg et al., 2000) but it could well be that strokes etiology in certain PXE individuals might not be related to ABCC6 deficiency as Hornstrup et al. could not statistically link cerebrovascular diseases with the most frequent ABCC6 mutation (p.R1141X). Login to comment

[hide] The vascular phenotype in Pseudoxanthoma elasticum... Front Genet. 2013 Feb 12;4:4. doi: 10.3389/fgene.2013.00004. eCollection 2013. Leftheriotis G, Omarjee L, Le Saux O, Henrion D, Abraham P, Prunier F, Willoteaux S, Martin L
The vascular phenotype in Pseudoxanthoma elasticum and related disorders: contribution of a genetic disease to the understanding of vascular calcification.
Front Genet. 2013 Feb 12;4:4. doi: 10.3389/fgene.2013.00004. eCollection 2013., [PMID:23408347]

Abstract [show]
Vascular calcification is a complex and dynamic process occurring in various physiological conditions such as aging and exercise or in acquired metabolic disorders like diabetes or chronic renal insufficiency. Arterial calcifications are also observed in several genetic diseases revealing the important role of unbalanced or defective anti- or pro-calcifying factors. Pseudoxanthoma elasticum (PXE) is an inherited disease (OMIM 264800) characterized by elastic fiber fragmentation and calcification in various soft conjunctive tissues including the skin, eyes, and arterial media. The PXE disease results from mutations in the ABCC6 gene, encoding an ATP-binding cassette transporter primarily expressed in the liver, kidneys suggesting that it is a prototypic metabolic soft-tissue calcifying disease of genetic origin. The clinical expression of the PXE arterial disease is characterized by an increased risk for coronary (myocardial infarction), cerebral (aneurysm and stroke), and lower limb peripheral artery disease. However, the structural and functional changes in the arterial wall induced by PXE are still unexplained. The use of a recombinant mouse model inactivated for the Abcc6 gene is an important tool for the understanding of the PXE pathophysiology although the vascular impact in this model remains limited to date. Overlapping of the PXE phenotype with other inherited calcifying diseases could bring important informations to our comprehension of the PXE disease.

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118 In the coronary arterial bed, the association with a heterozygous R1141X mutation in ABCC6 and ischemic vascular events including stroke, was not demonstrated in the general population (n = 66831 participants; Hornstrup et al., 2011), although a strong association was reported only with coronary artery disease (Koblos et al., 2010). Login to comment

[hide] ABCC6 Mutation in Patients with Angioid Streaks. Int J Biomed Sci. 2006 Feb;2(1):7-12. Mizutani Y, Nakayama T, Asai S, Shimada H, Yuzawa M
ABCC6 Mutation in Patients with Angioid Streaks.
Int J Biomed Sci. 2006 Feb;2(1):7-12., [PMID:23674961]

Abstract [show]
Angioid streaks (AS) are hereditary eye conditions caused by breaks in the elastic layer of Bruch's membrane. Patients with AS are also frequently affected with pseudoxanthoma elasticum (PXE). The locus of PXE has been reported to exist in chromosome 16p13.1, and the ABCC6 gene in this locus has been identified as the causal gene of PXE. In this study we investigated the association of the ABCC6 gene and AS. Elucidation of the causal gene of AS will be useful for gene diagnosis in the future. Many mutations in patients with PXE are found in exons 24 and 27 of the ABCC6 gene in previous reports. Therefore, we examined exons 24 and 27 of the ABCC6 gene using the single-strand conformation polymorphism technique. There was no mutation or polymorphism in exon 24. The base substitution of G3803A was identified in exon 27, with a change in the amino acid from CGG to CAG (R1268Q). The genotype frequencies in patients with AS were G/G 52% (23/44), G/A 32% (14/44) and A/A 16% (14/44). In control subjects, the genotype frequencies were G/G 69% (107/154), G/A 29% (44/154) and A/A 2% (3/154). Highly significant differences were observed in both genotype and allele frequencies of R1268Q between patients with AS and control subjects (p<0.001, p<0.002; chi-square test). In conclusion, the missense mutation R1268Q in the ABCC6 gene is not a specific marker of PXE, but is associated with the disease state of AS.

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101 However, some previous studies reported that R1141X mutation in exon 24 was the most frequent mutation in PXE (20, 21). Login to comment

[hide] Premature termination codon read-through in the AB... J Invest Dermatol. 2013 Dec;133(12):2672-7. doi: 10.1038/jid.2013.234. Epub 2013 May 23. Zhou Y, Jiang Q, Takahagi S, Shao C, Uitto J
Premature termination codon read-through in the ABCC6 gene: potential treatment for pseudoxanthoma elasticum.
J Invest Dermatol. 2013 Dec;133(12):2672-7. doi: 10.1038/jid.2013.234. Epub 2013 May 23., [PMID:23702584]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, with ~35% of all mutations being premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE-associated nonsense mutations, and was evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cytotoxicity in concentrations up to 20 mug ml(-1), and the facilitated read-through varied not only with dose but also with sequence context. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1,141 by glycine, tryptophan, or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish messenger RNA (mRNA) rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.

