New diagnostic pathways for mitochondrial disease

Abstract

Mitochondrial diseases collectively represent the most common cause of inherited metabolic disease. They are estimated to affect at least 1 in 8,000 adults and at least 1 in 250 adults carry a disease-causing genetic mutation. They comprise a heterogeneous group of disorders caused by mutations in either the nuclear or mitochondrial genome, which ultimately result in dysfunction of the critical cellular energy producing mitochondrial respiratory chain. Owing to the key role of mitochondria in energy production, mitochondrial disorders predominantly manifest in tissues with high metabolic demand. However, they demonstrate significant phenotypic and genotypic variability, often rendering the diagnostic process protracted and challenging. Since Luft's first description of mitochondrial disease nearly 60 years ago, substantial evolution in diagnostic techniques have simultaneously improved the diagnosis and understanding of mitochondrial disease and biology, but the standard diagnostic approach has failed to evolve at the same pace. Although sequencing technologies and analysis for the diagnosis of mitochondrial disease continue to evolve, advances to date, our expanding understanding of mitochondrial diseases and the increasing affordability of these new technologies justify a paradigm shift in the diagnostic approach. We review the progression, impact and challenges of diagnosing mitochondrial diseases and propose a minimally invasive "genetics first" approach incorporating stratification using non-invasive biomarkers, followed by non-targeted next-generation sequencing, such as whole genome sequencing. Such an approach may improve diagnostic yield and streamline diagnosis, leaving invasive investigations to address diagnostic challenges and functional validation of novel variants.

A benchmarking of human mitochondrial DNA haplogroup classifiers from whole-genome and whole-exome sequence data

“…However, the mean depth of mtDNA for WGS was around 30 times higher than that obtained by WES. For WGS, the depth reported in diverse studies ranged between 1,200-4,000X (Puttick et al, 2019; Raymond et al, 2018; Watson et al, 2020), fitting with the expected proportion of mtDNA copy number compared to the nuclear DNA, which theoretically differs by 10 to 100 times (Al-Nakeeb et al, 2017; Robin & Wong, 1988). With respect to WES, the observed depth fits in the range described in the literature (Abicht et al, 2018; Diroma et al, 2020; Griffin et al, 2014; Patowary et al, 2017).…”

The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

“…The usage of long reads for mitochondrial genome sequencing was reported for vertebrate species identification (Franco-Sierra and Díaz-Nieto, 2020), equine genetics (Dhorne-Pollet et al, 2020), the structural-wise grouping of plan mitochondrial genomes (Masutani et al, 2021), and clinical diagnostics (Wood et al, 2019). Nevertheless, despite the growing number of studies, long reads are still rarely applied as clinical panels (Orsini et al, 2018) due to low base-to-base quality, hampering their application for variant calling in comparison with short reads, for which protocols, standards, and recommendations are being continuously developed (DePristo et al, 2011;Koboldt et al, 2012;Van der Auwera et al, 2013;Koboldt, 2020;Watson et al, 2020).…”

“…Current interest in clinical aspects of mitochondrial genetics accelerates the development of diverse techniques to enhance the sequencing efficacy of mitochondrial DNA, applying capture-based approaches (Zhou et al, 2020) and preprocessing methods (Yao et al, 2019) and control region validation assay (Brandhagen et al, 2020). Nonetheless, accepted recommendations for NGS-driven analysis are focused on Illumina exclusively, and the diagnosing yield is discussed in the context of an adequate panel to be utilized (either target, whole-exome, or whole-genome ones) (Watson et al, 2020). The overwhelming majority of modern clinical studies, either identifying novel pathogenic variants or examining diseases' etiology regarding impairments in the mitochondrial genome, despite applying diverse sequencing approaches, still do not implement Nanopore data (Watson et al, 2020).…”

“…Nonetheless, accepted recommendations for NGS-driven analysis are focused on Illumina exclusively, and the diagnosing yield is discussed in the context of an adequate panel to be utilized (either target, whole-exome, or whole-genome ones) (Watson et al, 2020). The overwhelming majority of modern clinical studies, either identifying novel pathogenic variants or examining diseases' etiology regarding impairments in the mitochondrial genome, despite applying diverse sequencing approaches, still do not implement Nanopore data (Watson et al, 2020). The reason underlying this phenomenon could be explained by the fact that best practices for variant calling analysis for medical purposes have been developed and widely accepted for short reads only (Koboldt, 2020).…”

Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives

“…Pathogenic mutations in mtDNA and nuclear DNA (nDNA) can be used to confirm primary mitochondrial disease due to abnormal OXPHOS function. A genetics-first approach has been facilitated by the availability of exome and whole genome sequencing that has improved the identification of mitochondrial disease genes diagnosis [36][37][38]. Identification of nuclear mitochondrial disease genes are also used to diagnose mitochondrial dysfunctions [39][40][41][42].…”