Research Article| Volume 63, ISSUE 2, P494-502, August 2015

Targeted-bisulfite sequence analysis of the methylation of CpG islands in genes encoding PNPLA3, SAMM50, and PARVB of patients with non-alcoholic fatty liver disease

Published:March 14, 2015DOI:

      Background & Aims

      The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetic factors as well as by genetic variation.


      We performed targeted-bisulfite sequencing to determine the levels of DNA methylation of 4 CpG islands (CpG99, CpG71, CpG26, and CpG101) in the regulatory regions of PNPLA3, SAMM50, PARVB variant 1, and PARVB variant 2, respectively. We compared the levels of methylation of DNA in the livers of the first and second sets of patients with mild (fibrosis stages 0 and 1) or advanced (fibrosis stages 2 to 4) NAFLD and in those of patients with mild (F0 to F2) or advanced (F3 and F4) chronic hepatitis C infection. The hepatic mRNA levels of PNPLA3, SAMM50, and PARVB were measured using qPCR.


      CpG26, which resides in the regulatory region of PARVB variant 1, was markedly hypomethylated in the livers of patients with advanced NAFLD. Conversely, CpG99 in the regulatory region of PNPLA3 was substantially hypermethylated in these patients. These differences in DNA methylation were replicated in a second set of patients with NAFLD or chronic hepatitis C. PNPLA3 mRNA levels in the liver of the same section of a biopsy specimen used for genomic DNA preparation were lower in patients with advanced NAFLD compared with those with mild NAFLD and correlated inversely with CpG99 methylation in liver DNA. Moreover, the levels of CpG99 methylation and PNPLA3 mRNA were affected by the rs738409 genotype.


      Hypomethylation of CpG26 and hypermethylation of CpG99 may contribute to the severity of fibrosis in patients with NAFLD or chronic hepatitis C infection.

      Graphical abstract


      NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), GWAS (genome-wide association study), SNP (single-nucleotide polymorphism), PNPLA3 (patatin-like phospholipase domain containing 3), LD (linkage disequilibrium), SAMM50 (SAMM50 sorting and assembly machinery component), PARVB (parvin β), PCR (polymerase chain reaction), NAS (NAFLD activity score), HDL (high-density lipoprotein), SBP (systolic blood pressure), DBP (diastolic blood pressure), BMI (body mass index), qPCR (quantitative PCR), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), bp (base position), HbA1c (hemoglobin A1c), AST (aspartate aminotransferase), ALT (alanine aminotransferase)


