Serena Pelusi

Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease

Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very‐low‐density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more‐severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23‐2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20‐3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25‐0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39‐0.95). Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver. (Hepatology 2015;61:506‐514)

LPIN1 rs13412852 polymorphism in pediatric nonalcoholic fatty liver disease.

Objectives: The aim of the present study was to evaluate whether the lipin1 rs13412852 C>T polymorphism is associated with nonalcoholic steatohepatitis and fibrosis in pediatric Italian patients with nonalcoholic fatty liver disease (NAFLD). Methods: A total of 142 untreated, consecutive children and 115 adults with biopsy-proven NAFLD and 337 healthy controls without steatosis were studied. Liver histology was assessed by the NAFLD activity score and the rs13412852 polymorphism by a 5′ nuclease Taqman assay. Results: Homozygosity for the rs13412852 T allele was underrepresented in pediatric, but not adult, patients with NAFLD compared with healthy controls (7% vs 14%; odds ratio [OR] 0.58, 95% confidence interval [CI] 0.35–0.91), and it was associated with lower triglycerides both in pediatric patients and healthy controls (P ⩽ 0.01). Affected children carrying the rs13412852 TT genotype had a trend for a lower prevalence of nonalcoholic steatohepatitis, and significantly less severe liver damage, as indicated by NAFLD activity score severity (P = 0.026) and a lower prevalence of liver fibrosis (P = 0.012). The negative association between rs13412852 TT genotype and fibrosis was independent of Patatin-like phospholipase domain-containing-3 genotype and other clinical risk factors, including age, waist circumference, the presence of hyperglycemia, and alanine transaminase levels (OR 0.29; 95% CI 0.11–0.66), and it was confirmed at multivariate analysis in adults (OR 0.15; 95% CI 0.02–0.67). Conclusions: Lipin1 rs13412852 single nucleotide polymorphism is associated with the severity of liver damage and fibrosis progression in pediatric patients with histological NAFLD.

Risk of Obstructive Sleep Apnea with Daytime Sleepiness Is Associated with Liver Damage in Non-Morbidly Obese Patients with Nonalcoholic Fatty Liver Disease

Background A high prevalence of obstructive sleep apnea syndrome (OSAS) has been reported in severely obese patients with nonalcoholic fatty liver disease (NAFLD), but few studies have evaluated OSAS in non-morbidly obese NAFLD patients. Aims To determine the prevalence of risk for OSAS with or without daytime sleepiness in non-morbidly obese patients with NAFLD and evaluate the association with the severity of liver damage. Methods We considered 159 consecutive patients with histological NAFLD and body mass index (BMI) <35 Kg/m2, and 80 controls without ultrasonographic steatosis matched for age, sex, and BMI. OSAS risk was determined by positivity for Berlin questionnaire (BQ), and daytime sleepiness by the Sleepness Epworth Scale (ESS). Liver damage was evaluated according to the NAFLD activity score. Results In NAFLD patients, BQ alone was positive in 39 (25%), ESS in 8 (5%), and both in 13 (8%, OSAS with sleepines); p = ns vs. controls without steatosis. In NAFLD patients at risk for OSAS with (but not in those without) sleepiness, we observed a higher prevalence of nonalcoholic steatohepatitis (NASH; 11/13, 85% vs. 72/146, 49%; p = 0.018), and of clinically significant fibrosis (stage>1; 9/13, 69% vs. 39/146, 27%; p = 0.003). At multivariate logistic regression analysis, OSAS with sleepiness was strongly associated with NASH and fibrosis>1 independently of known clinical risk factors such as age, gender, BMI, diabetes, and ALT levels (OR 7.1, 95% c.i. 1.7–51, p = 0.005 and OR 14.0, 95% c.i. 3.5–70, p = 0.0002, respectively). Conclusions A proportion of NAFLD patients without severe obesity is at risk for OSAS with daytime sleepiness, which is associated with the severity of liver damage independently of body mass and other cofactors.

