ABCMdb

PMID: 22958180

Saison C, Helias V, Peyrard T, Merad L, Cartron JP, Arnaud L
The ABCB6 mutation p.Arg192Trp is a recessive mutation causing the Lan- blood type.
Vox Sang. 2012 Sep 10. doi: 10.1111/j.1423-0410.2012.01650.x., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCB6 p.Arg192Trp The ABCB6 mutation p.Arg192Trp is a recessive mutation causing the Lan) blood type C. Saison,1,† , V. Helias,1,† T. Peyrard,1,2 L. Merad,3 J.-P. Cartron1 & L. Arnaud1 1 National Institute of Blood Transfusion (INTS), Paris, France 2 National Reference Center for Blood Groups (CNRGS), Paris, France 3 Centre Europe Le Palatin, Hyères, France Received: 21 June 2012, revised 31 July 2012, accepted 31 July 2012 Background and Objective The membrane transporter ABCB6 has recently been shown to carry the high-frequency red-blood-cell (RBC) antigen Lan. Login to comment 6 ABCB6 p.Arg192Trp Results All the Lan) members of this family were homozygous for c.574C>T, p.Arg192Trp in ABCB6 while the Lan+ members were heterozygous for this missense mutation encoded by the SNP rs149202834. Login to comment 7 ABCB6 p.Arg192Trp Homozygosity for p.Arg192Trp was associated not only with absence of the Lan antigen, but also of the ABCB6 transporter in RBC membrane. Login to comment 8 ABCB6 p.Arg192Trp The complete absence of Lan expression resulting from p.Arg192Trp homozygosity was confirmed by the subsequent identification of five unrelated Lan) individuals who were homozygous for this mutation and who developed an anti-Lan. Login to comment 9 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser We also provide evidence that three other single amino acid mutations in ABCB6 (c.826C >T, p.Arg276Trp; c.85_87delTTC, p.Phe29del; c.1762G >A, p.Gly588Ser) may also define ABCB6 null alleles. Login to comment 10 ABCB6 p.Arg192Trp Conclusion p.Arg192Trp is the first ABCB6 missense mutation causing the Lan) blood type and appears to be a relatively frequent cause of this rare blood type. Login to comment 44 ABCB6 p.Arg192Trp PCR-RFLP assay for detecting the ABCB6 mutation c.574C>T (p.Arg192Trp) A 242 bp fragment corresponding to exon 2 of ABCB6 was amplified by PCR with the primers ABCB6-61 (5'GA- ATGTGTTCATGACACCAG) and ABCB6-62 (5'GTGTACCT GGCTCCTTTC) from genomic DNA isolated from whole blood. Login to comment 47 ABCB6 p.Arg192Trp The presence of the mutation p.Arg192Trp (c.574C>T) was detected by digestion of this PCR product with the restriction enzyme Fnu4H1 (the single Fnu4H1 site present in this 242 bp fragment is destroyed by c.574C>T) followed by electrophoresis in a 2% agarose gel with 1· TBE buffer. Login to comment 50 ABCB6 p.Arg192Trp ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser The pCEP5 plasmids corresponding to ABCB6 p.Arg192Trp, p.Arg648Ter, p.Arg276Trp, p.Gly588Ser and p.Phe29del were similarly constructed by using fully sequenced NotI / SbfI fragments corresponding to mutant coding sequences of ABCB6 cDNA generated by site-directed mutagenesis of the pCR4-ABCB6-dUTR plasmid. Login to comment 64 ABCB6 p.Arg192Trp p.Arg192Trp defines a novel ABCB6 null allele causing the Lan) blood type In order to identify the genetic basis of the Lan) blood type in the proband`s family, we first sequenced ABCB6, which encodes the Lan antigen, in all five members available at that time. Login to comment 68 ABCB6 p.Arg192Trp The minor allele of rs149202834 corresponds to the missense mutation p.Arg192Trp (in ABCB6 NP_005680.1), which is predicted by the SIFT and POLYPHEN softwares [5, 6] to have a 'damaging` effect on the folding of ABCB6. Login to comment 69 ABCB6 p.Arg192Trp Taken together, these data suggested that p.Arg192Trp might be a novel ABCB6 null mutation causing the Lan) blood type. Login to comment 70 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp From a genetic