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4 Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1,141 by glycine, tryptophan, or cysteine. Login to comment
20 Received 1 March 2013; revised 17 April 2013; accepted 27 April 2013; accepted article preview online 23 May 2013; published online 27 June 2013 Abbreviations: mRNA, messenger RNA; PXE, pseudoxanthoma elasticum 2672 Journal of Investigative Dermatology (2013), Volume 133 & 2013 The Society for Investigative Dermatology recurrent nonsense mutation is p.R1141X, which accounts for B30% of all pathogenic PXE mutations in Caucasian patient populations. Login to comment
26 In this study, we focused on seven of them, including the most common stop codon mutation, p.R1141X, in which the arginine at position 1,141 (codon CGA) has been replaced by a stop codon (TGA) (Table 1). Login to comment
38 Different pseudoxanthoma elasticum (PXE)- associated nonsense mutations tested for PTC124 read-through Mutation Nucleotide sequence1 Location (exon) Mutation frequency (%)2 p.R1141X TGA-A 24 54 p.R1164X TGA-C 24 10 p.R518X TGA-G 12 1.2 p.R1398X TGA-G 29 1.2 p.Q378X TAG-A 9 o1 p.Q1143X TAG-G 24 o1 p.R1275X TGA-C 27 o1 1 The sequence depicts the stop codon (bold) followed by the nucleotide shown. Login to comment
42 An expression vector (p.CMV6-Entry) containing full-length wild-type human ABCC6 cDNA (a) or a corresponding mutant cDNA harboring p.R1141X premature termination codon containing a 30 -end sequence that encodes a DDK reporter peptide (b, c) or p.R1275X (d) was transfected into HEK293 cells in culture without (a, b) or with PTC124 (10mg ml 1 for 72hours) (c, d). Login to comment
50 In case of the p.R1141X mutation, considering the codon redundancy and possible combinations of the corrected mutations from the stop codon (TGA) to an amino acid, in addition to arginine (CGA), this position could be occupied either by cysteine (TGC) or tryptophan (TGG) or glycine (GGA). Login to comment
71 In contrast, injection of human ABCC6 mRNA harboring the p.R1141X stop codon mutation failed to reverse the phenotype (Figure 5d and Table 2), suggesting that this mRNA rescue system can be used to check the pathogenicity of mutant human ABCC6 proteins, including those harboring the missense amino acids as a result of PTC124 read-through. Login to comment
87 However, injection of human ABCC6 mRNA, which harbors the p.R1141X mutation, does not result in phenotypic rescue (d). Login to comment
90 of embryos injected Lethality (%)2 Mo - 107 71.0 Scmo - 108 12.0 Mo&#fe; WT Arg 96 9.4 Mo&#fe; TGA STOP 102 85.3 Mo&#fe; TGG Trp 94 26.6 Mo&#fe; TGC Cys 134 7.5 Mo&#fe; GGA Gly 125 16.0 1 Zebrafish embryos were injected at day 0 with an abcc6a morpholino (Mo) alone or with human ABCC6 mRNA, either wild type (WT) harboring arginine at position 1,141, corresponding to the p.R1141X stop codon mutation (TGA), or containing a missense substitution of tryptophan (TGG), cysteine (TGC), or glycine (GGA) instead of arginine at position 1,141. Login to comment
93 ABCC6 with p.R1141X mutations in both alleles on the Abcc6 / background. Login to comment
95 It is conceivable that the presence of such an amino acid carried by a near-cognate transfer RNA (instead of the parent wild-type arginine in case of p.R1141X) could be pathogenic, essentially representing a missense mutation. Login to comment
132 Capped full-length human ABCC6 mRNA corresponding to wild-type, R1141X, or putative read-through substitutions of arginine1141 by cysteine, tryptophan, or glycine was transcribed from an expression vector pCMV-Tag4B using the T3 mMessage mMachine kit (Ambion, Austin, TX). Login to comment

[hide] New insights into the pathogenesis of pseudoxantho... Front Genet. 2013 Jun 19;4:114. doi: 10.3389/fgene.2013.00114. eCollection 2013. Hendig D, Knabbe C, Gotting C
New insights into the pathogenesis of pseudoxanthoma elasticum and related soft tissue calcification disorders by identifying genetic interactions and modifiers.
Front Genet. 2013 Jun 19;4:114. doi: 10.3389/fgene.2013.00114. eCollection 2013., [PMID:23802012]

Abstract [show]
Screening of the adenosine triphosphate binding cassette transporter protein subfamily C member 6 gene (ABCC6) in pseudoxanthoma elasticum (PXE) revealed a mutation detection rate of approximately 87%. Although 25% of the unidentified disease alleles underlie deletions/insertions, there remain several PXE patients with no clear genotype. The recent identification of PXE-related diseases and the high intra-familiar and inter-individual clinical variability of PXE led to the assumption that secondary genetic co-factors exist. Here, we summarize current knowledge of the genetics underlying PXE and PXE-related disorders based on human and animal studies. Furthermore, we discuss the role of genetic interactions and modifier genes in PXE and PXE-related diseases characterized by soft tissue calcification.

