Carlos J. Pirola

Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis

Objectives We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome. Methods The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls. Results Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity. Conclusions miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.

Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity

We explored the role of transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 C/T nonsynonymous (p.Glu167Lys) variant in genetic susceptibility to nonalcoholic fatty liver disease (NAFLD) and disease severity. A total of 361 individuals (135 control subjects and 226 patients with histologically proven NAFLD) were included in a sample with 97% power for the additive genetic model. A discrete trait analysis of NAFLD showed that rs58542926 was associated with a modest risk of fatty liver (P = 0.038; odds ratio [OR]: 1.37; 95% confidence interval [CI]: 1.02‐1.84); nevertheless, conditioning on patatin‐like phospholipase domain‐containing 3 (PNPLA3)‐rs738409 abolished this effect. We did not observe an interaction between rs738409 and rs58542926 variants on the risk of NAFLD. We observed a significant association of rs58542926 and disease severity (P = 0.027), but not lobular inflammation or fibrosis; rs58542926 was not associated with levels of liver enzymes. An allelic test showed that the T (Lys167) allele was significantly associated with disease progression (P = 0.021; OR, 1.66; 95% CI: 1.08‐2.55). A significant association was found with the histological degree of liver steatosis (β, 0.15; standard error: 0.06; P = 0.0299) that was independent of rs738409. Homozygous carriers of the C (Glu167) allele showed increased risk for cardiovascular disease. TM6SF2 protein expression was decreased markedly in liver of NAFLD patients, compared to controls. In addition, TM6SF2 immunoreactivity was reduced in subjects carrying at least one copy of the T allele, consistent with a difference in liver allele‐specific transcript abundance. Conclusion: rs58542926 is a low‐frequency variant with a modest effect on NAFLD, suggesting that carriers of the T allele are slightly more likely to accumulate fat in the liver and develop nonalcoholic steatohepatitis than those without. TM6SF2 appears to play a significant role in disease biology. (Hepatology 2015;61:515‐525)

Non-alcoholic fatty liver disease and risk of cardiovascular disease

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver diseases worldwide, causing considerable liver-related mortality and morbidity. During the past decade, it has also become increasingly evident that NAFLD is a multisystem disease that affects many extra-hepatic organ systems, including the heart and the vascular system. In this updated clinical review, we discuss the rapidly expanding body of clinical and epidemiological evidence that supports a strong association of NAFLD with cardiovascular diseases (CVDs) and other functional and structural myocardial abnormalities. We also discuss some recently published data that correlate NAFLD due to specific genetic polymorphisms with the risk of CVDs. Finally, we briefly examine the assessment tools for estimating the global CVD risk in patients with NAFLD as well as the conventional and the more innovative pharmacological approaches for the treatment of CVD risk in this group of patients.

The dual and opposite role of the TM6SF2‐rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: A meta‐analysis

The aim of this work was to estimate the strength of the effect of the TM6SF2 E167K (rs58542926 C/T) variant on blood lipid traits and nonalcoholic fatty liver disease (NAFLD) across different populations. We performed a systematic review by a meta‐analysis; literature searches identified 10 studies. The rs58542926 exerts a significant role in modulating lipid traits, including total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C), triglycerides (TG), and NAFLD. However, this influence on lipids and NAFLD is opposite between genotypes in the dominant model of inheritance. Pooled estimates of random effects in 101,326 individuals showed that carriers of the minor T allele (EK+KK individuals), compared with subjects homozygous for the ancestral C allele (EE genotype), are protected from cardiovascular disease (CVD), showing lower levels of TC, LDL‐C, and TG; the differences in mean ± standard error (mg/dL) are −8.38 ± 1.56, −3.7 ± 0.9, and −9.4 ± 2.1, respectively. The rs58542926 variant was not associated with high‐density lipoprotein cholesterol in a large sample (n = 91,937). In contrast, carriers of the T allele showed a moderate effect on the risk of NAFLD (odds ratio: 2.13; 95% confidence interval: 1.36–3.30; P = 0.0009; n = 3273) and approximately ∼2.2% higher lipid fat content when compared with homozygous EE (n = 3,413). Conclusions: The rs58542926 appears to be an important modifier of blood lipid traits in different populations. As a challenge for personalized medicine, the C‐allele, which has an overall frequency as high as 93%, is associated with higher blood lipids, whereas the T allele confers risk for NAFLD; in turn, CVD and NAFLD are strongly related outcomes. Although the variant confers protection against CVD at the expense of an increased risk of NAFLD, it does not explain the link between these two complex diseases.(Hepatology 2015;62:1742–1756)

