ABCMdb

PMID: 18692205

Chen HL, Liu YJ, Su YN, Wang NY, Wu SH, Ni YH, Hsu HY, Wu TC, Chang MH
Diagnosis of BSEP/ABCB11 mutations in Asian patients with cholestasis using denaturing high performance liquid chromatography.
J Pediatr. 2008 Dec;153(6):825-32. Epub 2008 Aug 9., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCB11 p.Arg487His ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Trp493* ABCB11 p.Arg303Lys Results Seven mutations in 4 of 16 patients with PFIC from different families were detected by DHPLC, including M183V, V284L, R303K, R487H, W493X, G1004D, and 1145delC. Login to comment 4 ABCB11 p.Gly1004Asp G1004D was found in a patient with BRIC. Login to comment 5 ABCB11 p.Leu827Ile L827I was found in another patient with neonatal cholestasis. Login to comment 7 ABCB11 p.Val444Ala ABCB11 p.Ala865Val Polymorphisms V444A and A865V, with an allele frequencies 75.6% and 0.6%, respectively, were found in our population. Login to comment 28 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly In a recent study analyzing patients of mostly European origin, 82 mutations that occurred throughout the protein were detected in 109 families.13 Some mutations were found in a number of affected families of European descent, such as E297G and D482G.4,13-14 No such hot spots have been found in patients with ABCB11 mutations in other ethnic backgrounds, especially in Asian populations. In this study, we developed a method to perform ABCB11 genomic analysis more efficiently by first amplifying all of the ABCB11 exons, and then subjecting the exons to denaturing high performance liquid chromatography (DHPLC) analysis, followed by confirmation using direct sequencing. Login to comment 46 ABCB11 p.Arg487His ABCB11 p.Gly1004Asp ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Arg303Lys Age of onset Bil T (␮mol/L) Bil D (␮mol/L) GGT (U/L) AST (U/L) ALT (U/L) Liver pathology Mutations found Exon Predicted effect Outcome 1 1 mo 169.3 121.4 56 518 244 IC, GCT c.850GϾC c.1145delC 9 11 p. Val284Leu p.Ala382AlafsX16 Death, 5 y 2 1 mo 100.9 51.3 30 88 66 IC, GCT, bridging fibrosis c.850GϾC c.1145delC 9 11 p. Val284Leu p.Ala382AlafsX16 Alive 5 y, chronic cholestasis, Failure to thrive 3 2 wk 119.7 58.1 36 500 341 IC, GCT c.547AϾG c.908GϾA 7 9 p.Met183Val p.Arg303Lys Death, 8 mo 4 1 mo 179.6 138.5 68 186 150 GCT, fibrosis c.1478GϾA c.3011GϾA 14 23 p.Trp493X p.Gly1004Asp Death, 2 yr 5 birth 237.7 155.6 47 1184 653 - c.1460GϾA 14 p.Arg487His Death, 1 y 6 2 mo 360.8 251.4 132 884 352 Lobular disarray NF Death, 9 mo 7 1 mo 119.7 109.4 25 54 49 IC, bridging fibrosis NF Alive, 7 y 8 2 mo 401.9 273.6 51 251 252 GCT, cell ballooning NF Death 1 y 6mo 9 3 mo 442.9 256.5 54 556 217 - NF Death, 7 mo 10 1 mo 157.3 90.6 20 606 828 GCT, hepatocyte ballooning, confluent necrosis NF Death, 9 mo 11 1 mo 193.2 136.8 52 523 345 GCT, focal necrosis NF Death 4 mo 12 2 mo 165.9 94.1 99 298 202 GCT, portal inflammation, ductal proliferation NF Death 8 mo 13 2 wk 277.0 194.9 64 1054 825 GCT, necrosis NF OLT, 6 mo 14 2 mo 413.8 229.1 51 625 665 GCT, biliary cirrhosis NF OLT, 9 mo 15 birth 371.1 271.9 68 584 400 GCT NF Alive, 10 mo 16 birth 176.1 145.4 57 352 249 GCT, bridging fibrosis NF Death, 9 mo 17 birth 136.8 46.2 47 29 117 GCT NF Alive, 5 y 18 32 y 143.6 63.3 24 105 91 Centrilobular canalicular cholestasis, hemosiderosis c.3011GϾA 23 p.Gly1004Asp Alive, 35 y, recurrent cholestasis IC, Intrahepatic