Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease

Abstract

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Keywords: Heteroplasmy; Mitochondrial genome; Multiple deletions; Single large-scale deletion; mtDNA mutation.

Figure 1.

Demographics for the study cohort A. Patient age groups. The numbers represent total patients in each group and percentage of the total; B. Number of samples in each tissue type and percentage of total. Other tissues included liver, brain, and gastrointestinal antrum biopsy samples; C. Overall diagnostic yield in this cohort. The inconclusive category includes variants of uncertain significance (VUS) and multiple large-scale mtDNA deletions at <10% heteroplasmy levels; D. Number of patients with positive findings. Multiple mtDNA deletions <10% heteroplasmy were not included in this category; E. The mtDNA test results distribution in two groups: a cohort with whole exome sequencing (ES) results available and a cohort in whom ES was not performed. The numbers represent patients with positive, inconclusive, or negative results in each group.

Figure 2.

Examples of large-scale mtDNA deletions (LSMD) with variable heteroplasmy levels quantified by ddPCR. A-B: Single LSMD (Corresponding to Table 2 - A: Patient 35 and B: Patient 36); C: Multiple LSMD (Patient 44 in Table 3). D: LSMD Negative control.