Hepcidin and HFE protein: Iron metabolism as a target for the anemia of chronic kidney disease

The anemia of chronic kidney disease and hemodialysis is characterized by chronic inflammation and release of cytokines, resulting in the upregulation of the iron hormone hepcidin, also increased by iron therapy and reduced glomerular filtration, with consequent reduction in iron absorption, recycling, and availability to the erythron. This response proves advantageous in the short-term to restrain iron availability to pathogens, but ultimately leads to severe anemia, and impairs the response to erythropoietin (Epo) and iron. Homozygosity for the common C282Y and H63D HFE polymorphisms influence iron metabolism by hampering hepcidin release by hepatocytes in response to increased iron stores, thereby resulting in inadequate inhibition of the activity of Ferroportin-1, inappropriately high iron absorption and recycling, and iron overload. However, in hemodialysis patients, carriage of HFE mutations may confer an adaptive benefit by decreasing hepcidin release in response to iron infusion and inflammation, thereby improving iron availability to erythropoiesis, anemia control, the response to Epo, and possibly survival. Therefore, anti-hepcidin therapies may improve anemia management in hemodialysis. However, HFE mutations directly favor hemoglobinization independently of hepcidin, and reduce macrophages activation in response to inflammation, whereas hepcidin might also play a beneficial anti-inflammatory and anti-microbic action during sepsis, so that direct inhibition of HFE-mediated regulation of iron metabolism may represent a valuable alternative therapeutic target. Genetic studies may offer a valuable tool to test these hypotheses and guide the research of new therapies.

Hepcidin levels in chronic hemodialysis patients: a critical evaluation.

Abstract Altered systemic iron metabolism is a key element of uremia, and functional iron deficiency mainly related to subclinical inflammation makes it difficult to maintain proper control of anemia in chronic hemodialysis patients (CHD). In the last decade, the hepatic hormone hepcidin has been progressively recognized as the master regulator of circulating iron levels through the modulation of cellular iron fluxes in response to iron stores, as well as to erythroid and inflammatory stimuli. Hepcidin is cleared by the kidney and progression of renal disease has been associated to increased serum hepcidin levels. This, in turn, reduces iron availability for erythropoiesis, suggesting anti-hepcidin strategies for improving anemia control. Moreover, hepcidin has been recently implicated in the pathogenesis of long-term complications of dialysis, like accelerated atherosclerosis. Initial studies almost invariably reported a sustained increase of serum hepcidin in chronic hemodialysis patients. Noteworthy, such studies included relatively few patients and controls that were poorly matched for major determinants of serum hepcidin at population level, i.e., age and gender. More recent data based on accurately matched larger series challenge the view that hepcidin is intrinsically increased in hemodialysis patients, showing a marked inter- and intra-individual variability of hormone levels. Here we take a critical look to the data published so far on hepcidin levels in CHD, analyze the reasons underlying the discrepancies in available studies and the hepcidin variability in CHD, and point out the need for further studies in large series of well-characterized CHD patients and controls.

PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis

Abstract Liver fibrosis is a pathological scarring response to chronic hepatocellular injury and hepatic stellate cells (HSCs) are key players in this process. PNPLA3 I148M is a common variant robustly associated with liver fibrosis but the mechanisms underlying this association are unknown. We aimed to examine a) the effect of fibrogenic and proliferative stimuli on PNPLA3 levels in HSCs and b) the role of wild type and mutant PNPLA3 overexpression on markers of HSC activation and fibrosis. Here, we show that PNPLA3 is upregulated by the fibrogenic cytokine transforming growth factor-beta (TGF-β), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-β-induced reduction in lipid droplets in primary human HSCs. Furthermore, we show that retinol release from human HSCs ex vivo is lower in cells with the loss-of-function PNPLA3 148M compared with 148I wild type protein. Stable overexpression of PNPLA3 148I wild type, but not 148M mutant, in human HSCs (LX-2 cells) induces a reduction in the secretion of matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), which is mediated by retinoid metabolism. In conclusion, we show a role for PNPLA3 in HSC activation in response to fibrogenic stimuli. Moreover, we provide evidence to indicate that PNPLA3-mediated retinol release may protect against liver fibrosis by inducing a specific signature of proteins involved in extracellular matrix remodelling.

Renin-Angiotensin System Inhibitors, Type 2 Diabetes and Fibrosis Progression: An Observational Study in Patients with Nonalcoholic Fatty Liver Disease.

