A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity

We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5–5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed chi2 19.9, degree of freedom (df): 2, P < 5 × 10−5, adjusted for HOMA and BMI. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% ± 3.9) in comparison with the CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (β 0.23 ± 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity. We explored the role of the adiponutrin (PNPLA3) nonsynonymous-rs738409 single nucleotide polymorphism (SNP) in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and whether this SNP contributes to the severity of histological disease. Two hundred sixty-six individuals were evaluated in a case-control association study, which included 172 patients with features of NAFLD and 94 control subjects. The rs738409 G allele was significantly associated with NAFLD (P < 0.001; OR 2.8 95%, CI 1.5–5.2), independent of age, sex, body mass index (BMI), and Homeostasis Model Assessment (HOMA) index. When we tested the hypothesis of a relation between the SNP and the histological spectrum of NAFLD, a significant association was observed chi2 19.9, degree of freedom (df): 2, P < 5 × 10−5, adjusted for HOMA and BMI. The degree of liver steatosis, as evaluated by liver biopsy, was significantly associated with the rs738409 G allele. Patients with CC genotype showed a lower steatosis score (14.9% ± 3.9) in comparison with the CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation attributed to rs738409 genotypes was 5.3% (β 0.23 ± 0.07; P < 0.002). Our data suggest that the rs738409 G allele is associated not only with fat accumulation in the liver but also with liver injury, possibly triggered by lipotoxicity. Nonalcoholic fatty liver disease (NAFLD) is an emerging epidemic disease with increasing prevalence worldwide. NAFLD not only affects the adult population (an estimated 20–40% of adults in Western countries have excess fat accumulation in the liver) (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton J.D. Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2905) Google Scholar), but also is the most common cause of pediatric liver disease (2Barshop N.J. Sirlin C.B. Schwimmer J.B. Lavine J.E. Review article: epidemiology, pathogenesis and potential treatments of paediatric non-alcoholic fatty liver disease.Aliment. Pharmacol. Ther. 2008; 28: 13-24Crossref PubMed Scopus (114) Google Scholar). The pathogenesis of NAFLD is multifactorial; as a complex disease, the disorder develops from the interplay between genetic susceptibility and the environment. NAFLD refers to a wide spectrum of liver diseases, ranging from fatty liver alone to nonalcoholic steatohepatitis (NASH) with evidence of liver cell injury, a mixed inflammatory lobular infiltrate, and variable fibrosis (3Neuschwander-Tetri B.A. Caldwell S.H. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference.Hepatology. 2003; 37: 1202-1219Crossref PubMed Scopus (1780) Google Scholar). Patients with simple steatosis usually have a benign prognosis for liver disease. In contrast, up to 20% of patients with NASH can progress to cirrhosis (4Sanyal A.J. AGA technical review on nonalcoholic fatty liver disease.Gastroenterology. 2002; 123: 1705-1725Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar). While it is well known that environmental risk factors influence the development and progression of NAFLD (5Suzuki A. Lindor K. St S.J. Lymp J. Mendes F. Muto A. Okada T. Angulo P. Effect of changes on body weight and lifestyle in nonalcoholic fatty liver disease.J. Hepatol. 2005; 43: 1060-1066Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 6Harrison S.A. Day C.P. Benefits of lifestyle modification in NAFLD.Gut. 2007; 56: 1760-1769Crossref PubMed Scopus (188) Google Scholar), the contribution of the individual genetic variation to disease predisposition is still uncertain, despite the fact that several genes in isolated studies have been suggested as potential candidates either for NAFLD susceptibility or disease progression (7Dong H. Wang J. Li C. Hirose A. Nozaki Y. Takahashi M. Ono M. Akisawa N. Iwasaki S. Saibara T. et al.The phosphatidylethanolamine N-methyltransferase gene V175M single nucleotide polymorphism confers the susceptibility to NASH in Japanese population.J. Hepatol. 2007; 46: 915-920Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 8Miele L. Beale G. Patman G. Nobili V. Leathart J. Grieco A. Abate M. Friedman S.L. Narla G. Bugianesi E. et al.The Kruppel-like factor 6 genotype is associated with fibrosis in nonalcoholic fatty liver disease.Gastroenterology. 2008; 135: 282-291Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Namikawa C. Shu-Ping Z. Vyselaar J.R. Nozaki Y. Nemoto Y. Ono M. Akisawa N. Saibara T. Hiroi M. Enzan H. et al.Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis.J. Hepatol. 2004; 40: 781-786Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 10Sookoian S. Castano G. Gemma C. Gianotti T.F. Pirola C.J. Common genetic variations in CLOCK transcription factor are associated with nonalcoholic fatty liver disease.World J. Gastroenterol. 2007; 13: 4242-4248Crossref PubMed Scopus (94) Google Scholar, 11Sookoian S. Castano G. Gianotti T.F. Gemma C. Pirola C.J. Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease.J. Nutr. Biochem. 