A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis

Abstract

Alcohol misuse is the leading cause of cirrhosis and the second most common indication for liver transplantation in the Western world1,2,3. We performed a genome-wide association study for alcohol-related cirrhosis in individuals of European descent (712 cases and 1,426 controls) with subsequent validation in two independent European cohorts (1,148 cases and 922 controls). We identified variants in the MBOAT7 (P = 1.03 × 10−9) and TM6SF2 (P = 7.89 × 10−10) genes as new risk loci and confirmed rs738409 in PNPLA3 as an important risk locus for alcohol-related cirrhosis (P = 1.54 × 10−48) at a genome-wide level of significance. These three loci have a role in lipid processing, suggesting that lipid turnover is important in the pathogenesis of alcohol-related cirrhosis.

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Figure 1: Genome-wide association meta-analysis of 712 cases with alcohol-related cirrhosis and 1,466 controls.
Figure 2: Forest plots of odds ratios and 95% confidence intervals for the susceptibility loci for alcohol-related cirrhosis in comparison to alcohol misusers and population controls.
Figure 3: Fine-mapping analysis of the MBOAT7 association signals.

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Acknowledgements

This study was supported by the German Ministry of Education and Research through the Virtual Liver Network (to J.H.), the PopGen 2.0 network biobank (grant 01EY1103) and institutional funds from the medical faculties of TU Dresden and Christian Albrechts University Kiel and by Swiss National Funds (grant 310030_138747 to F.S.). The Community Medicine Research network of the University of Greifswald, Germany, is funded by the Federal Ministry of Education and Research, the Ministry of Cultural Affairs and the Social Ministry of the Federal State of Mecklenburg–West Pomerania. M.M.L. and J.M. were supported by the Federal Ministry of Education and Research (BMBF GANI-MED 03152061A and BMBF 0314107), the European Union (EU-FP-7: EPC-TM and EU-FP-7-REGPOT-2010-1) and the EFRE–State Ministry of Economics (V-630-S-150-2012/132/133). S.C., M.M.N. and M. Rietschel were supported by the German Federal Ministry of Education and Research (BMBF) through the Integrated Networks IntegraMent and Sysmed Alcohol under the auspices of the e:Med Programme (grant 01ZX1314A to M.M.N. and S.C. and grant 01ZX1311A to M.M.N. and M. Rietschel). M.M.N. is a member of the DFG (Deutsche Forschungsgemeinschaft)-funded Excellence Cluster ImmunoSensation. Research by H.D.N. related to this project was funded by the Deutsche Krebshilfe (107865). The work of A.F. and D.E. was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept (SysInflame grant 01ZX1306A). This project received infrastructure support from DFG Excellence Cluster 306, 'Inflammation at Interfaces'. A.F. receives an endowment professorship from the Foundation for Experimental Medicine (Zurich, Switzerland). The UK research effort was funded by a PhD studentship award jointly funded by University College London and an anonymous donor. We thank colleagues from the following centers for obtaining samples from alcohol-dependent cases for genotyping: the Bexley Substance Misuse Service, South London and Maudsley National Health Service (NHS) Trust; the East Hertfordshire Community Drug Action Team; the Max Glatt Unit, Southall; Renfrew and Inverclyde Alcohol Services, Strathclyde; the Newcastle North Tyneside Drug and Alcohol Service, Tyne and Wear; and the Acute Admissions Unit and the Centre for Hepatology at the Royal Free Hospital, London. We also thank colleagues associated with the National Institute for Health Research (NIHR) Mental Health Research Network for their assistance in identifying cases, obtaining consent and collecting samples at the following NHS trusts: Sandwell Mental Health and Social Care; Northamptonshire Healthcare; Avon and Wiltshire Mental Health Partnership, Sheffield Health and Social Care; Tees Esk and Wear Valleys; Lincolnshire Partnership; Nottinghamshire Healthcare; Central and North West London; South Staffordshire and Shropshire Healthcare; Coventry and Warwickshire; and Dudley and Walsall Mental Health Partnership. We are grateful to J. Saini, K. Ruparelia, S. Montagnese, R. Kandaswamy, A. Jarram, G. Quadri and N. O'Brien for assisting with the collection and processing of samples and DNA extraction.

The Belgian research effort was supported by the Belgian Medical Genomics Initiative (BeMGI) funded by the phase VII Interuniversity Attraction Poles (IAP) program of the Belgian Federal Science Policy Office (BELSPO) and the Fund for Scientific Research–FNRS (F.R.S.-FNRS). E.T. is a Postdoctoral Researcher of the F.R.S.-FNRS, and D.F. is a Research Director of the F.R.S.-FNRS. We are grateful to O. Lemoine, D. Degré, A. Lemmers and M. Amrani for identifying cases and controls, obtaining consent and collecting samples at CUB Hôpital Erasme, Université Libre de Bruxelles. We also thank E. Quertinmont at the Laboratory of Experimental Gastroenterology, Université Libre de Bruxelles, for his help with the collection and processing of samples and DNA extraction.

We are also grateful to the Center for Information Services and High-Performance Computing (ZIH) at TU Dresden where the computations were performed on a PC cluster.