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22 The most frequent mutation found in PXE patients is a nonsense mutation in exon 24, p.R1141X (c.3421C>T, rs72653706), which is found in approximately 25% of the European patient population. Login to comment
40 Mother and maternal aunt were identified as compound heterozygous carriers of the recurrent ABCC6 nonsense mutation p.R1141X and a missense mutation in GGCX (p.S300F). Login to comment
118 Few studies investigated the correlation of ABCC6 mutations with cardiovascular disease and identified the frequent mutation p.R1141X as a strong genetic risk factor for CAD (Trip et al., 2002; Wegman et al., 2005; K&#f6;bl&#f6;s et al., 2010). Login to comment

[hide] Atorvastatin counteracts aberrant soft tissue mine... J Mol Med (Berl). 2013 Oct;91(10):1177-84. doi: 10.1007/s00109-013-1066-5. Epub 2013 Jun 27. Guo H, Li Q, Chou DW, Uitto J
Atorvastatin counteracts aberrant soft tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6(-)/(-)).
J Mol Med (Berl). 2013 Oct;91(10):1177-84. doi: 10.1007/s00109-013-1066-5. Epub 2013 Jun 27., [PMID:23807484]

Abstract [show]
Pseudoxanthoma elasticum (PXE), a multisystem heritable disorder with aberrant mineralization of arterial blood vessels, is caused by mutations in the ABCC6 gene. Previous studies have suggested that carriers of the ABCC6 mutations, particularly of p.R1141X, are at increased risk for coronary artery disease. In this study, we used Abcc6 (tm1Jfk) knock-out mice to determine the serum lipid profiles and examine the effects of atorvastatin on the aberrant mineralization in this model of PXE. First, serum lipid profiles at 12 weeks of age, after overnight fasting, revealed a statistically significant increase in total cholesterol and triglyceride levels in Abcc6 (tm1Jfk) mice compared to their wild-type littermates. Placing these mice at 4 weeks of age for 20 weeks on atorvastatin, either 0.01 % or 0.04 % of the diet (low statin and high statin groups, respectively), reduced the total triglyceride and cholesterol levels, which was accompanied with significantly reduced mineralization of the dermal sheath of vibrissae, a biomarker of the aberrant mineralization process in these mice. However, if the mice were placed on atorvastatin for 12 weeks at 12 weeks of age, at which time point significant mineralization had already taken place, no difference in the amount of mineralization was noted. These observations suggest that statins, particularly atorvastatin, can prevent, but not reverse, aberrant mineralization in this mouse model of PXE. For a clinical perspective, a survey of 1,747 patients with PXE was conducted regarding their present or past use of statins. The results indicated that about one third of all PXE patients are currently or have previously been on cholesterol-lowering drugs. Thus, a sizable number of patients with PXE could be subject to modulation of their mineralization processes by concomitant statin treatment. KEY MESSAGE: The Abcc6 (-/-) mice serve as a model system for PXE, an ectopic mineralization disorder Abcc6 (-/-) mice were shown to have elevated serum cholesterol and triglyceride levels Feeding of the Abcc6 (-/-) mice with atorvastatin prevented connective tissue mineralization A third of patients with PXE was found to be on cholesterol-lowering therapy Atorvastatin may potentially be beneficial for patients with PXE.

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1 Previous studies have suggested that carriers of the ABCC6 mutations, particularly of p.R1141X, are at increased risk for coronary artery disease. Login to comment
23 However, it has been suggested that heterozygous carrier status of loss-of-function mutations in the ABCC6 gene, particularly that of p.R1141X, the most common mutation in the Caucasian populations, is associated with a strong increase in the prevalence of coronary artery disease [13, 14]. Login to comment

[hide] Characterization of cardiovascular involvement in ... Arterioscler Thromb Vasc Biol. 2013 Nov;33(11):2646-52. doi: 10.1161/ATVBAHA.113.301901. Epub 2013 Aug 22. Campens L, Vanakker OM, Trachet B, Segers P, Leroy BP, De Zaeytijd J, Voet D, De Paepe A, De Backer T, De Backer J
Characterization of cardiovascular involvement in pseudoxanthoma elasticum families.
Arterioscler Thromb Vasc Biol. 2013 Nov;33(11):2646-52. doi: 10.1161/ATVBAHA.113.301901. Epub 2013 Aug 22., [PMID:23968982]

Abstract [show]
OBJECTIVE: Pseudoxanthoma elasticum (PXE) is an autosomal recessive connective tissue disorder with involvement of the skin, the retina, and the cardiovascular system. Cardiovascular involvement is mainly characterized by mineralization and fragmentation of elastic fibers of blood vessels and premature atherosclerosis. We conducted an ultrasound study to investigate the cardiovascular phenotype and to propose recommendations for the management of patients with PXE and heterozygous ABCC6 mutation carriers. APPROACH AND RESULTS: Thirty-two patients, 23 carriers, and 28 healthy volunteers underwent cardiac and vascular ultrasound studies. Cardiac imaging revealed left ventricular diastolic dysfunction in patients with PXE with a significantly prolonged deceleration time and lower septal early diastolic velocities of the mitral annulus compared with controls. Carriers also demonstrated significantly prolonged deceleration time. Carotid-to-femoral pulse wave velocity was significantly increased in patients with PXE when compared with carriers and controls. Vascular imaging revealed a high prevalence of peripheral artery disease in both patients and carriers and a significantly higher carotid intima-media thickness compared with controls. CONCLUSIONS: The results of this study clearly demonstrate impaired left ventricular diastolic function, impairment of the elastic properties of the aorta, and a high prevalence of peripheral artery disease in patients with PXE. Carriers also seem to exhibit a cardiovascular phenotype with mainly mild diastolic dysfunction and accelerated atherosclerosis. Increased awareness for cardiovascular events in both patients and heterozygous carriers is warranted.

