It is uncertain whether patients with nonalcoholic fatty liver disease (NAFLD) and normal alanine aminotransferase (ALT) have a milder disease and should undergo liver biopsy. We reviewed the histological data of 458 Italian patients with NAFLD in whom liver biopsy was indicated by altered liver enzymes (395 cases, 86%), or persistently elevated ferritin or long-lasting severe steatosis (63 cases). Factors associated with nonalcoholic steatohepatitis (NASH) and fibrosis ≥ 2 were identified by multivariate analysis. Patients with normal ALT were significantly older, had lower body mass index, fasting triglycerides, insulin resistance according to homeostasis model assessment (HOMA-IR), ALT, and gamma-glutamyltransferase, but a higher prevalence of hypertension. NASH was diagnosed in 59% and 74% of the patients with normal and increased ALT, respectively ( P = 0.01). In the overall series of patients, NASH was independently predicted by ALT (odds ratio [OR], 1.11; 95% confidence interval [CI], 1.04–1.19 per 10-IU/mL increase) and diabetes (OR, 1.5; 95% CI, 1.1–2.0). The same variables were selected in patients with increased ALT, whereas in those with normal ALT, HOMA-IR and ALT were independent predictors. Severe fibrosis was independently predicted by serum ferritin (OR, 1.04; 95% CI, 1.001–1.08 per 50-ng/mL increase), ALT (OR, 1.07; 95% CI, 1.02–1.14), and diabetes (OR, 1.8; 95% CI, 1.4–2.3) in the overall series, serum ferritin and diabetes in those with increased ALT, and only HOMA-IR (OR, 1.97; 95% CI, 1.2–3.7) in patients with normal ALT. Conclusion: Normal ALT is not a valuable criterion to exclude patients from liver biopsy. Alterations in glucose metabolism and insulin resistance in subjects with normal ALT should also be considered in the selection of NAFLD cases for histological assessment of disease severity and progression. (Hepatology 2008.)

Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C→G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine-to-methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real-time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8-6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis-related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04-1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12-2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09-2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes. Conclusion: In patients with NAFLD, adiponutrin rs738409 C→G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis. (Hepatology 2010;51:1209–1217)

Mutations in the hemochromatosis gene (HFE) (C282Y and H63D) lead to parenchymal iron accumulation, hemochromatosis, and liver damage. We investigated whether these factors also contribute to the progression of fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). We studied clinical, histologic (liver biopsy samples for hepatocellular iron accumulation), serologic (iron and enzyme levels), and genetic (HFE genotype) data from 587 patients from Italy with NAFLD and 184 control subjects. Iron accumulation predominantly in hepatocyes was associated with a 1.7-fold higher risk of a fibrosis stage greater than 1 (95% confidence interval [CI]: 1.2–2.3), compared with the absence of siderosis (after adjustment for age, body mass index, glucose tolerance status, and alanine aminotransferase level). Nonparenchymal/mixed siderosis was not associated with moderate/severe fibrosis (odds ratio, 0.72; 95% CI: 0.50–1.01). Hepatocellular siderosis was more prevalent in patients with HFE mutations than in those without; approximately one third of patients with HFE mutations had parenchymal iron accumulation (range, 29.8%–35.7%, depending on HFE genotype). Predominantly hepatocellular iron accumulation occurred in 52.7% of cases of patients with HFE mutations. There was no significant association between either the presence of HFE mutations or specific HFE genotypes and the severity of liver fibrosis. Iron deposition predominantly in hepatocyes is associated with more severe liver damage in patients with NAFLD. However, HFE mutations cannot be used to identify patients with hepatocellular iron accumulation.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in children. Genetic variability, which is a main player in NAFLD, is especially characterized by polymorphisms in genes involved in the development and progression of the disease to nonalcoholic steatohepatitis (NASH). Recently, the rs738409 C>G adiponutrin/patatin-like phospholipase domain-containing 3 ( PNPLA3 ) polymorphism, which encodes the I148M protein variant in the catalytic domain, has been associated with severe steatosis, NASH, and liver fibrosis in adults. In this study, we investigated the association between the rs738409 PNPLA3 gene polymorphism and NAFLD in 149 consecutive children and adolescents (age = 6-13 years) with biopsy-proven NAFLD. We analyzed the rs738409 polymorphism by a 5′-nuclease TaqMan assay and assessed its association with NASH: 41% of the subjects with NAFLD showed heterozygosity and 15% showed homozygosity for the at-risk G allele. The rs738409 genotype did not influence the body mass, adiposity, lipid levels, or insulin resistance and was not associated with alanine aminotransferase levels. Interestingly, the rs738409 G allele was strongly associated with the severity of steatosis ( P < 0.0001), the presence of NASH ( P < 0.0001), hepatocellular ballooning ( P < 0.0001), lobular inflammation ( P < 0.0001), and the presence of fibrosis ( P = 0.01) independently of confounders. Individuals carrying two minor G alleles almost always had severe steatosis and NASH, heterozygotes were at intermediate risk, and patients negative for G alleles had milder and often uncomplicated steatosis. Conclusion: The PNPLA3 rs738409 polymorphism is associated with steatosis severity, hepatocellular ballooning, lobular inflammation, and perivenular fibrosis in pediatric NAFLD. (Hepatology 2010)

