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Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study1

Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD... patient-oriented and epidemiological research Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study ,† § †† §§ Marcin Krawczyk,* Monika Rau, Jörn M. Schattenberg,** Heike Bantel, Anita Pathil, ††† §§§ Münevver Demir,*** Johannes Kluwe, Tobias Boettler, Frank Lammert,* and §,2 Andreas Geier for the NAFLD Clinical Study Group Department of Medicine II,* Saarland University Medical Center, Homburg, Germany; Laboratory of  Metabolic Liver Diseases, Department of General, Transplant and Liver Surgery,  Medical University of  Warsaw, Warsaw, Poland; Division of Hepatology, Department of Medicine II,  University Hospital Wuerzburg,  Wuerzburg, Germany; I. Department of Medicine,** University Medical Center Mainz, Johannes Gutenberg  †† University, Mainz, Germany; Department of Gastroenterology, Hepatology and Endocrinology,  Hannover  Medical School, Hannover, Germany; Department of Internal Medicine IV, Gastroenterology and  §§ Hepatology,  University of Heidelberg, Heidelberg, Germany; Clinic for Gastroenterology and  ††† Hepatology,*** University Hospital of Cologne, Cologne, Germany; Department of Medicine I, Hamburg §§§ University Medical Center, Hamburg, Germany; and Department of Medicine II, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany Abstract The PNPLA3 p.I148M, TM6SF2 p.E167K, and rs58542926, PNPLA3 rs738409, and MBOAT7 rs641738 vari- MBOAT7 rs641738 variants represent genetic risk factors for ants on NAFLD severity: A multicenter biopsy-based study. nonalcoholic fatty liver disease (NAFLD). Here we investi- J. Lipid Res. 2017. 58: 247–255. gate if these polymorphisms modulate both steatosis and fi- brosis in patients with NAFLD. We recruited 515 patients Supplementary key words  adiponutrin • fatty liver • fibrosis  • steatosis with NAFLD (age 16–88 years, 280 female patients). Liver biopsies were performed in 320 patients. PCR-based assays were used to genotype the PNPLA3, TM6SF2, and MBOAT7 Nonalcoholic fatty liver disease (NAFLD) affects more  variants. Carriers of the PNPLA3 and TM6SF2 risk alleles than 30% of adults in developed countries. Given the in- showed increased serum aspartate aminotransferase and ala- nine transaminase activities (P < 0.05). The PNPLA3 geno- creasing prevalence of environmental risk factors for this  type was associated with steatosis grades S2–S3 (P < 0.001) condition (e.g., hypercaloric diets and sedentary lifestyles)  and fibrosis stages F2–F4 (P < 0.001). The TM6SF2 genotype (1), the frequency of fatty liver is predicted to further in- was associated with steatosis (P = 0.003) but not with fibrosis crease in the coming years. In addition to environmental  (P > 0.05). The MBOAT7 variant was solely associated with triggers, genetic predisposition is known to modulate the  increased fibrosis (P = 0.046). In the multivariate model, degree of steatosis and liver injury (2). Conceptually, the  variants PNPLA3 (P = 0.004) and TM6SF2 (P = 0.038) were term “hepatic steatosis” refers to traits that are governed by  associated with steatosis. Fibrosis stages were affected by the multiple variants with modest effects. The major part of the  PNPLA3 (P = 0.042) and MBOAT7 (P = 0.021) but not by the genetic predisposition is, according to current knowledge,  TM6SF2 polymorphism (P > 0.05). The PNPLA3, TM6SF2, and MBOAT7 variants are associated with increased liver in- related to two common missense SNPs:  PNPLA3 p.I148M jury. The TM6SF2 variant seems to modulate predominantly and TM6SF2 p.E167K. These two polymorphisms, detected  hepatic fat accumulation, whereas the MBOAT7 polymor- in genome-wide (3) and exome-wide (4) association stud- phism is linked to fibrosis. The PNPLA3 polymorphism con- ies in patients with fatty livers, seem to impose different  fers risk of both increased steatosis and fibrosis.—Krawczyk,   M., M. Rau, J. M. Schattenberg, H. Bantel, A. Pathil, M.  Demir, J. Kluwe, T. Boettler, F. Lammert, A. Geier, NAFLD  Clinical  Study  Group.  Combined effects of the TM6SF2 Abbreviations:  ALT, alanine transaminase; AST, aspartate amino- transferase; GGT, gamma glutamyl transferase; HCV, hepatitis C virus;  NAFLD, nonalcoholic fatty liver disease; OR, odds ratio. This work was supported, in part, by Interdisciplinary Center for Clinical Re- 1  This study was presented in part at the AASLD Liver Meeting 2015,  search (IZKF) Würzburg and by Grant SFB 841 from the Deutsche Forschungsge- San Francisco, CA, November 13–17, 2015. meinschaft (J.K.). The authors declare no conflicts of interest.  To whom correspondence should be addressed.  Manuscript received 24 February 2016 and in revised form 13 October 2016.  e-mail: Geier_A2@ukw.de Published, JLR Papers in Press, November 11, 2016  The online version of this article (available at http://www.jlr.org)  DOI 10.1194/jlr.P067454 contains a supplement. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 58, 2017 247 This is an Open Access article under the CC BY license. written informed consent to participate in these studies. The  risks on their carriers. The PNPLA3 (patatin-like phospho- ethical committees at participating centers approved the study  lipase domain containing 3, also known as adiponutrin)  protocol. Ethanol intake (>20 g per day for women and >30 g for  p.I148M polymorphism is commonly regarded to be the men) was regarded as exclusion criterion. NAFLD was diagnosed  risk factor for both increased fat accumulation and fibrosis   either by imaging techniques (abdominal sonography, MRI, CT)  (5, 6). The association with steatosis was demonstrated in  or by liver biopsy. Liver biopsies were performed percutaneously  several candidate studies, whereas the link between PNPLA3 under ultrasound guidance or intraoperatively. Acquired liver  and liver scarring was substantiated by meta-analyses in pa- samples were evaluated by experienced local pathologists. The  tients with chronic hepatitis C virus (HCV) infection (7)  presence of acute and chronic liver diseases other than NAFLD  was excluded in all patients. All study subjects underwent a stan- and in alcoholics (8). The data concerning the involve- dardized clinical examination. Fasted venous blood samples were  ment of TM6SF2 (transmembrane 6 superfamily member  drawn for routine biochemical analyses, including liver function  2) in liver injury are less definitive.  So far, only a few studies  tests and DNA genotyping. Liver function tests were determined  investigating the TM6SF2 risk genotype in NAFLD (9) and  by clinical-chemical assays in the central laboratories of par- in HCV (10) have been published. Liu et al. (11) reported  ticipating centers. In a subgroup of 320 patients with NAFLD  that carriers of the minor allele are at risk of increased ste- with available histology, hepatic steatosis (grades S0–S3) and fi- atosis and fibrosis.  Interestingly, both PNPLA3 and TM6SF2 brosis (grades F0–F4) were quantified  according to the Kleiner  variants have been associated with “metabolically silent”  score (18). NAFLD; i.e., carriers of the risk genotypes seem to develop  Genotyping of the PNPLA3 (rs738409), TM6SF2 NAFLD and its severe forms even in the absence of charac- (rs58542926), and MBOAT7 (rs641738) variants teristics commonly associated with fatty liver (5, 12). In- Genotyping of the PNPLA3 (rs738409), TM6SF2 (rs58542926),  deed, numerous genetic studies failed to detect equivocal  and MBOAT7 (rs641738) variants was performed in a central lab- evidence for the association between TM6SF2 and PNPLA3 oratory (Homburg) by a technician blinded to the phenotype of  variants and traits such as obesity, insulin resistance, or hy- patients. DNA was extracted from peripheral blood mononuclear  perlipidemia. Most recently, the MBOAT7 polymorphism cells using the DNeasy Blood and Tissue Kit (Qiagen). DNA con- rs641738 was identified  as the new risk factor for NAFLD  centrations were measured using a NanoDrop spectrophotome- (13), also associated with severity of fibrosis  in alcoholic  ter. All variants were genotyped using TaqMan assays (19). The  liver disease (14) and in HCV infection (15). fluorescence  data were analyzed with allelic discrimination 7500  Liver  biopsy  represents  the  gold-standard  method  of  Software v.2.0.2. quantifying the degree of NAFLD (16). Although several  Statistical analysis noninvasive methods have been developed, liver biopsy  Unless stated otherwise, all statistical analyses were performed  represents the only reliable tool to distinguish between  with SPSS 20.0 (SPSS, Munich, Germany) or GraphPad Prism 5.0  nonalcoholic fatty liver and nonalcoholic steatohepatitis.  (GraphPad Software Inc., CA). Quantitative data were expressed  Analysis of liver specimens also provides exact data con- as medians and ranges. The association between the  PNPLA3, cerning steatosis, fibrosis,  and inflammation.  Hence, it is a  TM6SF2, and MBOAT7 variants and markers of liver injury was  powerful tool for quantifying the role of inherited predis- tested using ANOVA with post hoc tests. Exact tests were per- position in liver injury. formed  to  check  the  consistency  of  genotyping  results  in  To further elucidate the role of the genetic predisposi- with  Hardy-Weinberg  equilibrium  (http://ihg.gsf.de/cgi-bin/ hw/hwa1.pl). Genotype frequencies were compared in contin- tion in modulation of NAFLD, we performed genetic analy- gency tables. Power analysis was performed using PS: Power and  ses in a large cohort of patients with fatty liver to analyze  Sample Size Calculation v.3.0 (http://biostat.mc.vanderbilt.edu/ the signs of liver injury in combination with the carriage of  wiki/Main/PowerSampleSize).  