ABCMdb

PMID: 23300730

Amorosi CA, Myskova H, Monti MR, Argarana CE, Morita M, Kemp S, Dodelson de Kremer R, Dvorakova L, Oller de Ramirez AM
X-linked adrenoleukodystrophy: molecular and functional analysis of the ABCD1 gene in Argentinean patients.
PLoS One. 2012;7(12):e52635. doi: 10.1371/journal.pone.0052635. Epub 2012 Dec 31., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G.C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). Login to comment 6 ABCD1 p.His669Arg ABCD1 p.Ala19Ser In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). Login to comment 7 ABCD1 p.Ala19Ser In vitro studies suggest that p.Ala19Ser is a polymorphism. Login to comment 66 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro ABCD1 p.His669Arg ABCD1 p.His669Arg AMN 2 Phenotype 1 cDNA mutation Protein level Exon/Intron Polymorphisms Exon/Intron Protein level 1 AMN c.2006A.G p.His669Arg 10 c.55G.T 1 p.Ala19Ser c.1992-32C.T 9 2 CCALD c.1137dupC p.Glu380Argfs*21 3 c.1992-32C.T 9 c.2019C.T 10 p.Phe673Phe 3 AO c.1022C.A p.Ala341Asp 2 c.1634+14T.A 6 c.1992-32C.T 9 4 AO c.1081+5G.C Splice mutation c.1548G.A 6 p.Leu516Leu r.907_1494del p.Leu303_Glu498 IVS2 c.1992-32C.T 9 5 Asymptomatic c.1640A.G p.Tyr547Cys 7 6 CCALD c.1714_1725dek12bp p.Ser572_Asp575del 7 7 Adolescent cerebral ALD c.761delC p.Thr254Argfs*82 1 c.1634+14T.A 6 8 -- c.1259A.C p.His420Pro 1 9 AMN c.2006A.G p.His669Arg 10 c.1992-32C.T 9 10 CCALD c.852_853insACTC p.Ser284fs*16 1 1 Nucleotides numbered reflects cDNA numbering with +1 corresponding to the A of the ATG initiation codon in the reference sequence (NM000033), according to journal guidelines (www.hgvs.org/mutnomen). Login to comment 72 ABCD1 p.Tyr547Cys ABCD1 p.His420Pro ABCD1 p.Ala19Ser Briefly, different degenerate primer pair, introducing point mutation c.55 G.T (p.Ala19Ser), c.1259 A.G (p.His420Pro) or c.1640 A,G (p.Tyr547Cys), were used in a thermal cycling reaction (19 cycles). Login to comment 87 ABCD1 p.His669Arg We identified nine different mutations: one missense mutation was previously described (p.His669Arg) and eight were new ones (Table 1). Login to comment 88 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro These new changes included 3 frameshifts (p.Ser284fs*16; p.Glu380Argfs*21; p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del), a splicing mutation (c.1081+5G.C) and three single base pair substitutions (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). Login to comment 94 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro It was verified by functional studies that the missense substitutions p.Ala341Asp, p.His420Pro and p.Tyr547Cys are disease-causing mutations. Login to comment 95 ABCD1 p.Tyr547Cys When the construct encoding a substitution p.Tyr547Cys was transfected into X-ALD fibroblasts, ALDP was not detected by western blot analysis (Figure 1) and peroxisomal b-oxidation was deficient (Figure 2). Login to comment 96 ABCD1 p.His420Pro Similar results were obtained for the construct containing p.His420Pro substitution (data not show). Login to comment 97 ABCD1 p.Ala341Asp No protein was observed in western blot assays and b-oxidation was defective in fibroblast cultures of the patient with the p.Ala341Asp mutation (Figure 3 and 4). Login to comment 101 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro PolyPhen predicted that p.Ala341Asp is possibly damaging, and p.Tyr547Cys and p.His420Pro can be probably damaging (Table 2). Login to comment 106 ABCD1 p.Tyr547Cys ABCD1 p.Ala19Ser Each line was loaded with the same amount of total protein extracts, as verified with anti-b-actin protein. Line 1: healthy fibroblasts, line 2: X-linked adrenoleukodystrophy fibroblasts, line 3: fibroblats expressing wild type ALDP-GFP, line 4: fibroblats expressing ALDP-GFP (c.55G.T, p.Ala19Ser), line 5: fibroblats expressing ALDP-GFP (c.1640A.G, p.Tyr547Cys). Login to comment 114 ABCD1 p.His669Arg ABCD1 p.Ala19Ser In patient 1 (Table 1), we identified two different substitutions, one in exon 1 and other in exon 10 (p.Ala19Ser and p.His669Arg). Login to comment 115 ABCD1 p.Ala19Ser The last one has been identified previously as a disease causing mutation in the X-ALD database (http://www-x-ald.nl), but p.Ala19Ser has not been reported before. Login to comment 116 ABCD1 p.Ala19Ser When a construct