From Environment to Genome and Back : a Lesson from HFE Mutations

The environment and the human genome are closely entangled and many genetic variations that occur in human populations are the result of adaptive selection to ancestral environmental (mainly dietary) conditions. However, the selected mutations may become maladaptive when environmental conditions change, thus becoming candidates for diseases. Hereditary hemochromatosis (HH) is a potentially lethal disease leading to iron accumulation mostly due to mutations in the HFE gene. Indeed, homozygosity for the C282Y HFE mutation is associated with the primary iron overload phenotype. However, both penetrance of the C282Y variant and the clinical manifestation of the disease are extremely variable, suggesting that other genetic, epigenetic and environmental factors play a role in the development of HH, as well as, and in its progression to end-stage liver diseases. Alcohol consumption and dietary habits may impact on the phenotypic expression of HFE-related hemochromatosis. Indeed, dietary components and bioactive molecules can affect iron status both directly by modulating its absorption during digestion and indirectly by the epigenetic modification of genes involved in its uptake, storage and recycling. Thus, the premise of this review is to discuss how environmental pressures led to the selection of HFE mutations and whether nutritional and lifestyle interventions may exert beneficial effects on HH outcomes and comorbidities

UN¿AUMENTATA ESPRESSIONE DEL SUBSTRATO DEL RECETTORE DELL¿INSULINA 2 (IRS-2) È ASSOCIATA ALLA STEATOEPATITE E AL DISMETABOLISMO LIPIDICO IN PAZIENTI AFFETTI DA OBESITÀ GRAVE.

Design: Studio retrospettivo osservazionale. Soggetti: Abbiamo considerato 71 soggetti obesi (età compresa tra 20 e 68 anni; BMI>40 kg/m2 or BMI>35 kg/m2 in presenza di complicazioni metaboliche) classificati in tre gruppi secondo l’istologia epatica: controlli (n=12), steatosi semplice(n=27), e steatoepatite non alcolica (NASH; n=32). Abbiamo valutato i punti chiave del signalling insulinico e l’espressione genica delle molecole implicate nel pathway glucoregolatorio e nella DNL insulino dipendenti mediante PCR quantitativa real-time e Western blotting. RIASSUNTO 5 Risultati: I pazienti con steatosi semplice mostrano una ridotta fosforilazione della chinasi AKT1, responsabile della trasduzione del signalling insulinico, con conseguente sostenuta attività del fattore di trascrizione FOXO1 che media l’insulino resistenza a livello trascrizionale. Nonostante nessuna variazione significativa dell’espressione di insulin receptor substrate 1 (IRS1), i livelli proteici e di mRNA di IRS2, target di FOXO1, aumentano progressivamente con la severità della steatosi dai controlli alla NASH. L’espressione di IRS2 è correlata con la severità della steatosi, l’insulino resistenza e la dislipidemia. Nei pazienti con NASH, l’upregolazione di IRS2 è associata alla preservata attività di AKT2, che è il mediatore gli effetti stimolanti di insulina sulla DNL, e all’overespressione del suo target sterol regulatory element binding protein 1c (SREBP1c), che induce DNL a livello trascrizionale. L’overespressione sia di FOXO1 che di SREBP1c convergono sull’upregolazione della glucochinasi, che fornisce substrati alla DNL, nei pazienti con NASH. Conclusioni: La regolazione differenziale di IRS1 e IRS2 e dei loro effettori a valle AKT1 e AKT2 è coerente con l’upregolazione di FOXO1 e potrebbe giustificare lo stato paradossale di insulino resistenza a carico del pathway glucoregolatorio e l’aumentata insulino sensibilità di quello liporegolatorio tipico della steatosi e della dislipidemia in pazienti obesi con sindrome metabolica.Abstract Increased insulin receptor substrate 2 expression is associated with steatohepatitis and altered lipid metabolism in obese subjects Objective: The aim of this study was to evaluate whether dysregulation of molecules involved in FOXO1 dependent insulin signalling in the liver is associated with de novo lipogenesis (DNL) and altered lipid metabolism in severely obese subjects. Design: Observational retrospective study. Subjects: We considered 71 obese subjects (age 20-68 years; BMI>40 kg/m2 or BMI>35 kg/m2 in the presence of metabolic complications) classified into three groups according to liver histology: normal liver (n=12), simple steatosis (n=27), and non-alcoholic steatohepatitis (NASH; n=32). Key nodes in insulin signalling and gene expression of molecules implicated in insulin dependent glucoregulatory pathway and DNL were evaluated by quantitative real-time PCR and Western blotting. Results: Patients with steatosis had decreased phosphorylation of the insulin kinase AKT1, mediating insulin receptor signalling, and the transcription factor FOXO1, which was therefore more active mediating insulin resistance at transcriptional level. Despite no changes in insulin receptor substrate (IRS)1 mRNA levels, the mRNA and protein levels of the FOXO1 target IRS2 increased progressively with the severity of steatosis from normal liver to NASH. IRS2 expression was correlated with the severity of steatosis, dyslipidemia, and liver damage. In patients with NASH, upregulation of IRS2 was associated with preserved activation of AKT2, mediating the stimulating effect of insulin on DNL, and over-expression of its target sterol regulatory element binding protein 1c (SREBP1c), inducing DNL at transcriptional level. Both FOXO1 and SREBP1c overexpression converged on upregulation of glucokinase, providing substrates for DNL, in NASH patients. Conclusion: Differential regulation of IRS1 and IRS2 and of their downstream effectors AKT1 and AKT2 is consistent with upregulation of FOXO1 and may justify the paradoxical state of insulin resistance relative to the glucoregulatory pathway and augmented insulin sensitivity of the liporegulatory pathway typical of steatosis and the metabolic syndrome in obese patients

