ABCMdb

PMID: 24969679

Hu G, He P, Liu Z, Chen Q, Zheng B, Zhang Q
Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing.
Mol Med Rep. 2014 Sep;10(3):1264-74. doi: 10.3892/mmr.2014.2349. Epub 2014 Jun 20., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Arg928* ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Login to comment 11 ABCB11 p.Val444Ala Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. Login to comment 28 ABCB11 p.Val444Ala One is V444A (HGVS name: NM_003742.2:c.1331T>C; refSNP: rs2287622) in exon 13, which has been reported to be correlated with cholestasis (12) and chronic hepatititis C virus infection (13). Login to comment 79 ABCB11 p.Val444Ala To avoid false-negative results from HRM screening, confirmative direct sequencing was performed for exons 13 and 24 in which SNPs V444A (in exon 13) and A1028A (in exon 24) were common and rendered the interpretation of melting patterns difficult. Login to comment 105 ABCB11 p.Val444Ala Analysis of V444A and A1028A in patients and controls. Login to comment 107 ABCB11 p.Val444Ala These samples were tested using HRM analysis of exons 13 and 24, followed by direct sequencing to identify the V444A and A1028A polymorphisms. Login to comment 112 ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Arg928* ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro Results MutationsandSNPsdetectedinpatients.Amongthe20 patients with cholestasis, 14 types of variants were detected, including seven mutations in the coding region (p.Y337H, p.Y472C, p.R696W, p.R928X, p.Q931P, p.D1131V and p.H1198R) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Login to comment 113 ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro Missense mutations p.Y337H, p.R696W, p.Q931P, p.D1131V and p.H1198R were novel mutations identified in the study. Login to comment 118 ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Arg928* ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro Amino acid Carrier rate Variant Exon change RefSNP Patients and status in control (%) c.108T>C 4 D36D rs3815675 Heterozygous: P7, P11, P16 - c.270T>C 5 F90F rs4148777 Heterozygous: P6, P13 - c.807T>C 9 Y269Y rs2287616 Heterozygous: P7, P11, P16 - c.1009T>C 10 Y337H - Heterozygous: P5 0 c.1248C>A 12 I416I rs183390670 Heterozygous: P13 - c.1331T>C 13 V444A rs2287622 Heterozygous: P1, P5, P12, P16, P17, P19 94.5 Homozygous: P2, P3, P4, P6, P7, P8, P9, P10, P11, P14, P15, P18, P20 c.1415A>G 13 Y472C - Heterozygous: P3 0 c.2086C>T 18 R696W - Heterozygous: P11 0 c.2594C>T 21 A865V rs118109635 Heterozygous: P7, P17 - c.2782C>T 22 R928X - Heterozygous: P1 0 c.2792A>C 22 Q931P - Heterozygous: P4 0 c.3084A>G 24 A1028A rs497692 Heterozygous: P1, P8, P12, P13, P15, P16, P17, P20 90.5 Homozygous: P2, P3, P4, P5, P6, P7, P9, P10, P14, P18, P19 c.3392A>T 25 D1131V - Heterozygous: P3 0 c.3593A>G 26 H1198R - Heterozygous: P1 0 RefSNP refers to the reference SNP in the Single Nucleotide Polymorphism Database of NCBI. Login to comment 122 ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro The results based on comprehensive evaluation of SIFT, PolyPhen-2, SNPs&GO and evolution conservation indicated that p.Y337H, p.Y472C, p.R696W, p.D1131V and p.H1198R were likely damaging, p.Q931P and p.A865V were possibly damaging and p.V444A was predicted to be benign. Login to comment 123 ABCB11 p.Arg928* With the nonsense mutation, p.R928X is able to introduce a premature stop codon that results in premature protein truncation or failure of protein production, which had been reported previously in PFIC2 patients (26). Login to comment 128 ABCB11 p.Val444Ala ABCB11 p.Val444Ala The two SNPs of p.V444A and p.A1028A were also detected in the four patients, except that P11 only had p.V444A. Login to comment 135 ABCB11 p.Arg928* ABCB11 p.Arg928* ABCB11 p.His1198Arg ABCB11 p.His1198Arg Age of onset/ GGT TBA TBIL/DBIL ALT/AST Mutation Patient gender Symptoms (U/l) (&#b5;mol/l) (&#b5;mol/l) (U/l) Mutation origin P1 1 m/M Persistent jaundice, 49 101.3 162.5/130.4 432/606 Compound R928X, hepatosplenomegaly heterozygous maternal; p.R928X/p.H1198R H1198R, paternal. Login to comment 136 ABCB11 p.Tyr472Cys P3 2 d/F Persistent jaundice, 32 256.1 166.7/137.4 158/235 Compound Y472C, pruritus, heterozygous paternal. Login to comment 137 ABCB11 p.Tyr472Cys ABCB11 p.Asp1131Val ABCB11 p.Asp1131Val hepatosplenomegaly p.Y472C/p.D1131V D1131V, maternal. Login to comment 138 ABCB11 p.Arg696Trp ABCB11 p.Tyr337His P5 6 d/M Progressive 46 NA 99.7/72.6 165/211 Heterozygous NA jaundice p.Y337H P11 4 d/M Progressive 74 204.6 75.4/54.3 481/600 Heterozygous NA jaundice p.R696W GGT, gamma-glutamyltransferase; TBA, total bile acid; TBIL, total bilirubin; DBIL, direct bilirubin; ALT, alanine transaminase; AST, aspartate transaminase; M, male; F, female; NA, not available. Login to comment 141 ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro Variant SIFT PolyPhen-2 SNPs&GO EC/EN c.1009T>C (Y337H) 0.01 0.996 Disease EC c.1331T>C (V444A) 0.34 0.001 Neutral EC c.1415A>G (Y472C) 0 1.000 Disease EC c.2086C>T (R696W) 0.02 0.999 Disease EC c.2594C>T (A865V) 0.07 0.880 Disease EC c.2792A>C (Q931P) 0.02 0.178 Disease EN c.3392A>T (D1131V) 0 1.000 Disease EC c.3593A>G (H1198R) 0 1.000 Disease EC SIFT, Sorting Intolerant From Tolerant (mutation of residues with SIFT scores <0.05 are predicted to be deleterious); PolyPhen-2, Polymorphism Phenotyping version 2 (a score <0.2 denotes benign variants, between 0.2 and 0.85 is possibly damaging and >0.85 is highly likely damaging); SNPs&GO, a web tool to predict function of SNPs with a result of neutral or disease-related variants for human; EC, evolutionarily conserved; EN, evolutionarily non-conserved; SNP, single nucleotide polymorphism. Table VI. Login to comment 143 ABCB11 p.Val444Ala A, p.V444A (c.1331T>C) Variable PFIC2 (%) Cholestasis (non-PFIC2) (%) Control (%) Pa Pb Pc Polymorphism 0.847 0.493 0.580 TT 0 (0.0) 1 (6.3) 11 (5.5) TC 2 (50.0) 4 (25.0) 80 (40.0) CC 2 (50.0) 11 (68.7) 109 (54.5) Total no. Login to comment 147 ABCB11 p.Val444Ala The genotype distribution of control subjects was in HardyߛWeinberg equilibrium (V444A, P=0.46; A1028A, P=0.43). Login to comment 156 ABCB11 p.Arg928* ABCB11 p.Tyr472Cys ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro All the diseaseߛrelated mutations detected above were tested in 200 control subjects using HRM analysis to screen exon 10 (p.Y337H), exon 13 (p.Y472C), exon 18 (p.R696W), exon 22 (p.R928X), exon 22 (p.Q931P), exon 25 (p.D1131V) and exon 26 (p.H1198R). Login to comment 159 ABCB11 p.Val444Ala Two previously reported SNPs, p.V444A and p.A1028A, were identified in a number of the patients and control subjects. Login to comment 161 ABCB11 p.Val444Ala The distribution of p.V444A and p.A1028A polymorphisms, as well as allele frequencies, are revealed in Table VI. Login to comment 162 ABCB11 p.Val444Ala It was identified that V444A and A1028A were more prevalent polymorphisms in the control subjects, and had an allele frequency of 74.5 and 67.2%, respectively. Login to comment 163 ABCB11 p.Val444Ala This was consistent with previously reported data, which identified an allele frequency of 75.6% for V444A (19). Login to comment 164 ABCB11 p.Val444Ala However, neither V444A nor A1028A were associated with PFIC2 or cholestasis of undefined etiology in the patients. Login to comment 165 ABCB11 p.Val444Ala The distribution of alleles at the two SNPs in control group was in Hardy-Weinberg equilibrium (V444A, P=0.46; A1028A, P=0.43). Login to comment 179 ABCB11 p.Arg696Trp ABCB11 p.Asp1131Val ABCB11 p.Tyr337His ABCB11 p.His1198Arg ABCB11 p.Gln931Pro Among the seven mutations identified in the study, p.Y337H, p.R696W, p.Q931P, p.D1131V and p.H1198R are novel mutations according to data from The Human Gene Mutation Database (http://www. Login to comment 182 ABCB11 p.Tyr337His ABCB11 p.Tyr337His The missense mutation p.Y337H (c.1009T>C) occurred in the transmembrane domain (TMD) that led to an amino acid substitution from tyrosine to histidine at position 337 in BSEP. Login to comment 190 ABCB11 p.Gln931Pro These mutations were evolutionarily conserved and an comprehensive analysis of the results of SIFT, PolyPhen-2, SNPs&GO revealed that they may be deleterious with the exception of p.Q931P. Login to comment 192 ABCB11 p.Arg928* ABCB11 p.Tyr472Cys The missense mutation p.Y472C and nonsense mutation p.R928X, have been reported in PFIC2 patients of European populations and immunohistochemical staining for BSEP was undetectable (26-28,34). Login to comment 196 ABCB11 p.Arg928* In exon 22, the melting curve of the patient with heterozygous p.R928X (c.2782C>T) shifted away from those without mutations or with homozygous mutations. Login to comment 198 ABCB11 p.Arg928* Following direct sequencing, p.R928X was identified. Login to comment 202 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly Common mutations that have been reported in European populations, such as E297G and D482G, were not detected in Chinese subjects. Login to comment 204 ABCB11 p.Val444Ala V444A and A1028A are two highly prevalent polymorphisms. Login to comment 205 ABCB11 p.Val444Ala The allele frequency of V444A and A1028A has been reported in Japanese and Caucasian populations (44). Login to comment 206 ABCB11 p.Val444Ala According to the present study, the allele frequencies of V444A and A1028A were 74.5% and 67.2%, respectively, in mainland Chinese populations. Login to comment 207 ABCB11 p.Val444Ala V444A has previously been implicated in ICP and DIC with a higher allele frequency than normal controls suggesting that this polymorphism may become disease relevant in certain conditions, such as pregnancy and the use of ethinylestradiol and levonorgestrel (11,45). Login to comment 209 ABCB11 p.Val444Ala However, as the number of patients tested in the present study is small, it is not conclusive whether V444A or A1028A has a role in PFIC2 or cholestasis. Login to comment 214 ABCB11 p.Val444Ala V444A and A1028A were two highly prevalent SNPs found in ABCB11 exons in the study population; however, whether they are associated with pediatric cholestatic diseases remains unclear. Login to comment