Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease

Area of Science:

• Genetics
• Molecular Biology
• Clinical Diagnostics

Background:

• Mitochondrial disease diagnosis necessitates analyzing both nuclear and mitochondrial genomes.
• Identifying single-nucleotide variants (SNVs) and copy number alterations is crucial for accurate diagnosis.
• Phenotype correlation and analysis of maternal relatives are often required.

Purpose of the Study:

• To develop and validate a comprehensive mitochondrial DNA (mtDNA) sequencing test for diagnosing mitochondrial diseases.
• To accurately detect SNVs and large-scale mtDNA deletions (LSMD) with precise heteroplasmy quantification.
• To enhance diagnostic yield and pathogenicity assessment through integrated molecular and clinical data.

Main Methods:

• Development of a 'MitoGenome' test utilizing long-range PCR (LR-PCR) for full mtDNA genome amplification.
• Next-generation sequencing (NGS) for SNV detection and LSMD identification.
• Droplet digital PCR (ddPCR) for accurate quantification of LSMD heteroplasmy levels.

Main Results:

• The MitoGenome test was performed on 428 samples from 394 patients, yielding a 11% positive rate.
• Pathogenic SNVs were identified in 34 patients, with m.3243A>G being the most frequent.
• LSMDs were detected in 8 patients (single) and 3 patients (multiple), with heteroplasmy levels quantified by ddPCR.
• The assay demonstrated high accuracy for SNV detection down to 1% heteroplasmy and LSMD detection below 10%.

Conclusions:

• The LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for mtDNA variants and copy number alterations.
• Simultaneous detection of SNVs and LSMDs with precise heteroplasmy quantification improves diagnostic capabilities.
• Integration of maternal sample analysis and ddPCR for heteroplasmy assessment facilitates accurate pathogenicity interpretation.