cholestasis; GCT, giant-cell transformation; OLT, orthotopic liver transplantation; NF, mutations not found. Login to comment 75 ABCB11 p.Val444Ala ABCB11 p.Ala865Val Analysis of V444A and A865V in Patients with Neonatal Cholestasis and PFIC To investigate whether the 2 nonsynonymous polymorphisms found in our population have functional consequences and affect disease presentation, we analyzed the allele frequencies of polymorphisms in 21 patients with PFIC/BRIC, 23 patients with neonatal cholestasis other than PFIC, and 88 control subjects with chronic nonsymptomatic HBV. Login to comment 76 ABCB11 p.Val444Ala ABCB11 p.Ala865Val These samples were tested using DHPLC analysis of exons 13 and 21, followed by direct sequencing to identify the V444A and A865V polymorphisms. Login to comment 80 ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Arg303Lys RESULTS Mutations Detected in Patients with PFIC Of 17 patients with low-GGT PFIC, mutations in ABCB11 were found in 5 patients, including a sibling pair (patients 1 and 2) with compound heterozygous c.850GϾC (p.V284L) and c.1145delC (p.Ala382AlafsX16) mutations.12 Patient 3 had compound heterozygous c.547AϾG (p.M183V)/c.908GϾA (p.R303K) mutations. Login to comment 81 ABCB11 p.Arg487His ABCB11 p.Gly1004Asp ABCB11 p.Trp493* His next sibling was a heterozygous carrier and was later born healthy after prenatal diagnosis.18 Patient 4 had the compound missense and nonsense mutations c.3011GϾA (p.G1004D) and c.1478GϾA (p.W493X), whereas patient 5 had a heterozygous c.1460GϾA (p.R487H) mutation. Login to comment 84 ABCB11 p.Gly1004Asp One adult patient with BRIC was found to have a heterozygous G1004D mutation. Login to comment 88 ABCB11 p.Arg487His ABCB11 p.Arg303Lys R303K had been reported in 1 family of Central Asian/Arab background.13 R487H had previously been reported to occur in a Japanese patient.11 No mutational hot spots were identified in our patients. Login to comment 91 ABCB11 p.Val444Ala Polymorphism Analysis Two previously reported nonsynonymous polymorphisms at c.1331TϾC (p.V444A,) and c. Login to comment 92 ABCB11 p.Ala865Val 2720CϾT (p.A865V) were detected in a number of our patients and parents.19 We then analyzed these two polymorphic sites with DHPLC followed by sequencing analysis in 21 patients with PFIC/BRIC, 23 patients with neonatal cholestasis other than PFIC, and 88 control patients. Login to comment 93 ABCB11 p.Val444Ala The denaturing pattern for heterozygous TC in V444A (pattern A) was different from the TT and CC (pattern B, Figure 2). Login to comment 95 ABCB11 p.Val444Ala ABCB11 p.Val444Ala ABCB11 p.Ala865Val Distributions of V444A and A865V polymorphisms, as well as allele frequencies, are shown in Table III. We found that V444A was a more prevalent polymorphism in control patients, and had an allele frequency of 75.6%. Login to comment 96 ABCB11 p.Val444Ala However, V444A was not associated with PFIC or neonatal cholestasis in our patients using univariate analysis. Login to comment 97 ABCB11 p.Ala865Val There was a trend for a higher frequency of A865V in patients with PFIC/BRIC (9.5%) and neonatal cholestasis (8.7%) than in control subjects (1.1%, P ϭ .094 and .108, respectively) but without statistical significance. Login to comment 98 ABCB11 p.Val444Ala ABCB11 p.Ala865Val All of the patients with A865V also carried the homozygous V444A polymorphism. Login to comment 99 ABCB11 p.Ala865Val However, because