Background & Aims: Nonalcoholic fatty liver disease, which can range from fatty liver alone to nonalcoholic steatohepatitis and cirrhosis, is related to insulin resistance. Tumor necrosis factor α (TNF-α) may induce insulin resistance, and polymorphisms of its promoter have been associated with an increased release of this cytokine. We analyzed (1) the prevalence of insulin resistance, (2) the prevalence of the 238 and 308 TNF-α polymorphisms, and (3) the relationship among TNF-α polymorphisms, insulin resistance, and the occurrence of steatohepatitis in 99 patients with nonalcoholic fatty liver diagnosed by ultrasonography and confirmed by histologic analysis in the 53 who underwent biopsy. Methods: Insulin resistance was evaluated by the homeostatic metabolic assessment insulin resistance indices and TNF-α polymorphisms by polymerase chain reaction and restriction fragment length polymorphism analysis. Results: Insulin resistance was detected in almost all of the patients and was more severe in those with steatohepatitis. The prevalence of the 238, but not of the 308, TNF-α polymorphism was higher in subjects with nonalcoholic fatty liver than in controls (31% vs. 15%; P < 0.0001), and patients positive for TNF-α polymorphisms had higher insulin resistance indices, a higher prevalence of impaired glucose tolerance, and a lower number of associated risk factors for steatosis. Conclusions: TNF-α polymorphisms could represent a susceptibility genotype for insulin resistance, nonalcoholic fatty liver, and steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD), the most common of chronic liver disease in Western Country, is closely related to insulin resistance and oxidative stress and includes a wide spectrum of liver diseases ranging from steatosis alone, usually a benign and non-progressive condition, to nonalcoholic steatohepatitis (NASH), which may progress to liver fibrosis and cirrhosis. NAFLD is considered the hepatic manifestation of the metabolic syndrome with which shares several characteristics, however recent data suggest that NAFLD is linked to increased cardiovascular risk independently of the broad spectrum of risk factors of metabolic syndrome. Accumulating evidence suggests that the clinical burden of NAFLD is not restricted to liver-related morbidity and mortality, with the majority of deaths in NAFLD patients related to cardiovascular disease and cancer and not to the progression of liver disease. Retrospective and prospective studies provide evidence of a strong association between NAFLD and subclinical manifestation of atherosclerosis (increased intima-media thickness, endothelial dysfunction, arterial stiffness, impaired left ventricular function and coronary calcification). A general agreement emerging from these studies indicates that patients with NASH are at higher risk of cardiovascular diseases than those with simple steatosis, emphasizing the role of chronic inflammation in the pathogenesis of atherosclerosis of these patients. It is very likely that the different mechanisms involved in the pathogenesis of atherosclerosis in patients with NAFLD have a different relevance in the patients according to individual genetic background. In conclusion, in the presence of NAFLD patients should undergo a complete cardiovascular evaluation to prevent future atherosclerotic complications. Specific life-style modification and aggressive pharmaceutical modification will not only reduce the progression of liver disease, but also reduce morbidity for cardiovascular disease improving overall prognosis and survival.