Caratterizzazione molecolare dei difetti genici associati a Colestasi intraepatica familiare progressiva mediante Next Generation Sequencing

Progressive Familial Intrahepatic Cholestasis (PFIC) is a group of autosomal recessive diseases that affects especially newborns and children, with progression to liver failure in the first decades of life. PFIC is classified into five types based on the genetic defect involved in bile transport. It is caused by homozygous or compound heterozygous mutations in ATP8B1, ABCB11, ABCB4, TJP2 and NR1H4 genes. Other benign late-onset phenotypes and non-progressive forms (BRIC, LPAC, DIC and ICP) are caused by heterozygous mutations in the same gene pattern. Other genes have been recently involved in both progressive and non-progressive forms. The aim of the project is to develop and validate a broad, reliable, rapid and cost-saving NGS genetic test for PFIC patients. 96 patients were tested for the first described genes and 80 patients were sequenced with the latest discovered candidate genes for PFIC and other related benign phenotypes. Bioinformatic and statistic pipelines were applied. A total of 184 different variants has been identified in our cohort: 18 pathogenic, 46 VUS, 44 likely benign and 76 benign. P/LP mutations were found in 12% of patients: 2 in ATP8B1, 3 in ABCB11, ABCB4 and TJP2 each, one in ABCC2, JAG1, NOTCH2. Many patients had multiple variants in several genes. Patients had from 7 to 35 variants each and some SNPs were significantly associated with biochemical parameters and phenotypic features (e.g. liver fibrosis) that could better explain clinics and accelerate the progression to liver failure. Our detection rate is according to other studies proposing multi-gene panels. Our analysis may be useful for the molecular diagnostics of PFIC and a better characterization and understanding of the linking between molecular defects and different subtypes of the disease. The high SNPs prevalence let us to hypothesize a synergistic haplotype effect in determining different multifactorial cholestasis phenotypes and overlapping features

Molecular and Clinical Links between Drug-Induced Cholestasis and Familial Intrahepatic Cholestasis

Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs

Genetics in Familial Intrahepatic Cholestasis: Clinical Patterns and Development of Liver and Biliary Cancers: A Review of the Literature

The family of inherited intrahepatic cholestasis includes autosomal recessive cholestatic rare diseases of childhood involved in bile acids secretion or bile transport defects. Specific genetic pathways potentially cause many otherwise unexplained cholestasis or hepatobiliary tumours in a healthy liver. Lately, next-generation sequencing and whole-exome sequencing have improved the diagnostic procedures of familial intrahepatic cholestasis (FIC), as well as the discovery of several genes responsible for FIC. Moreover, mutations in these genes, even in the heterozygous status, may be responsible for cryptogenic cholestasis in both young and adults. Mutations in FIC genes can influence serum and hepatic levels of bile acids. Experimental studies on the NR1H4 gene have shown that high bile acids concentrations cause excessive production of inflammatory cytokines, resistance to apoptosis, and increased cell regeneration, all risk conditions for developing hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). NR1H4 gene encodes farnesoid X-activated receptor having a pivotal role in bile salts synthesis. Moreover, HCC and CCA can emerge in patients with several FIC genes such as ABCB11, ABCB4 and TJP2. Herein, we reviewed the available data on FIC-related hepatobiliary cancers, reporting on genetics to the pathophysiology, the risk factors and the clinical presentation

Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencing

Background: Mutations in ATP-transporters ATPB81, ABCB11, and ABCB4 are responsible for progressive familial intrahepatic cholestasis (PFIC) 1, 2 and 3, and recently the gene for tight junction protein-2 (TJP2) has been linked to PFIC4. Aim: As these four genes have been poorly studied in young people and adults, we investigated them in this context here. Methods: In patients with cryptogenic cholestasis, we analyzed the presence of mutations by high-throughput sequencing. Bioinformatics analyses were performed for mechanistic and functional predictions of their consequences on biomolecular interaction interfaces. Results: Of 108 patients, 48 whose cause of cholestasis was not established were submitted to molecular analysis. Pathogenic/likely pathogenic mutations were found in ten (21%) probands for 13 mutations: two in ATP8B1, six in ABCB11, two in ABCB4, three in TJP2. We also identified seven variants of uncertain significance: two in ATP8B1, one in ABCB11, two in ABCB4 and two in TJP2. Finally, we identified 11 benign/likely benign variants. Patients with pathogenic/likely pathogenic mutations had higher levels of liver stiffness (measured by FibroScan\uc2\uae) and bile acids, as well as higher rates of cholestatic histological features, compared to the patients without at least likely pathogenic mutations. The multivariate analysis showed that itching was the only independent factor associated with disease-causing mutations (OR 5.801, 95% CI 1.244\ue2\u80\u9327.060, p = 0.025). Conclusions: Mutations in the genes responsible for PFIC may be involved in both young and adults with cryptogenic cholestasis in a considerable number of cases, including in heterozygous status. Diagnosis should always be suspected, particularly in the presence of itching