Differences  in  anthropometric  the PNPLA3 p.I148M, TM6SF2 p.E67K, and MBOAT7 and clinical traits between patients with  PNPLA3 and MBOAT7 rs641738 variants The frequencies of these variants were  genotypes were compared using linear regression analysis under  related to i) results of liver biopsy,  ii) circulating levels of  an additive genetic model. Comparisons between carriers of the  markers of liver injury, and iii) metabolic traits. Analysis of  TM6SF2 genotypes were performed under a dominant genetic  genotype-phenotype interactions performed in this group model (due to the low number of homozygotes for the 167K mu- tant allele) using linear regression analysis. All models were ad- of patients demonstrated different effects of the PNPLA3, justed  for  confounding  factors  (age,  gender,  BMI,  diabetes  TM6SF2, and MBOAT7 variants on hepatic steatosis and -fi mellitus, and statin use, as appropriate). The effects of the studied  brosis, underscoring the notion that they play distinct roles variants, as well as additional risk factors, on hepatic steatosis and  in NAFLD progression. fibrosis  were analyzed in univariate and multivariate models using  logistic regression analysis. MATERIALS AND METHODS RESULTS Patients Patients for the study were recruited in eight German univer- Characteristics of the study cohort sity centers within the framework of the NAFLD Clinical Study  A total of 515 German patients with NAFLD (99.9%  Group (NAFLD CSG) project (17). In brief, the project was  white) were recruited.  Table 1 summarizes the baseline  started in 2012 as a multicentric study in Germany and was in- data of this study cohort, and Table 2 presents the results of tended to investigate triggers and modulators of NAFLD devel- opment, including common genetic variants. All patients gave  liver biopsies in 320 biopsied patients. More women (54%)  248 Journal of Lipid Research Volume 58, 2017 TABLE 1.  Baseline characteristics and genotype frequencies in the  PNPLA3 p.I148M and TM6SF2 p.E167K variants are study cohort associated with increased serum markers of liver injury Variables Entire cohort Biopsied patients The  PNPLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 variants were successfully genotyped in all pa- N (female/male) 515 (280/235) 320 (186/134) Age (years) 50 (16–88) 49 (16–88) tients. The genotype frequencies (Table 1) do not differ  BMI (kg/m ) 32 (17–70) 33 (17–69) from frequencies presented in previous publications and  ALT (U/l) 52 (12–279) 58 (13–279) a are localized on the Hardy-Weinberg equilibrium parabola  AST (U/l) 38 (5–397) 42 (4–397) (P > 0.05, exact test), which validates the genotyping qual- GGT (U/l) 61 (4–1,658) 67 (4–1,463) Triglycerides (mg/dl) 152 (45–770) 154 (49–770) ity. Relations of the studied variants to patient baseline  Total cholesterol (mg/dl) 204 (72–379) 206 (107–379) characteristics are presented in supplemental Table S1 (for  Glucose (mg/dl) 98 (55–367) 99 (63–286) PNPLA3 p.I148M), supplemental Table S2 (for  TM6SF2 Incidence of diabetes type 2 (%) 24.7 26.7 Statin use (%) 10.6 10.6 p.E167K), and supplemental Table S3 (for the  MBOAT7 TM6SF2 p.E167K genotypes (n) rs641738). As presented in the supplemental materials, the  [EE] 409 253 PNPLA3 and TM6SF2 variants were significantly  associated  [EK] 97 61 [KK] 9 6 with BMI (both P = 0.01). We did not detect any signifi- PNPLA3 p.I148M genotypes (n) cant association between clinical characteristics and the  [II] 215 126 MBOAT7 polymorphism (supplemental Table S3). In the  [IM] 222 138 entire cohort (i.e., 515 patients with NAFLD), the PNPLA3 [MM] 78 56 MBOAT7 rs641738 genotypes (n) p.I148M polymorphism was associated with increased se- [CC] 159 98 rum AST (ANOVA, P < 0.001) (Fig. 2A) and ALT (ANOVA,      [CT] 242 157 P = 0.002) (Fig. 2B) but not with GGT activities (ANOVA,      [TT] 114 65 P = 0.74) (Fig. 2C). Similarly, the TM6SF2 variant was asso- E, glutamic acid; I, isoleucine; K, lysine; M, methionine; MBOAT7, ciated with increased AST (P < 0.001) (Fig. 2D) and ALT  membrane bound O-acyltransferase domain containing 7; p, protein  (amino acid number);  PNPLA3, patatin-like phospholipase domain- (P = 0.011) (Fig. 2E) but not with GGT activities (P = 0.14)  containing protein 3; TM6SF2, transmembrane 6 superfamily member (Fig. 2F). We did not detect any significant  association  2. Values are given as medians (ranges), unless stated otherwise. between the  MBOAT7 polymorphism and liver function  P < 0.001 as compared with nonbiopsied individuals. tests (all  P > 0.05) (Fig. 2G–I). We detected a significant   (P < 0.0001) increase of serum AST activities with the incre- than men (46%) were included. The median age was  ment of risk alleles of either of the genotypes (Fig. 3A). We  50 years. In 320 patients who underwent liver biopsy,  also detected trends for increased ALT (P = 0.08) and GGT  57% had steatosis grades 2 or 3 (Table 2). Fibrosis stage  (P = 0.07) levels with increasing risk allele number (Fig. 3B, C). F2 or higher was present in 30% of patients. Patients un- dergoing  liver  biopsy  had  significantly   higher  alanine  PNPLA3 p.I148M and TM6SF2 p.E167K have different transaminase  (ALT)  and  aspartate  aminotransferase  effects on hepatic steatosis and fibrosis (AST) (both  P < 0.001) but not glutamyl transferase We performed separate analysis of the variants’ effects  (GGT) activities (P = 0.26) (Table 1). We did not detect  on the risk of developing hepatic steatosis and fibrosis  in  any differences in serum glucose, triglyceride, and cho- specimens acquired by liver biopsy. Overall, carriers of the  lesterol concentrations between biopsied and nonbiop- PNPLA3 risk allele (P = 0.043), but not TM6SF2 or MBOAT7 sied patients (all  P > 0.05). Individuals presenting with  variants (both  P > 0.05), were more frequently scheduled  steatosis grade 2 or 3 had significantly  higher serum glu- for liver biopsy. The  PNPLA3 polymorphism was signifi- cose (P = 0.002) and triglyceride (P = 0.025) concentra- cantly associated with the risk of developing steatosis grades  tions as compared with individuals with lower grades of  S2 and S3 [common odds ratio (OR) = 1.896;  P < 0.001] steatosis (Fig. 1A, B). There were no differences in terms  and fibrosis  stages F2–F4 (common OR, 2.348; P < 0.001)  of serum cholesterol in relation to hepatic steatosis (P >  (Tables 3 and 4). Analysis of TM6SF2 genotype frequencies  0.05) (Fig. 1C). (Tables 5 and 6) reveals that this variant was associated  with steatosis (common OR, 1.539; P = 0.003) but had no  major effects on fibrosis  (P > 0.05). Based on the frequency  TABLE 2.  Distribution of steatosis and fibrosis  in biopsied  of the minor allele among individuals with fibrosis  grade  individuals with NAFLD  <F2 (Table 6), this analysis had a power of 0.81 to detect  Biopsy results Distribution genetic  effects  with  OR  of  at  least  2.0.  Although  the  Grade of steatosis MBOAT7 polymorphism was not associated with hepatic    0/1 48% steatosis (all  P > 0.05), it was significantly  associated with  2 27% the development of liver fibrosis  (common OR, 1.446;  3 25% Grade of fibrosis P = 0.046) (Table 7). We also detected an increase in the    0/1 70% number of risk  PNPLA3, TM6SF2, and MBOAT7 alleles 2 16% with increasing hepatic fibrosis  (supplemental Fig. S1)  3 7% 4 7% and most of all steatosis (supplemental Fig. S2). In the uni- variate model,  PNPLA3 and TM6SF2 polymorphisms, but  Data available for 320 patients.  Data available for 295 patients. not MBOAT7, were associated with increased steatosis  TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 249 but not ALT (P = 0.17) (supplemental Fig. S3B) or GGT  (P = 0.13) (supplemental Fig. S3C). Notably, among indi- viduals scheduled for the liver biopsy, the  TM6SF2 poly- morphism was associated with increased AST (P = 0.005)  (supplemental Fig. S3D), ALT (P = 0.025) (supplemental  Fig. S3E), and GGT (P = 0.025) (supplemental Fig. S3F).  We did not detect any significant  association between liver  function test and the MBOAT7 polymorphism in biopsied patients (supplemental Fig. S3G-I).  Table 9 summarizes  the results of regression analyses for factors associated with  liver fibrosis  in biopsied patients. Of note, in the multivari- ate model we detect a significant  association for  PNPLA3 and MBOAT7 genotypes (both  P < 0.05) but not for the  TM6SF2 polymorphism (P > 0.05). DISCUSSION In the current study we analyzed a thoroughly pheno- typed cohort of patients with NAFLD. According to current  knowledge (2, 5), the three variants that we chose to geno- type might play major roles in the development of hepatic  steatosis. We demonstrate that both PNPLA3 and TM6SF2 polymorphisms are associated with increased aminotrans- ferase activities, which might mirror enhanced liver injury  in NAFLD. However, the analysis of biopsy samples under- scored that the deleterious effects conferred by the tested variants  are  apparently  related  to  distinct  mechanisms:  whereas the PNPLA3 genotype modulates the progression of both fibrosis  and steatosis, the TM6SF2 variant seems to  be predominantly associated with steatosis. The  MBOAT7 polymorphism is likely to be, in turn, associated with the  risk of liver scarring. Our observations with respect to the PNPLA3 variant are  in line with the majority of previous studies in patients with  NAFLD (6). Patients with PNPLA3-associated steatohepati- tis are known to be at risk of progressive liver fibrosis,  cir- rhosis,  and  eventually  hepatocellular  carcinoma  (20).  