Experiences from dual genome next-generation sequencing panel testing for mitochondrial disorders: a comprehensive molecular diagnosis

Abstract

Introduction: The molecular diagnosis of mitochondrial disorders is complicated by phenotypic variability, genetic heterogeneity, and the complexity of mitochondrial heteroplasmy. Next-generation sequencing (NGS) of the mitochondrial genome in combination with a targeted panel of nuclear genes associated with mitochondrial disease provides the highest likelihood of obtaining a comprehensive molecular diagnosis. To assess the clinical utility of this approach, we describe the results from a retrospective review of patients having dual genome panel testing for mitochondrial disease.

Methods: Dual genome panel testing by NGS was performed on a cohort of 1,509 unrelated affected individuals with suspected mitochondrial disorders. This test included 163 nuclear genes associated with mitochondrial diseases and the entire mitochondrial genome. A retrospective review was performed to evaluate diagnostic yield, disease-gene contributions, and heteroplasmy levels of pathogenic/likely pathogenic (P/LP) mitochondrial DNA (mtDNA) variants.

Results: The overall diagnostic yield was 14.6%, with 7.7% from the nuclear genome and 6.9% from the mtDNA genome. P/LP variants in nuclear genes were enriched in both well-established genes (e.g., POLG) and more recently described genes (e.g., FBXL4), highlighting the importance of keeping the panel design updated.

Conclusion: Variants in nuclear and mitochondrial genomes equally contributed to a 14.6% diagnostic yield in this patient cohort. Dual genome NGS testing provides a comprehensive framework for diagnosing mitochondrial disorders, offering clinical utility that can be considered as first-tier approach compared to single genome testing. Characterizing disease-causing genes, variants, and mtDNA heteroplasmy enhances understanding of mitochondrial disorders. Testing alternative tissues can further increase diagnostic yield.

Keywords: NGS; dual-genome; functional group analysis; heteroplasmy; mitochondria.

FIGURE 1

Contribution of observed nuclear genes/variants in patient cohort. (A) Distribution of causative defects in nuclear genes calculated from 117 solved cases. The size of the colored sectors represents the relative percentage of each causative nuclear gene. (B) Distribution of 614 unique pathogenic/likely pathogenic variants in nuclear genes regardless of causative (cases solved) or not (cases not solved) status. The size of the colored sector represents the relative percentage of unique pathogenic/likely pathogenic variant counts in genes annotated accordingly. There are 73 genes with a percentage of less than 1% of unique pathogenic/likely pathogenic variant, which are combined under “Others”.

FIGURE 2

Observed defects in mitochondrial genome in the patient cohort. (A) Distribution of heteroplasmy level for observed pathogenic/likely pathogenic variants. Sample types were blood (78.5%), muscle (14.2%), extracted DNA (5.2%) and others (2.2%). (B) Distribution of pathogenic/likely pathogenic mitochondrial variants detected in our patient cohort. The size of the colored sectors represents the relative percentage of the number of pathogenic/likely pathogenic variants in genes annotated accordingly.