Novel genetic causes of Familial Intrahepatic Cholestasis (FIC): new genes and variants

Familial Intrahepatic Cholestasis (FIC) is a group of rare genetic disorders characterized by bile flow impairment. Clinical phenotypes range from Progressive Familial Intrahepatic Cholestasis (PFIC), characterized by pediatric onset and a more severe phenotype, to Adult-Onset Cholestatic Disease (AOCD), which encompasses a variety of cholestatic manifestations presenting in the adult population with variable severity and progression. 13 PFIC types have been currently described, caused by mutations in genes involved in major processes of bile handling, and over the years mutations in the same PFIC-associated genes have been also often identified in patients with AOCD. A non-negligible portion of cases still eludes genetic diagnosis, and novel genetic etiologies continue to be discovered, providing additional molecular diagnoses and trying to fill this gap in disease understanding. The aim of this project is to identify novel causative genes of FIC, understand the molecular mechanisms by which these genes are involved in the development of the disease, and broaden the range of clinical phenotypes related to cholestasis. 5 patients with different cholestatic phenotypes and genetic backgrounds were selected among the study cohort of patients with cholestasis. All presented with no mutations in cholestasis-associated genes. Genomic DNA isolated from blood was subjected to WES and bioinformatic analysis to identify putative disease-causing mutations. Sanger sequencing was performed to confirm mutations and assess segregation within the family. Identified mutations were further analyzed through appropriate functional tests to evaluate their pathogenic effect. 5 novel PLEC variants were identified in 3 unrelated patients. Patient 1, a young woman with recurrent cholestasis since adolescence, carried compound heterozygous variants, and immunofluorescence staining revealed a significantly reduced and abnormal expression of PLEC, K8 and BSEP proteins. Patient 2, an adult woman with a milder and later-onset cholestasis, harbored in cis heterozygous variants, and liver biopsy exhibited less pronounced protein alterations. Patient 3, a young man with severe transient cholestasis triggered by an environmental factor, presented one heterozygous variant. Functional studies demonstrated specific patterns of mislocalization and architectural disruption that correlate with clinical severity and variant configuration, offering mechanistic insights into how PLEC variants may lead to cholestasis and providing biological context for variant interpretation beyond computational predictions. These findings represent the first evidence linking heterozygous PLEC variants to adult-onset cholestasis and establish a dose-dependent model of PLEC-associated disease. Furthermore, 2 novel NEK8 variants were described in one patient with severe neonatal-onset cholestasis, and functional studies demonstrated a strong negative impact of the variants on kinase activity, supporting a potential role in the development of cholestasis and extending the range of clinical phenotypes associated with NEK8 mutations. Finally, one heterozygous variant in the LSR gene was found in patient 5, an adult man who developed acute cholestatic liver injury after treatment with valproic acid. TEM analysis on liver tissue revealed widened intracellular spaces and disrupted morphology of junctional complexes, consistent with impaired integrity of tricellular tight junctions. This represents the first case linking heterozygous LSR variants to DIC susceptibility, suggesting a model wherein genetic predisposition synergizes with environmental triggers to precipitate clinical cholestasis. In conclusion, this study provided significant insights on genetic and molecular characterization of FIC, presenting evidence for both pediatric and adult-onset cholestatic manifestations.