Liver transcriptome sequencing contributes to the molecular diagnosis of genetic liver diseases

Background & Aims: Since DNA sequencing alone faces challenges in variant interpretation during genetic diagnosis, RNA sequencing has recently gained attention in resolving these diagnostic gaps. This study aimed to evaluate the advantages of liver tissue RNA sequencing in the diagnosis of genetic liver diseases. Approach & Result: Liver tissue RNA sequencing was performed on 147 patients with prior DNA sequencing. We evaluated the role of RNA sequencing by analyzing aberrant gene expression, splicing, allele-specific expression, transcript-level similarity and mosaic variants. Liver RNA-seq supported the molecular diagnoses in 56 patients diagnosed by DNA sequencing alone. Among 91 previously undiagnosed patients, incorporating RNA sequencing established a diagnosis in 17 (18.68%) patients. Among the 33 patients with indicative clinical phenotypes or prioritized variants, diagnosis was established in 15 (45.45%) patients under the help of RNA sequencing. This improvement was primarily (16/17) driven by the detection of aberrant splicing and allele-specific expression, instead of aberrant expression. RNA sequencing revealed ±50bp of cryptic splicing sites as hotspot regions, characterized allele-specific expression at both the gene and variant levels and revealed shared transcriptomic features in low GGT cholestasis. Conclusions: While DNA sequencing demonstrates superior sensitivity in detecting clinically relevant variants, liver RNA sequencing significantly enhances genetic diagnosis mainly by revealing aberrant splicing and allele-specific expression. These findings suggest that RNA sequencing is an essential complement to DNA sequencing.