Activated FGFR2 signalling as a biomarker for selection of intrahepatic cholangiocarcinoma patients candidate to FGFR targeted therapies

Abstract FGFR inhibitors have been developed to inhibit FGFR activation and signal transduction; notwithstanding, currently the selection of intrahepatic cholangiocarcinoma (iCCA) patients for these drugs only relies on the detection of FGFR2 genetic alterations (GAs) in tumor tissues or circulating tumor DNAs, without concomitant assessment of FGFR2 signalling status. Accordingly, we performed multi-omic analyses of FGFR2 genes and FGFR2 signalling molecules in the tissue samples from 36 iCCA naïve patients. Gain-of-function FGFR2 GAs were detected in 7 patients, including missense mutations (n = 3; p.F276C, p.C382R and p.Y375C), translocations (n = 1) and copy number gain (n = 4; CNV ≥ 4). In contrast, among 29 patients with wild-type FGFR2, 4 cases showed activation of FGFR2 signalling, as they expressed the FGFR2 ligand FGF10 and phosphorylated FGFR2/FRS2α proteins; the remaining 25 cases resulted negative for activated FGFR2 signalling, as they lacked FGFR2 (n = 8) or phosphorylated FRS2α (n = 17) expression. Overall, we found that activation of FGFR2 signalling occurs not only in iCCA naïve patients with FGFR2 GAs, but also in a subgroup carrying wild-type FGFR2. This last finding entails that also this setting of patients could benefit from FGFR targeted therapies, widening indication of these drugs for iCCA patients beyond current approval. Future clinical studies are therefore encouraged to confirm this hypothesis

Genetic Variants Within Key Nodes of the Cascade of Antipsychotic Mechanisms: Effects on Antipsychotic Response and Schizophrenia Psychopathology in a Naturalistic Treatment Setting in Two Independent Korean and Italian Samples

Introduction: Schizophrenia (SCZ) is one of the most disabling psychiatric disorders. Genetic factors play an important role in both SCZ liability and its treatment outcome. In the present paper, we investigated the effects of several single nucleotide polymorphisms (SNPs) within ten strong candidate genes involved with antipsychotics (APs) mechanisms of action. Methods: Two independent samples were investigated in the present study. Totals of 176 SCZ subjects and 326 controls of Korean ancestry, and 83 SCZ subjects and 194 controls of Italian ancestry were recruited and genotyped. SCZ risk and other parameters were also investigated. Results: Concerning APs response, only a nominal association with HOMER1 rs3822568 in the Korean sample was found. In the haplotype analysis, rs9801117 C\ue2\u80\u93rs12668837 C\ue2\u80\u93rs4621754 A haplotype within ESYT2 and NCAPG2 genes was associated with APs response in the same sample. As for secondary outcomes, rs7439 within PKDCC and rs12668837 within NCAPG2 were associated with SCZ risk in the Italian sample. In the haplotype analysis, rs2788478 G\ue2\u80\u93rs2657375 T\ue2\u80\u93rs1039621 A within the region between WDR60 and ESYT genes and rs2013 C (ESYT2)\ue2\u80\u93rs6459896 A (NCAPG2) haplotypes were associated with SCZ in the same sample. No association was found in the Korean sample. Finally, our exploratory data suggest a possible modulation of HOMER1, ARC, BDNF, TXNRD2, WDR60, and ESYT2 genes in the APs response to specific symptom clusters. Conclusion: Our results did not support a primary role for the genes investigated in the APs response. On the other hand, our secondary results suggest a possible involvement of NACPG2 and PKDCC in SCZ liability. Finally, our exploratory findings may deserve further investigations in specific studies

Genetic Variants Within Key Nodes of the Cascade of Antipsychotic Mechanisms: Effects on Antipsychotic Response and Schizophrenia Psychopathology in a Naturalistic Treatment Setting in Two Independent Korean and Italian Samples

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Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

BackgroundWe previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15-20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in similar to 80% of cases.MethodsWe report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded.ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5-528.7, P=1.1x10(-4)) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70[95%CI 1.3-8.2], P=2.1x10(-4)). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR=19.65[95%CI 2.1-2635.4], P=3.4x10(-3)), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR=4.40[9%CI 2.3-8.4], P=7.7x10(-8)). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10(-5)).ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old