Previously published results concerning the  TM6SF2 vari- ant are less consistent. The association between this variant  and fibrosis  postulated by Liu et al. (11) was not replicated  a biopsy-based study from Argentina (21). In this study, the  authors analyzed a total of 361 patients, among them 226  with biopsy-proven mostly mild NAFLD, and found a ge- netic association with steatosis but not with fibrosis.  These  results are in line with our study. In contrast, Sookoian  et al. (21) did not detect any major effects of the TM6SF2 polymorphism on liver function tests. Interestingly, the  analysis of patients with chronic HCV infection (10) pro- vided hints that the presence of variant TM6SF2 enhances Fig. 1.  Relation between steatosis grade at liver biopsy and meta- liver fibrogenesis  in this setting. Also, alcoholics carrying  bolic traits. Increased steatosis was associated with higher serum  the susceptible TM6SF2 genotype seem to be at risk of liver  glucose (A) and triglyceride (B) levels, but it did not affect total  cirrhosis (14). Recently, Eslam et al. (22) analyzed the ef- cholesterol (C). fects of this variant on metabolic traits and liver status in a  (Table 8). The association remained significant  for these  cohort of 3,260 individuals, among which a total of 502  two genotypes in the multivariate analysis (Table 8). In the  presented with NAFLD. In this study, variant TM6SF2 was  analyses of liver function tests restricted to biopsied pa- overrepresented in patients with NAFLD, among whom  tients, the PNPLA3 polymorphism was associated with sig- presence of the minor TM6SF2 allele was associated with  nificantly  increased AST (P = 0.013) (supplemental Fig. S3A)  increased fibrosis  and lower serum triglycerides. It did not  250 Journal of Lipid Research Volume 58, 2017 Fig. 2.  Box-and-whisker plots illustrating liver function tests in carriers of distinct PNPLA3, TM6SF2, and MBOAT7 variants. Carriers of ei- ther PNPLA3 or TM6SF2 risk alleles present with increased AST and ALT activities (A and B for PNPLA3; D and E for TM6SF2). We did not  detect any major effects of these variants on the GGT activities (C and F). The MBOAT7 polymorphism did not affect liver function tests. All  tests were performed using ANOVA with post hoc tests or with Mann-Whitney U as appropriate. *P < 0.05 in post hoc tests. affect other metabolic traits, NAFLD activity score (NAS)  associated with severe NAFLD by Mancina et al. (13). Most  or transaminase activities. Overall, these data might suggest  recently, it was demonstrated that its presence is associated  that the TM6SF2 polymorphism is associated with advanced  with an increased fibrosis  risk in patients with HCV (15).  liver fibrosis  in the presence of additional nongenetic fac- The association between fibrosis  and  MBOAT7 in our co- tors (e.g., alcohol or viral hepatitis). However, these addi- hort is, hence, in line with the previous studies.  tional factors that might promote fibrogenesis  in patients  We did not detect a link between the MBAOT7 genotype with NAFLD carrying the TM6SF2 risk genotype are yet  and increased steatosis. This lack of association is in line  to be defined. The  MBOAT7 polymorphism has lately with our recent results in patients undergoing bariatric  emerged as a new risk factor for severe liver diseases. First  surgery (23) but might be also related to an insufficient   detected by Buch et al. (14) as a genetic determinant of an  power of our cohort, which included fewer subjects than  increased cirrhosis risk in alcoholics, it was subsequently  the above-mentioned studies (13–15). Furthermore, the  TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 251 TABLE 3.  Distribution of alleles and genotypes for PNPLA3 p.I148M and association tests in respect to steatosis grade Count of alleles/genotypes Steatosis grade <S2 Steatosis S2–S3  PNPLA3 p.I148M allele/genotype (2N = 306) (2N = 334) [I] 211 (0.69) 197 (0.54) [M] 95 (0.31) 155 (0.46) [II] 73 (0.48) 53 (0.31) [IM] 65 (0.42) 73 (0.44) [MM] 15 (0.10) 41 (0.25) Association test OR P value   Armitage’s trend test 1.896 <0.001 OR statistics OR (95% CI) P value [M] ↔ [I] 1.923 (1.391–2.659) <0.001 [MM] ↔ [II] 3.765 (1.890–7.499) <0.001 [MM] ↔ [IM + II] 2.994 (1.580–5.671) <0.001 [MM + IM] ↔ [II] 1.936 (1.246–3.091) 0.003 I, isoleucine; M, methionine; p, protein (amino acid number);  PNPLA3, adiponutrin. [M] represents the steatosis risk allele. Allele  and genotype frequency differences were assessed by chi test or by Armitage’s trend test as appropriate (https://ihg.gsf.de/cgi-bin/hw/ hwa1.pl). Metabolic syndrome is believed to be the major trigger  of hepatic steatosis. The presence of steatosis was associ- ated with increased serum glucose and triglyceride concen- trations in our cohort as well (Fig. 1). No major association  between the risk of  PNPLA3, TM6SF2, or MABOT7 geno- types and distorted metabolic status has been described. Because PNPLA3- and TM6SF2-driven steatosis might even  be “metabolically silent” (12), the inclusion of these two  genotypes  in  the  diagnostic  work-up  of  patients  with  NAFLD could help, together with a detailed analysis of en- vironmental determinants of fatty liver, to identify individ- uals at increased risk of liver injury even in the absence of  the full ensemble of metabolic traits commonly associated with fatty liver disease. By adding the  MBOAT7 polymor- phism as the third genetic factor to the clinical work-up of  the patients with NAFLD, one could further improve the  chance of detecting patients who are at risk of liver fibrosis.   According to a recent study (24), fibrosis  represents the  TABLE 4.  Distribution of alleles and genotypes for PNPLA3 p.I148M and association tests in respect to fibrosis  grade Count of alleles/genotypes PNPLA3 p.I148M allele/ Fibrosis grade <F2  Fibrosis grade F2–F4  genotype (2N = 410) (2N = 180) [I] 211 (0.69) 197 (0.54) Fig. 3. Combined analysis of the PNPLA3 p.I148M, TM6SF2 [M] 95 (0.31) 155 (0.46) p.E167K, and MBOAT7 rs641738 risk alleles on liver function tests.  [II] 95 (0.46) 18 (0.20) [IM] 83 (0.41) 44 (0.49) The graphs demonstrate median AST (A), ALT (B), and GGT (C)  [MM] 27 (0.13) 28 (0.31) by the number of risk alleles in either of the tested genes. Analyses  Association test OR P value were performed using trend test. The following frequencies of car-   Armitage’s trend test 2.348 <0.001 riers of risk alleles were detected: zero risk alleles, n = 56; one risk  OR statistics OR (95% CI) P value allele, n = 142; two risk alleles, n = 170; three risk alleles, n = 117;  [M] ↔ [I] 2.491 (1.740–3.565) <0.001 four risk alleles, n = 27; five  risk alleles, n = 3. [MM] ↔ [II] 5.473 (2.637–11.361) <0.001 [MM] ↔ [IM + II] 2.977 (1.630–5.439) <0.001 [MM + IM] ↔ [II] 3.455 (1.925–6.200) <0.001 currently studied cohort encompassed well-characterized  patients from eight centers, but each biopsy was evaluated  CI, confidence  interval; I, isoleucine; M, methionine; p, protein  (amino acid number); PNPLA3, adiponutrin. [M] represents the steatosis only by local pathologists, so interobserver discrepancies in  risk allele. Allele and genotype frequency differences were assessed  defining  fibrosis  and steatosis and their effects of the asso- by chi  test or by Armitage’s trend test as appropriate (https://ihg.gsf. ciation tests could not be excluded. de/cgi-bin/hw/hwa1.pl). 252 Journal of Lipid Research Volume 58, 2017 TABLE 5.  Distribution of alleles and genotypes for TM6SF2 p.E167K TABLE 7.  Distribution of alleles and genotypes for MBOAT7 and association tests with respect to steatosis grade rs641738 and association tests in respect to fibrosis  grade Count of alleles/genotypes Count of alleles/genotypes TM6SF2 p.E167K allele/  Steatosis grade <S2 Steatosis S2–S3  MBOAT7 rs641738 allele/ Fibrosis ade F0  Fibrosis grade F1–F4  genotype (2N = 306) (2N = 334) genotype (2N = 206) (2N = 384) [E] 280 (0.92) 287 (0.86) [C] 122 (0.59) 194 (0.51) [K] 26 (0.08) 47 (0.14) [T] 44 (0.41) 190 (0.49) [EE] 130 (0.85) 123 (0.74) [CC] 34 (0.33) 53 (0.28) [EK] 20 (0.13) 41 (0.25) [CT] 54 (0.52) 88 (0.46) [KK] 3 (0.02) 3 (0.01) [TT] 15 (0.15) 51 (0.26) Association test OR P value Association test OR P value   Armitage’s trend test 1.539 0.003   Armitage’s trend test 1.446 0.046 OR statistics OR (95% CI) P value OR statistics OR (95% CI) P value [K] ↔ [E] 1.764 (1.063–2.927) 0.026   [T] ↔ [C] 1.422 (1.010–2.003) 0.043 [KK] ↔ [EE] 1.057 (0.209–5.336) 0.946   [TT] ↔ [CC] 2.181 (1.063–4.476) 0.031 [KK] ↔ [EK + EE] 0.915 (0.182–4.601) 0.913   [TT] ↔ [CT + CC] 2.122 (0.251–9.319) 0.012 [KK + EK] ↔ [EE] 2.022 (1.153–3.544) 0.001   [TT + CT] ↔ [CC] 1.292 (0.770–2.170) 0.350 CI,  confidence   interval;  E,  glutamic  acid;  K,  lysine;  p,  protein  CI, confidence  interval; p, protein (amino acid number); MBOAT7, (amino acid number); TM6SF2, transmembrane 6 superfamily member membrane  bound  O-acyltransferase  domain  containing  7.  [T]  2. [K] represents the steatosis risk allele. Allele and genotype frequency  represents the risk allele. Allele and genotype frequency differences  2 2 differences were assessed by chi  test or by Armitage’s trend test as  were assessed by chi  test or by Armitage’s trend test as appropriate  appropriate (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). most important factor affecting the long-term survival in  fibrosis,   in  our  cohort  the  TM6SF2  polymorphism  was  patients with NAFLD. Currently, liver biopsy is mostly  linked solely to the grade of steatosis. Importantly, the pres- recommended in patients with signs of severe steatohep- ence of variant TM6SF2 might even represent a protective  atitis or fibrosis,  which might lead to a selection bias in  factor against metabolic challenges (9, 12, 26). Based on  genetic studies. Hence, combined analyses of invasive and  our current results, one can still argue that, in comparison noninvasive markers of liver injury might be required in  to the TM6SF2 and MBOAT7 genotypes, the PNPLA3 the future to elucidate the risks conferred by the PNPLA3, p.I148M variant plays a more important role as the deter- TM6SF2, and MBOAT7 variants. minant of severe hepatic phenotypes ranging from steato- Recently, it has been suggested that increased serum sis to fibrosis  and cirrhosis. This is in line with our latest  aminotransferase activities in patients with NAFLD might  controlled-attenuation, parameter-based study in patients indicate metabolic adaptation of the liver to the fat over- with chronic liver diseases (19). In this analysis, we did not  load rather than hepatic injury (25). Hence, it is not  identify any major effects of the  TM6SF2 variant on liver  surprising that although both PNPLA3 and TM6SF2 poly- injury, which, however, were detected in carriers of the  morphisms were associated in our patients with NAFLD  PNPLA3 minor allele. These discrepancies might be re- with increased liver functions tests, analyses of liver biopsy  lated to substantially higher frequencies of the  PNPLA3 results demonstrate their different involvement in steatosis  risk allele in the general population as compared with the  and fibrosis.  Although the PNPLA3 and MBOAT7 risk vari- ants display a clear association with NAFLD-driven liver  TABLE 8.  Risk factors for developing hepatic steatosis Factor OR 95% CI P value TABLE 6.  Distribution of alleles and genotypes for TM6SF2 p.E167K and association tests with respect to fibrosis  grade Univariate analysis PNPLA3 p.I148M 2.418 1.323–4.419 0.004 Count of alleles/genotypes TM6SF2 p.E167K 4.622 1.077–19.831 0.039 MBOAT7 rs641738 1.260 0.749–2.119 0.384 TM6SF2 p.E167K allele/  Fibrosis grade <F2  Fibrosis grade F2–F4  genotype (2N = 410) (2N = 180) Glucose 1.015 0.994–1.037 0.168 BMI 0.966 0.933–1.001 0.055 [E] 366 (0.89) 156 (0.87) Age (years) 1.005 0.979–1.033 0.692 [K] 44 (0.11) 24 (0.13) Sex 2.080 0.933–4.634 0.073 [EE] 164 (0.80) 68 (0.76) Presence of diabetes 1.224 0.504–2.973 0.656 [EK] 38 (0.19) 20 (0.22) Triglycerides 1.002 0.996–1.007 0.594 [KK] 3 (0.01) 2 (0.02) Cholesterol 0.997 0.988–1.007 0.539 Association test OR P value Multivariate analysis   Armitage’s trend test 1.269 0.370 PNPLA3 p.I148M 2.424 1.326–4.419 0.004 OR statistics OR (95% CI) P value TM6SF2 p.E167K 4.725 1.093–20.429 0.038 [K] ↔ [E] 1.280 (0.752–2.177) 0.362 CI, confidence  interval; E, glutamic acid; I, isoleucine; K, lysine; M,  [KK] ↔ [EE] 1.608 (0.269–9.838) 0.608 methionine;  MBOAT7,  membrane  bound  O-acyltransferase  domain  [KK] ↔ [EK + EE] 1.530 (0.251–9.319) 0.642 containing 7; p, protein (amino acid number);  PNPLA3, adiponutrin;  [KK + EK] ↔ [EE] 1.294 (0.717–2.335) 0.391 TM6SF2, transmembrane 6 superfamily member 2. The relationships  between steatosis  PNPLA3, TM6SF2, and MBOAT7 variants as well as  CI,  confidence   interval;  E,  glutamic  acid;  K,  lysine;  p,  protein  other potentially prosteatotic factors were assessed by univariate and  (amino acid number); TM6SF2, transmembrane 6 superfamily member multivariate logistic regression analysis. Genetic analyses were calculated  2. [K] represents the steatosis risk allele. Allele and genotype frequency   test or by Armitage’s trend test as  by using either additive (for  PNPLA3 and MBOAT7) or dominant (for  differences were assessed by chi appropriate (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). TM6SF2) models. TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 253 TABLE 9.  Risk factors for developing hepatic fibrosis   5. Krawczyk,  M.,  P.  Portincasa,  and  F.  Lammert.  2013.  PNPLA3- associated steatohepatitis: toward a gene-based classification  of fatty  Factor OR 95% CI P value liver disease. Semin. Liver Dis. 33: 369–379.   6. Sookoian, S., and C. J. Pirola. 2011. Meta-analysis of the influence   Univariate analysis of I148M variant of patatin-like phospholipase domain containing  PNPLA3 p.I148M 1.679 1.192–2.367 0.003 3 gene (PNPLA3) on the susceptibility and histological severity of  TM6SF2 p.E167K 1.060 0.587–1.914 0.846 nonalcoholic fatty liver disease. Hepatology. 53: 1883–1894. MBOAT7 rs641738 1.410 1.003–1.982 0.048  7. Fan, J. H., M. Q. Xiang, Q. L. Li, H. T. Shi, and J. J. Guo. 2016.  Glucose 1.020 1.008–1.033 0.002 PNPLA3 rs738409 polymorphism associated with hepatic steatosis  BMI 0.989 0.965–1.015 0.413 and advanced fibrosis  in patients with chronic hepatitis C virus: a  Age (years) 1.020 1.002–1.039 0.027 meta-analysis. Gut Liver. 10: 456–463. Sex 1.088 0.671–1.763 0.732  8. Salameh, H., E. Raff, A. Erwin, D. Seth, H. D. Nischalke, E. Falleti,  Presence of diabetes 2.092 1.136–3.852 0.018 M. A. Burza, J. Leathert, S. Romeo, A. Molinaro, et al. 2015. PNPLA3  Triglycerides 1.003 1.000–1.007 0.083 gene polymorphism is associated with predisposition to and severity  Cholesterol 0.997 0.991–1.003 0.314 of alcoholic liver disease. Am. J. Gastroenterol. 110: 846–856. Multivariate analysis  9. Pirola, C. J., and S. Sookoian. 2015. The dual and opposite role of  PNPLA3 p.I148M 1.676 1.019–2.757 0.042 the TM6SF2-rs58542926 variant in protecting against cardiovascu- MBOAT7 rs641738 1.766 1.089–2.864 0.021 lar disease and conferring risk for nonalcoholic fatty liver: a meta- analysis. Hepatology. 62: 1742–1756. CI, confidence  interval; E, glutamic acid; I, isoleucine; K, lysine; M,   10. Milano, M., A. Aghemo, R. M. Mancina, J. Fischer, P. Dongiovanni,  methionine;  MBOAT7,  membrane  bound  O-acyltransferase  domain  S. De Nicola, A. L. Fracanzani, R. D’Ambrosio, M. Maggioni,  containing 7; p, protein (amino acid number);  PNPLA3, adiponutrin;  R. De Francesco, et al. 2015. Transmembrane 6 superfamily mem- TM6SF2, transmembrane 6 superfamily member 2 The relationships  ber 2 gene E167K variant impacts on steatosis and liver damage in  between steatosis  PNPLA3, TM6SF2, and MBOAT7 variants as well as  chronic hepatitis C patients. Hepatology. 62: 111–117. other potentially profibrotic  factors were assessed by univariate and   11. Liu, Y. L., H. L. Reeves, A. D. Burt, D. Tiniakos, S. McPherson, J. B.  multivariate logistic regression analysis. Genetic analyses were calculated  Leathart, M. E. Allison, G. J. Alexander, A. C. Piguet, R. Anty, et al.  using either additive (for  PNPLA3 and MBOAT7) and dominant (for  2014. TM6SF2 rs58542926 influences  hepatic fibrosis  progression  TM6SF2) models. in patients with non-alcoholic fatty liver disease.  Nat. Commun. 5:  12. Zhou,  Y.,  G.  Llaurado,  M.  Oresic,  T.  Hyotylainen,  M.  Orho- TM6SF2 minor allele. Indeed, the first  one is carried by  Melander, and H. Yki-Jarvinen. 2015. Circulating triacylglycerol sig- 10% of Europeans in the homozygous form, whereas  natures and insulin sensitivity in NAFLD associated with the E167K  <1% of individuals are homozygous carriers of the TM6SF2 variant in TM6SF2. J. Hepatol. 62: 657–663. p.167K allele. Hence, as in the case of MBOAT7, larger co-  13. Mancina, R. M., P. Dongiovanni, S. Petta, P. Pingitore, M. Meroni,  R. Rametta, J. Boren, T. Montalcini, A. Pujia, O. Wiklund, et al.  horts of patients might be required to fully elucidate the  2016. The MBOAT7–TMC4 variant rs641738 increases risk of non- involvement of the  TM6SF2 polymorphism in hepatic in- alcoholic fatty liver disease in individuals of European descent.  jury.  Indeed,  as  described  by  Mancina  et  al.  (13),  the  Gastroenterology. 150: 1219–1230 e6.  14. Buch,  S.,  F.  Stickel,  E.  Trepo,  M.  Way,  A.  Herrmann,  H.  D.  PNPLA3 variant has larger impact on the whole spectrum  Nischalke, M. Brosch, J. Rosendahl, T. Berg, M. Ridinger, et al.  of liver disease than TM6SF2. Therefore, we could not ex- 2015. A genome-wide association study confirms  PNPLA3 and iden- clude the possibility that the lack of association may be re- tifies  TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.  Nat. 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TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 255 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Lipid Research American Society for Biochemistry and Molecular Biology

Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study1

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References (29)

Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2017 Elsevier Inc.