The role of insulin resistance in nonalcoholic steatohepatitis and liver disease development : a potential therapeutic target?

Insulin resistance (IR) is defined by the inability of insulin to exert its metabolic actions, due to impaired activation of intracellular insulin signaling. This condition is caused by genetic defects or by environmental conditions, among which the most common is obesity. Systemic IR determines the development of hepatic fat accumulation, which can progress to nonalcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma, and is a major determinant of liver disease independently of coexisting factors. Therefore, insulin-sensitizing drugs are currently under evaluation to improve steatohepatitis. Indeed, manipulation of nuclear hormone receptors is already under scrutiny for liver disease prevention by amelioration of IR, whereas NOTCH signaling inhibition represents a novel approach. Nevertheless, further research is warranted to better understand the mechanism linking IR to progressive fibrogenesis in the absence of inflammation and to identify novel drug targets

Virtualization of set-top-box devices in next generation SDN-NFV networks: the INPUT project perspective

Due to the emergence of Software Defined Networking (SDN) and Network Functions Virtualization (NFV) paradigms, coupled with a hyper-connectivity communication paradigm, the \u201csoftwarisation\u201d of the Internet infrastructure and of its network management framework is gaining increasing popularity. This is the target of the INPUT platform, a novel infrastructure and paradigm supporting Future Internet personal cloud services in a more scalable and sustainable way, and with innovative addedvalue capabilities. The INPUT technologies enable next-generation cloud applications to go beyond classical service models, and even replace physical Smart Devices, usually placed in users\u2019 homes (e.g., set-top boxes), with their virtual images, providing them to users \u201cas a Service\u201d. In this paper we present the Virtual set-top box from both architectural and functional points of view, demonstrating the feasibility of the softwarized SDN/NFV paradigm joined with the fog-computing approach to support personal cloud services

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

A novel alpha1-antitrypsin null variant (PiQ0Milano)

Alpha1-antitrypsin deficiency is an autosomal recessive disease characterized by reduced serum levels of alpha1-antitrypsin (AAT) due to mutations in the SERPINA1 gene causing early onset pulmonary emphysema and, occasionally, chronic liver disease. We report an incidental finding of a novel null AAT allele, Q0Milano, consisting of a 17 nucleotides deletion in exon 3 of SERPINA1 gene, in an Italian child with persistently increased liver enzymes, a mild decrease in circulating AAT levels and without any pulmonary disease. Q0Milano variant results in an unfunctional protein lacking of AAT active site, as the resultant protein is truncated near PiS locus involved in AAT protein stability

Patatin-like phospholipase domain containing-3 gene I148M polymorphism, steatosis, and liver damage in hereditary hemochromatosis

