ABCMdb

PMID: 17264802

Lang C, Meier Y, Stieger B, Beuers U, Lang T, Kerb R, Kullak-Ublick GA, Meier PJ, Pauli-Magnus C
Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury.
Pharmacogenet Genomics. 2007 Jan;17(1):47-60., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr ABCB4 p.Leu1082Gln ABCB4 p.Ile764Leu Results Four highly conserved nonsynonymous mutations were specific for drug-induced liver injury [ABCB11: D676Y (drug-induced cholestasis) and G855R (drug-induced cholestasis); ABCB4: I764L (drug-induced cholestasis) and L1082Q (drug-induced hepatocellular injury)]. Login to comment 6 ABCB11 p.Val444Ala Furthermore, a polymorphism in exon 13 of ABCB11 (V444A), which is associated with decreased hepatic BSEP expression was significantly more frequent in drug-induced cholestasis patients than in drug-induced hepatocellular injury patients and healthy controls (76 versus 50 and 59% in drug-induced cholestasis patients, drug-induced hepatocellular injury patients and healthy controls, respectively; P < 0.05). Login to comment 7 ABCB11 p.Val444Ala ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr The in-vitro transport activity of the V444A and the D676Y BSEP constructs was similar, whereas the G855R mutation was nonfunctional. Login to comment 39 ABCB11 p.Val444Ala Furthermore, genotype distribution at position 1331T > C (V444A) was determined in 110 additional Caucasian individuals recently described by Meier and coworkers [24] (total: 95 + 110; n = 205). Login to comment 62 ABCB11 p.Met677Val ABCB11 p.Asp676Tyr Amino acid exchanges were introduced by site-directed mutagenesis, using the Quickchange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, California, USA) with the following mutagenesis primer pairs: (i) replacement of alanine to valine at position 444: forward 50 -CTAAAT GACCTCAACATGGTCATTAAACCAGGGG-30 and reverse 50 -CCCCTGGTTTAATGACCATGTTGAGGTCAT TTAG-30 ; (ii) replacement of aspartic acid to tyrosine at position 676: forward 50 -GGATGCAACTGAAGATTA CATGCTTGCGAGG-30 and reverse 50 -CCTCGCAAG CATGTAATCTTCAGTTGCATCC-30 ; (iii) replacement of methionine to valine at position 677: forward 50 -GCAACTGAAGATGACGTGCTTGCGAGGACC-30 and reverse 50 -GGTCCTCGCAAGCACGTCATCTTCAGT TGC-30 . Login to comment 63 ABCB11 p.Gly855Arg The mutation G855R was introduced into the complete ABCB11 DNA sequence using the following primers: forward 50 -CCTCAGAAATAGCCCTAGAGCATT GACAACAAGAC-30 and reverse 50 -GTCTTGTTGTCA ATGCTCTAGGGCTATTTCTGAGG-30 . Login to comment 72 ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Transport assays ATP-dependent transport of labeled taurocholic acid was determined for reference ABCB11 and the A444V, M677 V, D676Y and G855R variants by a rapid filtration assay as described [26]. Login to comment 78 ABCB11 p.Asp676Tyr Furthermore, transport activity of taurocholic acid and its inhibition by fluvastatin was measured in reference BSEP and the D676Y construct in the presence and absence of ATP, allowing the estimation of IC50 values. Login to comment 82 ABCB11 p.Met677Val ABCB11 p.Asp676Tyr All calculations Table 3 ABCB11 (BSEP) variant sites in drug-induced liver injury