ISSN
0022-2275
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1539-7262
DOI
10.1194/jlr.p067454
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Abstract

patient-oriented and epidemiological research Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study ,† § †† §§ Marcin Krawczyk,* Monika Rau, Jörn M. Schattenberg,** Heike Bantel, Anita Pathil, ††† §§§ Münevver Demir,*** Johannes Kluwe, Tobias Boettler, Frank Lammert,* and §,2 Andreas Geier for the NAFLD Clinical Study Group Department of Medicine II,* Saarland University Medical Center, Homburg, Germany; Laboratory of  Metabolic Liver Diseases, Department of General, Transplant and Liver Surgery,  Medical University of  Warsaw, Warsaw, Poland; Division of Hepatology, Department of Medicine II,  University Hospital Wuerzburg,  Wuerzburg, Germany; I. Department of Medicine,** University Medical Center Mainz, Johannes Gutenberg  †† University, Mainz, Germany; Department of Gastroenterology, Hepatology and Endocrinology,  Hannover  Medical School, Hannover, Germany; Department of Internal Medicine IV, Gastroenterology and  §§ Hepatology,  University of Heidelberg, Heidelberg, Germany; Clinic for Gastroenterology and  ††† Hepatology,*** University Hospital of Cologne, Cologne, Germany; Department of Medicine I, Hamburg §§§ University Medical Center, Hamburg, Germany; and Department of Medicine II, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany Abstract The PNPLA3 p.I148M, TM6SF2 p.E167K, and rs58542926, PNPLA3 rs738409, and MBOAT7 rs641738 vari- MBOAT7 rs641738 variants represent genetic risk factors for ants on NAFLD severity: A multicenter biopsy-based study. nonalcoholic fatty liver disease (NAFLD). Here we investi- J. Lipid Res. 2017. 58: 247–255. gate if these polymorphisms modulate both steatosis and fi- brosis in patients with NAFLD. We recruited 515 patients Supplementary key words  adiponutrin • fatty liver • fibrosis  • steatosis with NAFLD (age 16–88 years, 280 female patients). Liver biopsies were performed in 320 patients. PCR-based assays were used to genotype the PNPLA3, TM6SF2, and MBOAT7 Nonalcoholic fatty liver disease (NAFLD) affects more  variants. Carriers of the PNPLA3 and TM6SF2 risk alleles than 30% of adults in developed countries. Given the in- showed increased serum aspartate aminotransferase and ala- nine transaminase activities (P < 0.05). The PNPLA3 geno- creasing prevalence of environmental risk factors for this  type was associated with steatosis grades S2–S3 (P < 0.001) condition (e.g., hypercaloric diets and sedentary lifestyles)  and fibrosis stages F2–F4 (P < 0.001). The TM6SF2 genotype (1), the frequency of fatty liver is predicted to further in- was associated with steatosis (P = 0.003) but not with fibrosis crease in the coming years. In addition to environmental  (P > 0.05). The MBOAT7 variant was solely associated with triggers, genetic predisposition is known to modulate the  increased fibrosis (P = 0.046). In the multivariate model, degree of steatosis and liver injury (2). Conceptually, the  variants PNPLA3 (P = 0.004) and TM6SF2 (P = 0.038) were term “hepatic steatosis” refers to traits that are governed by  associated with steatosis. Fibrosis stages were affected by the multiple variants with modest effects. The major part of the  PNPLA3 (P = 0.042) and MBOAT7 (P = 0.021) but not by the genetic predisposition is, according to current knowledge,  TM6SF2 polymorphism (P > 0.05). The PNPLA3, TM6SF2, and MBOAT7 variants are associated with increased liver in- related to two common missense SNPs:  PNPLA3 p.I148M jury. The TM6SF2 variant seems to modulate predominantly and TM6SF2 p.E167K. These two polymorphisms, detected  hepatic fat accumulation, whereas the MBOAT7 polymor- in genome-wide (3) and exome-wide (4) association stud- phism is linked to fibrosis. The PNPLA3 polymorphism con- ies in patients with fatty livers, seem to impose different  fers risk of both increased steatosis and fibrosis.—Krawczyk,   M., M. Rau, J. M. Schattenberg, H. Bantel, A. Pathil, M.  Demir, J. Kluwe, T. Boettler, F. Lammert, A. Geier, NAFLD  Clinical  Study  Group.  Combined effects of the TM6SF2 Abbreviations:  ALT, alanine transaminase; AST, aspartate amino- transferase; GGT, gamma glutamyl transferase; HCV, hepatitis C virus;  NAFLD, nonalcoholic fatty liver disease; OR, odds ratio. This work was supported, in part, by Interdisciplinary Center for Clinical Re- 1  This study was presented in part at the AASLD Liver Meeting 2015,  search (IZKF) Würzburg and by Grant SFB 841 from the Deutsche Forschungsge- San Francisco, CA, November 13–17, 2015. meinschaft (J.K.). The authors declare no conflicts of interest.  To whom correspondence should be addressed.  Manuscript received 24 February 2016 and in revised form 13 October 2016.  e-mail: Geier_A2@ukw.de Published, JLR Papers in Press, November 11, 2016  The online version of this article (available at http://www.jlr.org)  DOI 10.1194/jlr.P067454 contains a supplement. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 58, 2017 247 This is an Open Access article under the CC BY license. written informed consent to participate in these studies. The  risks on their carriers. The PNPLA3 (patatin-like phospho- ethical committees at participating centers approved the study  lipase domain containing 3, also known as adiponutrin)  protocol. Ethanol intake (>20 g per day for women and >30 g for  p.I148M polymorphism is commonly regarded to be the men) was regarded as exclusion criterion. NAFLD was diagnosed  risk factor for both increased fat accumulation and fibrosis   either by imaging techniques (abdominal sonography, MRI, CT)  (5, 6). The association with steatosis was demonstrated in  or by liver biopsy. Liver biopsies were performed percutaneously  several candidate studies, whereas the link between PNPLA3 under ultrasound guidance or intraoperatively. Acquired liver  and liver scarring was substantiated by meta-analyses in pa- samples were evaluated by experienced local pathologists. The  tients with chronic hepatitis C virus (HCV) infection (7)  presence of acute and chronic liver diseases other than NAFLD  was excluded in all patients. All study subjects underwent a stan- and in alcoholics (8). The data concerning the involve- dardized clinical examination. Fasted venous blood samples were  ment of TM6SF2 (transmembrane 6 superfamily member  drawn for routine biochemical analyses, including liver function  2) in liver injury are less definitive.  So far, only a few studies  tests and DNA genotyping. Liver function tests were determined  investigating the TM6SF2 risk genotype in NAFLD (9) and  by clinical-chemical assays in the central laboratories of par- in HCV (10) have been published. Liu et al. (11) reported  ticipating centers. In a subgroup of 320 patients with NAFLD  that carriers of the minor allele are at risk of increased ste- with available histology, hepatic steatosis (grades S0–S3) and fi- atosis and fibrosis.  Interestingly, both PNPLA3 and TM6SF2 brosis (grades F0–F4) were quantified  according to the Kleiner  variants have been associated with “metabolically silent”  score (18). NAFLD; i.e., carriers of the risk genotypes seem to develop  Genotyping of the PNPLA3 (rs738409), TM6SF2 NAFLD and its severe forms even in the absence of charac- (rs58542926), and MBOAT7 (rs641738) variants teristics commonly associated with fatty liver (5, 12). In- Genotyping of the PNPLA3 (rs738409), TM6SF2 (rs58542926),  deed, numerous genetic studies failed to detect equivocal  and MBOAT7 (rs641738) variants was performed in a central lab- evidence for the association between TM6SF2 and PNPLA3 oratory (Homburg) by a technician blinded to the phenotype of  variants and traits such as obesity, insulin resistance, or hy- patients. DNA was extracted from peripheral blood mononuclear  perlipidemia. Most recently, the MBOAT7 polymorphism cells using the DNeasy Blood and Tissue Kit (Qiagen). DNA con- rs641738 was identified  as the new risk factor for NAFLD  centrations were measured using a NanoDrop spectrophotome- (13), also associated with severity of fibrosis  in alcoholic  ter. All variants were genotyped using TaqMan assays (19). The  liver disease (14) and in HCV infection (15). fluorescence  data were analyzed with allelic discrimination 7500  Liver  biopsy  represents  the  gold-standard  method  of  Software v.2.0.2. quantifying the degree of NAFLD (16). Although several  Statistical analysis noninvasive methods have been developed, liver biopsy  Unless stated otherwise, all statistical analyses were performed  represents the only reliable tool to distinguish between  with SPSS 20.0 (SPSS, Munich, Germany) or GraphPad Prism 5.0  nonalcoholic fatty liver and nonalcoholic steatohepatitis.  (GraphPad Software Inc., CA). Quantitative data were expressed  Analysis of liver specimens also provides exact data con- as medians and ranges. The association between the  PNPLA3, cerning steatosis, fibrosis,  and inflammation.  Hence, it is a  TM6SF2, and MBOAT7 variants and markers of liver injury was  powerful tool for quantifying the role of inherited predis- tested using ANOVA with post hoc tests. Exact tests were per- position in liver injury. formed  to  check  the  consistency  of  genotyping  results  in  To further elucidate the role of the genetic predisposi- with  Hardy-Weinberg  equilibrium  (http://ihg.gsf.de/cgi-bin/ hw/hwa1.pl). Genotype frequencies were compared in contin- tion in modulation of NAFLD, we performed genetic analy- gency tables. Power analysis was performed using PS: Power and  ses in a large cohort of patients with fatty liver to analyze  Sample Size Calculation v.3.0 (http://biostat.mc.vanderbilt.edu/ the signs of liver injury in combination with the carriage of  wiki/Main/PowerSampleSize).  Differences  in  anthropometric  the PNPLA3 p.I148M, TM6SF2 p.E67K, and MBOAT7 and clinical traits between patients with  PNPLA3 and MBOAT7 rs641738 variants The frequencies of these variants were  genotypes were compared using linear regression analysis under  related to i) results of liver biopsy,  ii) circulating levels of  an additive genetic model. Comparisons between carriers of the  markers of liver injury, and iii) metabolic traits. Analysis of  TM6SF2 genotypes were performed under a dominant genetic  genotype-phenotype interactions performed in this group model (due to the low number of homozygotes for the 167K mu- tant allele) using linear regression analysis. All models were ad- of patients demonstrated different effects of the PNPLA3, justed  for  confounding  factors  (age,  gender,  BMI,  diabetes  TM6SF2, and MBOAT7 variants on hepatic steatosis and -fi mellitus, and statin use, as appropriate). The effects of the studied  brosis, underscoring the notion that they play distinct roles variants, as well as additional risk factors, on hepatic steatosis and  in NAFLD progression. fibrosis  were analyzed in univariate and multivariate models using  logistic regression analysis. MATERIALS AND METHODS RESULTS Patients Patients for the study were recruited in eight German univer- Characteristics of the study cohort sity centers within the framework of the NAFLD Clinical Study  A total of 515 German patients with NAFLD (99.9%  Group (NAFLD CSG) project (17). In brief, the project was  white) were recruited.  Table 1 summarizes the baseline  started in 2012 as a multicentric study in Germany and was in- data of this study cohort, and Table 2 presents the results of tended to investigate triggers and modulators of NAFLD devel- opment, including common genetic variants. All patients gave  liver biopsies in 320 biopsied patients. More women (54%)  248 Journal of Lipid Research Volume 58, 2017 TABLE 1.  Baseline characteristics and genotype frequencies in the  PNPLA3 p.I148M and TM6SF2 p.E167K variants are study cohort associated with increased serum markers of liver injury Variables Entire cohort Biopsied patients The  PNPLA3 p.I148M, TM6SF2 p.E167K, and MBOAT7 rs641738 variants were successfully genotyped in all pa- N (female/male) 515 (280/235) 320 (186/134) Age (years) 50 (16–88) 49 (16–88) tients. The genotype frequencies (Table 1) do not differ  BMI (kg/m ) 32 (17–70) 33 (17–69) from frequencies presented in previous publications and  ALT (U/l) 52 (12–279) 58 (13–279) a are localized on the Hardy-Weinberg equilibrium parabola  AST (U/l) 38 (5–397) 42 (4–397) (P > 0.05, exact test), which validates the genotyping qual- GGT (U/l) 61 (4–1,658) 67 (4–1,463) Triglycerides (mg/dl) 152 (45–770) 154 (49–770) ity. Relations of the studied variants to patient baseline  Total cholesterol (mg/dl) 204 (72–379) 206 (107–379) characteristics are presented in supplemental Table S1 (for  Glucose (mg/dl) 98 (55–367) 99 (63–286) PNPLA3 p.I148M), supplemental Table S2 (for  TM6SF2 Incidence of diabetes type 2 (%) 24.7 26.7 Statin use (%) 10.6 10.6 p.E167K), and supplemental Table S3 (for the  MBOAT7 TM6SF2 p.E167K genotypes (n) rs641738). As presented in the supplemental materials, the  [EE] 409 253 PNPLA3 and TM6SF2 variants were significantly  associated  [EK] 97 61 [KK] 9 6 with BMI (both P = 0.01). We did not detect any signifi- PNPLA3 p.I148M genotypes (n) cant association between clinical characteristics and the  [II] 215 126 MBOAT7 polymorphism (supplemental Table S3). In the  [IM] 222 138 entire cohort (i.e., 515 patients with NAFLD), the PNPLA3 [MM] 78 56 MBOAT7 rs641738 genotypes (n) p.I148M polymorphism was associated with increased se- [CC] 159 98 rum AST (ANOVA, P < 0.001) (Fig. 2A) and ALT (ANOVA,      [CT] 242 157 P = 0.002) (Fig. 2B) but not with GGT activities (ANOVA,      [TT] 114 65 P = 0.74) (Fig. 2C). Similarly, the TM6SF2 variant was asso- E, glutamic acid; I, isoleucine; K, lysine; M, methionine; MBOAT7, ciated with increased AST (P < 0.001) (Fig. 2D) and ALT  membrane bound O-acyltransferase domain containing 7; p, protein  (amino acid number);  PNPLA3, patatin-like phospholipase domain- (P = 0.011) (Fig. 2E) but not with GGT activities (P = 0.14)  containing protein 3; TM6SF2, transmembrane 6 superfamily member (Fig. 2F). We did not detect any significant  association  2. Values are given as medians (ranges), unless stated otherwise. between the  MBOAT7 polymorphism and liver function  P < 0.001 as compared with nonbiopsied individuals. tests (all  P > 0.05) (Fig. 2G–I). We detected a significant   (P < 0.0001) increase of serum AST activities with the incre- than men (46%) were included. The median age was  ment of risk alleles of either of the genotypes (Fig. 3A). We  50 years. In 320 patients who underwent liver biopsy,  also detected trends for increased ALT (P = 0.08) and GGT  57% had steatosis grades 2 or 3 (Table 2). Fibrosis stage  (P = 0.07) levels with increasing risk allele number (Fig. 3B, C). F2 or higher was present in 30% of patients. Patients un- dergoing  liver  biopsy  had  significantly   higher  alanine  PNPLA3 p.I148M and TM6SF2 p.E167K have different transaminase  (ALT)  and  aspartate  aminotransferase  effects on hepatic steatosis and fibrosis (AST) (both  P < 0.001) but not glutamyl transferase We performed separate analysis of the variants’ effects  (GGT) activities (P = 0.26) (Table 1). We did not detect  on the risk of developing hepatic steatosis and fibrosis  in  any differences in serum glucose, triglyceride, and cho- specimens acquired by liver biopsy. Overall, carriers of the  lesterol concentrations between biopsied and nonbiop- PNPLA3 risk allele (P = 0.043), but not TM6SF2 or MBOAT7 sied patients (all  P > 0.05). Individuals presenting with  variants (both  P > 0.05), were more frequently scheduled  steatosis grade 2 or 3 had significantly  higher serum glu- for liver biopsy. The  PNPLA3 polymorphism was signifi- cose (P = 0.002) and triglyceride (P = 0.025) concentra- cantly associated with the risk of developing steatosis grades  tions as compared with individuals with lower grades of  S2 and S3 [common odds ratio (OR) = 1.896;  P < 0.001] steatosis (Fig. 1A, B). There were no differences in terms  and fibrosis  stages F2–F4 (common OR, 2.348; P < 0.001)  of serum cholesterol in relation to hepatic steatosis (P >  (Tables 3 and 4). Analysis of TM6SF2 genotype frequencies  0.05) (Fig. 1C). (Tables 5 and 6) reveals that this variant was associated  with steatosis (common OR, 1.539; P = 0.003) but had no  major effects on fibrosis  (P > 0.05). Based on the frequency  TABLE 2.  Distribution of steatosis and fibrosis  in biopsied  of the minor allele among individuals with fibrosis  grade  individuals with NAFLD  <F2 (Table 6), this analysis had a power of 0.81 to detect  Biopsy results Distribution genetic  effects  with  OR  of  at  least  2.0.  Although  the  Grade of steatosis MBOAT7 polymorphism was not associated with hepatic    0/1 48% steatosis (all  P > 0.05), it was significantly  associated with  2 27% the development of liver fibrosis  (common OR, 1.446;  3 25% Grade of fibrosis P = 0.046) (Table 7). We also detected an increase in the    0/1 70% number of risk  PNPLA3, TM6SF2, and MBOAT7 alleles 2 16% with increasing hepatic fibrosis  (supplemental Fig. S1)  3 7% 4 7% and most of all steatosis (supplemental Fig. S2). In the uni- variate model,  PNPLA3 and TM6SF2 polymorphisms, but  Data available for 320 patients.  