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86 Patients with PXE generally have a normal life span but are prone to vascular disease, such as CAD, PAD, and cerebrovascular disease.3 Conversely, a large population-based study showed an association between premature CAD and a frequent mutation in the ABCC6 gene (R1141X).12 As such, PXE can serve as a genetic model for the study of the pathophysiology of common cardiovascular diseases. Login to comment

[hide] Mutations in the ABCC6 gene as a cause of generali... J Invest Dermatol. 2014 Mar;134(3):658-65. doi: 10.1038/jid.2013.370. Epub 2013 Sep 5. Li Q, Brodsky JL, Conlin LK, Pawel B, Glatz AC, Gafni RI, Schurgers L, Uitto J, Hakonarson H, Deardorff MA, Levine MA
Mutations in the ABCC6 gene as a cause of generalized arterial calcification of infancy: genotypic overlap with pseudoxanthoma elasticum.
J Invest Dermatol. 2014 Mar;134(3):658-65. doi: 10.1038/jid.2013.370. Epub 2013 Sep 5., [PMID:24008425]

Abstract [show]
Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by congenital calcification of large- and medium-sized arteries, associated with early myocardial infarction, heart failure, and stroke, and premature death. Most cases of GACI are caused by mutations in the ENPP1 gene. We first studied two siblings with GACI from a non-consanguineous family without mutations in the ENPP1 gene. To search for disease-causing mutations, we identified genomic regions shared between the two affected siblings but not their unaffected parents or brother. The ABCC6 gene, which is mutated in pseudoxanthoma elasticum (PXE), resided within a small region of homozygosity shared by the affected siblings. Sequence analysis of ABCC6 revealed that the two affected siblings were homozygous for the missense mutation p.R1314W. Subsequently, ABCC6 mutations were identified in five additional GACI families with normal ENPP1 sequences. Genetic mutations in ABCC6 in patients with PXE are associated with ectopic tissue mineralization in the skin and arterial blood vessels. Thus, our findings provide additional evidence that the ABCC6 gene product inhibits calcification under physiologic conditions and confirm a second locus for GACI. In addition, our study emphasizes the potential utility of shared homozygosity mapping to identify genetic causes of inherited disorders.

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36 Family D 1 2 1 Family E c.3692insTT +/- 1 2 1 2 3 Family F I II g.del23-29 +/- p.R760W +/- 1 2 1 2 3 4 1 2 1 2 1 2 1 Family A Family B Family C I II p.R1314W +/- p.R1314W +/- c.2787+1G>T c.3736-1G>A +/- p.R391G +/- p.R391G +/- p.R1141X +/- c.346-6G>A +/- +/+ 1 2 3 4 p.R1141X +/+ +/- c.346-6G>A +/- 1 2 -/- -/- +/- +/+ +/+ +/+ +/- +/+ Figure 1. Login to comment
43 The proband in Family D (patient 5) had compound heterozygous mutations c.346-6G4A and p.R1141X in intron 3 and exon 24 of ABCC6. Login to comment

[hide] Analysis of pseudoxanthoma elasticum-causing misse... J Invest Dermatol. 2014 Apr;134(4):946-53. doi: 10.1038/jid.2013.482. Epub 2013 Nov 11. Pomozi V, Brampton C, Fulop K, Chen LH, Apana A, Li Q, Uitto J, Le Saux O, Varadi A
Analysis of pseudoxanthoma elasticum-causing missense mutants of ABCC6 in vivo; pharmacological correction of the mislocalized proteins.
J Invest Dermatol. 2014 Apr;134(4):946-53. doi: 10.1038/jid.2013.482. Epub 2013 Nov 11., [PMID:24352041]

Abstract [show]
Mutations in the ABCC6 gene cause soft-tissue calcification in pseudoxanthoma elasticum (PXE) and, in some patients, generalized arterial calcification of infancy (GACI). PXE is characterized by late onset and progressive mineralization of elastic fibers in dermal, ocular, and cardiovascular tissues. GACI patients present a more severe, often prenatal arterial calcification. We have tested 10 frequent disease-causing ABCC6 missense mutants for the transport activity by using Sf9 (Spodoptera frugiperda) cells, characterized the subcellular localization in MDCKII (Madin-Darby canine kidney (cell line)) cells and in mouse liver, and tested the phenotypic rescue in zebrafish. We aimed at identifying mutants with preserved transport activity but with improper plasma membrane localization for rescue by the chemical chaperone 4-phenylbutyrate (4-PBA). Seven of the mutants were transport-competent but mislocalized in mouse liver. The observed divergence in cellular localization of mutants in MDCKII cells versus mouse liver underlined the limitations of this 2D in vitro cell system. The functionality of ABCC6 mutants was tested in zebrafish, and minimal rescue of the morpholino-induced phenotype was found. However, 4-PBA, a drug approved for clinical use, restored the plasma membrane localization of four ABCC6 mutants (R1114P, S1121W, Q1347H, and R1314W), suggesting that allele-specific therapy may be useful for selected patients with PXE and GACI.