2008; (Epub ahead of print. October 14, 2008.)PubMed Google Scholar, 12Sookoian S. Castano G. Gianotti T.F. Gemma C. Rosselli M.S. Pirola C.J. Genetic variants in STAT3 are associated with nonalcoholic fatty liver disease.Cytokine. 2008; 44: 201-206Crossref PubMed Scopus (49) Google Scholar, 13Tokushige K. Takakura M. Tsuchiya-Matsushita N. Taniai M. Hashimoto E. Shiratori K. Influence of TNF gene polymorphisms in Japanese patients with NASH and simple steatosis.J. Hepatol. 2007; 46: 1104-1110Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 14Wilfred de Alwis N.M. Day C.P. Genes and nonalcoholic fatty liver disease.Curr. Diab. Rep. 2008; 8: 156-163Crossref PubMed Scopus (59) Google Scholar). Advances in genome analysis (including the development of comprehensive sets of informative genetic markers, improved physical mapping methods, and improvements in high throughput genotyping technology) have strongly contributed to the understanding of the pathogenesis of complex diseases. In fact, genome-wide association studies (GWAS) using a dense map of single nucleotide polymorphisms (SNP) enable scientists to detect common genetic variants that influence susceptibility to complex diseases, illuminating both disease mechanisms and the translation of this knowledge for diagnosis, prognosis, and therapy. A remarkable example of the impact of the use of GWAS in the understanding of the genetic architecture of human diseases is given by the recent three type 2 diabetes GWAS as meta-analyzed by Zeggini et al. (15Zeggini E. Scott L.J. Saxena R. Voight B.F. Marchini J.L. Hu T. de Bakker P.I. Abecasis G.R. Almgren P. Andersen G. et al.Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes.Nat. Genet. 2008; 40: 638-645Crossref PubMed Scopus (1515) Google Scholar). Although factors promoting NAFLD include obesity and type 2 diabetes, and NAFLD is now considered the hepatic manifestation of the metabolic syndrome (16Marchesini G. Brizi M. Bianchi G. Tomassetti S. Bugianesi E. Lenzi M. McCullough A.J. Natale S. Forlani G. Melchionda N. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.Diabetes. 2001; 50: 1844-1850Crossref PubMed Scopus (1962) Google Scholar), there have been no studies using this approach until recently when researchers of the Dallas Heart Study performed a GWAS for liver fat content in 2,111 individuals from different ancestry groups (17Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2168) Google Scholar). Interestingly, this study examined 9,222 nonsynonymous variants and showed strong evidence of association of NAFLD, evaluated by proton magnetic resonance spectroscopy, with the rs738409 G allele of the patatin-like phospholipase domain containing 3 gene (PNPLA3), also known as adiponutrin (ADPN gene) (17Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2168) Google Scholar). In addition, a significant association was also observed between the G allele and the elevation of alanine aminotransferase (ALT) levels, at least in the Hispanic population. Another population-based GWAS of plasma liver-enzyme levels in the Caucasian population also reported that the PNPLA3 locus is strongly associated with ALT levels (18Yuan X. Waterworth D. Perry J.R. Lim N. Song K. Chambers J.C. Zhang W. Vollenweider P. Stirnadel H. Johnson T. et al.Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.Am. J. Hum. Genet. 2008; 83: 520-528Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar), supporting an inflammatory effect of this gene on the liver. While the functional impact of the reported SNP is still unknown, data from these two studies are very consistent and point out the role of the PNPLA3 rs738409 G/C, also known as Met148Ile, in the susceptibility to NAFLD. None of the above-mentioned studies examined the relation between PNPLA3 SNP and NAFLD severity. The main reason seems to be the population-based nature of both studies; none of them included patients with NAFLD diagnosed by liver biopsy. We performed a hospital-based adult case-control association study to replicate for the first time the association between the rs738409 and NAFLD susceptibility and to further evaluate the association between the SNP and the histological disease severity. We performed a cross-sectional study on NAFLD in a county hospital of the city of Buenos Aires. The study involved a total of 266 unrelated individuals (80 males and 186 females), of which 172 patients had features of NAFLD and 94 were control subjects. The screening criteria was liver ultrasonographic (US) examination indicative of fatty infiltration (19Mendler M.H. Bouillet P. Le Sidaner A. Lavoine E. Labrousse F. Sautereau D. Pillegand B. Dual-energy CT in the diagnosis and quantification of fatty liver: limited clinical value in comparison to ultrasound scan and single-energy CT, with special reference to iron overload.J. Hepatol. 