the number of patients carrying A865V was small, it is not conclusive whether these differences make any clinical significance. Login to comment 100 ABCB11 p.Ala865Val ABCB11 p.Leu827Ile While analyzing patients for A865V polymorphism, another novel missense mutation c.2479CϾA (p.L827I), located in exon 21, was found in 1 patient with neonatal cholestasis with a favorable outcome. Login to comment 102 ABCB11 p.Arg487His ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Arg303Lys ABCB11 p.Leu827Ile Analysis of Mutations in Normal Controls All the missense mutations detected above were tested in 80 normal control subjects (160 alleles), using DHPLC for M183V (exon 7), V284L (exons 9), R303K (exon 9), R487H (exon 14), L827I (exon 21), and G1004D (exon 23). Login to comment 104 ABCB11 p.Val444Ala ABCB11 p.Ala865Val Comparing our results with previously reported large scale genetic analysis of ABCB11 variations in healthy Caucasians, African Americans, and Japanese subjects,19 the missense mutations found in our study were not reported in all the 3 populations. In the same study, V444A and A865V were the only nonsynonymous polymorphisms found in Japanese population, which was consistent with our findings. Login to comment 105 ABCB11 p.Arg487His ABCB11 p.Arg303Lys R303K and R487H had previously been reported to occur in patients with PFIC, further supporting their disease-causing roles.11,13 Figure 1. Login to comment 108 ABCB11 p.Val444Ala ABCB11 p.Ala865Val The SIFT results indicated that all of the missense mutations (except polymorphisms V444A and A865V) may affect protein function. Login to comment 109 ABCB11 p.Arg487His ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Arg303Lys ABCB11 p.Leu827Ile The PolyPhen results indicated that V284L and G1004D are possibly damaging, and M183V, R303K, R487H, and L827I were predicted to be benign (Table IV). Login to comment 119 ABCB11 p.Asp482Gly ABCB11 p.Glu297Gly E297G and D482G were present in 58% of European families with PFIC, and the 2 mutations were proposed to originate from Northern Europe and Central/Eastern Europe, respectively.13 From our present data, there is no evidence that these mutations have spread to east Asia. Login to comment 121 ABCB11 p.Arg487His R487H was found in 1 Japanese and 1 Taiwanese family. Login to comment 122 ABCB11 p.Gly1004Asp ABCB11 p.Arg303Lys In our study, G1004D was found in 1 patient with PFIC and in 1 patient with BRIC.12 R303K, which was found in our patient, had been reported in a family of central Asian/Arab background.13 Further investigations are therefore required to confirm whether these mutations are found more commonly in Asian patients. Login to comment 123 ABCB11 p.Val444Ala ABCB11 p.Val444Ala ABCB11 p.Ala865Val ABCB11 p.Ala865Val Interestingly, although our patients had mutations distinct from patients in Western countries, there Table III. Distribution of V444A and A865V polymorphisms and allele frequencies in Taiwanese patients and control subjects PFIC/BRIC Neonatal cholestasis (non-PFIC) Control P1 P2 P3 V444A n ϭ 21 n ϭ 23 n ϭ 88 0.798 0.508 0.806 TT 2 (9.5%) 2 (8.7%) 5 (5.7%) TC 8 (38.1%) 11 (47.8%) 33 (37.5%) CC 11 (52.4%) 10 (43.5%) 50 (56.8%) Allele frequency T % 12 (28.6%) 15 (32.6%) 43 (24.4%) 0.693 0.264 0.818 C % 30 (71.4%) 31 (67.4%) 133 (75.6%) A865V n ϭ 21 n ϭ 23 n ϭ 88 0.094 0.108 1.000 CC 19 (90.5%) 21 (91.3%) 87 (98.9%) CT 2 (9.5%) 2 (8.7%) 1 (1.1%) TT 