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39 In addition to 10 missense mutants, R1141X was also used as a negative control in certain experiments. Login to comment
50 The nonsense R1141X mutant was not expressed in Sf9 cells. Login to comment
51 Subcellular localization in vitro Each mutant (with the exception of R1141X) was individually expressed in MDCKII cells, which were then grown either on plastic wells as nonpolarized cells or on Transwell filters, which ensures development of monolayers of polarized cells. Login to comment
67 The R1141X mutant was not expressed in the mouse liver. Login to comment
72 Next, the injection of the nonsense R1141X mRNA showed that this mutant was ineffective in rescuing the morpholino-mediated phenotype (4.8%). Login to comment
133 (d) Animals injected with morpholino and with ABCC6 R1141X mRNA. Login to comment
150 Summary of the characterization and rescue of disease-causing ABCC6 mutants Localization in mouse liver Localization in MDCKII cell line Nonpolarized Polarized ABCC6 variant Sf9 transport activity Without treatment After 4-PBA treatment Without treatment After 4-PBA treatment Without treatment After 4-PBA treatment Zebrafish &#fe; mRNA rescue (%) Wild type Active PM1 PM PM PM PM PM 90.6 R1114P Active IC4PM PM (rescue) ICoPM PM (rescue) PM PM 0.0 S1121W Active IC4PM PM (rescue) PM PM PM PM 7.9 R1138Q Active IC4PM IC4PM (no effect) IC PM (rescue) PM PM 1.8 V1298F o20% PM ND PM PM PM ND 32.0 T1301I Active IC4PM IC4PM (no effect) IC4PM PM (rescue) PM PM 5.1 R1314W1 Active IC4PM PM (rescue) IC PM (rescue) IC4PM PM (rescue) 0.0 G1321S o20% IC ND IC4PM IC4PM (no effect) IC IC (no effect) 0.0 R1339C Not stable IC IC (no effect) IC IC (no effect) IC IC (no effect) 0.0 Q1347H Active IC4PM PM (rescue) IC4PM PM (rescue) IC &#bc; PM IC&#bc; PM (no effect) 0.8 R1459C Active PM ND IC &#bc; PM PM (rescue) ICoPM ICoPM (no effect) 0.0 delABCC6 o20% IC IC IC IC IC IC ND R1141X Stop ND ND ND ND ND ND 4.8 Abbreviations: IC, intracellular; ND, not determined; PM, plasma membrane. Login to comment

[hide] Regulation of ABCC6 trafficking and stability by a... PLoS One. 2014 May 19;9(5):e97360. doi: 10.1371/journal.pone.0097360. eCollection 2014. Xue P, Crum CM, Thibodeau PH
Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence.
PLoS One. 2014 May 19;9(5):e97360. doi: 10.1371/journal.pone.0097360. eCollection 2014., [PMID:24840500]

Abstract [show]
Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.

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430 Wegman JJ, Hu X, Tan H, Bergen AA, Trip MD, et al. (2005) Patients with premature coronary artery disease who carry the ABCC6 R1141X mutation have no Pseudoxanthoma Elasticum phenotype. Login to comment

[hide] ABCC6- a new player in cellular cholesterol and li... Lipids Health Dis. 2014 Jul 27;13:118. doi: 10.1186/1476-511X-13-118. Kuzaj P, Kuhn J, Dabisch-Ruthe M, Faust I, Gotting C, Knabbe C, Hendig D
ABCC6- a new player in cellular cholesterol and lipoprotein metabolism?
Lipids Health Dis. 2014 Jul 27;13:118. doi: 10.1186/1476-511X-13-118., [PMID:25064003]

Abstract [show]
BACKGROUND: Dysregulations in cholesterol and lipid metabolism have been linked to human diseases like hypercholesterolemia, atherosclerosis or the metabolic syndrome. Many ABC transporters are involved in trafficking of metabolites derived from these pathways. Pseudoxanthoma elasticum (PXE), an autosomal-recessive disease caused by ABCC6 mutations, is characterized by atherogenesis and soft tissue calcification. METHODS: In this study we investigated the regulation of cholesterol biosynthesis in human dermal fibroblasts from PXE patients and healthy controls. RESULTS: Gene expression analysis of 84 targets indicated dysregulations in cholesterol metabolism in PXE fibroblasts. Transcript levels of ABCC6 were strongly increased in lipoprotein-deficient serum (LPDS) and under serum starvation in healthy controls. For the first time, increased HMG CoA reductase activities were found in PXE fibroblasts. We further observed strongly elevated transcript and protein levels for the proprotein convertase subtilisin/kexin type 9 (PCSK9), as well as a significant reduction in APOE mRNA expression in PXE. CONCLUSION: Increased cholesterol biosynthesis, elevated PCSK9 levels and reduced APOE mRNA expression newly found in PXE fibroblasts could enforce atherogenesis and cardiovascular risk in PXE patients. Moreover, the increase in ABCC6 expression accompanied by the induction of cholesterol biosynthesis supposes a functional role for ABCC6 in human lipoprotein and cholesterol homeostasis.