point of view, the hypothesis that p.Arg192Trp defines a recessive ABCB6 allele causing the Lan) blood type was further supported by two pieces of evidence: (1) the analysis of the Lan phenotype and ABCB6 genotype that was performed later in the 4-month-old daughter of the proband and her husband (Fig. 1a, II.4 and III.3), and (2) the subsequent identification of five unrelated Lan) patients (AZO, KEM, CAL, PAO and BOJ) who were homozygous for p.Arg192Trp, with no other silencing mutations in ABCB6. Login to comment 73 ABCB6 p.Arg192Trp We also observed by flow cytometry analysis of RBCs labelled with the monoclonal anti-Lan OSK43 that the levels of the Lan antigen in individuals heterozygous for p.Arg192Trp were reduced to around 50% of wild type individuals (Fig. 1c). Login to comment 74 ABCB6 p.Arg192Trp This strongly suggested that p.Arg192Trp prevents the expression of the Lan antigen, which was 6 3 2 ND 5 ND 1 1 2 I II III 192 192 192 192 192 4 3 d. Login to comment 75 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp 7 days 1 ND 2 ND 192 192 bp p.Arg192Trp WT HO HT 1 3 2 4 Fnu4HI RFLP Uncut PCR 300 400 200 100 300 400 200 100 116 200 97 66 55 36 55 kDa 1 3 2 WB1 ABCB6 WB2 p55 p.Arg192Trp WT HO HT 0 500 1000 Fluorescence intensity p.Arg192Trp WT HO HT (a) (b) (c) (d) Fig. 1 Identification and characterization of p.Arg192Trp as a novel ABCB6 null mutation causing the Lan) blood type. Login to comment 76 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp (a) Lan phenotyping and ABCB6 genotyping in the family of the proband; the Lan phenotype is indicated by a colour code (Lan) in black, Lan+ in white and ND for 'no data`), the genotype for the mutation c.574C>T, p.Arg192Trp (R192W) is indicated by annotated extracts of ABCB6 sequencing data ['X` designates an 'unspecified` residue (R or W) and 'Y` a 'pyrimidine` base (C or T) according to the current HGVS recommendations] and the proband is indicated by an arrow. Login to comment 77 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp (b) PCR-RFLP assay for detecting the mutation p.Arg192Trp; exon 2 of ABCB6 was amplified by PCR from the genomic DNA of individuals, who were either wild type (lane 2), homozygous (lane 3) or heterozygous (lane 4) for this mutation, and was analysed before (top panel) and after (bottom panel) digestion with the restriction enzyme Fnu4H1, whose single site in exon 2 of ABCB6 is destroyed by c.574C>T, p.Arg192Trp. Login to comment 79 ABCB6 p.Arg192Trp The geometric mean fluorescence intensity of OSK43-labelled RBCs of individuals homozygous for p.Arg192Trp is virtually identical to that of unlabelled RBCs. Login to comment 83 ABCB6 p.Arg192Trp We then performed a Western blot analysis of ABCB6 in RBC membrane lysates to determine whether p.Arg192Trp affected also the expression of ABCB6. Login to comment 84 ABCB6 p.Arg192Trp We used a polyclonal antibody directed against a peptide corresponding to residues 405-415 of this transporter, and thus should recognize equally well wild type and mutant p.Arg192Trp ABCB6. Login to comment 85 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp As shown in Fig. 1d, we detected no ABCB6 in individuals homozygous for p.Arg192Trp (lane 2) and reduced levels of ABCB6 in individuals heterozygous for p.Arg192Trp (lane 3). Login to comment 86 ABCB6 p.Arg192Trp From all these data, we concluded that the missense mutation p.Arg192Trp defines a novel ABCB6 null allele causing the Lan) blood type. Login to comment 87 ABCB6 p.Arg192Trp p.Arg192Trp in ABCB6 prevents the expression of the Lan antigen in a heterologous system Non-synonymous SNPs may be deleterious to gene function by altering the structure and / or function of the gene product. Login to comment 89 ABCB6 p.Arg192Trp In order to determine whether the deleterious effect of p.Arg192Trp