Genetic predisposition in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease whose prevalence has reached global epidemic proportions. Although the disease is relatively benign in the early stages, when severe clinical forms, including nonalcoholic steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma, occur, they result in worsening the long-term prognosis. A growing body of evidence indicates that NAFLD develops from a complex process in which many factors, including genetic susceptibility and environmental insults, are involved. In this review, we focused on the genetic component of NAFLD, with special emphasis on the role of genetics in the disease pathogenesis and natural history. Insights into the topic of the genetic susceptibility in lean individuals with NAFLD and the potential use of genetic tests in identifying individuals at risk are also discussed.

Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells

PNPLA3 was recently associated with the susceptibility to nonalcoholic fatty liver disease, a common cause of chronic liver disease characterized by abnormal triglyceride accumulation. Although it is established that PNPLA3 has both triacylglycerol lipase and acylglycerol O-acyltransferase activities, is still unknown whether the gene has any additional role in the modulation of the human liver metabolome. To uncover the functional role of PNPLA3 on liver metabolism, we performed high-throughput metabolic profiling of PNPLA3 siRNA-silencing and overexpression of wild-type and mutant Ile148Met variants (isoleucine/methionine substitution at codon 148) in Huh-7 cells. Metabolomic analysis was performed by using GC/MS and LC/MS platforms. Silencing of PNPLA3 was associated with a global perturbation of Huh-7 hepatoma cells that resembled a catabolic response associated with protein breakdown. A significant decrease in amino- and γ-glutamyl-amino acids and dipeptides and a significant increase in cysteine sulfinic acid, myo-inositol, lysolipids, sphingolipids, and polyunsaturated fatty acids were observed. Overexpression of the PNPLA3 Met148 variant mirrored many of the metabolic changes observed during gene silencing, but in the opposite direction. These findings were replicated by the exploration of canonical pathways associated with PNPLA3 silencing and Met148 overexpression. Overexpression of the PNPLA3 Met148 variant was associated with a 1.75-fold increase in lactic acid, suggesting a shift to anaerobic metabolism and mitochondrial dysfunction. Together, these results suggest a critical role of PNPLA3 in the modulation of liver metabolism beyond its classical participation in triacylglycerol remodeling.

PNPLA3, the history of an orphan gene of the potato tuber protein family that found an organ: The liver