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228 Pisciotta et al. examined a hypercholesterolemic PXE patient, who was compound-heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and for further mutations in candidate genes causing autosomal co-dominant hypercholesterolemia [54]. Login to comment

[hide] Efficiency of exome sequencing for the molecular d... J Invest Dermatol. 2015 Apr;135(4):992-8. doi: 10.1038/jid.2014.421. Epub 2014 Sep 29. Hosen MJ, Van Nieuwerburgh F, Steyaert W, Deforce D, Martin L, Leftheriotis G, De Paepe A, Coucke PJ, Vanakker OM
Efficiency of exome sequencing for the molecular diagnosis of pseudoxanthoma elasticum.
J Invest Dermatol. 2015 Apr;135(4):992-8. doi: 10.1038/jid.2014.421. Epub 2014 Sep 29., [PMID:25264593]

Abstract [show]
The molecular etiology of pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in ATP-binding cassette family C member 6 (ABCC6) but also ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and gamma-glutamyl carboxylase (GGCX) can cause resembling phenotypes. Identification of modifier genes, such as vascular endothelial growth factor A, has further contributed to the molecular heterogeneity of PXE. In such heterogeneous diseases, next-generation sequencing (NGS) allows to perform mutation screening of several genes in a single reaction. We explored whole-exome sequencing (WES) as an efficient diagnostic tool to identify the causal mutations in ABCC6, GGCX, ENPP1, and vitamin K epoxide reductase complex, subunit 1 (VKORC1) in 16 PXE patients. WES identified a causal ABCC6 mutation in 30 out of 32 alleles and one GGCX mutation, whereas no causal mutations in ENPP1 or VKORC1 were detected. Exomes with insufficient reads (20 depth) for the four genes and patients with single mutations were further evaluated by Sanger sequencing (SS), but no additional mutations were found. The potential of WES compared with targeted NGS is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative. Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.

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89 List of mutations found by WES and SS Gene Nucleotide change Protein change Patient ID Hom/Het WES SS Known/PUR Reference ABCC6 c.C118T p.(P40S) P10 Het O O PUR ABCC6 c.998 &#fe; 2 998 &#fe; 3del TG P8 Het O O PUR ABCC6 c.T1484A p.(L495H) P7 Het O O Known Miksch et al., 2005 ABCC6 c.G1553A p.(R518Q) P11 Hom O O Known Uitto et al., 2001 ABCC6 c.G1553A p.(R518Q) P12, P13, P14 Het O O Known Uitto et al., 2001 ABCC6 c.G2263A p.(G755R) P11 Het O O Known Pfendner et al., 2007 ABCC6 c.G2294A p.(R765Q) P3 Het O O Known Le Saux et al., 2001 ABCC6 del2860_2865 P12, P13,14 Het O O PUR ABCC6 c.T2911C p.(W971R) P11 Het O O PUR ABCC6 Ex23_24del P2 Hom O O Known Ringpfeil et al., 2001 ABCC6 c.T3032C p.(L1011P) P9 Hom O O PUR ABCC6 c.C3190T p.(A1064T) P7 Het O O Known Miksch et al., 2005 ABCC6 c.G3413A p.(R1138Q) P11 Het O O Known Le Saux O, 2011 ABCC6 c.C3421T p.(R1141X) P4 Hom O O Known Bergen et al., 2000 ABCC6 c.C3421T p.(R1141X) P52 , P8, P162 Het O O Known Bergen et al., 2000 ABCC6 c.C3490T p.(R1164X) P6, P15 Hom O O Known Struk et al., 2000 ABCC6 c.G4198A p.(E1400K) P10 Het O O Known Chassaing et al., 2004 ABCC6 c.C4216A p.(Q1406K) P3 Het O O PUR GGCX c.C1321T p.(R441C) P7 Het O O PUR Het, heterozygous; Hom, homozygous; PUR, previously unreported; SS, Sanger sequencing; WES, whole-exome sequencing. Login to comment
99 In some of these, the p.(R1141X) mutation has been correlated with the presence of coronary artery disease (Trip et al., 2002; Sherer et al., 2001). Login to comment
100 In our cohort, two heterozygous patients, P5 and P16, carry the p.(R1141X) mutation (Table 2). Login to comment

[hide] Large-scaled metabolic profiling of human dermal f... PLoS One. 2014 Sep 29;9(9):e108336. doi: 10.1371/journal.pone.0108336. eCollection 2014. Kuzaj P, Kuhn J, Michalek RD, Karoly ED, Faust I, Dabisch-Ruthe M, Knabbe C, Hendig D
Large-scaled metabolic profiling of human dermal fibroblasts derived from pseudoxanthoma elasticum patients and healthy controls.
PLoS One. 2014 Sep 29;9(9):e108336. doi: 10.1371/journal.pone.0108336. eCollection 2014., [PMID:25265166]

Abstract [show]
Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.

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19 The prevalence of the autosomal recessive disease PXE is estimated to be between 1:25.000 and 1:100.000 [4], and to date up to 350 ABCC6 mutations were described [7] with p.R1141X (20-30%) and c.EX23_EX29del (515%) being the most frequent in European PXE patients [8]. Login to comment
159 Interestingly, fibroblasts from PXE patient 2 showing the lowest protein content, were found to carry two heterozygous ABCC6 mutations c.3421C.T (p.R1141X) and c.2787+1G.T. Login to comment
160 Furthermore, PXE patient 5 with 69% protein level detected, exhibit a genetic variation within the promotor region (c.-90ins14) in addition to the frequently observed c.3421C.T (p.R1141X) mutation. Login to comment
342 Hu X, Peek R, Plomp A, ten Brink J, Scheffer G, et al. (2003) Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum. Login to comment

[hide] Heart transplant and 2-year follow up in a child w... Eur J Pediatr. 2014 Dec;173(12):1735-40. doi: 10.1007/s00431-014-2447-7. Epub 2014 Nov 1. Giovannoni I, Callea F, Travaglini L, Amodeo A, Cogo P, Secinaro A, Bizzarri C, Cutrera R, El Hachem M, Francalanci P
Heart transplant and 2-year follow up in a child with generalized arterial calcification of infancy.
Eur J Pediatr. 2014 Dec;173(12):1735-40. doi: 10.1007/s00431-014-2447-7. Epub 2014 Nov 1., [PMID:25367056]