on the Lan antigen was pre-or post-translational, we used a heterologous expression system. Login to comment 92 ABCB6 p.Arg192Trp When we transfected an ABCB6 cDNA harbouring p.Arg192Trp, we observed the same result as with p.Arg648Ter, that is, no expression of the Lan antigen (Fig. 2c). Login to comment 93 ABCB6 p.Arg192Trp We concluded from this result that the single amino acid change resulting from p.Arg192Trp is by itself responsible for the lack of expression of the Lan antigen, most likely by generating a misfolding of ABCB6 that induces its degradation. Login to comment 95 ABCB6 p.Arg192Trp In fact, these three donors (BAR, LIN and GAR) expressed half the normal levels of the Lan antigen, suggesting that they carried only one functional allele of ABCB6, as the individuals heterozygous for p.Arg192Trp (Fig. 1c). Login to comment 97 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser Sequencing of ABCB6 revealed that donor BAR was heterozygous for p.Arg276Trp (c.826C>T), donor GAR was heterozygous for p.Phe29del (c.85_87delTTC) and donor LIN was heterozygous for p.Gly588Ser (c.1762G>A). Login to comment 98 ABCB6 p.Arg192Trp ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser Of note, p.Arg276Trp and p.Gly588Ser corresponded to the minor alleles of rs57467915 and rs145526996, respectively, and both were predicted to be as 'damaging` as p.Arg192Trp by the SIFT and POLYPHEN softwares [5, 6]. Login to comment 99 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser Towards demonstrating that p.Arg276Trp, p.Phe29del and p.Gly588Ser were responsible for the partial lack of expression of the Lan antigen in these three blood donors, we used the aforementioned heterologous expression system. Surprisingly, only p.Phe29del prevented the expression of the Lan antigen (Fig. 3b). Login to comment 100 ABCB6 p.Arg192Trp ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser This result indicated that the single amino acid change resulting from p.Arg276Trp and p.Gly588Ser, contrary to p.Arg192Trp and p.Phe29del, could not by themselves account for reduced levels of the Lan antigen or the ABCB6 transporter. Login to comment 101 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser While c.826C>T (p.Arg276Trp) and c.1762G>A (p.Gly588Ser) might induce degradation of ABCB6 mRNA or a splicing defect, we could not rule out that these two mutations were completely unrelated to the lack of expression of the Lan antigen that we observed in the corresponding blood donors. Login to comment 102 ABCB6 p.Arg192Trp ABCB6 p.Gly588Ser ABCB6 p.Gly588Ser However, one of the Lan) individuals recently referred to us (HAU) appeared to be heterozygous for p.Gly588Ser and p.Arg192Trp, which corroborated that p.Gly588Ser corresponded to an ABCB6 null allele. Login to comment 103 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser Though incomplete, all these data suggested that p.Arg276Trp, p.Phe29del and p.Gly588Ser also define null alleles of ABCB6. Login to comment 105 ABCB6 p.Arg192Trp Discussion In-depth analysis of the family of the proband allowed us to identify p.Arg192Trp as a novel recessive ABCB6 mutation causing the Lan) blood type. Login to comment 107 ABCB6 p.Arg192Trp It turned out that the (Lan)) mother of the proband was homozygous for p.Arg192Trp, while the (Lan+) father of the proband was heterozygous for this mutation. Login to comment 108 ABCB6 p.Arg192Trp While this peculiar situation was a hurdle, it suggested either a close consanguinity of the proband`s parents (undeclared) or the high frequency of p.Arg192Trp in their ethnicity (unknown). Login to comment 109 ABCB6 p.Arg192Trp Apart from this Lan) family, we also found p.Arg192Trp responsible for the Lan) blood type in six unrelated Lan) individuals (five homozygous and one double heterozygous). Login