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        • Angulo P.
        Nonalcoholic fatty liver disease.
        N Engl J Med. 2002; 346: 1221-1231
        • Farrell G.C.
        Non-alcoholic steatohepatitis: what is it, and why is it important in the Asia-Pacific region?.
        J Gastroenterol Hepatol. 2003; 18: 124-138
        • Okanoue T.
        • Umemura A.
        • Yasui K.
        • Itoh Y.
        Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in Japan.
        J Gastroenterol Hepatol. 2011; 1: 153-162
        • Ludwig J.
        • Viggiano T.R.
        • McGill D.B.
        • Oh B.J.
        Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.
        Mayo Clin Proc. 1980; 55: 434-438
        • Wilfred de Alwis N.M.
        • Day C.P.
        Genes and nonalcoholic fatty liver disease.
        Curr Diab Rep. 2008; 8: 156-163
        • Romeo S.
        • Kozlitina J.
        • Xing C.
        • Pertsemlidis A.
        • Cox D.
        • Pennacchio L.A.
        • et al.
        Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
        Nat Genet. 2008; 40: 1461-1465
        • Chalasani N.
        • Guo X.
        • Loomba R.
        • Goodarzi M.O.
        • Haritunians T.
        • Kwon S.
        • et al.
        Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
        Gastroenterology. 2010; 139: 1567-1576
        • Speliotes E.K.
        • Yerges-Armstrong L.M.
        • Wu J.
        • Hernaez R.
        • Kim L.J.
        • Palmer C.D.
        • et al.
        Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
        PLoS Genet. 2011; 7: e1001324
        • Kawaguchi T.
        • Sumida Y.
        • Umemura A.
        • Matsuo K.
        • Takahashi M.
        • Takamura T.
        • et al.
        Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese.
        PLoS One. 2012; 7: e38322
        • Kitamoto T.
        • Kitamoto A.
        • Yoneda M.
        • Hyogo H.
        • Ochi H.
        • Nakamura T.
        • et al.
        Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan.
        Hum Genet. 2013; 132: 783-792
        • Hotta K.
        • Yoneda M.
        • Hyogo H.
        • Ochi H.
        • Mizusawa S.
        • Ueno T.
        • et al.
        Association of the rs738409 polymorphism in PNPLA3 with liver damage and the development of nonalcoholic fatty liver disease.
        BMC Med Genet. 2010; 11: 172
        • Sookoian S.
        • Pirola C.J.
        Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
        Hepatology. 2011; 53: 1883-1894
        • Zain S.M.
        • Mohamed R.
        • Mahadeva S.
        • Cheah P.L.
        • Rampal S.
        • Basu R.C.
        • et al.
        A multi-ethnic study of a PNPLA3 gene variant and its association with disease severity in non-alcoholic fatty liver disease.
        Hum Genet. 2012; 131: 1145-1152
        • Kitamoto T.
        • Kitamoto A.
        • Yoneda M.
        • Hyogo H.
        • Ochi H.
        • Mizusawa S.
        • et al.
        Targeted next-generation sequencing and fine linkage disequilibrium mapping reveals association of PNPLA3 and PARVB with the severity of nonalcoholic fatty liver disease.
        J Hum Genet. 2014; 59: 241-246
        • Ray K.
        NAFLD: Profiling NAFLD-liver gene expression and DNA methylation patterns to characterize disease severity.
        Nat Rev Gastroenterol Hepatol. 2013; 10: 565
        • Sookoian S.
        • Rosselli M.S.
        • Gemma C.
        • Burgueño A.L.
        • Fernández Gianotti T.
        • Castaño G.O.
        • et al.
        Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor coactivator 1 promoter.
        Hepatology. 2010; 52: 1992-2000
        • Ahrens M.
        • Ammerpohl O.
        • von Schönfels W.
        • Kolarova J.
        • Bens S.
        • Itzel T.
        • et al.
        DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
        Cell Metab. 2013; 18: 296-302
        • Murphy S.K.
        • Yang H.
        • Moylan C.A.
        • Pang H.
        • Dellinger A.
        • Abdelmalek M.F.
        • et al.
        Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
        Gastroenterology. 2013; 145: 1076-1087
        • Paul D.S.
        • Guilhamon P.
        • Karpathakis A.
        • Butcher L.M.
        • Thirlwell C.
        • Feber A.
        • et al.
        Assessment of RainDrop BS-seq as a method for large-scale, targeted bisulfite sequencing.
        Epigenetics. 2014; 9: 678-684
        • Sanyal A.J.
        American Gastroenterological Association: AGA technical review on nonalcoholic fatty liver disease.
        Gastroenterology. 2002; 123: 1705-1725
        • Matteoni C.A.
        • Younossi Z.M.
        • Gramlich T.
        • Boparai N.
        • Liu Y.C.
        • McCullough A.J.
        Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.
        Gastroenterology. 1999; 116: 1413-1419
        • Teli M.R.
        • James O.F.
        • Burt A.D.
        • Bennett M.K.
        • Day C.P.
        The natural history of nonalcoholic fatty liver: a follow-up study.
        Hepatology. 1995; 22: 1714-1719
        • Brunt E.M.
        Nonalcoholic steatohepatitis: definition and pathology.
        Semin Liver Dis. 2001; 21: 3-16
        • Kleiner D.E.
        • Brunt E.M.
        • Van Natta M.
        • Behling C.
        • Contos M.J.
        • Cummings O.W.
        • et al.
        Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
        Hepatology. 2005; 41: 1313-1321
        • Bedossa P.
        • Poynard T.
        An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.
        Hepatology. 1996; 24: 289-293
        • Arai H.
        • Yamamoto A.
        • Matsuzawa Y.
        • Saito Y.
        • Yamada N.
        • Oikawa S.
        • et al.
        Prevalence of metabolic syndrome in the general Japanese population in 2000.
        J Atheroscler Thromb. 2006; 13: 202-208
      1. Examination Committee of Criteria for ‘Obesity Disease’ in Japan; Japan Society for the Study of Obesity: New criteria for ‘obesity disease’ in Japan.
        Circ J. 2002; 66: 987-992
        • Krueger F.
        • Andrews S.R.
        Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.
        Bioinformatics. 2011; 27: 1571-1572
        • Langmead B.
        • Salzberg S.L.
        Fast gapped-read alignment with Bowtie 2.
        Nat Methods. 2012; 9: 357-359
        • Li H.
        • Handsaker B.
        • Wysoker A.
        • Fennell T.
        • Ruan J.
        • Homer N.
        • et al.
        The Sequence Alignment/Map format and SAMtools.
        Bioinformatics. 2009; 25: 2078-2079
        • Robinson J.T.
        • Thorvaldsdóttir H.
        • Winckler W.
        • Guttman M.
        • Lander E.S.
        • Getz G.
        • et al.
        Integrative genomics viewer.
        Nat Biotechnol. 2011; 29: 24-26
        • Schroeder A.
        • Mueller O.
        • Stocker S.
        • Salowsky R.
        • Leiber M.
        • Gassmann M.
        • et al.
        The RIN: an RNA integrity number for assigning integrity values to RNA measurements.
        BMC Mol Biol. 2006; 7: 3
        • Viechtbauer W.
        Conducting meta-analyses in R with the metafor package.
        J Stat Softw. 2010; 36: 1-48
        • Storey J.D.
        • Tibshirani R.
        Statistical significance for genomewide studies.
        Proc Natl Acad Sci U S A. 2003; 100: 9440-9445
        • Krueger F.
        • Kreck B.
        • Franke A.
        • Andrews S.R.
        DNA methylome analysis using short bisulfite sequencing data.
        Nat Methods. 2012; 9: 145-151
        • Seki Y.
        • Williams L.
        • Vuguin P.M.
        • Charron M.J.
        Minireview: Epigenetic programming of diabetes and obesity: animal models.
        Endocrinology. 2012; 153: 1031-1038
        • Fujii H.
        • Kawada N.
        Inflammation and fibrogenesis in steatohepatitis.
        J Gastroenterol. 2012; 47: 215-225
        • Wang J.
        • Zhuang J.
        • Iyer S.
        • Lin X.Y.
        • Greven M.C.
        • Kim B.H.
        • et al. a Wiki-based database for transcription factor-binding data generated by the ENCODE consortium.
        Nucleic Acids Res. 2013; 41: D171-D176
        • Sun L.
        • Mathews L.A.
        • Cabarcas S.M.
        • Zhang X.
        • Yang A.
        • Zhang Y.
        • et al.
        Epigenetic regulation of SOX9 by the NF-κB signaling pathway in pancreatic cancer stem cells.
        Stem Cells. 2013; 31: 1454-1466