Amplicon DNA position cDNA position Nucleotide reference Nucleotide variant AA change AF DC (n = 46) AF DH (n = 26) AF controls (n = 190) Amplicon - 1 Intron - 1 - 2080 CTCT delCTCT 0.02 0.00 0.10 Amplicon - 1 Intron - 1 - 1952 T C 0.43 0.54 0.48 Amplicon - 1 Intron - 1 - 1820 G A 0.09 0.04 0.10 Amplicon - 1 Intron - 1 - 1746 G A 0.09 0.04 0.10 Amplicon - 1 Intron - 1 - 1275 G A 0.07 0.04 0.03 Amplicon - 1 Intron - 1 - 1239 G A 0.24 0.35 0.30 Amplicon - 1 Intron - 1 - 1155 T C 0.57 0.65 0.62 Amplicon - 1 Intron - 1 - 1009 T C 0.09 0.04 0.02 Amplicon - 1 Intron - 1 - 837 A G 0.02 0.00 0.02 Amplicon 2 Intron 1 - 50 G A 0.09 0.04 0.02 Amplicon 4 Intron 3 - 20 T C 0.09 0.08 0.07 Amplicon 5 Exon 5 270 T C syn 0.09 0.04 0.03 Amplicon 5 Intron 5 8 G A 0.00 0.13 0.05 Amplicon 9 Exon 9 851 T C V284A_c 0.02 0.00 0.01 Amplicon 10 Intron 9 - 69 C T 0.00 0.04 0.05 Amplicon 10 Intron 9 - 31 C T 0.00 0.04 0.05 Amplicon 10 Intron 9 - 17 G A 0.00 0.04 0.05 Amplicon 10 Intron 9 - 15 A G 0.77 0.65 0.70 Amplicon 10 Exon 10 957 A G syn 0.00 0.04 0.05 Amplicon 13 Exon 13 1331 T C V444A_c 0.76 0.50 0.59 Amplicon 13 Intron 13 70 C T 0.76 0.50 0.59 Amplicon 14 Intron 14 32 T C 0.75 0.54 0.60 Amplicon 17 Exon 17 2026 G T D676Y 0.02 0.00 0.00 Amplicon 17 Exon 17 2029 A G M677V 0.00 0.04 0.04 Amplicon 18 Exon 18 2093 G A R698H_c 0.02 0.00 0.01 Amplicon 18 Exon 18 2134 T C syn 0.02 0.00 0.01 Amplicon 19 Intron 18 - 17 C A 0.52 0.62 0.43 Amplicon 20 Intron 19 - 17 T C 0.66 0.75 0.69 Amplicon 21 Exon 21 2563 G A G855R_c 0.02 0.00 0.00 Amplicon 24 Exon 24 3084 G A syn 0.50 0.62 0.46 Amplicon 28 Intron 27 - 34 G A 0.48 0.62 0.45 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_003742.2. Login to comment 97 ABCB11 p.Val444Ala ABCB11 p.Met677Val ABCB11 p.Arg698His ABCB11 p.Val284Ala ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Chemical classification of all causative drugs revealed that the most prominent structures were the b-lactam ring of antibacterials in Fig. 2 Extracellular V284A V444A G855R R698H D676Y M677V Cytoplasm Secondary structure of bile salt export pump (BSEP) with nonsynonymous coding region variants. Login to comment 100 ABCB11 p.Val444Ala ABCB11 p.Val444Ala Table 4 Genotype distribution of ABCB11 SNP 1331T > C (V444A) in DC and DH patients and healthy controls DCall a DCØmut a DHa Healthy controls Variant siteb n (%) 95% CIc n (%) 95% CI n (%) 95% CI n (%) 95% CI Total 23 (100%) 20 (100) 13 (100) 205 (100) 1331T > C (V444A) TT (VV) 2 (8.7%) 1.0-28.1 2 (10.0) 1.2-31.7 4 (30.8) 9.0-61.5 38 (18.5) 13.4-24.6 TC (VA) 7 (30.4%) 13.2-53.0 4 (20.0) 5.7-43.7 5 (38.5) 13.7-68.5 101 (49.3) 42.2-56.4 CC (AA) 14 (60.9%) 38.5-80.3 14 (70.0) 45.7-88.2 4 (30.8) 9.0-61.5 66 (32.2) 25.8-39.1 a All drug-induced cholestasis patients (DCall), DC without disease associated nonsynonymous ABCB11 and ABCB4 mutations (DCØmut). Login to comment 118 ABCB11 p.Val444Ala ABCB11 p.Met677Val Of the coding region changes, eight have been previously described in healthy Caucasians [23], including two frequent nonsynonymous polymorphisms (exon 13: 1331T > C-V444A and exon 17: 2029A > G- M677V). Login to comment 119 ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Two