Data available for 295 patients. not MBOAT7, were associated with increased steatosis  TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 249 but not ALT (P = 0.17) (supplemental Fig. S3B) or GGT  (P = 0.13) (supplemental Fig. S3C). Notably, among indi- viduals scheduled for the liver biopsy, the  TM6SF2 poly- morphism was associated with increased AST (P = 0.005)  (supplemental Fig. S3D), ALT (P = 0.025) (supplemental  Fig. S3E), and GGT (P = 0.025) (supplemental Fig. S3F).  We did not detect any significant  association between liver  function test and the MBOAT7 polymorphism in biopsied patients (supplemental Fig. S3G-I).  Table 9 summarizes  the results of regression analyses for factors associated with  liver fibrosis  in biopsied patients. Of note, in the multivari- ate model we detect a significant  association for  PNPLA3 and MBOAT7 genotypes (both  P < 0.05) but not for the  TM6SF2 polymorphism (P > 0.05). DISCUSSION In the current study we analyzed a thoroughly pheno- typed cohort of patients with NAFLD. According to current  knowledge (2, 5), the three variants that we chose to geno- type might play major roles in the development of hepatic  steatosis. We demonstrate that both PNPLA3 and TM6SF2 polymorphisms are associated with increased aminotrans- ferase activities, which might mirror enhanced liver injury  in NAFLD. However, the analysis of biopsy samples under- scored that the deleterious effects conferred by the tested variants  are  apparently  related  to  distinct  mechanisms:  whereas the PNPLA3 genotype modulates the progression of both fibrosis  and steatosis, the TM6SF2 variant seems to  be predominantly associated with steatosis. The  MBOAT7 polymorphism is likely to be, in turn, associated with the  risk of liver scarring. Our observations with respect to the PNPLA3 variant are  in line with the majority of previous studies in patients with  NAFLD (6). Patients with PNPLA3-associated steatohepati- tis are known to be at risk of progressive liver fibrosis,  cir- rhosis,  and  eventually  hepatocellular  carcinoma  (20).  Previously published results concerning the  TM6SF2 vari- ant are less consistent. The association between this variant  and fibrosis  postulated by Liu et al. (11) was not replicated  a biopsy-based study from Argentina (21). In this study, the  authors analyzed a total of 361 patients, among them 226  with biopsy-proven mostly mild NAFLD, and found a ge- netic association with steatosis but not with fibrosis.  These  results are in line with our study. In contrast, Sookoian  et al. (21) did not detect any major effects of the TM6SF2 polymorphism on liver function tests. Interestingly, the  analysis of patients with chronic HCV infection (10) pro- vided hints that the presence of variant TM6SF2 enhances Fig. 1.  Relation between steatosis grade at liver biopsy and meta- liver fibrogenesis  in this setting. Also, alcoholics carrying  bolic traits. Increased steatosis was associated with higher serum  the susceptible TM6SF2 genotype seem to be at risk of liver  glucose (A) and triglyceride (B) levels, but it did not affect total  cirrhosis (14). Recently, Eslam et al. (22) analyzed the ef- cholesterol (C). fects of this variant on metabolic traits and liver status in a  (Table 8). The association remained significant  for these  cohort of 3,260 individuals, among which a total of 502  two genotypes in the multivariate analysis (Table 8). In the  presented with NAFLD. In this study, variant TM6SF2 was  analyses of liver function tests restricted to biopsied pa- overrepresented in patients with NAFLD, among whom  tients, the PNPLA3 polymorphism was associated with sig- presence of the minor TM6SF2 allele was associated with  nificantly  increased AST (P = 0.013) (supplemental Fig. S3A)  increased fibrosis  and lower serum triglycerides. It did not  250 Journal of Lipid Research Volume 58, 2017 Fig. 2.  Box-and-whisker plots illustrating liver function tests in carriers of distinct PNPLA3, TM6SF2, and MBOAT7 variants. Carriers of ei- ther PNPLA3 or TM6SF2 risk alleles present with increased AST and ALT activities (A and B for PNPLA3; D and E for TM6SF2). We did not  detect any major effects of these variants on the GGT activities (C and F). The MBOAT7 polymorphism did not affect liver function tests. All  tests were performed using ANOVA with post hoc tests or with Mann-Whitney U as appropriate. *P < 0.05 in post hoc tests. affect other metabolic traits, NAFLD activity score (NAS)  associated with severe NAFLD by Mancina et al. (13). Most  or transaminase activities. Overall, these data might suggest  recently, it was demonstrated that its presence is associated  that the TM6SF2 polymorphism is associated with advanced  with an increased fibrosis  risk in patients with HCV (15).  liver fibrosis  in the presence of additional nongenetic fac- The association between fibrosis  and  MBOAT7 in our co- tors (e.g., alcohol or viral hepatitis). However, these addi- hort is, hence, in line with the previous studies.  tional factors that might promote fibrogenesis  in patients  We did not detect a link between the MBAOT7 genotype with NAFLD carrying the TM6SF2 risk genotype are yet  and increased steatosis. This lack of association is in line  to be defined. The  MBOAT7 polymorphism has lately with our recent results in patients undergoing bariatric  emerged as a new risk factor for severe liver diseases. First  surgery (23) but might be also related to an insufficient   detected by Buch et al. (14) as a genetic determinant of an  power of our cohort, which included fewer subjects than  increased cirrhosis risk in alcoholics, it was subsequently  the above-mentioned studies (13–15). Furthermore, the  TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 251 TABLE 3.  Distribution of alleles and genotypes for PNPLA3 p.I148M and association tests in respect to steatosis grade Count of alleles/genotypes Steatosis grade <S2 Steatosis S2–S3  PNPLA3 p.I148M allele/genotype (2N = 306) (2N = 334) [I] 211 (0.69) 197 (0.54) [M] 95 (0.31) 155 (0.46) [II] 73 (0.48) 53 (0.31) [IM] 65 (0.42) 73 (0.44) [MM] 15 (0.10) 41 (0.25) Association test OR P value   Armitage’s trend test 1.896 <0.001 OR statistics OR (95% CI) P value [M] ↔ [I] 1.923 (1.391–2.659) <0.001 [MM] ↔ [II] 3.765 (1.890–7.499) <0.001 [MM] ↔ [IM + II] 2.994 (1.580–5.671) <0.001 [MM + IM] ↔ [II] 1.936 (1.246–3.091) 0.003 I, isoleucine; M, methionine; p, protein (amino acid number);  PNPLA3, adiponutrin. [M] represents the steatosis risk allele. Allele  and genotype frequency differences were assessed by chi test or by Armitage’s trend test as appropriate (https://ihg.gsf.de/cgi-bin/hw/ hwa1.pl). Metabolic syndrome is believed to be the major trigger  of hepatic steatosis. The presence of steatosis was associ- ated with increased serum glucose and triglyceride concen- trations in our cohort as well (Fig. 1). No major association  between the risk of  PNPLA3, TM6SF2, or MABOT7 geno- types and distorted metabolic status has been described. Because PNPLA3- and TM6SF2-driven steatosis might even  be “metabolically silent” (12), the inclusion of these two  genotypes  in  the  diagnostic  work-up  of  patients  with  NAFLD could help, together with a detailed analysis of en- vironmental determinants of fatty liver, to identify individ- uals at increased risk of liver injury even in the absence of  the full ensemble of metabolic traits commonly associated with fatty liver disease. By adding the  MBOAT7 polymor- phism as the third genetic factor to the clinical work-up of  the patients with NAFLD, one could further improve the  chance of detecting patients who are at risk of liver fibrosis.   According to a recent study (24), fibrosis  represents the  TABLE 4.  Distribution of alleles and genotypes for PNPLA3 p.I148M and association tests in respect to fibrosis  grade Count of alleles/genotypes PNPLA3 p.I148M allele/ Fibrosis grade <F2  Fibrosis grade F2–F4  genotype (2N = 410) (2N = 180) [I] 211 (0.69) 197 (0.54) Fig. 3. Combined analysis of the PNPLA3 p.I148M, TM6SF2 [M] 95 (0.31) 155 (0.46) p.E167K, and MBOAT7 rs641738 risk alleles on liver function tests.  [II] 95 (0.46) 18 (0.20) [IM] 83 (0.41) 44 (0.49) The graphs demonstrate median AST (A), ALT (B), and GGT (C)  [MM] 27 (0.13) 28 (0.31) by the number of risk alleles in either of the tested genes. Analyses  Association test OR P value were performed using trend test. The following frequencies of car-   Armitage’s trend test 2.348 <0.001 riers of risk alleles were detected: zero risk alleles, n = 56; one risk  OR statistics OR (95% CI) P value allele, n = 142; two risk alleles, n = 170; three risk alleles, n = 117;  [M] ↔ [I] 2.491 (1.740–3.565) <0.001 four risk alleles, n = 27; five  risk alleles, n = 3. [MM] ↔ [II] 5.473 (2.637–11.361) <0.001 [MM] ↔ [IM + II] 2.977 (1.630–5.439) <0.001 [MM + IM] ↔ [II] 3.455 (1.925–6.200) <0.001 currently studied cohort encompassed well-characterized  patients from eight centers, but each biopsy was evaluated  CI, confidence  interval; I, isoleucine; M, methionine; p, protein  (amino acid number); PNPLA3, adiponutrin. [M] represents the steatosis only by local pathologists, so interobserver discrepancies in  risk allele. Allele and genotype frequency differences were assessed  defining  fibrosis  and steatosis and their effects of the asso- by chi  test or by Armitage’s trend test as appropriate (https://ihg.gsf. ciation tests could not be excluded. de/cgi-bin/hw/hwa1.pl). 252 Journal of Lipid Research Volume 58, 2017 TABLE 5.  Distribution of alleles and genotypes for TM6SF2 p.E167K TABLE 7.  Distribution of alleles and genotypes for MBOAT7 and association tests with respect to steatosis grade rs641738 and association tests in respect to fibrosis  grade Count of alleles/genotypes Count of alleles/genotypes TM6SF2 p.E167K allele/  Steatosis grade <S2 Steatosis S2–S3  MBOAT7 rs641738 allele/ Fibrosis ade F0  Fibrosis grade F1–F4  genotype (2N = 306) (2N = 334) genotype (2N = 206) (2N = 384) [E] 280 (0.92) 287 (0.86) [C] 122 (0.59) 194 (0.51) [K] 26 (0.08) 47 (0.14) [T] 44 (0.41) 190 (0.49) [EE] 130 (0.85) 123 (0.74) [CC] 34 (0.33) 53 (0.28) [EK] 20 (0.13) 41 (0.25) [CT] 54 (0.52) 88 (0.46) [KK] 3 (0.02) 3 (0.01) [TT] 15 (0.15) 51 (0.26) Association test OR P value Association test OR P value   Armitage’s trend test 1.539 0.003   Armitage’s trend test 1.446 0.046 OR statistics OR (95% CI) P value OR statistics OR (95% CI) P value [K] ↔ [E] 1.764 (1.063–2.927) 0.026   [T] ↔ [C] 1.422 (1.010–2.003) 0.043 [KK] ↔ [EE] 1.057 (0.209–5.336) 0.946   [TT] ↔ [CC] 2.181 (1.063–4.476) 0.031 [KK] ↔ [EK + EE] 0.915 (0.182–4.601) 0.913   [TT] ↔ [CT + CC] 2.122 (0.251–9.319) 0.012 [KK + EK] ↔ [EE] 2.022 (1.153–3.544) 0.001   [TT + CT] ↔ [CC] 1.292 (0.770–2.170) 0.350 CI,  confidence   interval;  E,  glutamic  acid;  K,  lysine;  p,  protein  CI, confidence  interval; p, protein (amino acid number); MBOAT7, (amino acid number); TM6SF2, transmembrane 6 superfamily member membrane  bound  O-acyltransferase  domain  containing  7.  [T]  2. [K] represents the steatosis risk allele. Allele and genotype frequency  represents the risk allele. Allele and genotype frequency differences  2 2 differences were assessed by chi  test or by Armitage’s trend test as  were assessed by chi  test or by Armitage’s trend test as appropriate  appropriate (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). most important factor affecting the long-term survival in  fibrosis,   in  our  cohort  the  TM6SF2  polymorphism  was  patients with NAFLD. Currently, liver biopsy is mostly  linked solely to the grade of steatosis. Importantly, the pres- recommended in patients with signs of severe steatohep- ence of variant TM6SF2 might even represent a protective  atitis or fibrosis,  which might lead to a selection bias in  factor against metabolic challenges (9, 12, 26). Based on  genetic studies. Hence, combined analyses of invasive and  our current results, one can still argue that, in comparison noninvasive markers of liver injury might be required in  to the TM6SF2 and MBOAT7 genotypes, the PNPLA3 the future to elucidate the risks conferred by the PNPLA3, p.I148M variant plays a more important role as the deter- TM6SF2, and MBOAT7 variants. minant of severe hepatic phenotypes ranging from steato- Recently, it has been suggested that increased serum sis to fibrosis  and cirrhosis. This is in line with our latest  aminotransferase activities in patients with NAFLD might  controlled-attenuation, parameter-based study in patients indicate metabolic adaptation of the liver to the fat over- with chronic liver diseases (19). In this analysis, we did not  load rather than hepatic injury (25). Hence, it is not  identify any major effects of the  TM6SF2 variant on liver  surprising that although both PNPLA3 and TM6SF2 poly- injury, which, however, were detected in carriers of the  morphisms were associated in our patients with NAFLD  PNPLA3 minor allele. These discrepancies might be re- with increased liver functions tests, analyses of liver biopsy  lated to substantially higher frequencies of the  PNPLA3 results demonstrate their different involvement in steatosis  risk allele in the general population as compared with the  and fibrosis.  Although the PNPLA3 and MBOAT7 risk vari- ants display a clear association with NAFLD-driven liver  TABLE 8.  Risk factors for developing hepatic steatosis Factor OR 95% CI P value TABLE 6.  Distribution of alleles and genotypes for TM6SF2 p.E167K and association tests with respect to fibrosis  grade Univariate analysis PNPLA3 p.I148M 2.418 1.323–4.419 0.004 Count of alleles/genotypes TM6SF2 p.E167K 4.622 1.077–19.831 0.039 MBOAT7 rs641738 1.260 0.749–2.119 0.384 TM6SF2 p.E167K allele/  Fibrosis grade <F2  Fibrosis grade F2–F4  genotype (2N = 410) (2N = 180) Glucose 1.015 0.994–1.037 0.168 BMI 0.966 0.933–1.001 0.055 [E] 366 (0.89) 156 (0.87) Age (years) 1.005 0.979–1.033 0.692 [K] 44 (0.11) 24 (0.13) Sex 2.080 0.933–4.634 0.073 [EE] 164 (0.80) 68 (0.76) Presence of diabetes 1.224 0.504–2.973 0.656 [EK] 38 (0.19) 20 (0.22) Triglycerides 1.002 0.996–1.007 0.594 [KK] 3 (0.01) 2 (0.02) Cholesterol 0.997 0.988–1.007 0.539 Association test OR P value Multivariate analysis   Armitage’s trend test 1.269 0.370 PNPLA3 p.I148M 2.424 1.326–4.419 0.004 OR statistics OR (95% CI) P value TM6SF2 p.E167K 4.725 1.093–20.429 0.038 [K] ↔ [E] 1.280 (0.752–2.177) 0.362 CI, confidence  interval; E, glutamic acid; I, isoleucine; K, lysine; M,  [KK] ↔ [EE] 1.608 (0.269–9.838) 0.608 methionine;  MBOAT7,  membrane  bound  O-acyltransferase  domain  [KK] ↔ [EK + EE] 1.530 (0.251–9.319) 0.642 containing 7; p, protein (amino acid number);  PNPLA3, adiponutrin;  [KK + EK] ↔ [EE] 1.294 (0.717–2.335) 0.391 TM6SF2, transmembrane 6 superfamily member 2. The relationships  between steatosis  PNPLA3, TM6SF2, and MBOAT7 variants as well as  CI,  confidence   interval;  E,  glutamic  acid;  K,  lysine;  p,  protein  other potentially prosteatotic factors were assessed by univariate and  (amino acid number); TM6SF2, transmembrane 6 superfamily member multivariate logistic regression analysis. Genetic analyses were calculated  2. [K] represents the steatosis risk allele. Allele and genotype frequency   test or by Armitage’s trend test as  by using either additive (for  PNPLA3 and MBOAT7) or dominant (for  differences were assessed by chi appropriate (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). TM6SF2) models. TM6SF2, PNPLA3, and MBOAT7 variants and liver injury in NAFLD 253 TABLE 9.  Risk factors for developing hepatic fibrosis   5. Krawczyk,  M.,  P.  Portincasa,  and  F.  Lammert.  2013.  PNPLA3- associated steatohepatitis: toward a gene-based classification  of fatty  Factor OR 95% CI P value liver disease. Semin. Liver Dis. 33: 369–379.   6. Sookoian, S., and C. J. Pirola. 2011. Meta-analysis of the influence   Univariate analysis of I148M variant of patatin-like phospholipase domain containing  PNPLA3 p.I148M 1.679 1.192–2.367 0.003 3 gene (PNPLA3) on the susceptibility and histological severity of  TM6SF2 p.E167K 1.060 0.587–1.914 0.846 nonalcoholic fatty liver disease. Hepatology. 53: 1883–1894. MBOAT7 rs641738 1.410 1.003–1.982 0.048  7. Fan, J. H., M. Q. Xiang, Q. L. Li, H. T. Shi, and J. J. Guo. 2016.  Glucose 1.020 1.008–1.033 0.002 PNPLA3 rs738409 polymorphism associated with hepatic steatosis  BMI 0.989 0.965–1.015 0.413 and advanced fibrosis  in patients with chronic hepatitis C virus: a  Age (years) 1.020 1.002–1.039 0.027 meta-analysis. Gut Liver. 10: 456–463. Sex 1.088 0.671–1.763 0.732  8. Salameh, H., E. Raff, A. Erwin, D. Seth, H. D. Nischalke, E. Falleti,  Presence of diabetes 2.092 1.136–3.852 0.018 M. A. Burza, J. Leathert, S. Romeo, A. Molinaro, et al. 2015. PNPLA3  Triglycerides 1.003 1.000–1.007 0.083 gene polymorphism is associated with predisposition to and severity  Cholesterol 0.997 0.991–1.003 0.314 of alcoholic liver disease. Am. J. Gastroenterol. 110: 846–856. 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Journal

Journal of Lipid Research – American Society for Biochemistry and Molecular Biology

Published: Jan 1, 2017

Keywords: adiponutrin; fatty liver; fibrosis; steatosis

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