ABCMdb

PMID: 22922874

Coelho D, Kim JC, Miousse IR, Fung S, du Moulin M, Buers I, Suormala T, Burda P, Frapolli M, Stucki M, Nurnberg P, Thiele H, Robenek H, Hohne W, Longo N, Pasquali M, Mengel E, Watkins D, Shoubridge EA, Majewski J, Rosenblatt DS, Fowler B, Rutsch F, Baumgartner MR
Mutations in ABCD4 cause a new inborn error of vitamin B(12) metabolism.
Nat Genet. 2012 Oct;44(10):1152-5. doi: 10.1038/ng.2386. Epub 2012 Aug 26., [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCD4 p.Tyr319Cys Subject 1 carried a missense mutation, c.956A>G (p.Tyr319Cys) (NM_005050.3), predicted to be probably damaging with a score of 0.984 using the PolyPhen-2 program5, and a dinucleotide insertion, c.1746_1747insCT (p.Glu583Leufs*9), resulting in a frameshift and the introduction of a premature stop codon leading to removal of 14 amino acids from the C terminus. Login to comment 41 ABCD4 p.Gly486Cys Subject 2 carried two mutations that are predicted to disrupt consensus splice sites: c.542+1G>T, located at the 5' splice donor site of intron 5, and c.1456G>T (p.Gly486Cys), located at the last nucleotide of exon 14. Login to comment 71 ABCD4 p.Tyr319Cys Among the four mutations detected in our affected subjects, two (encoding p.Asp143_Ser181del and p.Tyr319Cys) occur in the predicted transmembrane domain, whereas the other two (encoding p.Gly443_ Ser485del and p.Glu583Leufs*9) are located in the predicted NBD11. Login to comment 72 ABCD4 p.Asp548Asn Expression of an ABCD4 construct -encoding a mutant Walker B motif (p.Asp548Asn) in cells from subject 1 led to very low levels of Cbl cofactor synthesis (Fig. 4b; P < 0.00001 for AdoCbl and 0.0001 for MeCbl synthesis compared with wild-type ABCD4 construct), indicating that this Walker B motif aspartate is necessary for ABCD4 function. Login to comment 73 ABCD4 p.Lys427Leu ABCD4 p.Lys427Leu ABCD4 p.Glu549Gln ABCD4 p.Glu549Gln Moreover, expression of constructs with selective changes affecting the Walker A motif (p.Lys427Leu) and the putative catalytic site (p.Glu549Gln) also led to reduced synthesis of both Cbl cofactors (Fig. 4b; p.Lys427Leu: P = 0.003 for AdoCbl and 0.007 for MeCbl synthesis; p.Glu549Gln: P = 0.001 for AdoCbl and 0.0021 for MeCbl synthesis). Login to comment 75 ABCD4 p.Tyr319Cys Among the four mutations detected in our affected subjects, two (encoding p.Asp143_Ser181del and p.Tyr319Cys) occur in the predicted transmembrane domain, whereas the other two (encoding p.Gly443_ Ser485del and p.Glu583Leufs*9) are located in the predicted NBD11. Login to comment 76 ABCD4 p.Asp548Asn Expression of an ABCD4 construct -encoding a mutant Walker B motif (p.Asp548Asn) in cells from subject 1 led to very low levels of Cbl cofactor synthesis (Fig. 4b; P < 0.00001 for AdoCbl and 0.0001 for MeCbl synthesis compared with wild-type ABCD4 construct), indicating that this Walker B motif aspartate is necessary for ABCD4 function. Login to comment 77 ABCD4 p.Lys427Leu ABCD4 p.Lys427Leu ABCD4 p.Glu549Gln ABCD4 p.Glu549Gln Moreover, expression of constructs with selective changes affecting the Walker A motif (p.Lys427Leu) and the putative catalytic site (p.Glu549Gln) also led to reduced synthesis of both Cbl cofactors (Fig. 4b; p.Lys427Leu: P = 0.003 for AdoCbl and 0.007 for MeCbl synthesis; p.Glu549Gln: P = 0.001 for AdoCbl and 0.0021 for MeCbl synthesis). Login to comment 82 ABCD4 p.Tyr319Cys The position of the p.Tyr319Cys and p.Glu583Leufs*9 alterations (detected in subject 1) are represented with a red dot, and the polypeptide fragments deleted in the p.Asp143_Ser181del and p.Gly443_Ser485del protein products (detected in subject 2) are highlighted in red. Login to comment 86 ABCD4 p.Tyr319Cys ABCD4 p.Asp548Asn ABCD4 p.Lys427Leu ABCD4 p.Glu549Gln Mutant alleles encoding changes to highly conserved amino-acid residues known to be involved in ATPase activity, for example, p.Lys427Leu in the Walker A site, p.Asp548Asn in the Walker B site and p.Glu549Gln in the putative catalytic domain of ABCD4, were transfected transiently into immortalized fibroblasts from subject 1 by electroporation and were assayed for rescue of AdoCbl and MeCbl synthesis. Login to comment 89 ABCD4 p.Tyr319Cys ABCD4 p.Asp548Asn ABCD4 p.Lys427Leu a Intralysosomal Membrane Cytosolic H2N 37 54 81 98 Asp143 Gly443 p.Gly443_Ser485del Ser485 536 421 ATP binding (Walker B) COOH 606 p.Glu583Leufs*9 p.Tyr319Cys 35 30 ATP binding (Walker A) p.Asp143_Ser181del 160 177 Ser181 206 189 293 313 330276 b 25 20 15 10 5 0 PercentageoftotalCbl AdoCbI MeCbI Vectoronly ABC D 4-p.Lys427Leu ABC D 4-p.G lu549G ln ABC D 4-p.Asp548Asn ABC D 4-w t Figure 5 Subcellular localization of ABCD4 detected by fluorescence and confocal microscopy. Login to comment 90 ABCD4 p.Asp548Asn ABCD4 p.Lys427Leu ABCD4 p.Glu549Gln Mutant alleles encoding changes to highly conserved amino-acid residues known to be involved in ATPase activity, for example, p.Lys427Leu in the Walker A site, p.Asp548Asn in the Walker B site and p.Glu549Gln in the putative catalytic domain of ABCD4, were transfected transiently into immortalized fibroblasts from subject 1 by electroporation and were assayed for rescue of AdoCbl and MeCbl synthesis. Login to comment 93 ABCD4 p.Tyr319Cys a Intralysosomal Membrane Cytosolic H2N 37 54 81 98 Asp143 Gly443 p.Gly443_Ser485del Ser485 536 421 ATP binding (Walker B) COOH 606 p.Glu583Leufs*9 p.Tyr319Cys 35 30 ATP binding (Walker A) p.Asp143_Ser181del 160 177 Ser181 206 189 293 313 330 276 b 25 20 15 10 5 0 Percentage of total Cbl AdoCbI MeCbI V e c t o r o n l y A B C D 4 - p . Login to comment