Nonalcoholic fatty liver disease

Transcription

1 TM6SF2: Catch-22 in the Fight Against Nonalcoholic Fatty Liver Disease and Cardiovascular Disease? Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most common cause of chronic liver disease worldwide. 1,2 NAFLD includes a spectrum of disease ranging from the accumulation of fat in the liver (steatosis) to histologic evidence of necroinflammation (nonalcoholic steatohepatitis [NASH]), then fibrosis and cirrhosis, in the absence of excessive alcohol ingestion. 2,3 Approximately one-third of the US population has radiologic evidence of NAFLD. Although the majority (70% 90%) will have relatively benign simple steatosis, 4 6 NAFLD may progress to cirrhosis or hepatocellular carcinoma, and can lead to liver failure in the absence of lifestyle change or effective pharmacological treatments. 2,7 Although morbidity and mortality from liver disease are increased greatly in patients with NAFLD, morbidity and mortality from cardiovascular disease is even more prevalent in this population. 8 NAFLD, a complex trait influenced by interpatient genetic variations and environmental influences, is widely considered to be the hepatic manifestation of the metabolic syndrome. 1,2 It correlates epidemiologically with the presence of features of the metabolic syndrome, which consists of having 3 of the following: impaired fasting glucose, central obesity, dyslipidemia (low high-density lipoprotein cholesterol, high low-density lipoprotein [LDL], or both) and hypertension. 8 There is substantial interpatient variation in which features of the metabolic syndrome are exhibited, and which clinical sequelae an individual may develop. In some cases the metabolic syndrome predisposes to NAFLD 9 and in others to cardiovascular disease. 10 Determining why this occurs is critical to making clinical recommendations based on personal rather than population risk. Although the exact cause of NAFLD or NASH has not been elucidated, we are now able to begin to understand the genetic basis for the disease. NAFLD clusters in families, 11 and its heritability is estimated to be 26% 27% for CT-measured hepatic steatosis. 12 Genome-wide association studies (GWAS) and candidate gene studies have provided important insights into the genetic contribution to NAFLD. 13 The nonsynonymous single nucleotide polymorphism (SNP), rs (c.444 C>G, I148M) in patatin-like phospholipase domaincontaining 3 (PNPLA3) was associated significantly with 1 H-magnetic resonance spectroscopy measured hepatic triglyceride (TG) content 14 and has subsequently been validated globally as a major genetic determinant of not only steatosis, but also severity of NASH, stage of hepatic fibrosis/ cirrhosis, and occurrence of NAFLDassociated HCC. 12,15,16 The largest GWAS meta-analysis for NAFLD to date, using 2.4 million SNPs imputed to HapMap in 7176 individuals of European ancestry, identified common variants at 5 loci. These variants are in or near the genes PNPLA3, GCKR, LYPLAL1, and PPP1R3B, and a region on chromosome 19 (19p13.11) that contains multiple genes and has variously been called NCAN/TM6SF2/ CILP2/PBX4 in the literature. 12 The variants at most of these loci are also associated with NASH/fibrosis (all but those at PPP1R3B). 12 Variants at the PNPLA3 locus conferred the strongest effect, predisposing to advanced liver disease with an odds ratio (OR) of 3.26 (95% CI, ), with the variants at the 19p13.11 locus being the second strongest at 1.65 (95% CI, ) per mutant allele. 12 The PNPLA3 and chromosome 19p13.11 locus also associated with histologic NASH/fibrosis in an independent sample of bariatric surgery patients. 17 Interestingly, NAFLDassociated variants did not result in uniform abnormalities of serum lipids or glycemic and anthropometric traits. 12 In particular, variants at the chromosome 19p13.11 locus associated with increased hepatic steatosis/ NASH/fibrosis were also associated with decreased serum LDL cholesterol and lower serum TGs, 12,18,19 which was contrary to the usually observed epidemiologic pattern of these traits. Variants at 19p13.11 were not associated significantly with serum glucose or measures of insulin resistance in the best powered analyses at the time. 12,18,19 Variants at PNPLA3 and LYPLAL1 did not affect serum lipid or glycemic traits, whereas those at GCKR and PPP1R3B were associated with increased serum LDL cholesterol and lower serum glucose levels. 12 These results suggested a heterogeneous etiology to NAFLD and the existence of multiple genetic metabolic disease subtypes that may requiremoreprecisiontreatmentinthe future. 