nonsynonymous changes were specific for the collective of patients with DC (exon 17: 2026G > T-D676Y and exon 21: 2563G > A-G855R). Login to comment 120 ABCB11 p.Asp676Tyr The D676Y mutation was observed in a heterozygous patient taking fluvastatin (no. Login to comment 121 ABCB11 p.Gly855Arg 23) and the G855R mutation was observed in a heterozygous patient taking ethinylestradiol/gestagen for hormonal contraception (no. Login to comment 125 ABCB11 p.Val444Ala Fig. 3 DCØmutDCall (n=46) (n=40) (n=26) (n=410) DH Healthy controls P = 0.015; OR: 4 (1.3; 11.9) P = 0.01; OR: 2.4 (1.2; 4.9) NS P = 0.04; OR: 3.2 (1.1; 8.9) Allelicfrequencypercentage(95%CI) P = 0.004; OR: 3 (1.4; 6.8) T-allele C-allele 100 90 80 70 60 50 40 30 20 10 0 Allelic frequency of the T-allele (white panel) and C-allele (black panel) of the ABCB11 1331T > C (V444A) polymorphism: 23 DC patients (DCall, n = 46 alleles), 20 DC patients with no additional nonsynonymous ABCB4 or ABCB11 mutations (DCØmut, n = 40), 13 with DH (n = 24), and 205 Caucasians (n = 410). Login to comment 129 ABCB11 p.Gly855Arg Sequencing of the grandmother revealed heterozygosity for the ABCB11 mutation at position G855R, which was also found in our index patient. Login to comment 131 ABCB11 p.Val444Ala In addition, the V444A polymorphism was observed significantly more frequently in patients with DC than in patients with DH and healthy controls, with the CC genotype being encountered in 61% of DC patients compared with 31 and 32% in DH and healthy controls, respectively (Table 4). Login to comment 142 ABCB4 p.Arg652Gly ABCB4 p.Ile764Leu With the exception of the I764L, which was located in the eighth transmembrane domain of MDR3, all other nonsynonymous variants were Table 6 ABCB4 (MDR3)1 variant sites in drug-induced liver injury Amplicon DNA position cDNA position Nucleotide reference Nucleotide variant AA change AF DC (n = 46) AF DH (n = 26) AF controls (n = 112) Amplicon - 3 Intron - 4 - 394 T G 0.02 0.00 0.05 Amplicon - 3 Intron - 4 - 197 G T 0.00 0.04 0.00 Amplicon - 3 Exon - 3 - 410 T C Noncoding 0.02 0.00 0.05 Amplicon - 2 Exon - 2 - 228 C T Noncoding 0.04 0.08 0.11 Amplicon - 1 Intron - 2 - 221 C T 0.04 0.08 0.11 Amplicon - 1 Intron - 2 - 204 A G 0.04 0.08 0.13 Amplicon 1 Intron - 1 - 301 C G 0.07 0.08 0.17 Amplicon 1 Intron - 1 - 220 C T 0.02 0.00 0.05 Amplicon 1 Intron - 1 - 201 C G 0.04 0.08 0.13 Amplicon 1 Intron - 1 - 184 T C 0.33 0.46 0.26 Amplicon 4 Exon 4 175 C T syn 0.05 0.08 0.13 Amplicon 5 Intron 4 - 61 C T 0.11 0.08 0.03 Amplicon 5 Intron 5 113 A G 0.07 0.08 0.16 Amplicon 6 Intron 5 - 62 AGAAA delAGAAA 0.05 0.04 0.08 Amplicon 6 Intron 5 - 50 GAAA delGAAA 0.02 0.00 0.00 Amplicon 6 Exon 6 504 C T syn 0.62 0.46 0.57 Amplicon 6 Exon 6 523 A G T175A_c 0.02 0.00 0.03 Amplicon 8 Exon 8 711 A T syn 0.07 0.08 0.15 Amplicon 11 Intron10 - 43 T C 0.02 0.00 0.00 Amplicon 12 Intron 11 - 88 T delT 0.05 0.00 0.07 Amplicon 12 Intron 11 - 81 T delT 0.04 0.04 0.00 Amplicon 12 Exon 12 1314 G A syn 0.00 0.04 0.00 Amplicon 12 Intron 12 130 G T 0.08 0.04 0.06 Amplicon 13 Intron 12 - 40 G A 0.02 0.00 0.05 Amplicon 15 Intron 14 - 39 A G 0.02 0.08 