Comparison of the severity of metabolic, liver and cardiovascular damage in NAFLD patients attending the hepatology clinic over the last three decades.

BACKGROUND AND AIMS: NAFLD prevalence is increasing worldwide. AIM: to assess whether severity of hepatic, metabolic and cardiovascular (CV) disease changed over time. METHODS: 422 NAFLD patients (388 biopsy proven and 34 clinical cirrhosis) diagnosed between 1990 and 2021 and subdivided according to decade of presentation. Metabolic parameters, early atherosclerosis (carotid plaques at Doppler ultrasound), severity of liver damage (NAS score, NASH, significant fibrosis (≥2) and cirrhosis) and PNPLA3 genotyping were assessed. RESULTS: No difference in age, sex and prevalence of dyslipidemia and hypertension was found across decades (p for trend), whereas a higher prevalence of diabetes (p = 0.02), obesity (p<0.001), histological severe steatosis (p<0.001), NASH (p<0.001), fibrosis ≥2 (p<0.001), cirrhosis (p<0.001) and carotid plaques (p = 0.05) was observed in the last decade compared to the others. A higher prevalence of PNPLA3 GG polymorphism was found over time (p = 0.02). In the whole cohort, age, metabolic alterations and PNPLA3 G homozygosity were independent risk factors for hepatic fibrosis and carotid plaques, independently of the decade considered. CONCLUSION: Over the past 10 years compared to previous decades, NAFLD patients presented to observation with more severe liver disease and subclinical atherosclerosis, paralleling the spread of diabetes and obesity. PNPLA3 unfavorable genotype became more prevalent over time.

Juvenile hemochromatosis associated with heterozygosity for novel hemojuvelin mutations and with unknown cofactors.

BACKGROUND & AIMS: Juvenile hemochromatosis (JH) is a rare autosomal recessive disorder characterized by severe early-onset iron overload, caused by mutations in hemojuvelin (HJV), hepcidin (HAMP), or a combination of genes regulating iron metabolism. Here we describe two JH cases associated with simple heterozygosity for novel HJV mutations and unknown genetic factors. Case 1: A 12 year-old male from Central Italy with beta-thalassemia trait, increased aminotransferases, ferritin 9035 ng/ml and transferrin saturation 84%, massive hepatocellular siderosis and hepatic bridging fibrosis. Case 2: A 12 year-old female from Northern Italy with ferritin 467 ng/ml, transferrin saturation 87-95%, and moderate hepatic iron overload. MATERIAL AND METHODS: Direct sequencing of hemochromatosis genes (HFE-TfR2-HJV-HAMP-FPN-1) was performed in the children and siblings. RESULTS: In case 1, we detected heterozygosity for a novel HJV mutation (g.3659_3660insG), which was inherited together with the beta thalassemia trait from the father, who (as well as the mother) had normal iron parameters. In case 2, we detected another novel HJV mutation (g.2297delC) in heterozygosity, which was inherited from the mother, affected by mild iron deficiency. The father had normal iron stores. Both mutations are frameshifts determining premature stop codons. No other disease causing variant was detected. CONCLUSION: Although beta-thalassemia trait was a possible cofactor of iron overload in case 1, iron overload cannot be explained by simple heterozygosity for HJV mutations in both cases. Other genetic factors should be investigated, and further studies are needed to understand genotype-phenotype correlations in JH.

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.

A randomized trial of iron depletion in patients with nonalcoholic fatty liver disease and hyperferritinemia.

AIM: To compare iron depletion to lifestyle changes alone in patients with severe nonalcoholic fatty liver disease (NAFLD) and hyperferritinemia, a frequent feature associated with more severe liver damage, despite at least 6 mo of lifestyle changes. METHODS: Eligible subjects had to be 18-75 years old who underwent liver biopsy for ultrasonographically detected liver steatosis and hyperferritinemia, ferritin levels ≥ 250 ng/mL, and NAFLD activity score > 1. Iron depletion had to be achieved by removing 350 cc of blood every 10-15 d according to baseline hemoglobin values and venesection tolerance, until ferritin < 30 ng/mL and/or transferrin saturation (TS) < 25%. Thirty-eight patients were randomized 1:1 to phlebotomy (n = 21) or lifestyle changes alone (n = 17). The main outcome of the study was improvement in liver damage according to the NAFLD activity score at 2 years, secondary outcomes were improvements in liver enzymes [alanine aminotransferases (ALT), aspartate aminotransferase (AST), and gamma-glutamyl-transferases (GGT)]. RESULTS: Phlebotomy was associated with normalization of iron parameters without adverse events. In the 21 patients compliant to the study protocol, the rate of histological improvement was higher in iron depleted vs control subjects (8/12, 67% vs 2/9, 22%, P = 0.039). There was a better improvement in steatosis grade in iron depleted vs control patients (P = 0.02). In patients followed-up at two years (n = 35), ALT, AST, and GGT levels were lower in iron-depleted than in control patients (P < 0.05). The prevalence of subjects with improvement in histological damage or, in the absence of liver biopsy, ALT decrease ≥ 20% (associated with histological improvement in biopsied patients) was higher in the phlebotomy than in the control arm (P = 0.022). The effect of iron depletion on liver damage improvement as assessed by histology or ALT decrease ≥ 20% was independent of baseline AST/ALT ratio and insulin resistance (P = 0.0001). CONCLUSION: Iron depletion by phlebotomy is likely associated with a higher rate of improvement of histological liver damage than lifestyle changes alone in patients with NAFLD and hyperferritinemia, and with amelioration of liver enzymes.