Background The clinical determinants of fibrosis progression in nonalcoholic fatty liver disease (NAFLD) are still under definition. Aim To assess the clinical determinants of fibrosis progression rate (FPR) in NAFLD patients with baseline and follow-up histological evaluation, with a special focus on the impact of pharmacological therapy. Methods In an observational cohort of 118 Italian patients from tertiary referral centers, liver histology was evaluated according to Kleiner. Independent predictors of FPR were selected by a stepwise regression approach. Results Median follow-up was 36 months (IQR 24–77). Twenty-five patients (18%) showed some amelioration, 63 (53%) had stability, 30 (25%) had progression of fibrosis. Patients with nonalcoholic steatohepatitis (NASH) had similar demographic and anthropometric features, but a higher prevalence of type 2 diabetes (T2D; p = 0.010), and use of renin-angiotensin axis system (RAS) inhibitors (p = 0.005). Fibrosis progression was dependent of the length of follow-up, and was associated with, but did not require, the presence of NASH (p<0.05). Both fibrosis progression and faster FPR were independently associated with higher APRI score at follow-up, absence of treatment with RAS inhibitors, and T2D diagnosis at baseline (p<0.05). There was a significant interaction between use of RAS inhibitors and T2D on FPR (p = 0.002). RAS inhibitors were associated with slower FPR in patients with (p = 0.011), but not in those without (p = NS) T2D. Conclusions NASH is not required for fibrosis progression in NAFLD, whereas T2D seems to drive fibrogenesis independently of hepatic inflammation. Use of RAS inhibitors may contrast fibrosis progression especially in high-risk patients affected by T2D.

Hepcidin resistance in dysmetabolic iron overload

Dysmetabolic iron overload syndrome (DIOS) is a frequent condition predisposing to metabolic, cardiovascular and hepatic damage, whose pathogenesis remains poorly defined. Aim of this study was to characterize iron metabolism in DIOS.

Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver

Nonalcoholic fatty liver disease is epidemiologically associated with hepatic and metabolic disorders. The aim of this study was to examine whether hepatic fat accumulation has a causal role in determining liver damage and insulin resistance.

Telomerase reverse transcriptase germline mutations and hepatocellular carcinoma in patients with nonalcoholic fatty liver disease

In an increasing proportion of cases, hepatocellular carcinoma (HCC) develops in patients with nonalcoholic fatty liver disease (NAFLD). Mutations in telomerase reverse transcriptase (hTERT) are associated with familial liver diseases. The aim of this study was to examine telomere length and germline hTERT mutations as associated with NAFLD‐HCC. In 40 patients with NAFLD‐HCC, 45 with NAFLD‐cirrhosis and 64 healthy controls, peripheral blood telomere length was evaluated by qRT‐PCR and hTERT coding regions and intron–exon boundaries sequenced. We further analyzed 78 patients affected by primary liver cancer (NAFLD‐PLC, 76 with HCC). Enrichment of rare coding mutations (allelic frequency <0.001) was evaluated by Burden test. Functional consequences were estimated in silico and by over‐expressing protein variants in HEK‐293 cells. We found that telomere length was reduced in individuals with NAFLD‐HCC versus those with cirrhosis (P = 0.048) and healthy controls (P = 0.0006), independently of age and sex. We detected an enrichment of hTERT mutations in NAFLD‐HCC, that was confirmed when we further considered a larger cohort of NAFLD‐PLC, and was more marked in female patients (P = 0.03). No mutations were found in cirrhosis and local controls, and only one in 503 healthy Europeans from the 1000 Genomes Project (allelic frequency = 0.025 vs. <0.001; P = 0.0005). Mutations with predicted functional impact, including the frameshift Glu113Argfs*79 and missense Glu668Asp, cosegregated with liver disease in two families. Three patients carried missense mutations (Ala67Val in homozygosity, Pro193Leu and His296Pro in heterozygosity) in the N‐terminal template‐binding domain (P = 0.037 for specific enrichment). Besides Glu668Asp, the Ala67Val variant resulted in reduced intracellular protein levels. In conclusion, we detected an association between shorter telomeres in peripheral blood and rare germline hTERT mutations and NAFLD‐HCC.