0.01 Amplicon 15 Exon 15 1769 G A R590Q_c 0.02 0.00 0.01 Amplicon 16 Exon 16 1954 A G R652G 0.09 0.04 0.07 Amplicon 16 Intron 16 55 A G 0.09 0.04 0.05 Amplicon 17 Intron 17 16 T C 0.03 0.00 0.05 Amplicon 18 Exon 18 2290 A C I764L_c 0.02 0.00 0.00 Amplicon 20 Intron 20 40 A G 0.11 0.00 0.06 Amplicon 23 Intron 23 70 G T 0.00 0.04 0.00 Amplicon 25 Exon 25 3245 T A L1082Q_c 0.00 0.04 0.00 Amplicon 27 Intron 26 - 16 T C 0.35 0.58 0.92 Amplicon 28 Intron 27 - 72 T C 0.11 0.00 0.07 cDNA numbers are relative to the ATG site and based on the cDNA sequence from GenBank accession number NM_000443.2. Login to comment 149 ABCB4 p.Arg590Gln ABCB4 p.Thr175Ala ABCB4 p.Arg652Gly Of the coding region changes, eight have previously been described in healthy Caucasians, including three synonymous and two nonsynonymous changes (synonymous: exon 4: 175C > T, exon 6: 504C > T, exon 8: 711A > T; nonsynonymous: exon 6: 523A > G-T175A, exon 15: 1769G > A-R590Q and exon 16: 1954A > G-R652G). Login to comment 150 ABCB4 p.Leu1082Gln ABCB4 p.Ile764Leu Six variant sites were specific for patients with drug-induced liver injury, including three intronic and three coding region changes (synonymous: exon 12: 1314G > A; nonsynonymous: exon 18: 2290A > C-I764L and exon 25: 3245T > A-L1082Q). Login to comment 151 ABCB4 p.Ile764Leu The heterozygous I764L mutation was observed in a patient with DC taking risperidone (no. Login to comment 152 ABCB4 p.Leu1082Gln 18) and the L1082Q mutation in a patient with DH taking amoxicillin/clavulanic acid (no. Login to comment 158 ABCB11 p.Met677Val ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Comparable protein amounts were detected for the alanine and valine containing constructs in position 444 (Fig. 5a), while expression levels of the M677V, D676Y and G855R variants amounted to 40, 60 and 20% of expression levels of reference BSEP (Fig. 6a), respectively. Login to comment 161 ABCB11 p.Met677Val ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr Furthermore, adjusted taurocholate transport activities for M677V and D676Y were similar to reference BSEP, whereas hardly any transport activity was seen with the G855R construct (Fig. 6c). Login to comment 162 ABCB11 p.Asp676Tyr In addition, fluvastatin could be shown to inhibit transport activity of reference BSEP and of the D676Y construct. Login to comment 163 ABCB4 p.Arg590Gln ABCB4 p.Thr175Ala ABCB4 p.Arg652Gly ABCB4 p.Leu1082Gln ABCB4 p.Ile764Leu The estimated IC50 was 50 and 60 mmol/l for reference BSEP Fig. 4 Extracellular I764L T175A R652G R590Q Cytoplasm L1082Q Secondary structure of multidrug resistance protein 3 (MDR3) with nonsynonymous coding region variants. Login to comment 166 ABCB11 p.Asp676Tyr ABCB11 p.Asp676Tyr 56 Pharmacogenetics and Genomics 2007, Vol 17 No 1 and D676Y, respectively (log IC50 1.70 and 1.78 for reference BSEP and D676Y, respectively) (Fig. 7). Login to comment 168 ABCB11 p.Val444Ala Very interestingly, a common ABCB11 polymorphism in exon 13 (1331T > C-V444A) was observed significantly more frequently in patients with DC than in healthy Caucasian controls and patients with DH. Login to comment 172 ABCB11 p.Met677Val