12 Work from several groups has now extended these findings. It has recently been reported that a nonsynonymous genetic variant within a gene of unknown function called TM6SF2, transmembrane 6 superfamily member 2, (rs c.449 C>T) at the 19p13.11 locus was associated with hepatic steatosis in 2,736 individuals genotyped using a human exome chip. 20 This variant is in strong linkage disequilibrium (r 2 ¼ 0.798) with variants in the chromosome 19p13.11 locus previously reported to be NAFLD risk factors, 12 making it likely that the new and old signals are the same. Indeed, conditional analyses indicate that TM6SF2 rs may be the causal variant driving the association at this locus. 20,21 The TM6SF2 rs variant has subsequently been associated with severity of NAFLD-associated hepatic fibrosis/ cirrhosis (OR, 1.88 [95% CI, ] for advanced fibrosis per each copy of the minor allele carried), independent of confounding factors including age, diabetes, obesity, or PNPLA3 genotype, in a cohort of >1000 histologically characterized patients, 21 consistent with previous associations at this Gastroenterology 2015;148:

2 locus. 12 This association has itself been validated independently 21,22 and other studies have also associated the TM6SF2 variant with decreased levels of serum LDL cholesterol and TG 20,23 (consistent with the earlier reports 12 ). Studies have also described associations of the minor allele at this locus with increased serum alanine transaminase, aspartate transaminase, 20 and increased risk of diabetes, 24 with decreased serum alkaline phosphatase and lower risk of myocardial infarction, 23 atherosclerosis, and cardiovascular disease 22 (Table 1). Because the association signal in the chromosome 19p13.11 region covers >20 genes and the SNPs across this region are in high LD with one another (highly correlated) and include both known (in TM6SF2, NCAN, CILP2) and yet to be discovered nonsynonymous SNPs, it remains to be proven whether all of these phenotypes are owing to 1 or possibly multiple correlated functional variants/ genes in the region. Toward this end, several groups have started to carry out functional experiments with the genes under the GWAS signal and their variants to narrow down the causal gene(s) and variant(s). TM6SF2 rs c.449 C>T heterozygous variant is a nonsynonymous change producing a glutamate to lysine amino acid substitution at residue 167 (E167K), with the glutamate being highly conserved across mammals. 20 In mice, overexpression of human wildtype TM6SF2 in liver resulted in higher total cholesterol, LDL cholesterol, TGs and lower high-density lipoprotein cholesterol, whereas knockdown of mouse Tm6sf2 in liver resulted in decreased serum total cholesterol. 23 Because the E167K allele results in lower total cholesterol in humans, these data suggest that the E167K mutation in TM6SF2 was a loss of function allele. 23 Kozlitina et al 20 showed that expression of TM6SF2 (E167K) protein is lower than wild type, consistent with this being a loss of function allele, and showed that knockdown of mouse Tm6sf2 resulted in increased liver lipid accumulation and decreased serum TG and LDL. They also found that knockdown of mouse Tm6sf2 resulted in decreased Table1.Association of Variants at chr 19p13.11 Locus With Manifestations of the Metabolic Syndrome Association With Other Traits Fatty liver ALP/ALT/AST NASH/Fibrosis Fasting Glucose HOMA-IR LDL-C TG TC T2D CAD/MI DBP SBP LD (r 2 ) With rs SNP Annotation Gene: Protein Change SNP (Minor Allele, MAF) exonic: NCAN:P92S NA NA NA NA NS a NS a Y Y NA [ NA/NA/NA [ rs (T, 0.07) intronic: SUGP [ Y NS a NS a NS a NA Y Y Y NA NA/NA/NA NA intronic: SUGP [ NA NA NA NS a NS a Y Y Y NA NA/NA/NA NA exonic:tm6sf2:e167k Same SNP NA Y NA NA NA NA Y Y Y NA NA/[/NA NA exonic:tm6sf2:e167k Same SNP NA NA NA NA NA NA Y Y NA [ Y/[/[ [ exonic:tm6sf2:e167k Same SNP NA NA NA NA NA NA NA NA NA [ NA/NA/NA [ rs (C, 0.09) rs (C, 0.08) rs (T, 0.084) rs (T, 0.07) rs (T, 0.12) ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate transaminase; CAD/MI, coronary artery disease/myocardial infarction; DBP, diastolic blood pressure; HOMA-IR, homeostatic model assessment for insulin resistance; LDL-C, low-density lipoprotein cholesterol; MAF, minor allele frequency in Europeans; NA, not assessed in the study referenced; NASH, nonalcoholic steatohepatitis; SBP, systolic blood pressure; T2D, type 2 diabetes; TC, total cholesterol; TG, triglycerides; [, significant increase; Y, significant decrease. a Based on P value after correcting for multiple tests. 680

