Use of Next-Generation Sequencing and Whole-Exome Sequencing in the Diagnosis of Adult-Onset Familial Intrahepatic Cholestasis: Challenges in Interpreting Variants of Uncertain Significance

Abstract

Progressive Familial Intrahepatic Cholestasis (PFIC) is a rare liver disorder that, although typically present in childhood, can also occur in adulthood. Early diagnosis is crucial for appropriate clinical management, prognostic assessment, and therapeutic decision-making; however, it remains challenging due to phenotypic variability and the frequent identification of genetic variants of uncertain significance (VUS). Advances in next-generation sequencing (NGS) and whole-exome sequencing (WES) have improved the detection of disease-associated variants, revealing that the same genetic variants—often in a heterozygous state—may lead to adult-onset cholestatic disease, with milder or atypical presentations, while still conferring a significant risk of progressive liver injury and related complications. To assess the diagnostic yield and clinical utility of NGS panels and WES in adults with suspected PFIC or unexplained cholestatic liver disease, focusing on variant interpretation, emerging disease-associated genes, and clinical significance of VUS. A literature review was conducted to assess molecular diagnostics in adult cryptogenic cholestasis, with a focus on studies employing targeted NGS panels and WES. Selected studies were examined for diagnostic yield, variant classification, interpretive challenges, including VUS, and integration with clinical data. Data on patient demographics, sequencing techniques, and variant interpretation strategies were extracted to evaluate the current capabilities and limitations of genomic testing. From an initial search of 211 publications, 15 studies met the inclusion criteria, comprising five large adult cohorts and ten single-patient or trio-based reports. Across 72 patients, sequencing technologies identified 75 pathogenic or likely pathogenic variants, predominantly missense mutations, demonstrating high genetic heterogeneity. The most implicated genes included ABCB4, ABCB11, ATP8B1, TJP2, and USP53, with diagnostic yields ranging from 13.2% in large heterogeneous cohorts to substantially higher rates in carefully selected individual cases. VUS were identified in up to 67% of cases, highlighting the need for multidisciplinary interpretation integrating clinical, biochemical, and genetic data. Emerging evidence also implicated ciliopathy-associated genes such as DCDC2, NPHP3, PKHD1, TULP3, and TTC21B, expanding the genetic spectrum of adult cholestasis. Clinically, presentations ranged from mild biochemical abnormalities to progressive cholestasis, with pruritus being a common feature across studies. Comprehensive genetic testing significantly enhances diagnostic accuracy, informs prognosis, and guides individualized management in adults with cryptogenic cholestasis. Emerging evidence suggests that ciliopathy-associated genes may contribute to the genetic architecture of adult cholestatic disorders, further expanding the spectrum of disease-associated genes. However, the high prevalence of VUS remains a major challenge, highlighting the need for functional studies, longitudinal follow-up, and improved variant interpretation frameworks. As genomic technologies and analytical approaches continue to evolve, genetic testing is expected to play an increasingly central role in precision hepatology.