The A736V TMPRSS6 polymorphism influences hepatic iron overload in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Hepatic iron accumulation due to altered trafficking is frequent in patients with nonalcoholic fatty liver disease (NAFLD), and is associated with more severe liver damage and hepatocellular carcinoma. The p.Ala736Val TMPRSS6 variant influences iron metabolism regulating the transcription of the hepatic hormone hepcidin, but its role in the pathogenesis of iron overload disorders is controversial. Aim of this study was to evaluate the whether the TMPRSS6 p.Ala736Val variant influences hepatic iron accumulation in a well-characterized series of Italian patients with histological NAFLD. METHODS: 216 patients with histological NAFLD. TMPRSS6 and HFE variants were assessed by allele specific PCR, liver histology by the NAFLD activity score and Perls' staining for iron. RESULTS: Homozygosity for the p.736Val allele previously linked to higher hepcidin did not influence transferrin saturation (TS), but was associated with lower hepatic iron stores (p = 0.01), and ferritin levels (median 223 IQR 102-449 vs. 308 IQR 141-618 ng/ml; p = 0.01). Homozygosity for TMPRSS6 p.736Val was nearly associated with lower ballooning (p = 0.05), reflecting hepatocellular damage related to oxidative stress. The influence of TMPRSS6 on hepatic iron accumulation was more marked in patients negative for HFE genotypes predisposing to iron overload (p.Cys282Tyr + and p.His63Asp +/+; p = 0.01), and the p.736Val variant was negatively associated with hepatic iron accumulation independently of age, gender, HFE genotype, and beta-thalassemia trait (OR 0.59, 0.39-0.88). CONCLUSIONS: The p.Ala736Val TMPRSS6 variant influences secondary hepatic iron accumulation in patients with NAFLD.

IL28B, HCV core mutations, and hepatocellular carcinoma: does host genetic make-up shape viral evolution in response to immunity?

Mutations in the core sequence of the HCV genome have been reported to influence treatment response, fibrosis progression, and hepatocarcinogenesis in Asian patients with genotype-1 chronic hepatitis C (CHC). In this issue, Miura et al. report data consistent with a causal relationship between the R70 â†’ Q70 core variant and hepatocellular carcinoma (HCC) risk in CHC genotype-1b patients, by the prospective evaluation of changes in the consensus sequence in the entire open reading frame between treatment failure and HCC development or end of follow-up, and validation of the initial findings in a confirmatory cohort. Furthermore, they observed an association between the IL28B genotype, which is believed to influence the immune response to viral infection, and the direction of time-dependent changes in core residue 70, with unfavorable IL28B genotypes linked to a preferential shift to the 70Q associated with HCC. Although this association needs to be validated in independent cohorts, and IL28B variants did not influence HCC risk, these results suggest that IL28B genotype might not only influence the behavior of the innate immune system in the presence of HCV genotype-1 infection but also shape the resultant viral evolution, with possible consequences on major clinical outcomes, such as HCC development, and treatment response.

Unusual presentation in a case of primary hyperparathyroidism.

This report describes a case of classic severe primary hyperparathyroidism (PH) with clinical presentation that is very infrequent nowadays, which was osteitis fibrosa cystica. As bone scintigraphy demonstrated multiple areas of increasing uptake associated with hypercalcemia, a thorough investigation was conducted to exclude the neoplasms which most frequently are responsible for bone secondarisms. A fludeoxyglucose (FDG) positron emission tomography/CT demonstrated diffuse and multiple foci of increased FDG uptake and a focal uptake at the left thyroid region. Parathyroid function was studied, revealing unexpectedly high parathyroid hormone (PTH) levels. Further tests confirmed the diagnosis of PH and localized a parathyroid adenoma in the lower left side.

The hand arthropathy of hereditary hemochromatosis is strongly associated with iron overload.