1998; 28: 785-794Abstract Full Text PDF PubMed Scopus (131) Google Scholar), which was carried out by the same operator and performed in all the participants. Secondary causes of steatosis, including alcohol abuse (≥30 g/d alcohol for men and ≥20 g for women), total parenteral nutrition, hepatitis B and hepatitis C virus infection, and the use of drugs known to precipitate steatosis were excluded. In addition, patients with any of the following diseases were excluded from participation in this study: autoimmune liver disease, metabolic liver disease, Wilson's disease, and α-1-antitrypsin deficiency. For the purpose of exploring the hypothesis of a relation between the rs738409 and NAFLD, we included in the analysis all the NAFLD patients (n and NAFLD was considered as a For the purpose of the hypothesis of a relation between the rs738409 and the histological severity of NAFLD, we included in the analysis only patients that had evidence of fatty liver disease, either simple steatosis or on liver performed this study (n in the liver were from patients hospital for the NAFLD patients and In to the all the control individuals were to liver were included in the study not have evidence of fatty or control were not to have any of the features of the metabolic syndrome as by the of The of The on of In 2001; PubMed Scopus Google and not abuse The and the were the same study from the same population of patients and all of them the same of and examination included a on and mass index was as and as the index for and were also was from had been for at least total and plasma and liver were by clinical Homeostasis Model Assessment (HOMA) was to evaluate an index and was as × plasma was evaluated only in NAFLD patients using the performed with g of to was as or of body fat content was performed using a at and The body fat content was by a that age, and fat was as body fat weight × as by the Patients were to have liver in the of at least ALT aminotransferase as and the performed in this study were in with the of the of from individuals was in with the by the of of liver was on of liver for be the and of the A. J. on the use of liver in clinical of PubMed Scopus Google Scholar). NAFLD patients were a liver showed either liver or or value (4Sanyal A.J. AGA technical review on nonalcoholic fatty liver disease.Gastroenterology. 2002; 123: 1705-1725Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar, L. Bugianesi E. M. A. E. C. E. D. G. et of liver disease in nonalcoholic fatty liver disease with aminotransferase a role for and 2008; PubMed Scopus Google Scholar). patients that showed features of liver steatosis as well as and of were not included in the histological was performed with and on an were in in and with and and for The same liver was to all the the were at least 2 in and a of The degree of steatosis was to the by et al. B.A. Nonalcoholic steatohepatitis: a for and the histological J. Gastroenterol. PubMed Scopus Google recently by et al. N.M. C. M.S. A. et and of a histological for nonalcoholic fatty liver 2005; PubMed Scopus Google on the of containing fat as no of containing fat 2, of containing fat and of containing fat NASH was as steatosis mixed inflammatory cell and and any of including fibrosis (3Neuschwander-Tetri B.A. Caldwell S.H. Nonalcoholic steatohepatitis: summary of an AASLD Single Topic Conference.Hepatology. 2003; 37: 1202-1219Crossref PubMed Scopus (1780) Google Scholar). The severity of was on a as 2 and 3 as by et al. B.A. Nonalcoholic steatohepatitis: a for and the histological J. Gastroenterol. PubMed Scopus Google Scholar). The severity of fibrosis was on a as fibrosis in 2 3 and The genetic were on from by a as from and A to and Google Scholar). of the PNPLA3 rs738409 was performed by a genotyping of with that for as Y. Hu S. SNP genotyping by with 2001; PubMed Scopus Google Scholar). genotyping we included as of known and with a a control were not The analysis was estimated by a to the same the of the the genotypes for the we had only genotype the observed is estimated to be genotype was in the we a of and at different loci in and and the data with the 2 M. P. of population using genotype PubMed Scopus Google Scholar). We no evidence of in the and the showed and were with a to by the with no further in the by up to of was for data were as ± For and to any variable and between groups were by the or for two or the association between genotypes and disease we and a analysis for an as the with disease severity as the variable simple steatosis, and NASH as and 2, HOMA and as and genotypes as analysis was included for the of the association between genotypes and histological disease severity. the association between genotypes with NAFLD or as ALT and we and or for as age, We the for the and at diagnosis in NAFLD patients and are in NAFLD patients were and showed most of the risk factors of the metabolic and and HOMA individuals had type 2 diabetes, of them The of the patients were to the of type 2 diabetes with a of weight and physical and of the population to disease and histological features of patients with of ± ± × ± ± × ± ± × ± ± ± ± fat content ± ± × plasma ± ± × plasma ± ± × ± ± × ± ± ± ± ± ± ± ± ± ± × ± ± × ± ± × ± ± × ± ± × features of NAFLD steatosis of of are as ± for using are in not of of containing fat 2, of containing fat and of containing fat 2 and 3 fibrosis in 2 3 and in a are as ± for using are in not of of containing fat 2, of containing fat and of containing fat 2 and 3 fibrosis in 2 3 and on histological patients were to the simple steatosis and were included in the NASH The histological features of patients liver are in In the the of the G allele and the C allele were and and the of the genotypes was in not analysis of NAFLD in the population showed that the rs738409 G allele was associated with a in the risk for NAFLD P < The association for in the the rs738409 was strongly associated with NAFLD (P < 0.001; OR G CI independent of age, sex, and HOMA index. The