ABCMdb

PMID: 22898925

von Kampen O, Buch S, Nothnagel M, Azocar L, Molina H, Brosch M, Erhart W, von Schonfels W, Egberts J, Seeger M, Arlt A, Balschun T, Franke A, Lerch MM, Mayerle J, Kratzer W, Boehm BO, Huse K, Schniewind B, Tiemann K, Jiang ZY, Han TQ, Mittal B, Srivastava A, Fenger M, Jorgensen T, Schirin-Sokhan R, Tonjes A, Wittenburg H, Stumvoll M, Kalthoff H, Lammert F, Tepel J, Puschel K, Becker T, Schreiber S, Platzer M, Volzke H, Krawczak M, Miquel JF, Schafmayer C, Hampe J
Genetic and functional identification of the likely causative variant for cholesterol gallstone disease at the ABCG5/8 lithogenic locus.
Hepatology. 2012 Aug 16. doi: 10.1002/hep.26009., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCG8 p.Asp19His ABCG8 p.Arg50Cys Subsequent mutation detection and genotyping yielded two disease-associated variants ABCG5-R50C (p=4.94×10-9 ) and ABCG8-D19H (p=1.74×10-10 ) in high pair-wise LD (r2 =0.95). Login to comment 28 ABCG8 p.Asp19His However, although coding variant ABCG8-D19H has often been used as the major tagging SNP in both, the discovery and the replication studies in humans, its causative role is by no means clear. Login to comment 47 ABCG8 p.Asp19His ABCG8 p.Arg50Cys The different versions of both transporters, defined by the respective alleles of R50C and D19H, were introduced by site-directed mutagenesis (QuickChange Lightning Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, USA) according to the Page 10 manufacturer`s protocol. HEK cells were transfected using Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer`s protocol. Login to comment 49 ABCG8 p.Asp19His ABCG8 p.Arg50Cys In brief, HEK cells transiently expressing different allelic variants of ABCG5-R50C and ABCG8-D19H were incubated in 24 well cell culture plates with 0.5 ml DMEM supplemented with 10 mM HEPES, pH 7.4, 30 mg/ml cholesterol, 0.5 mCi/ml [³H]-cholesterol and 0.2% fatty acid free BSA for 24 h. Login to comment 67 ABCG8 p.Asp19His In a single point analysis (Supplementary Table 1), the most significant disease associations were observed in an allele-based test for variants rs11887534 (ABCG8-D19H, p=1.7×10-10 ), rs72875462 (ABCG8 intronic, p=2.1×10-10 ) and rs56132765 (ABCG8-V151V p=3.9×10-10 ). Login to comment 68 ABCG5 p.Arg50Cys SNP rs6756629 (ABCG5-R50C) yielded the second most significant association (p=4.9×10-9 ) of all previously known, non-synonymous variants in ABCG5/8. Login to comment 79 ABCG8 p.Asp19His ABCG8 p.Arg50Cys ABCG8-D19H and ABCG5-R50C are the only functional candidates in the disease interval Owing to the absence of allelic imbalance and allele-dependent splicing, disease mechanisms such as differential transcription efficiency (caused by promoter polymorphisms or variable intronic enhancers) as well as differential transcript structure or stability could be safely ruled out. Login to comment 84 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Arg50Cys ABCG8 p.Arg50Cys ABCG8-D19H, but not ABCG5-R50C leads to increased cholesterol transport Of all coding SNPs investigated in the disease-associated region, only rs6756629 (ABCG5-R50C; ORallelic=1.96, 95% CI: 1.56-2.47) and rs11887534 (ABCG8-D19H; ORallelic=2.07, 95% CI: 1.65-2.60) were found to be significantly associated with cholelithiasis in our Page 14 mapping panel. Login to comment 85 ABCG5 p.Gln604Glu The significance of their disease associations was approximately seven orders of magnitude higher than that of the next "best" SNP (rs6720173, ABCG5-Q604E, pallelic=0.0024) which, moreover, is located well outside the disease-associated region defined above. Login to comment 96 ABCG8 p.Asp19His Whether ABCG5-50R or ABCG5-50C was present in the construct did not influence cholesterol efflux, given the respective