RNA Sequencing Resolves Cryptic Pathogenic Variants in Mitochondrial Disease

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biochemistry

Background:

• Mitochondrial diseases are common inherited metabolic disorders with significant clinical and genetic heterogeneity.
• Current DNA sequencing methods leave up to 50% of patients undiagnosed.
• Accurate molecular diagnosis is essential for understanding and managing mitochondrial diseases.

Purpose of the Study:

• To evaluate the efficacy of RNA sequencing (RNA-seq) in enhancing molecular diagnostic rates for mitochondrial diseases in undiagnosed Chinese pediatric patients.
• To identify cryptic RNA splicing and regulatory events missed by DNA sequencing.
• To improve the diagnostic yield in cases with variants of uncertain significance (VUS) and in unsolved genetic cohorts.

Main Methods:

• RNA sequencing (RNA-seq) was performed on skin fibroblasts from 140 undiagnosed pediatric patients with suspected mitochondrial disease.
• Patients were stratified into candidate (n=28) and unsolved (n=112) groups based on whole exome sequencing (WES) findings.
• Integrative analysis of RNA-seq, WES, and whole genome sequencing (WGS) data was used to identify pathogenic variants and aberrant RNA events.

Main Results:

• A genetic diagnosis was achieved in 25% of patients overall (71% in candidate group, 13% in unsolved group) using integrated multi-omics analysis.
• Aberrant RNA splicing was a major diagnostic contributor, revealing pathogenic variants missed by in silico predictors.
• The study identified various variant types, including synonymous mutations, with a recurrent East Asian founder mutation in ECHS1 identified in seven cases.

Conclusions:

• RNA sequencing significantly enhances molecular diagnosis of mitochondrial disease by uncovering cryptic splicing and regulatory events.
• Transcriptome analysis is an essential tool for comprehensive genomic diagnostics in neurometabolic disorders.
• These findings advocate for the routine inclusion of RNA-seq in genetic testing workflows for undiagnosed mitochondrial diseases.