ABCMdb

ABCB6 p.Arg192Trp

ClinVar:	c.575G>A , p.Arg192Gln ? , Uncertain significance
Predicted by SNAP2:	A: D (80%), C: D (75%), D: D (91%), E: D (85%), F: D (91%), G: D (85%), H: D (75%), I: D (85%), K: D (59%), L: D (85%), M: D (80%), N: D (71%), P: D (91%), Q: D (80%), S: D (75%), T: D (80%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] The ABCB6 mutation p.Arg192Trp is a recessive muta... Vox Sang. 2012 Sep 10. doi: 10.1111/j.1423-0410.2012.01650.x. 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., [PMID:22958180]

Abstract [show]
Background and Objective The membrane transporter ABCB6 has recently been shown to carry the high-frequency red-blood-cell (RBC) antigen Lan. All the Lan- individuals genotyped so far have inherited two recessive null mutations in ABCB6. The finding of a family with the Lan- blood type occurring in two successive generations prompted this study. Methods Mutations in ABCB6 were searched by Sanger sequencing of exons and flanking intronic regions. Expression analysis of the Lan antigen was carried out by serology and flow cytometry. PCR-RFLP genotyping and Western blot analysis were also applied. 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. Homozygosity for p.Arg192Trp was associated not only with absence of the Lan antigen, but also of the ABCB6 transporter in RBC membrane. 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. 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. 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. Like the previously reported frameshift, nonsense and essential splice-site mutations in ABCB6, this missense mutation is recessive and defines an ABCB6 null allele. Other single amino acid mutations in ABCB6 may also cause the Lan- blood type.

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No. Sentence Comment
0 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 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 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 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
10 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 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 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 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 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 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 Taken together, these data suggested that p.Arg192Trp might be a novel ABCB6 null mutation causing the Lan) blood type. Login to comment
70 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 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 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 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 (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 (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 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 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 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 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 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 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 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 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 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 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
98 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
100 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
102 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
105 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 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 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 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 Hence, p.Arg192Trp is so far the most common mutation causing the Lan) blood type. Login to comment
112 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 The mutation p.Arg192Trp is the first ABCB6 missense mutation causing the Lan) blood type. Login to comment
115 Nevertheless, all these mutations including p.Arg192Trp are null mutations since they result in complete absence of ABCB6 in RBCs. Login to comment
116 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 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
123 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 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 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 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
130 However, we managed to demonstrate that p.Arg192Trp defines a novel ABCB6 null allele causing the Lan) blood type. Login to comment