3 very low-density lipoprotein (VLDL) cholesterol secretion. 20 In other independent work 25 using confocal microscopy, investigators observed localization of green fluorescent protein tagged TM6SF2 to the endoplasmic reticulum in 2 human hepatoma cell lines. Additionally, knockdown of TM6SF2 in both cell lines resulted in reduced secretion of TG-rich lipoproteins, apolipoprotein B, and increased cellular TG levels. At the subcellular level, a marked increase in lipid droplet area, which could be attributed to increases in both the number and average size of lipid droplets, was reported. Conversely, overexpression of TM6SF2 caused a decrease in the number and average size of lipid droplets. 25 In parallel experiments, Kozlitina et al 20 also observed an increase in the size and number of lipid droplets in the livers of mice after knockdown of Tm6sf2. These results suggest a role for TM6SF2 in regulating the supply of lipids for the synthesis of TG-rich lipoproteins. Finally, RNAi knockdown of both TM6SF2 and NCAN in HeLa cells resulted in increased free cholesterol, 26 suggesting that the product of >1 gene in this region may be involved in hepatic lipid handling. Not all the studies have unequivocal findings. Neither Kozlitina et al 20 nor Holmen et al 23 observed changes with serum alkaline phosphatase in the mouse experiments described, contrary to the results observed in humans. Kozlitina et al 20 reported increased accumulation of hepatic TGs in Tm6sf2 knockdown mice, whereas Holmen et al 23 found no evidence of TG accumulation in the livers of Tm6sf2 knockdown mice. Kozlitina et al used adeno-associated virus knockdown, whereas Holmen used adenovirus knockdown of TM6SF2 in liver in C57BL/6J mice. These differences in hepatic steatosis may be owing to differences between humans and mice, to the short duration of gene-altering studies in mouse livers, or to speciesor tissue-specific effects of TM6SF2. Targeting just the liver for knockdown/overexpression in mice, for example, may result in phenotypes related to just this organ, whereas TM6SF2, which is expressed more widely, may exert some of its effects via its role in extrahepatic organs. TM6SF2 has highest expression levels in the intestine, where alkaline phosphatase is also expressed (in addition to the liver) and thus TM6SF2 may influence on serum alkaline phosphatase via intestinal effects. The association between TM6SF2 rs and the NAFLD phenotype spectrum from steatosis to advanced fibrosis/cirrhosis has now been robustly demonstrated in several large independent cohorts 12,20 22,25 ; however, 2 studies 1 from China 27 and 1 from South America 28 have been unable to replicate these associations. This may, in part, be owing to the generally low minor allele frequency of NAFLD associating variants in the 19p13.11 locus and interethnic variations in their frequency of carriage. Specifically, the minor allele at rs (T), has a frequency of 0.07 in Europeans, 0.04 in Hispanics, and 0.02 in African Americans. 29 Thus, the liver disease promoting allele at TM6SF2 will affect more individuals of European ancestry than Hispanic or African ancestry. Inadequate statistical power to detect an effect on histologic markers of disease progression is a particular issue in the South American study, 28 where only 226 NAFLD cases with histologically mild disease were included. Of these cases, only 130 had anything more than bland steatosis, but these cases exhibited a mean fibrosis stage of only 1.4 out of 4, further degrading the sensitivity of the study. It is, therefore, not surprising that the authors were unable to detect an association with features of steatohepatitis or fibrosis, whereas groups with larger cohorts have been successful. 12,21,22 The above functional studies in vivo 20,23 and in vitro 20,23,25 suggest that the TM6SF2 gene affects hepatic lipid efflux, with its deletion or mutation resulting in a reduction of lipoprotein secretion (VLDL, TG, and apolipoprotein B) coincident with increased hepatocellular lipid droplet size and TG content. The 19p13.11 locus overall, and the TM6SF2 rs variant in particular, Corroborating this finding, even though only about 0.5% of individuals of European ancestry in the population are homozygous for the minor allele of TM6SF2, 1.5% of individuals selected for having histologic NASH/fibrosis have this genotype, suggesting that carrying 2 copies of the T allele predisposes to developing advanced liver disease. 21 In contrast, carriage of the C-allele is associated with multiple classic characteristics of the metabolic syndrome, including dyslipidemia and cardiovascular disease. 23 Thus, carrying the T allele protects against development of dyslipidemia and cardiovascular disease. 23 Whether the effects of variants at the 167 position will be strong enough to overshadow the effects of many other environmental or genetic variants on the risk of liver and cardiovascular disease at an individual level, and so merit distinct medical recommendations for patients carrying this allele, remains to be determined. Nevertheless, studying the effects of NAFLD-associated human genetic variants on related metabolic traits promises to teach us much about how these traits interrelate to one another pathophysiologically. provide new insights into the mechanistic basis of progressive NAFLD, and the association between NAFLD and cardiovascular disease. They suggest that TM6SF2 is an important determinant of clinical outcome across metabolic syndrome related endorgan damage. Indeed, current evidence suggests that TM6SF2 may act as a switch with TM6SF2 rs T-allele mediating hepatic retention of TG and cholesterol predisposing to NAFLD-fibrosis, whereas C-allele carriage promotes VLDL excretion, protecting the liver at the expense of increased risk of atherosclerosis and cardiovascular disease (Figure 1). Thus, although in the general population, NAFLD is associated with an increased risk of cardiovascular disease, these can become dissociated: individuals carrying the minor (T) allele of TM6SF2 rs (167K) may be prone to experience liver-related rather than cardiovascular morbidity and mortality. 2,30,31 681

