Use of Whole-Genome Sequencing for Mitochondrial Disease Diagnosis

Abstract

Background and objectives: Mitochondrial diseases (MDs) are the commonest group of heritable metabolic disorders. Phenotypic diversity can make molecular diagnosis challenging, and causative genetic variants may reside in either mitochondrial or nuclear DNA. A single comprehensive genetic diagnostic test would be highly useful and transform the field. We applied whole-genome sequencing (WGS) to evaluate the variant detection rate and diagnostic capacity of this technology with a view to simplifying and improving the MD diagnostic pathway.

Methods: Adult patients presenting to a specialist MD clinic in Sydney, Australia, were recruited to the study if they satisfied clinical MD (Nijmegen) criteria. WGS was performed on blood DNA, followed by clinical genetic analysis for known pathogenic MD-associated variants and MD mimics.

Results: Of the 242 consecutive patients recruited, 62 participants had "definite," 108 had "probable," and 72 had "possible" MD classification by the Nijmegen criteria. Disease-causing variants were identified for 130 participants, regardless of the location of the causative genetic variants, giving an overall diagnostic rate of 53.7% (130 of 242). Identification of causative genetic variants informed precise treatment, restored reproductive confidence, and optimized clinical management of MD.

Discussion: Comprehensive bigenomic sequencing accurately detects causative genetic variants in affected MD patients, simplifying diagnosis, enabling early treatment, and informing the risk of genetic transmission.

Figure 1. WGS Provides Deep, Uniform Coverage of Both the Mitochondrial and Nuclear Genome

The average depth of sequencing coverage across the (A) nuclear and (B) mitochondrial genomes from a cohort of adult participants with suspected mitochondrial disease (n = 242). (C) m.3243A>G variant heteroplasmy assessed by WGS and pyrosequencing for DNA extracted from blood (black) from n = 50 patients diagnosed with the m.3243A>G variant and multiple autopsy tissues (n = 10; colored points) from patients E9 and E59. Heteroplasmy was highly correlated between WGS and pyrosequencing (R² = 0.994). Colors in (C) are consistent with those in Figure 2 for the different tissues. MT = mitochondrial; WGS = whole-genome sequencing.

Figure 2. Different Tissues Provide High Depth of Coverage of the Mitochondrial Genome and High Levels of Heteroplasmy

(A and C) Depth of sequencing coverage across the mitochondrial genome and (B and D) m.3243A>G heteroplasmy varied between autopsy tissues from 2 patients, being considerably higher in solid tissues compared with blood. Colored lines in A and C are consistent with colored bars in B and D. Colors in A–D are consistent with those in Figure 1C for the different tissues.

Figure 3. The Diagnostic Performance of WGS for Nuclear DNA–Based Mitochondrial Diseases

(A) The number of diagnoses made by WGS in the nuclear or mitochondrial genomes, from n = 242 patients with suspected mitochondrial disease. (B) The frequency of diagnoses made from mitochondrial disease genes in the nuclear genome. (C) WGS identified a hemizygous 16.4 megabase pair (Mbp) deletion on chromosome 4 in a patient with seizures, mild ophthalmoparesis, optic atrophy, cerebellar ataxia, myopathy, diabetes, recurrent pseudo-obstruction of the bowel, and liver dysfunction. (D) WGS identified a homozygous 946 base pair deletion in SPG7 in a patient with hereditary spastic paraplegia complicated by cerebellar ataxia, ophthalmoplegia, and sensory neuropathy. MT = mitochondrial; WGS = whole-genome sequencing.

Figure 4. The Diagnostic Performance of WGS for Mitochondrial DNA–Based Mitochondrial Diseases

(A) The number of causative mtDNA variants detected by WGS and (B) their corresponding variant heteroplasmy. (C) Two mitochondrial DNA single deletions were identified in blood, both at heteroplasmy below 1%. One mitochondrial deletion was found in autopsy muscle at a much higher heteroplasmy of 15.7%, shown in more detail in (D), which highlights the sequencing coverage and reads spanning the deletion. mtDNA = mitochondrial DNA; WGS = whole-genome sequencing.

Figure 5. Molecular and Diagnostic Heterogeneity Within Different MD Phenotypes

Patients were grouped into 6 clinical phenotypes (CPEO, stroke-like episodes, optic atrophy, MIDD, complex, and oligosymptomatic) to highlight the frequency of pathogenic nuclear gene and mitochondrial DNA variants identified by WGS. Precise molecular diagnoses are shown, and the number of undiagnosed cases in each subgroup is shown in gray. CPEO = chronic progressive external ophthalmoplegia; MD = mitochondrial disease; MIDD = maternally inherited deafness and diabetes; WGS = whole-genome sequencing.

Figure 6. Cerebral MRI in α-Methylacyl-CoA Racemase Deficient Patients

(A) Representative axial T1 weighted images showing focal areas of abnormal signal intensity in the right parieto-occipital region after episodes of encephalopathy in patient E19. Note there are also small high signal intensities scattered throughout the subcortical white matter. (B) Magenta arrows indicate high signal areas in the thalami of patient B54, consistent with what is seen in adult-onset Leigh syndrome.