Abstract [show]
Generalized arterial calcification of infancy (GACI, OMIM 208000) and pseudoxanthoma elasticum (PXE, OMIM 264800) are rare autosomal-recessive disorders which represent the opposite ends of the same spectrum of pathologies characterized by progressive ectopic calcification and degeneration of elastic fibers at skin, eyes, and cardiovascular level. Patients with GACI suffer from hypertension, severe myocardial ischemia, and congestive heart failure and often die within 6 months of life. On the other end, PXE is associated with considerable morbidity, rarely with mortality. GACI and PXE are associated with biallelic mutations in ENPP1 and in ABCC6. We report the case of a 4-year-old Italian child submitted to heart transplant, at 18 months old, for end-stage heart failure due to extensive myocardial infarction of the left ventricle and diffuse coronary calcifications. The histology showed generalized arterial calcification and the molecular analysis identified mutations in ABCC6. Two years after transplantation, the child shows good clinical conditions and growth with no recurrence of calcium deposits in the heart. CONCLUSION: Bisphosphonate therapy at present is the treatment of choice for systemic arterial involvement in GACI, and heart transplant has proven to be the definitive treatment in case with extensive myocardial infarction, as in our. Molecular analysis is mandatory for a complete diagnosis and familial counseling.

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56 A compound heterozygosity was identified in ABCC6: the mutations were located at nucleotide position c.1132C>T (p.Q378X) and c.3421C>T (p.R1141X). Login to comment
58 Segregation analysis showed a compound heterozygous mutations with c.1132C>T (p.Q378X) and del_24-27 in the mother and a double heterozygous mutations c.1132C>T (p.Q378X) and c.3421C>T (p.R1141X) in the father. Login to comment
85 Fig. 2 Pedigree and haplotype analysis with markers from the ABCC6 regions: a double mutation on the same allele in cis (p.Q378X, p.R1141X) was found in the father and a mutation on the other allele in trans (p.Q378X, del_24-27) was found in the mother, affected by PXE (asterisk); the patient showed a compound heterozygous mutations of p.Q378X, p.R1141X, and del_24-27 Conclusions In this child with GACI and end-stage myocardial ischemia, the transplant solved the heart failure as observed throughout the 2-year follow-up and no new calcifications have been observed at the cardiac level (no coronary deposits). Login to comment

[hide] PSEUDOXANTHOMA ELASTICUM: DIAGNOSTIC FEATURES, CLA... Expert Opin Orphan Drugs. 2014 Jun 1;2(6):567-577. Uitto J, Jiang Q, Varadi A, Bercovitch LG, Terry SF
PSEUDOXANTHOMA ELASTICUM: DIAGNOSTIC FEATURES, CLASSIFICATION, AND TREATMENT OPTIONS.
Expert Opin Orphan Drugs. 2014 Jun 1;2(6):567-577., [PMID:25383264]

Abstract [show]
INTRODUCTION: Pseudoxanthoma elasticum (PXE), a multisystem orphan disease, clinically affects the skin, the eyes, and the cardiovascular system with considerable morbidity and mortality. The clinical manifestations reflect the underlying pathology consisting of ectopic mineralization of peripheral connective tissues. AREAS COVERED: The diagnostic criteria of PXE include characteristic clinical findings, together with histopathology of accumulation of pleiomorphic elastic structures in the dermis with progressive mineralization, and the presence of mutations in the ABCC6 gene. PXE-like cutaneous changes can also be encountered in other ectopic mineralization disorders, including generalized arterial calcification of infancy (GACI) caused by mutations in the ENPP1 gene. In some cases, overlapping clinical features of PXE/GACI, associated with mutations either in ABCC6 or ENPP1, have been noted. PXE demonstrates considerable inter- and intrafamilial heterogeneity, and consequently, accurate diagnosis is required for appropriate classification with prognostic implications. There is no effective and specific treatment for the systemic manifestations of PXE, but effective therapies to counteract the ocular complications are in current clinical use. EXPERT OPINION: A number of observations in the murine model, the Abcc6-/- mouse, have indicated that the mineral composition of diet, particularly the magnesium content, can influence the severity of the mineralization phenotype. These observations suggest that appropriate dietary interventions, coupled with lifestyle modifications, including smoking cessation, might alleviate the symptoms and improve the quality of life of individuals affected with this, currently intractable, orphan disease.

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49 In particular, heterozygous carriers of the loss-of-function mutation p.R1141X, the most common mutation in the Caucasion populations, is associated with a strong increase in the prevalence of coronary artery disease [17-19] Uitto et al. Page 3 3. Login to comment
149 Approximately 40% of all ABCC6 gene mutations in patients with PXE consist of PTCs, including p.R1141X, which accounts ~25% of all mutations in PXE [8]. Login to comment
231 Frequent mutation in the ABCC6 gene (R1141X) is associated with a strong increase in the prevalence of coronary artery disease. Login to comment
235 K&#f6;bl&#f6;s G, Andrikovics H, Proh&#e1;szka Z, et al. The R1141X loss-of-function mutation of the ABCC6 gene is a strong genetic risk factor for coronary artery disease. Login to comment