association was still significant the type 2 patients (P < OR independent of age, sex, and HOMA index. In the analysis of the association of rs738409 and NAFLD progression from simple steatosis to we observed a significant association between the rs738409 genotypes and histological disease severity In fact, we observed significantly of disease severity in individuals with the GG genotype ± in comparison with with the genotype ± and the CC genotype ± P < genotypes to histological features of NAFLD patients a liver OR with simple steatosis (n with NASH (n for using of freedom in a for using of freedom for potential we a analysis for an with by the histological that from to NASH control simple steatosis patients by liver and NASH patients by liver as and 2, this the association 19.9, degree of freedom P < 5 for HOMA and as independent this the association between NAFLD and the we performed a analysis the NASH the simple steatosis (including only the patients that had liver We still observed a significant association between the rs738409 genotypes and the histological disease severity G CI P < both the groups included type 2 for type 2 diabetes, we observed that the association still CI P < from the analysis the type 2 the association was significant and for HOMA index not The degree of liver steatosis evaluated by liver was significantly associated with the rs738409 G as with CC genotype showed lower steatosis (14.9% ± 3.9) in comparison with CG genotype (26.3% ± 3.5) and GG genotype (33.3% ± 4.0) (P < 0.005). The proportion of the total variation for the rs738409 for age, sex, and was 5.3% (β ± 0.23 ± 0.07; P < 0.002). the rs738409 G allele at PNPLA3 was significantly associated with ALT and for age, and plasma analysis of liver and as variable and PNPLA3 rs738409 age, and plasma as independent ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a A association study from the population-based Dallas Heart Study an association between a nonsynonymous in PNPLA3 gene and liver fat as and by proton magnetic resonance (17Romeo S. Kozlitina J. Xing C. Pertsemlidis A. Cox D. Pennacchio L.A. Boerwinkle E. Cohen J.C. Hobbs H.H. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.Nat. Genet. 2008; 40: 1461-1465Crossref PubMed Scopus (2168) Google Scholar). we have performed for the first time a replication study in a hospital-based population to evaluate not only the between the SNP and the of fatty liver but also a association with NAFLD as by liver biopsy. In the we observed that the rs738409 was significantly associated with fatty that of the G allele at G allele CI to have NAFLD in comparison with independent of age, sex, and HOMA index. association was also observed type 2 patients from the In addition, we observed a significant association between the histological spectrum of NAFLD steatosis and and the rs738409 G independent of the effect of the or of G allele showed of histological severity and liver steatosis the of G allele the degree of liver steatosis by association with histological disease severity for type 2 The of association between the and type 2 diabetes observed in study that the mechanisms by which the rs738409 are independent of type 2 the rs738409 G allele was significantly and associated with levels of both ALT and a that also the of a recently GWAS on several loci influencing plasma levels of liver (18Yuan X. Waterworth D. Perry J.R. Lim N. Song K. Chambers J.C. Zhang W. Vollenweider P. Stirnadel H. Johnson T. et al.Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.Am. J. Hum. Genet. 2008; 83: 520-528Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Our on patients the G allele of liver steatosis on liver be by a relation between a of the PNPLA3 and an in hepatic triglyceride data that the the liver fat the liver is by K. The pathogenesis of nonalcoholic steatohepatitis and fatty liver a including the role of and hepatic in the progression to 2004; PubMed Scopus (162) Google Scholar). the significant association we have observed between the SNP and the histological disease severity and liver the in in genetic association the of the rs738409 in not only hepatic triglyceride population in disease but also in inflammatory to liver lipotoxicity. While no functional studies the role of the have been the fact that rs738409 is a nonsynonymous SNP a functional impact for to a of the as the is in the and domain of the Although rs738409 SNP the and for Single Polymorphisms Wang H.H. A. an and for SNP analysis and PubMed Scopus Google showed that it a high score risk for with protein the protein were observed using of the impact of In on the knowledge human genetic variation by the GWAS are on the of the common diseases. In of the of these studies is the of of disease pathogenesis the of genes or unrelated to a disease. is the of the PNPLA3 associated with the risk of NAFLD. GWAS are are still and and that include genotyping of of in of subjects. genes influencing fatty liver disease predisposition and progression to be the replication of the data by in but be In this studies of as disease severity are adiponutrin alanine aminotransferase aminotransferase body mass index degree of freedom genome-wide association studies Homeostasis Model Assessment nonalcoholic fatty liver disease nonalcoholic steatohepatitis patatin-like phospholipase domain containing 3 gene single nucleotide polymorphism ultrasonographic