to comment 110 ABCB6 p.Arg192Trp Hence, p.Arg192Trp is so far the most common mutation causing the Lan) blood type. Login to comment 112 ABCB6 p.Arg192Trp Due to the apparent frequency of p.Arg192Trp, especially when compared with the low frequency of the Lan) blood type, the PCR-RFLP assay that we have developed to detect this mutation may be used in first intention genotyping. Login to comment 113 ABCB6 p.Arg192Trp The mutation p.Arg192Trp is the first ABCB6 missense mutation causing the Lan) blood type. Login to comment 115 ABCB6 p.Arg192Trp Nevertheless, all these mutations including p.Arg192Trp are null mutations since they result in complete absence of ABCB6 in RBCs. Login to comment 116 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp While the Lan) proband of the aforementioned family did not develop an anti-Lan, the five other Lan) individuals who we found homozygous for p.Arg192Trp did so, providing in vivo evidence that p.Arg192Trp defines a null allele of ABCB6, and not a weak allele. Login to comment 117 ABCB6 p.Arg192Trp Using a heterologous expression system, we showed that p.Arg192Trp is by itself responsible for the lack of expression of the Lan antigen at the cell surface. Login to comment 120 ABCB6 p.Gly588Ser During the course of this study, we also identified three other single amino variations in ABCB6 (p.Arg276, p.Phe29del and p.Gly588Ser) that we suspect define ABCB6 null alleles. Login to comment 123 ABCB6 p.Arg192Trp ABCB6 p.Arg192Trp Thus, ABCB6 wild type ABCB6 p.Arg648Ter ABCB6 p.Arg192Trp (a) (b) (c) Fig. 2 Study of the ABCB6 mutation p.Arg192Trp in a heterologous expression system for the Lan antigen. Login to comment 124 ABCB6 p.Arg192Trp K-562 cells were stably transfected with expression constructs corresponding to ABCB6 wild type (a), p.Arg648Ter (b) or p.Arg192Trp (c), and cell surface expression of the Lan antigen was analysed by flow cytometry with the monoclonal anti-Lan OSK43. Login to comment 126 ABCB6 p.Arg192Trp ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser p.Arg276Trp, p.Phe29del and p.Gly588Ser are reminiscent of p.Arg192Trp that we have characterized as an ABCB6 null mutation. Login to comment 127 ABCB6 p.Arg192Trp By using a heterologous expression system, we showed that p.Phe29del, like p.Arg192Trp, can by itself account for the lack of expression of the Lan antigen. Login to comment 128 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser In contrast, we observed no effect of p.Arg276Trp and p.Gly588Ser on the expression of the Lan antigen in this heterologous system, suggesting that another molecular mechanism, yet unknown, is involved. Login to comment 130 ABCB6 p.Arg192Trp However, we managed to demonstrate that p.Arg192Trp defines a novel ABCB6 null allele causing the Lan) blood type. Login to comment 131 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser We also provide partial evidence that p.Arg276Trp, p.Phe29del and p.Gly588Ser may also define ABCB6 null alleles, expecting that other laboratories genotyping Lan) individuals may confirm our results in the future. Login to comment 149 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser G l y 5 8 8 S e r WB1 ABCB6 WB2 p55 ABCB6 p.Gly588Ser ABCB6 p.Phe29del ABCB6 p.Arg276Trp (a) (b) Fig. 3 Characterization of three ABCB6 mutations potentially responsible for the Lan) blood type. Login to comment 150 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser (a) Western blot analysis of ABCB6 in blood donors who were heterozygous for p.Arg276Trp (lane 1), for p.Phe29del (lane 3), for p.Gly588Ser (lane 5) or wild type (lanes 2 and 4). Login to comment 152 ABCB6 p.Arg276Trp ABCB6 p.Gly588Ser (b) Study of p.Arg276Trp, p.Phe29del and p.Gly588Ser in the heterologous expression system for the Lan antigen as in Fig. 2. Login to comment