ABCMdb

PMID: 12714611

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., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Haplotypes of 16 patients with the R1141X mutation were determined with eight polymorphic markers spanning the ABCC6 locus. Login to comment 5 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* All R1141X alleles were associated with a common haplotype, covering at least three intragenic ABCC6 markers. Login to comment 11 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* A specific founder effect for the R1141X mutation exists in Dutch patients with PXE. Login to comment 15 ABCC6 p.Arg1141* The R1141X mutation induces instability of the aberrant mRNA. Login to comment 42 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Sixteen Dutch families and patients with the R1141X mutation were included in the study. Login to comment 65 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* We constructed (phase-known) haplotypes of all informative alleles from patients with the R1141X mutation. Login to comment 83 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* The R1141X mutation was caused by a C3T substitution at nucleotide 3421 in the putative eighth intracellular loop. Login to comment 90 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* ABCC6 p.Gln749* 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 ABCC6 p.Arg1141* ABCC6 p.Gln749* The last patient, P26240, inherited the R1141X mutation and another nonsense mutation, Q749X. Login to comment 101 ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 104 ABCC6 p.Arg1268Gln ABCC6 p.His632Gln ABCC6 p.Ala830Gly Three of these were used in this study to construct the following haplotypes: (1) 1896 C3A in exon 15 predicting an H632Q substitution, (2) 2490 C3G in exon 19 predicting an A830G substitution, and (3) 3803 G3A in exon 27 predicting a R1268Q substitution. Login to comment 112 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Gln749* 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 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Next, we analyzed the amount of mRNA from alleles with the R1141X mutation. Login to comment 122 ABCC6 p.Arg1141* RT-PCR analyses of cultured PXE fibroblasts with the R1141X in homozygous form did not contain detectable ABCC6 mRNA. Login to comment 123 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* The latter result indicates that the R1141X mutation affects the abundance of the mutant mRNA. Login to comment 125 ABCC6 p.Arg1141* 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 126 ABCC6 p.Arg1459Cys As a control for the latter analysis, we also determined this ratio in blood cells from a healthy donor and from a patient with PXE heterozygous for a missense mutation R1459C in exon 30 of ABCC6. Login to comment 127 ABCC6 p.Arg1141* 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 128 ABCC6 p.Arg1459Cys In contrast, the relative amount of mRNA carrying the R1459C mutation (48%) was very similar to that of the wild-type mRNA (52%; Table 3), whereas the cells of a healthy donor contained only wild-type mRNA. Login to comment 129 ABCC6 p.Arg1141* In parallel experiments, we determined the expression of the R1141X truncated protein in cells of patients with PXE. Login to comment 130 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Identical haplotypes for the R1141X mutation are shaded. Login to comment 137 ABCC6 p.Arg1141* Underline shows homozygosity for the R1141X mutation in the proband(s). Login to comment 148 ABCC6 p.Arg1141* The presence of the R1141X mutation was confirmed by digestion with BsiYI of PCR products containing exon 24. Login to comment 151 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* These results suggest that the R1141X mutation in ABCC6 does not lead to detectable amounts of truncated protein. Login to comment 154 ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* ABCC6 p.Arg1141* DISCUSSION R1141X Mutation Analysis We detected the R1141X mutation in homozygous, heterozygous, and compound heterozygous forms. Login to comment 162 ABCC6 p.Arg1141* In nine patients the R1141X mutation was present in a homozygous form or a compound heterozygous form. Login to comment 164 ABCC6 p.Arg1141* In seven patients, we detected R1141X in heterozygous form. Login to comment 166 ABCC6 p.Arg1141* However, despite extensive screening, we have not yet found another mutation or deletion in the second, non-R1141X, ABCC6 allele. Login to comment 168 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Nonetheless, potential mild expression of the disease in carriers of the R1141X mutation warrants further investigation. Login to comment 170 ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* To test this hypothesis, we analyzed the R1141X mutation in more detail in this study. Login to comment 172 ABCC6 p.Arg1141* The majority of our R1141X mutant alleles (17/19) shared a common haplotype spanning at least one ABCC6 flanking marker. Login to comment 179 ABCC6 p.Arg1141* Top: RT-PCR analysis of ABCC6 expression in cultured dermal fibroblasts from patients heterozygous or homozygous for the R1141X mutation. Login to comment 183 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* ABCC6 p.Arg1141* ABCC6 p.Arg1459Cys ABCC6 p.Arg1459Cys ABCC6 p.Arg1459Cys 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* Consistent with this observation, no ABCC6 protein was detected in cultured dermal fibroblasts of a patient homozygous for R1141X. Login to comment 190 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* 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 ABCC6 p.Arg1141* No clear correlation between the R1141X genotype and phenotype could be established in the cohort studied. Login to comment