ABCG8-D19H allele. Login to comment 98 ABCG8 p.Asp19His ABCG8 p.Arg50Cys ABCG8-D19H but not ABCG5-R50C captures the genetic risk across populations In addition to the functional experiments, nested logistic regression analyses were performed to evaluate statistically the causative role of individual variants in the disease-associated region. Login to comment 99 ABCG8 p.Asp19His In the German mapping panel, none of the coding SNPs listed in Supplementary Table 1 significantly improved the allelic risk model over the inclusion of ABCG8-D19H alone. Login to comment 100 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Arg50Cys When ABCG5-R50C was included as a mandatory explanatory variable, ABCG8-D19H significantly improved the model fit (p=0.0175) thereby suggesting again that ABCG8-D19H is the major causative variant in the region. Login to comment 101 ABCG8 p.Asp19His ABCG8 p.Arg50Cys To confirm this result, rs6756629 (ABCG5-R50C) and rs11887534 (ABCG8-D19H) were also genotyped in additional case-controls samples of German, Chilean, Danish, Indian and Chinese origin (Table 2). Login to comment 103 ABCG8 p.Asp19His ABCG8 p.Arg50Cys In each of the samples, ABCG8-D19H consistently showed an equal or higher allelic odds ratio as compared to ABCG5-R50C, thereby supporting the above conclusion. Login to comment 104 ABCG8 p.Asp19His ABCG8 p.Asp19His Moreover, logistic regression analysis containing ABCG8-D19H and other tagging SNPs (Supplementary Page 16 Table 1) were performed in the German and Chilean mapping panels also revealing no significant improvement over ABCG8-D19H alone (all p>0.1). Login to comment 113 ABCG8 p.Asp19His More specifically, we could show that the histidine-encoding allele of rs11887534 (ABCG8-D19H) is associated with an increased allelic cholesterol transport efficiency of the ABCG5/8 heterodimer, thereby contributing to biliary cholesterol hypersecretion and supersaturation and, most likely, to a predisposition to gallstone formation. Login to comment 114 ABCG8 p.Asp19His This increased biliary cholesterol transport may also contribute to the lower serum cholesterol and phytosterol levels observed previously in carriers of ABCG8-D19H (44,45). Login to comment 121 ABCG5 p.Arg50Cys ABCG8 p.Asp19His After extensive genotyping, only two candidate variants remained: ABCG5-R50C and ABCG8-D19H, both with similar allelic odds ratios (Supplementary Table 1, Table 2). Login to comment 122 ABCG8 p.Asp19His Since the two SNPs were in high LD, formal genetic differentiation of the two variants was achieved only in three out of the six investigated patient samples from Germany, Denmark, Chile, India and China implicating rs11887534 (ABCG8-D19H) as the likely risk factor. Login to comment 124 ABCG8 p.Asp19His In line with these statistical results, only ABCG8-D19H was found to yield a functional effect in in vitro co-expression experiments. Login to comment 126 ABCG8 p.Asp19His However, in a logistic regression analysis, none of the known common tagging SNPs improved the model fit significantly over rs11887534 (ABCG8-D19H) which implies that there is probably not much "missing heritability" left at this locus. Login to comment 128 ABCG8 p.Asp19His In this report, we genetically establish rs11887534 (ABCG8-D19H) through thorough transcript mapping, mutation detection and association analysis in ethnically different populations as the likely causative variant for gallstone susceptibility. Login to comment 129 ABCG8 p.Asp19His Further, we show that rs11887534 (ABCG8-D19H) increases the transport efficiency of cholesterol of the sterolin heterodimer, thereby drawing a link between the known basic pathomechanism of cholesterol gallstone formation to this specific genetic variant. Login to comment