0 (0.0%) 0 (0.0%) 0 (0.0%) Allele frequency C % 40 (95.2%) 44 (95.7%) 175 (99.4%) 0.096 0.110 1.000 T % 2 (4.8%) 2 (4.3%) 1 (0.6%) P1: Comparisons between PFIC/BRIC vs control. Login to comment 128 ABCB11 p.Val444Ala ABCB11 p.Arg487His ABCB11 p.Ala865Val ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu ABCB11 p.Met183Val ABCB11 p.Arg303Lys ABCB11 p.Leu827Ile Prediction of functional consequences of nonsynonymous mutations and polymorphisms in ABCB11 found in Asian patients Amino acid change SIFT PolyPhen (PSIC score) EC/EU M183V 0.02 1.45 EC V284L 0.02 1.87 EC R303K 0.00 0.38 EC V444A 0.76 0.60 EC R487H 0.01 0.65 EC L827I 0.01 1.23 EC A865V 0.10 0.76 EC G1004D 0.00 1.97 EC SIFT, Sorting intolerant from tolerant (SIFT scores Ͻ0.05 indicate evolutionarily conserved amino acids, and mutation of these residues are predicted to be deleterious); PolyPhen, polymorphism phenotyping (a PSIC score Ͻ0.5 denotes benign variants, between 1.5 and 2 is possibly damaging, and Ͼ2 is probably damaging); EC, evolutionarily conserved; EU, evolutionarily unconserved. Login to comment 130 ABCB11 p.Arg487His ABCB11 p.Trp493* ABCB11 p.Arg487Pro For example, R487H and W493X occurred in 1 of the 2 highly conserved nucleotide-binding domains (residues 414-610 and 1072-1321) in which 60% of missense mutations clustered; the 1-bp deletion at nucleotide 1145 occurred at the "deletion hot spot" located between nucleotides 1100 and 1146.13 Different mutations that occurred at the same sites, R487P and c.1145_1165del, were reported, suggesting sequence instability or mutagen interaction in these regions. Login to comment 135 ABCB11 p.Gly1004Asp ABCB11 p.Val284Leu Although the PolyPhen analysis only predicted 2 missense mutations (V284L and G1004D) to be possibly deleterious, other mutations had borderline scores. Login to comment 140 ABCB11 p.Val444Ala V444A is a highly prevalent polymorphism. Login to comment 146 ABCB11 p.Val444Ala (80.4%) than in Caucasians (59.5%).19 V444A has previously been implicated in BRIC-2 and severe intrahepatic cholestasis during pregnancy, when present in combination with other ABCB11 or ABCB4 mutations.7,10 This polymorphism has also been implicated in drug-induced cholestasis. Login to comment 147 ABCB11 p.Val444Ala ABCB11 p.Val444Ala An in vitro study demonstrated that the V444A polymorphism leads to a lower BSEP transporter activity.9 However, ABCB11 sequence variations were found to be less important in the development of pregnancy-associated cholesatsis.22 In our study, homozygous V444A mutations were highly prevalent in PFIC parents and in control patients lacking a cholestatic phenotype. Login to comment 148 ABCB11 p.Val444Ala ABCB11 p.Ala865Val Because the number of patients tested in this study is small, it is not conclusive whether V444A or A865V plays roles in PFIC or neonatal cholestasis. Login to comment 149 ABCB11 p.Val444Ala It is possible that the functional consequence of V444A is affected by many other factors, such as concurrent mutations in ABCB11 or other genes, drugs or toxins, and different ethnic backgrounds. Login to comment 150 ABCB11 p.Val444Ala A protective effect of V444A in certain populations is also possible. Login to comment 165 ABCB11 p.Val444Ala ABCB11 p.Ala865Val V444A and A865V are nonsynonymous polymorphisms found in ABCB11 exons in our population, and their association with pediatric cholestatic diseases is not clear. Login to comment