Genetic characterization of adult-onset cryptogenic cholestasis: a prospective multicentre study of 233 patients

Background and Aims: Inherited cholestatic liver diseases are rare disorders that impact the production or transport of bile, traditionally regarded as conditions of childhood. With the implementation of Next-Generation Sequencing (NGS) panels in clinical practice, there is growing interest in assessing the role of mutations in these genes in Adult-Onset Cryptogenic Cholestasis (AOCC) as well. We investigated a multigene panel, as these genes have been insufficiently studied in both young individuals and adults. Method: From February 2017 to May 2023, all outpatients over 14 years old with AOCC were consecutively enrolled at four tertiary referral centres in Italy. We analysed the presence of mutations through high-throughput sequencing, including 36 genes: the detected variants were classified according to the American College of Medical Genetics and Genomics criteria. Results: 233 patients with AOCC underwent molecular analysis. Among these, 108 (46.4%) were male, with a median age at presentation of 35.6 (± 15.2) years: Serum bile acids (16.2 ± 2.8 µmol/L) and alkaline phosphatase (154,6 ± 95,2 UI/L) were moderately elevated, and the patients exhibited liver stiffness consistent with mild liver fibrosis (6.7 ± 2.4 kPa). Out of 99 liver biopsies, 41 (41.4%) showed cholestasis on histology, including reduced expression of MDR3 and BSEP. Pathogenic (P) or likely pathogenic (LP) mutations were identified in 45 (19.3%) probands, either as single or compound heterozygosity. Including variants of uncertain significance (VUS), mutations occurred in 106 patients (45.5%). Considering all P/LP/VUS, the most recurrent genes were: ABCB4 (27 cases), TJP2 (18), SLC01B3 (10), ABCB11 (9), MYO5B (8), ABCC2 (7), VPS33B (6), and ATP8B1 (5). We compared the features of patients with P/LP mutations to those of others in the cohort study. No differences were observed in liver stiffness, itching history, choleretic drugs, or blood tests, except for bile acids (BAs). Female sex, BAs, age at testing, intrahepatic cholestasis of pregnancy (ICP), family history of liver disease, juvenile cholelithiasis, and low phospholipid-associated cholelithiasis occurred more frequently in patients with P/LP (p < 0.05). The multivariate analysis indicated that the only independent factors associated with disease-causing mutations were ICP and juvenile cholelithiasis. Conclusion: Mutations in the genes responsible for inherited cholestasis were found to recur in AOCC in numerous cases with mild liver fibrosis, particularly in individuals with a history of juvenile cholestasis and ICP. The ABCB4 gene was the most frequently involved