Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing.

OBJECTIVES: The aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH). DESIGN, SETTING AND SUBJECTS: A total of 77 subjects with a Dutch Lipid Clinic Network score of ≥ 3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined. RESULTS: A total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6-intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband. CONCLUSIONS: Using a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene.

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 signaling 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; body mass index (BMI)>40 kg m(-2) or BMI>35 kg m(-2) in the presence of metabolic complications) classified into three groups according to liver histology: normal liver (n=12), simple steatosis (n=27) and nonalcoholic steatohepatitis (NASH; n=32). Key nodes in insulin signaling 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 signaling, 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 overexpression 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.

Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Increased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD. METHODS AND RESULTS: Vascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, hemochromatosis gene (HFE) mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin (p=0.048). Carotid plaques were independently associated with age, ferritin, glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and serum hepcidin-25 was independently associated with carotid plaques (p=0.05). CONCLUSION: Ferritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages.

Implications of PNPLA3 polymorphism in chronic hepatitis C patients receiving peginterferon plus ribavirin.

BACKGROUND: Homozygosity for the PNPLA3 p.I148M polymorphism influences steatosis and fibrogenesis in chronic hepatitis C (CHC). AIM: To evaluate the effect of p.148M/M on sustained virological response (SVR) and viral kinetics in patients who underwent antiviral therapy with peg-interferon and ribavirin, stratified according to viral genotype and fibrosis severity, and secondarily, the interaction with interleukin-28B ( IL28B ) genotype on liver damage. METHODS: In this observational study, we considered 602 treatment-naïve consecutive patients from tertiary referral centres in Milan and Vienna [61% genotype 1 (G1), 30% advanced fibrosis, 33% IL28B rs12979860 CC]. RESULTS: The p.148M/M genotype, detected in 8% of patients, did not influence SVR in the overall series (P = 0.29), but it was associated with SVR (3/17, 17% vs. 56/121, 46%; P = 0.034) and complete early viral response (4/17, 23% vs. 68/121, 56%; P = 0.018) in G1/4 patients with advanced fibrosis. After adjustment for age, viral load, IL28B CC genotype, treatment dose, and steatosis, p.148M/M remained a predictor of SVR in G1/4 patients with advanced fibrosis (OR 0.23, 95% CI 0.04-0.87). The p.148M/M genotype was associated with more advanced fibrosis in the overall series (P = 0.049), whereas the rs12979860 IL28B CC genotype only in patients negative for p.148M/M (P = 0.017), independently of age, BMI and alanine transaminase levels (OR 1.51, 95% CI 1.01-2.27). CONCLUSIONS: PNPLA3 p.148M/M genotype was negatively associated with SVR and early viral kinetics independently of steatosis, albeit only in difficult-to-cure G1/4 patients with advanced fibrosis, whereas stratification for the p.148M/M PNPLA3 genotype unmasked an association between IL28B CC genotype and more severe liver fibrosis.