carrying substitution p.Ala19Ser was transfected into X-ALD fibroblasts, ALDP was detected by western blot analysis (Figure 1) and peroxisomal b-oxidation was restored to control levels (Figure 2). Login to comment 117 ABCD1 p.Ala19Ser ABCD1 p.Ala19Ser This demonstrates that the p.Ala19Ser may represent a SNP. As expected, SIFT predicted that p.Ala19Ser is tolerated with a score of 0.05 (Table 2). Login to comment 118 ABCD1 p.Ala19Ser PolyPhen predicted that p.Ala19Ser can be a benign variant although the alanine residue at position 19 it was highly conserved between different species (Figure 5). Login to comment 119 ABCD1 p.Ala19Ser Finally, five polymorphisms were detected: 3 novel intronic changes (c.1634+14T.A, c.1992-32C.T and c.55G.T or p.Ala19Ser) and 2 previously reported (c.1548G.A, p.Leu516Leu and c.2019C.T, p.Phe673Phe). Login to comment 126 ABCD1 p.Ala341Asp Each line was loaded with the same amount of total protein extracts, as verified with anti-b-actin protein. Line 1: healthy fibroblasts, line 2: X-linked adrenoleukodystrophy fibroblasts, line 3: c.1081+5G.C, line 4: p.Ala341Asp. Login to comment 132 ABCD1 p.His669Arg The majority of X-ALD patients in our study group had non-recurrent (89%) mutations, except two patient (one of them Argentinean and the other of Italian origin) that had the same mutation p.His669Arg. Login to comment 139 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro For the p.Ala341Asp, p.Tyr547Cys and p.His420Pro alleles, we did not observe protein in western blots and the level of b-oxidation was deficient (Figures 1, 2, 3, 4 and data not shown). Login to comment 140 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro These results confirm that p.Ala341Asp, p.Tyr547Cys and p.His420Pro are the causing disease mutations in each patient. Login to comment 147 ABCD1 p.Ala341Asp D3-C16:0 levels in normal, X-ALD (mock) and X-ALD fibroblast from patients; c.1081+5G.C and c.1022C.A (p.Ala341Asp). Login to comment 150 ABCD1 p.Ala341Asp ABCD1 p.Tyr547Cys ABCD1 p.His420Pro ABCD1 p.His669Arg ABCD1 p.Ala19Ser Missense Change p.Ala19Ser p.Tyr547Cys p.His420Pro p.His669Arg p.Ala341Asp Multiple sequence alignment MSA Highly conserved Highly conserved Relatively conserved Conserved Highly conserved http://www.ebi.ac.uk/clustalw2/ SIFT Tolerated Non-tolerated Tolerated Tolerated Non-tolerated http://blocks.fhcrc.org/sift/SIFT.html PolyPhen Benign Probably damaging Probably damaging Benign Possibly damaging http://genetics.bwh.harvard.edu/pph doi:10.1371/journal.pone.0052635.t002 showed that the mutant transcript is not translated (Figure 3); therefore the levels of b-oxidation are deficient (Figure 4). Login to comment 154 ABCD1 p.Ala19Ser The bioinformatics studies are predictive; however the results were consistent in most cases with functional studies, except for p.Ala19Ser. Login to comment 155 ABCD1 p.His669Arg ABCD1 p.Ala19Ser In patient 1 (Table 1) we identified two different one-base substitution, one new in exon 1 (c.55G.T) and one known in exon 10 (c.2006A.G, http://www-x-ald.nl), both of them causing an amino acid change (p.Ala19Ser and p.His669Arg, respectively). Login to comment 156 ABCD1 p.Ala19Ser The p.Ala19Ser allele had similar expression and b-oxidation levels to wild type ALDP in the experiments of transient expression in X-ALD fibroblast (Figures 1 and 2). Login to comment 157 ABCD1 p.His669Arg On the other hand according to the literature when the change p.His669Arg is present, ALDP was not observed in western blot. Login to comment 158 ABCD1 p.His669Arg ABCD1 p.Ala19Ser Besides the fact that p.His669Arg has been found as the only mutation in other patients where no protein was detected in western blot also suggests that p.Ala19Ser is a SNP. Login to comment 159 ABCD1 p.Ala19Ser However p.Ala19Ser is highly conserved in all species unlike p.His669 (Figure 6) and both were predicted to be ''bening`` by the Polyphen program and ''tolerated`` by the SIFT program (Table 2). Login to comment 170 ABCD1 p.His420Pro Therefore, an alternative would be that p.His420Pro mutant might be misfolded on the peroxisomal membranes and unable to interact correctly with each ALDP or other peroxisomal ABC proteins such PMP70 or ALDRP [23-27] and are recruited to proteasomes for degradation. Login to comment