[hide] The prevalence of pseudoxanthoma elasticum-like co... Oral Surg Oral Med Oral Pathol Oral Radiol. 2015 Apr;119(4):441-50. doi: 10.1016/j.oooo.2014.12.003. Epub 2014 Dec 13. Harrington C, Beck FM, Allen CM, Kalmar JR
The prevalence of pseudoxanthoma elasticum-like connective tissue changes in an oral biopsy service and review of the literature.
Oral Surg Oral Med Oral Pathol Oral Radiol. 2015 Apr;119(4):441-50. doi: 10.1016/j.oooo.2014.12.003. Epub 2014 Dec 13., [PMID:25640304]

Abstract [show]
OBJECTIVE: The objective of this pilot study is to determine the prevalence of pseudoxanthoma elasticum (PXE)-like connective changes in an oral biopsy service and compare it with the estimated prevalence of PXE as well as to the prevalence of the mutated PXE gene ABCC6. STUDY DESIGN: This prevalence study utilized 500 oral mucosal biopsy specimens received from the biopsy service of the Oral Pathology Consultants at the Ohio State University. Each specimen was microscopically evaluated using hematoxylin and eosin, Verhoeff-van Gieson and von Kossa stains. RESULTS: A prevalence of 9.8% was identified for PXE-like changes in the connective tissue of oral biopsy specimens submitted to this service. CONCLUSIONS: The overall prevalence of PXE-like connective tissue changes found in routine oral mucosal biopsy specimens (9.8%) was much higher than either the suspected prevalence of PXE (0.001%-0.004%) or the estimated prevalence of the mutated gene ABCC6 (0.625%-1.25%).

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71 Mutation analysis of the ABCC6 gene in these families clarified and confirmed an autosomal recessive mode of inheritance.4 Two-generation involvement is explained by pseudodominance.1,4,5,16 Consanguinity is strongly associated with PXE but may be difficult to confirm either because of the reluctance of current family members to discuss or disclose such a possibility or because a confident determination is simply not possible with past generations.9 ABCC6, the affected gene in PXE, was discovered in 2000,16,20-22 which led to the conclusion that PXE is a primary metabolic disorder with secondary connective tissue manifestations.23-25 Over 300 mutations of the ABCC6 gene have been identified,19 with R1141X being the most common.14,33,40 ABCC6, also known as "multidrug resistanceeassociated protein 6" (MRP6), is a member of the C-family of adenosine triphosphate (ATP)e binding cassette proteins located on chromosome 16.20,41 ABCC6 is found mainly in the liver but is also found in the kidneys and serves as a transporter of Table I. Prevalence of positive findings by decade, gender and overall (N &#bc; 500) Gender Decade First Second Third Fourth Fifth Sixth Seventh Eighth Ninth All Female Estimate 0.0 0.0 0.0 8.8 12.0 12.5 16.2 27.6 20.0 10.1 (LCB.95) 0.0 0.0 0.0 1.9 2.6 4.7 6.2 12.7 2.5 6.8 (UCB.95) 0.0 0.0 0.0 23.7 31.2 25.3 32.0 47.2 55.6 14.3 Male Estimate 0.0 0.0 3.5 11.8 8.0 6.5 28.1 8.3 66.7 9.4 (LCB.95) 0.0 0.0 0.1 3.3 0.1 0.7 13.8 0.2 9.4 5.9 (UCB.95) 0.0 0.0 17.8 27.5 26.0 21.4 46.8 38.5 99.2 14.0 All Estimate 0.0 0.0 1.9 10.3 10.0 10.1 21.7 22.0 30.8 9.8 (LCB.95) 0.0 0.0 0.1 4.4 3.3 4.5 12.7 10.6 9.1 7.3 (UCB.95) 0.0 0.0 9.9 20.0 21.8 19.0 33.3 37.6 61.4 12.8 LCB, lower confidence bound; UCB, upper confidence bound. Login to comment

[hide] [Pseudodominant inheritance of pseudoxanthoma elas... Ophthalmologe. 2015 Aug;112(8):686-90. doi: 10.1007/s00347-014-3231-9. Charbel Issa P, Gliem M, Holz FG, Knabbe C, Hendig D
[Pseudodominant inheritance of pseudoxanthoma elasticum].
Ophthalmologe. 2015 Aug;112(8):686-90. doi: 10.1007/s00347-014-3231-9., [PMID:25735631]

Abstract [show]
Pseudoxanthoma elasticum (PXE) is a system disease due to mutations in the ABCC6 gene with characteristic alterations in the eyes, the skin and the cardiovascular system. Herein, we report on two families with PXE in two subsequent generations due to genetically confirmed pseudodominance. A literature review revealed that PXE due to mutations in ABCC6 follows an autosomal recessive inheritance and that disease manifestation in two subsequent generations is due to pseudodominance.

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78 Hornstrup LS,Tybjaerg-Hansen A, Haase CL et al (2011) Heterozygosity for R1141X in ABCC6 and risk of ischemic vascular disease. Login to comment
80 K&#f6;bl&#f6;s G, Andrikovics H, Prohaszka Z et al (2010) The R1141X loss-of-function mutation of the ABCC6 gene is a strong genetic risk factor for coronary artery disease. Login to comment
92 Trip MD, SmuldersYM,Wegman JJ et al (2002) Frequent mutation in the ABCC6 gene (R1141X) is associated with a strong increase in the prevalence of coronary artery disease. Login to comment