ABCMdb

PMID: 16039748

Noe J, Kullak-Ublick GA, Jochum W, Stieger B, Kerb R, Haberl M, Mullhaupt B, Meier PJ, Pauli-Magnus C
Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis.
J Hepatol. 2005 Sep;43(3):536-43., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Results: The PFIC2 patient was compound heterozygous for a splicing mutation in intron 4 ((C3)AOC) combined with an early stop codon at position 930 (R930X), while the BRIC2 patient was compound heterozygous for two nonsynonymous mutations in exon 9 (E297G) and exon 12 (R432T), respectively. Login to comment 6 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr In BRIC2, taurocholate transport was decreased to 13% and 20% of reference levels for R432T and E297G, respectively. Login to comment 7 ABCB11 p.Arg432Thr Conclusions: The intron 4 (C3)AOC, R930X and R432T represent previously undescribed mutations of the ABCB11 gene that confer a PFIC2 and a BRIC2 phenotype, respectively. Login to comment 29 ABCB11 p.Asp482Gly However, the D482G mutation, which was found to exhibit decreased function by Wang, showed preserved transport activity when cloned into mouse Abcb11, indicating the possible impact of species differences on protein function [13]. Login to comment 32 ABCB11 p.Cys336Ser For instance, the C336S mutation in ABCB11, which was detected in two different patients with a PFIC2 phenotype [8] affected neither trafficking nor in-vitro taurocholate transport, suggesting that this mutation alone is not sufficient to cause cholestasis. Login to comment 61 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr The single nucleotide polymorphisms 890AOG (codon GAGOGGG) and 1294GOC (codon AGAOACA), which result in the nonsynonymous mutations E297G and R432T, respectively, were introduced into the reference h ABCB11 cDNA. Login to comment 68 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Functional transport studies ATP-dependent transport of labeled bile salts was determined for the E297G and the R432T mutations by a rapid filtration assay as described [16]. Login to comment 81 ABCB11 p.Lys930* The parents of the patient had normal liver enzyme levels and no clinical signs of hepatopathy. 3.1.1. Sequencing Sequence analysis in the index patient detected a heterozygous splicing mutation in intron 4 (C3)AOC combined with a heterozygous insertion of five basepairs (GAGAT) in exon 21 predicting a frameshift with the introduction of an early stop codon at amino acid position 930 (K930X). Login to comment 82 ABCB11 p.Lys930* Analysis of the patient`s parents indicated that the intron 4 (C3)AOC mutation was inherited from the mother, while the K930X mutation was inherited from the father (Fig. 1). Login to comment 100 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr The parents of the patient had normal liver enzyme levels in the absence of clinical signs of hepatopathy. 3.2.1. Sequencing Sequence analysis indicated a nonsynonymous mutation in exon 9 (891GOA) predicting the substitution of a glutamate by a glycine in position 297 (E297G) combined with a nonsynonymous mutation in exon 12 (1296GOC), predicting a substitution of an arginine by a threonine in position 432 (R432T). Login to comment 101 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Analysis of the patient`s parents indicated that the E297G mutation was inherited from the mother, whereas the R432T mutation was inherited from the father (Fig. 1). Login to comment 110 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Transport studies Western blot analysis detected comparable protein amounts for R432T, E297G and reference BSEP in SF-9 cell vesicles (Fig. 3). Login to comment 111 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Transport capacity of the mutated constructs amounted to about 13 and 20% of reference activity for the R432T and the E297G construct, respectively (Fig. 3). Login to comment 112 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Initial taurocholate transport by reference BSEP as well as by R432T and E297G mutants exhibited saturability with increasing substrate concentrations (Fig. 4). Login to comment 114 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr The Km values for R432T and the E297G mutant were 5.6 mmol/L and 22 mmol/L, respectively, which is comparable to the Km value of reference BSEP. Login to comment 115 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr In contrast, the maximum taurocholate transport velocity for the BSEP mutants was greatly reduced, with Vmax values of 53 pmol/L mg proteinK1 minK1 , and 111 pmol/ L mg proteinK1 minK1 for R432T and E297G, respectively, compared to 686 pmol/L mg proteinK1 minK1 for reference BSEP (Fig. 4). Login to comment 124 ABCB11 p.Lys930* Compound heterozygosity for a splicing mutation in intron 4 (C3)AOC combined with a frameshift mutation in exon 22 (K930X) resulted in a PFIC2 phenotype with early onset progressive cholestasis. Login to comment 127 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Compound heterozygosity for two nonsynonymous variants in exon 9 (E297G) and in exon 12 (R432T) was encountered in the patient exhibiting a BRIC phenotype. Login to comment 128 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr While the E297G site had already been associated with inherited intrahepatic cholestasis, the R432T mutation was previously undescribed in cholestatic disease. Login to comment 130 ABCB11 p.Glu297Gly Out of the detected variants, the E297G mutation had been functionally characterized by Wang and Hayashi [12,17], who found this mutation to result in defective trafficking of the protein to the apical membrane of transfected MDCK cells, whereas in-vitro transport studies yielded discrepant results. Login to comment 133 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr However, it cannot completely be excluded that in our patient the R432T allele is compensating for defective BSEP expression associated with the E297G mutation. Login to comment 134 ABCB11 p.Glu297Gly In previous studies the E297G site has been associated with different clinical phenotypes as it was found in patients with PFIC2 and BRIC2 syndrome as well as in phenotypically normal subjects, indicating variable penetrance of this mutation [5-8,11]. Login to comment 137 ABCB11 p.Glu297Gly ABCB11 p.Glu297Gly As our data indicate residual BSEP function for the E297G variant, it can be suspected that the E297G variant alone is not responsible for the observed phenotype in patients with progressive intrahepatic cholestasis. Login to comment 146 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Kinetics of ATP-dependent transport of taurocholate by reference BSEP, R432T BSEP mutant and E297G BSEP mutant in membrane vesicles isolated from Sf9 cells. Login to comment 147 ABCB11 p.Glu297Gly ABCB11 p.Arg432Thr Initial uptake rates for taurocholate by reference BSEP, E297G mutant (60 s) and R432T mutant (90 s) were determined in the presence and absence of ATP (5 mmol/L). Login to comment 150 ABCB11 p.Glu297Gly The E297G example demonstrates that homo- or heterozygosity for a nonsynonymous ABCB11 mutation does not suffice to explain a PFIC2 or BRIC2 phenotype as long as its consequences on BSEP expression and function are not established. Login to comment 154 ABCB11 p.Glu297Gly Differences in experimental design might also explain the recent discrepancies observed with in-vitro phenotyping of the E297G mutation by different groups [12,17]. Login to comment 160 ABCB11 p.Glu297Gly Initial data suggest that alleles with residual function such as the E297G mutant are predictive for a positive outcome of bile duct diversion [18] and the functional characterization of ABCB11 mutants might therefore help to determine patients, who will profit from such an intervention. Login to comment