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Contributions

S. Buch performed genotyping, meta-analysis and in silico analysis, and drafted and revised the manuscript. F.S. conceptualized the study, recruited subjects, and wrote and revised the manuscript. E.T. recruited subjects, validated the study, provided replication data, and wrote and revised the manuscript. M.W. recruited subjects, performed genotyping for the validation study and revised the manuscript. A.H. performed bioinformatics work. H.D.N. recruited and phenotyped subjects. M.B. performed expression analysis. J.R. and T.B. recruited subjects. M. Ridinger, M. Rietschel, A.M., J.F. and F.K. recruited subjects and performed phenotyping and recruitment of alcoholic controls. S.S. provided technical support and critically revised the manuscript. W.L. helped with population genetic statistics. M.S. recruited subjects and phenotyped alcoholic controls. N.S., E.A., C.D., R.S., S. Brückner, S. Zeissig and A.-M.S. recruited subjects. N.W. recruited subjects and performed phenotyping of alcoholic controls. J.D., N.C., C. Sarrazin, F.L., T.G. and P.D. recruited and phenotyped subjects. H.V. recruited the population cohort. M.M.L., J.M., F.E. and C. Schafmayer recruited and phenotyped subjects. S.C. and M.M.N. performed phenotyping and recruitment of alcoholic controls. M.N. supervised and reviewed statistical analysis. D.E. assisted with bioinformatics analysis. K.H. performed expression analysis. A.F. gave conceptual advice and bioinformatics support. S. Zopf, C.H. and C.M. recruited subjects. D.F. and M.Y.M. recruited subjects and drafted and critically revised the manuscript. J.H. conceptualized the study and analytical design, and drafted and revised the manuscript. All authors critically revised and contributed to the final manuscript.

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Correspondence to Felix Stickel.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantile-quantile plot of the primary meta-analysis of the German and UK data sets.

Samples are as outlined in Table 1. After GC adjustment, a λ value of 1.005 was obtained for the combined analysis.

Supplementary Figure 2 Manhattan plot of the adjusted meta-analysis of the German and UK data sets.

Samples are as outlined in Supplementary Table 2.

Supplementary Figure 3 Quantile-quantile plot of the adjusted meta-analysis of the German and UK data sets.

After GC adjustment, a λ value of 0.985 was obtained for the combined analysis.

Supplementary Figure 4 Fine-mapping analysis of the TM6SF2 association signals.

The –log10 (P values) are plotted against SNP genomic position based on NCBI Build 37. The known coding variant of likely functional significance, rs58542926 is highlighted in purple. The squares denote genotyped SNPs; the circles denote imputed SNPs (using 1000 Genomes Project–based imputation). SNPs are colored to reflect correlation with the most significant SNP, with red denoting the highest LD (r2 >0.8) to the lead SNP. Estimated recombination rates from 1000 Genomes Project (hg19/genomes March 2012 EUR) are plotted in blue to reflect the local LD structure. Gene annotations were obtained from the UCSC Genome Browser. The plot was generated using LocusZoom.

Supplementary Figure 5 Tissue expression of MBOAT7 and TMC4 mRNA.

Expression was tested by PCR in the human cDNA tissue panel from Takara Clontech (636742 and 636743) using transcript-specific primer pairs (MBOAT7: 5′-TCCTTGTGTCTTTCGCTCC-3′ and 5′-TACACACGGTGACCTGTCA-3′; TMC4: 5′-TGAGACCACCCAGAATTTCC-3′ and 5′-CTAGGCTTACAATGGGCCTG-3′). MBOAT7 shows a ubiquitous expression pattern in the tissues tested, albeit with lower expression in skeletal muscle. Expression of TMC4 is more selective, with no expression detected in brain or skeletal muscle.

Supplementary Figure 6 Genotype-specific relative transcript abundance in liver tissue from patients with alcohol-related cirrhosis.

No significant change in transcript abundance was seen for TMC4 across genotypes, whereas a significant increase in transcript abundance was detected for MBOAT7 in the homozygous mutant genotype (Mann-Whitney U test P = 0.0087).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 2, 3 and 5–7. (PDF 887 kb)

Supplementary Table 1: Results of the primary GWAS meta-analysis (Germany/UK).

Results of the GWAS meta-analysis of 712 cases with alcohol-related cirrhosis and 1,466 controls. Variants that entered replication genotyping are marked "SNP1" to "SNP10". SNPs are ranked by combined P value, and data are provided for the top variants through SNP10. (XLSX 534 kb)

Supplementary Table 4: Results of the secondary GWAS meta-analysis (Germany/UK) adjusted for sex, age, BMI and type 2 diabetes status.

Results of the GWAS meta-analysis adjusted for age, sex, BMI and type 2 diabetes status. Variants that entered replication genotyping are marked "SNP1" to "SNP10". SNPs are ranked by combined P value, and data are provided for the top variants through SNP10. Adjusted meta-analysis results for the replicating variants of the primary analysis are as follows: TM6SF2 (rs10401969), Pmeta = 0.000667, ORmeta = 1.87 (1.30–2.69); MBOAT7 (rs626283), Pmeta = 0.0173, ORmeta = 1.28 (1.04–1.56). These rank below SNP10 in this analysis. (XLSX 153 kb)

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Buch, S., Stickel, F., Trépo, E. et al. A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis. Nat Genet 47, 1443–1448 (2015). https://doi.org/10.1038/ng.3417

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