T he patatin-like phospholipase domain containing 3 gene (PNPLA3) was an orphan gene looking for a disease until 2008 when the first genome-wide association study (GWAS) on nonalcoholic fatty liver disease (NAFLD) showed the association between the risk of liver fat accumulation and the nonsynonymous rs738409 C/G variant. Months later, the variant was significantly associated with NAFLD disease severity and alanine aminotransferase (ALT) levels, and the role of PNPLA3 as a modifier of the natural history of NAFLD was unequivocally replicated in different populations around the world, from children to adults. The enthusiasm about PNPLA3 led to the search for other biologically plausible liver disease associations, for instance, alcoholic liver disease (ALD) and an increased risk of steatosis in patients with chronic hepatitis C (CHC) and B. The variant was not only associated with cirrhosis and the occurrence of hepatocellular carcinoma (HCC), but the G allele was also identified as an independent risk factor for death and poor prognosis. At last, PNPLA3 found an organ: the liver. In this issue, Tr epo et al. explore the strength of the association between the rs738409 and the prevalence of HCC in cirrhosis patients by means of a meta-analysis of 2,503 individual participant data (IPD) from Europe. The authors found that the risk allele G was significantly associated with HCC (odds ratio [OR] per allele 5 1.77), after adjusting for sex, age, and obesity. Although the association was also found in patients with HCV-related cirrhosis (OR 5 1.55), in which the variant showed a small but statistically significant effect on the risk of HCC, the effect was more pronounced among patients with ALD (OR 5 2.20), suggesting that the variant influence is stronger, if not exclusive, on fat-derived HCC. Altogether, these results suggest some reflections: 1) rs738409 is the most consistent genetic modifier of the natural history of common chronic liver diseases in which inflammation and fibrogenesis are induced by environmental factors (dietary fat, alcohol, or viruses). 2) rs738409 is consistently associated with disease progression in liver diseases in which liver fat accumulation matters (NAFLD, ALD, and also CHC). Hence, instead of being “the cause” of the disease in terms of traditional monogenic disorders, the variant seems to be an important link between environmental stressors and the susceptibility of the liver to develop a more aggressive phenotype. 3) From a theoretical perspective, it can be argued that rs738409 has per se a substantial role in hepatocarcinogenesis because the variant is involved in related histological outcomes, such as steatosis and inflammation. Because the report of Tr epo et al. is a cross-sectional study, we cannot rule out the possibility that patients enrolled in this meta-analysis have had fatty liver prior to developing cirrhosis; thus, we do not know whether carriers of the G allele that developed HCC were those who had increased susceptibility to steatosis (in other terms, adjustment by steatosis). Considering that IPD is a unique opportunity to have full access to patients’ records, it would have been interesting to know whether the association between the variant and HCVHCC remained significant after adjusting for wellAbbreviations: ALD, alcoholic liver disease; ALT, alanine-aminotransferase; AUC, area under the curve; CHC, chronic hepatitis C; GWAS, genome-wide association study; HCC, hepatocellular carcinoma; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PNPLA3, patatin-like phospholipase domain containing 3; ROC, receiver operating characteristic; SNP, single nucleotide polymorphism Received October 1, 2013; accepted October 10, 2013. Partially supported by grants PICT 2008-1521 and PICT 2010-0441 (Agencia Nacional de Promoci on Cient ıfica y Tecnol ogica), and UBACYT CM04 (Universidad de Buenos Aires). S.S. and C.J.P. belong to Consejo Nacional de Investigaciones Cient ıficas, y Tecnicas, (CONICET). Address reprint requests to: Silvia Sookoian, M.D., Ph.D., or Carlos J. Pirola, Ph.D., Instituto de Investigaciones M edicas A. Lanari-CONICET, Combatiente de Malvinas 3150, Buenos Aires (1427), Argentina. E-mail: sookoian.silvia@lanari.fmed.uba.ar or pirola.carlos@lanari.fmed.uba.ar; fax: 54-11-4523-8947. Copyright VC 2014 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI: 10.1002/hep.26895 Potential conflict of interest: Nothing to report.

Nonalcoholic steatohepatitis is associated with a state of betaine‐insufficiency

Nonalcoholic fatty liver disease (NAFLD) develops from a complex process, which includes changes in the liver methylome. Betaine plays a pivotal role in the regulation of methylogenesis. We performed a two‐stage case–control study, which included patients with biopsy‐proven NAFLD to explore circulating levels of betaine and its association with the histological spectrum. We also explored the association between a missense rs1805074, p.Ser646Pro variant in DMGDH (dimethylglycine dehydrogenase mitochondrial) and NAFLD severity (n=390).

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Fil: Sookoian, Silvia Cristina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Medicas; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

Mitochondrial genome architecture in non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) is associated with mitochondrial dysfunction, a decreased liver mitochondrial DNA (mtDNA) content, and impaired energy metabolism. To understand the clinical implications of mtDNA diversity in the biology of NAFLD, we applied deep‐coverage whole sequencing of the liver mitochondrial genomes. We used a multistage study design, including a discovery phase, a phenotype‐oriented study to assess the mutational burden in patients with steatohepatitis at different stages of liver fibrosis, and a replication study to validate findings in loci of interest. We also assessed the potential protein‐level impact of the observed mutations. To determine whether the observed changes are tissue‐specific, we compared the liver and the corresponding peripheral blood entire mitochondrial genomes. The nuclear genes POLG and POLG2 (mitochondrial DNA polymerase‐γ) were also sequenced. We observed that the liver mtDNA of patients with NAFLD harbours complex genomes with a significantly higher mutational (1.28‐fold) rate and degree of heteroplasmy than in controls. The analysis of liver mitochondrial genomes of patients with different degrees of fibrosis revealed that the disease severity is associated with an overall 1.4‐fold increase in mutation rate, including mutations in genes of the oxidative phosphorylation (OXPHOS) chain. Significant differences in gene and protein expression patterns were observed in association with the cumulative number of OXPHOS polymorphic sites. We observed a high degree of homology (∼98%) between the blood and liver mitochondrial genomes. A missense POLG p.Gln1236His variant was associated with liver mtDNA copy number. In conclusion, we have demonstrated that OXPHOS genes contain the highest number of hotspot positions associated with a more severe phenotype. The variability of the mitochondrial genomes probably originates from a common germline source; hence, it may explain a fraction of the ‘missing heritability’ of NAFLD. Copyright