OBJECTIVE: To analyze the clinical characteristics and genetic background associated with the presence of hand arthropathy, as determined by radiological findings, in Italian patients with hereditary hemochromatosis (HHC). METHODS: In 88 consecutive unselected patients with phenotypically expressed HHC, joint involvement was systematically evaluated in plain radiographs of hands, wrists, lumbar spine, pelvis, and knees. Risk factors considered were age, sex, body mass index, alcohol abuse, organ involvement at other sites, and indices of iron overload, including ferritin, transferrin saturation, and iron removed to reach depletion. HFE genotype was also considered. The independent role of risk factors was tested by logistic regression analysis. RESULTS: Thirty-two subjects (36%) showed signs of metacarpophalangeal (MCP) arthropathy. Intercarpal, radiocarpal, and chondrocalcinosis were less frequent and occurred in association with MCP arthropathy. At multivariate analysis MCP arthropathy was independently associated with older age [odds ratio (OR) 1.20, 95% confidence interval (CI) 1.1-1.33/yr; p = 0.0001], higher ferritin levels at diagnosis (OR 4.17, 95% CI 1.60-13.9 for values > 1000 ng/ml; p = 0.008), the presence of the C282Y +/+ and C282Y/H63D HFE genotypes (OR 2.69, 95% CI 1.09-7.87; p = 0.04), and higher percentage transferrin saturation (OR 1.05, 95% CI 1-1.1; p = 0.05). The severity of arthropathy was independently associated with older age (p = 0.03) and higher ferritin values (p = 0.05). CONCLUSION: MCP arthropathy together with a typical pattern of joint involvement is observed in one-third of unselected patients with HHC, and is influenced by the duration and degree of the iron overload.

Relative contribution of iron genes, dysmetabolism and hepatitis C virus (HCV) in the pathogenesis of altered iron regulation in HCV chronic hepatitis.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) chronic hepatitis predisposes to iron overload, which negatively influences the prognosis of this infection. Since the underlying mechanisms of this iron overload are undefined, we analyzed the prevalence of altered iron parameters, and the relative contribution of viral, metabolic, and genetic factors in Italian patients. DESIGN AND METHODS: We studied the metabolic and biochemical characteristics of 143 previously untreated, biopsied patients with HCV who were not alcohol abusers. Hepatic iron was determined according to Deugnier, HFE genotype by restriction analysis, hepcidin, hemojuvelin, ferroportin-1, and transferrin receptor-2 mutations by denaturing high performance liquid chromatography and sequencing. RESULTS: Increased transferrin saturation was observed in 20%, hyperferritinemia in 22%, and histological iron deposition in 32% of patients. Ferritin was independently correlated with iron stores and host metabolic parameters, whereas hepatic iron deposition was correlated with ferritin and histological severity of hepatitis. Sinusoidal iron deposition was associated with metabolic alterations, including body mass index, insulin resistance, and LDL cholesterol. Conversely, the prevalence of HFE mutations and serum ferritin values increased with the severity of steatosis. The prevalence of HFE and beta-globin mutations was not different from that of controls (31% and 2%, respectively). No tranferrin receptor-2, hemojuvelin, or ferroportin-1 mutations were detected, but two patients carried the -72C>T hepcidin promoter mutation. The C282Y HFE mutation, hepcidin and beta-globin mutations influenced iron stores. Both carriers of the -72C>T Hepcidin mutation had beta-thalassemia trait, moderate iron overload, and liver cirrhosis. INTERPRETATION AND CONCLUSIONS: Iron genes influence iron overload and steatosis development, but the major burden is related to HCV itself and host metabolic factors.

TNFalpha genotype affects TNFalpha release, insulin sensitivity and the severity of liver disease in HCV chronic hepatitis.

BACKGROUND/AIMS: TNFalpha induces insulin resistance and promoter polymorphisms of TNFalpha gene affect the release of this cytokine, implicated in the pathogenesis of HCV-related diabetes and fatty liver. The aim was to define whether in patients with HCV chronic hepatitis TNFalpha genotype influences TNFalpha activity, insulin resistance, and the severity of the disease. METHODS: 186 patients, 65% with steatosis, 17% with diabetes. TNFalpha and sTNFR2 were determined by ELISA, insulin resistance by HOMA-R index and TNFalpha -238, -308, and -863 polymorphisms by restriction analysis. RESULTS: TNFalpha, sTNFR2, and insulin resistance were higher in patients than in 89 controls. TNFalpha pathway activity was correlated with LDL cholesterol, steatosis, and insulin resistance, which, in turn, was correlated with the severity of liver damage. Patients subdivided according to TNFalpha genotype significantly differed for TNFalpha release, insulin sensitivity and the prevalence of cirrhosis. The -308 and -238 TNFalpha polymorphisms, characterized by increased promoter activity, were associated with higher TNFalpha activity, insulin resistance and severity of the disease, whereas the -863 polymorphism, characterized by reduced promoter activity, with lower TNFalpha activity, and higher insulin sensitivity. CONCLUSIONS: TNFalpha genotype modulates the activity of the TNFalpha pathway, influences insulin sensitivity and the severity of HCV chronic hepatitis.