4 Figure 1.Effects of TM6SF2. TM6SF2 plays a role in VLDL export from liver to serum which results in increased serum lipids and myocardial infarction, and decreased risk of liver steatosis. chol, cholesterol; LDL, low-density lipoprotein cholesterol; IHTG, intrahepatic triglyceride; NASH, nonalcoholic steatohepatitis; TG, triglyceride; VLDL, very low-density lipoprotein. Although we have learned many things already from these discoveries, some interesting questions remain. The T allele of TM6SF2 rs has been associated with decreased alkaline phosphatase levels, but how the E167K change in TM6SF2 actually results in this biochemical effect remains to be determined. Interestingly, knocking down hepatic TM6SF2 expression did not result in changes in serum alkaline phosphatase. 20 Because TM6SF2 is expressed not only in liver but also in intestine where alkaline phosphatase is also present at high levels, the authors suggest that intestine may be where the E167K change may exert its effect to cause lower levels of alkaline phosphatase. 20 This theory remains to be tested. Another unresolved question relates to the role of TM6SF2 in the development of type 2 diabetes mellitus. Although one large meta-analysis suggests that TM6SF2 T-allele carriage is associated with an increased risk of type 2 diabetes, 24 its effect on serum glucose and on insulin resistance is not significant 24 and direct evidence of an effect on insulin sensitivity is currently lacking, with clamp studies finding no reduction in whole body, hepatic, or adipose tissue insulin sensitivity. 31 Further research, perhaps utilizing stable global and tissue-specific knockouts of TM6SF2 in mice, may help to clarify these questions. Even though there is evidence that the E167K mutation of TM6SF2 results in less VLDL in serum, the exact mechanism by which this occurs is not known. Because VLDL formation requires a multistep process where TGs are combined with apolipoproteins that are then secreted, altered, and eventually taken up again by the liver as VLDL remnants, how if at all this cycle is disrupted by the E167K variant at TM6SF2 remains to be tested. Because chylomicron assembly and circulation in intestine parallels VLDL assembly and circulation in liver and because TM6SF2 is also expressed in intestine, it remains to be determined whether chylomicron biology is also affected by the E167K TM6SF2 change in humans and mice. Through further study of TM6SF2 and other NAFLD-related loci, we will be able to better understand the mechanisms by which metabolic disease develops so that we can hopefully better tailor clinical recommendations for disease treatment in the future. Indeed, although genetic testing for common diseases is currently limited, this may change with a better understanding of how this information can be used to inform patient care. BRATATI KAHALI University of Michigan Ann Arbor, Michigan YANG-LIN LIU ANN K. DALY CHRISTOPHER P. DAY QUENTIN M. ANSTEE Newcastle University Newcastle-upon-Tyne, UK ELIZABETH K. SPELIOTES University of Michigan Ann Arbor, Michigan References 1. Dietrich P, Hellerbrand C. Nonalcoholic fatty liver disease, obesity and the metabolic syndrome. Best Pract Res Clin Gastroenterol 2014; 28: Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes 682

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6 Reprint requests Address requests for reprints to: Elizabeth K. Speliotes, MD, PhD, MPH, Division of Gastroenterology and Hepatology, University of Michigan Health System, 3912 Taubman Center, 1500 E. Medical Center Drive, SPC 5362, Ann Arbor, MI umich.edu; Quentin M. Anstee, BSc, MBBS, PhD, MRCP(UK), Institute of Cellular Medicine, The Medical School, Newcastle University, 4th Floor, William Leech Building, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK. Conflicts of interest The authors disclose no conflicts. Funding Bratati Kahali and Elizabeth K. Speliotes were supported by the Doris Duke Medical Foundation, and the University of Michigan Internal Medicine Department, Division of Gastroenterology, and Biological Sciences Scholars Program. Quentin M. Anstee was supported by a Clinical Senior Lectureship Award from the Higher Education Funding Council for England (HEFCE) by the AGA Institute /$