In contrast, no differences as to taurocholate transport activity or hepatic BSEP expression were observed for the second frequent ABCB11 polymorphism in exon 17 (M677V) [24]. Login to comment 174 ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr The affected patients were heterozygous carriers of these mutations and developed cholestasis under intake of fluvastatin (D676Y) and ethinylestradiol/gestodene (G855R), respectively. Login to comment 175 ABCB11 p.Gly855Arg No taurocholate transport activity could be detected for the G855R-containing construct and, very interestingly, the carrier of this mutant had a family history of hormonal cholestasis in two female family members. Login to comment 179 ABCB11 p.Gly855Arg While the heterozygous state for the G855R mutation is probably sufficient to maintain bile salt secretion under normal conditions, our data readily explain the functional failure under certain circumstances, such as drug intake or hormonal challenges. Login to comment 187 ABCB11 p.Asp676Tyr BSEP and MDR3 mutations in drug-induced liver injury Lang et al. 57 In contrast, functional characterization of taurocholate transport activity showed unaltered transport for the D676Y construct, which could be inhibited by fluvastatin in a concentration-dependent manner. Login to comment 189 ABCB11 p.Asp676Tyr As taurocholate transport activity and BSEP inhibition characteristics of the D676Y mutation were similar to those of reference BSEP, however, the occurrence of cholestasis in this patient is not explained by our findings. Login to comment 191 ABCB11 p.Asp676Tyr Fig. 6 150 kDa Ref. BSEP M 677V D676Y G 855R M ock transf. Login to comment 192 ABCB11 p.Asp676Tyr 0 10 20 30 40 50 60(b) (a) (c) Taurocholateuptake (pmol/mgprotein×min) Ref. BSEP D676Y G855RM677V Mock transf. Login to comment 194 ABCB11 p.Met677Val ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr ABCB11 p.Asp676Tyr Normalizedtransportactivity (in%ofref.BSEP) A 0 20 40 60 80 100 120 Ref. BSEP D676Y G855RM677V 140 Bile salt export pump (BSEP) expression and taurocholate transport in Sf9 membrane vesicles, expressing reference BSEP and the D676Y, M677V and the G855R constructs. Login to comment 195 ABCB11 p.Met677Val ABCB11 p.Gly855Arg ABCB11 p.Asp676Tyr (a) Expression levels of the D676Y, the M677V and the G855R constructs amounted to 60, 40 and 20% of reference BSEP, respectively. Login to comment 200 ABCB4 p.Ile764Leu The conserved I764L site encountered in a heterozygous patient with risperidone-induced cholestasis was located in the transmembrane domain of MDR3, a region that exhibits only very little variation in members of the ABC superfamily of transporters [29]. Login to comment 203 ABCB4 p.Leu1082Gln The L1082Q site was encountered in a heterozygous patient with amoxicillin/clavulanic acid-induced hepatocellular injury. Login to comment 210 ABCB11 p.Gly855Arg Third, the family history in the patient with the ABCB11 G855R mutation supports a common pathomechanism for certain forms of DC and intrahepatic cholestasis of pregnancy, which has already been observed for ABCB4-related forms of cholestasis [21]. Login to comment