AIM: To investigate whether the patatin-like phospholipase domain containing-3 gene (PNPLA3) I148M polymorphism is associated with steatosis, fibrosis stage, and cirrhosis in hereditary hemochromatosis (HH). METHODS: We studied 174 consecutive unrelated homozygous for the C282Y HFE mutation of HH (C282Y+/+ HH) patients from Northern Italy, for whom the presence of cirrhosis could be determined based on histological or clinical criteria, without excessive alcohol intake (< 30/20 g/d in males or females) or hepatitis B virus and hepatitis C virus viral hepatitis. Steatosis was evaluated in 123 patients by histology (n = 100) or ultrasound (n = 23). The PNPLA3 rs738409 single nucleotide polymorphism, encoding for the p.148M protein variant, was genotyped by a Taqman assay (assay on demand, Applied Biosystems). The association of the PNPLA3 I148M protein variant (p.I148M) with steatosis, fibrosis stage, and cirrhosis was evaluated by logistic regression analysis. RESULTS: PNPLA3 genotype was not associated with metabolic parameters, including body mass index (BMI), the presence of diabetes, and lipid levels, but the presence of the p.148M variant at risk was independently associated with steatosis [odds ratio (OR) 1.84 per p.148M allele, 95% confidence interval (CI): 1.05-3.31; P = 0.037], independently of BMI and alanine aminotransaminase (ALT) levels. The p.148M variant was also associated with higher aspartate aminotransferase (P = 0.0014) and ALT levels (P = 0.017) at diagnosis, independently of BMI and the severity of iron overload. In patients with liver biopsy, the 148M variant was independently associated with the severity (stage) of fibrosis (estimated coefficient 0.56 \ub1 0.27, P = 0.041). In the overall series of patients, the p.148M variant was associated with cirrhosis in lean (P = 0.049), but not in overweight patients (P = not significant). At logistic regression analysis, cirrhosis was associated with BMI 65 25 (OR 1.82, 95% CI: 1.02-3.55), ferritin > 1000 ng/mL at diagnosis (OR 19.3, 95% CI: 5.3-125), and with the G allele in patients with BMI < 25 (OR 3.26, 95% CI: 1.3-10.3). CONCLUSION: The PNPLA3 I148M polymorphism may represent a permissive factor for fibrosis progression in patients with C282Y+/+ HH

The A736V TMPRSS6 Polymorphism Influences Hepatic Iron Overload in Nonalcoholic Fatty Liver Disease

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\u200a=\u200a0.01), and ferritin levels (median 223 IQR 102-449 vs. 308 IQR 141-618 ng/ml; p\u200a=\u200a0.01). Homozygosity for TMPRSS6 p.736Val was nearly associated with lower ballooning (p\u200a=\u200a0.05), 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\u200a=\u200a0.01), 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

The TM6SF2 E167K genetic variant induces lipid biosynthesis and reduces apolipoprotein B secretion in human hepatic 3D spheroids

There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human in vitro disease model to understand mechanisms linked to genetic risk variants associated with NAFLD. The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD

The i148m Pnpla3 polymorphism influences serum adiponectin in patients with fatty liver and healthy controls

BACKGROUND: Reduced adiponectin is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), and the I148M Patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism predisposes to NAFLD and liver damage progression in NASH and chronic hepatitis C (CHC) by still undefined mechanisms, possibly involving regulation of adipose tissue function. Aim of this study was to evaluate whether the I148M PNPLA3 polymorphism influences serum adiponectin in liver diseases and healthy controls. METHODS: To this end, we considered 144 consecutive Italian patients with NAFLD, 261 with CHC, 35 severely obese subjects, and 257 healthy controls with very low probability of steatosis, all with complete clinical and genetic characterization, including adiponectin (ADIPOQ) genotype. PNPLA3 rs738409 (I148M) and ADIPOQ genotypes were evaluated by Taqman assays, serum adiponectin by ELISA. Adiponectin mRNA levels were evaluated by quantitative real-time PCR in the visceral adipose tissue (VAT) of 35 obese subjects undergoing bariatric surgery. RESULTS: Adiponectin levels were independently associated with the risk of NAFLD and with the histological severity of the disease. Adiponectin levels decreased with the number of 148\u2009M PNPLA3 alleles at risk of NASH both in patients with NAFLD (p\u2009=\u20090.03), and in healthy subjects (p\u2009=\u20090.04). At multivariate analysis, PNPLA3 148\u2009M alleles were associated with low adiponectin levels (<6\u2009mg/ml, median value) independently of NAFLD diagnosis, age, gender, BMI, and ADIPOQ genotype (OR 1.67, 95% c.i. 1.07-2.1 for each 148\u2009M allele). The p.148\u2009M PNPLA3 variant was associated with decreased adiponectin mRNA levels in the VAT of obese patients (p\u2009<\u20090.05) even in the absence of NASH. In contrast, in CHC, characterized by adiponectin resistance, low adiponectin was associated with male gender and steatosis, but not with PNPLA3 and ADIPOQ genotypes and viral features. CONCLUSIONS: The I148M PNPLA3 variant is associated with adiponectin levels in patients with NAFLD and in healthy subjects, but in the presence of adiponectin resistance not in CHC patients. The I148M PNPLA3 genotype may represent a genetic determinant of serum adiponectin levels. Modulation of serum adiponectin might be involved in mediating the susceptibility to steatosis, NASH, and hepatocellular carcinoma in carriers of the 148\u2009M PNPLA3 variant without CHC, with potential therapeutic implications