Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Abstract

1-3Pathogenic mtDNA mutations, ranging from point mutations to large deletions, are often present in mixed proportions with wildtype mtDNA molecules within the same cell, a biological phenomenon termed heteroplasmy. The degree of mtDNA mutant heteroplasmy can vary significantly across different tissues of the same individual, and the percentage of a mutation is an important contributor to the clinical phenotype.4,5 Determination of the heteroplasmic status of any mtDNA mutation is clinically important to providing a family with informative genetic counseling regarding recurrence risk. Therefore, accurate measurement of heteroplasmy is an essential component of the molecular diagnostic scheme for mtDNA-related disorders.The diagnosis of an individual with suspected mtDNA-related disorders begins with a thorough clinical evaluation and assessment of family history.6 If a disorder with matrilineal inheritance can be established, common mtDNA point mutations and large deletions are typically analyzed using PCR-based targeted assays and Southern blotting methodology. If the screening for common point mutations and large deletions is negative, then the entire mitochondrial genome is usually analyzed by a conventional PCR-based Sanger sequencing approach with multiple pairs of primers. When a mutation is identified, analysis of the degree of heteroplasmy is carried out using various methods, including the commonly used allele refractory mutation system-based quantitative PCR (ARMS qPCR) for quantitative measurement.7 Large deletions are usually confirmed by PCR using primers encompassing the deletion break points followed by sequence analysis to map the break points, or, alternatively, by oligonucleotide array comparative genome hybridization.8 These sequential approaches are time consuming and costly. In an effort to improve efficiency and sensitivity of mtDNA mutation detection, we recently developed a comprehensive one-step long range/massively parallel sequencing (LR-PCR/MPS)-based approach that uses a single pair of PCR primers to amplify the entire mitochondrial genome. This method allows the simultaneous detection of point mutations with quantified heteroplasmy as well as large mtDNA deletions with accurately mapped break points. Purpose: The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome. Methods:The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers. Results:Our results demo...

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants

“…7,10,22,23 The expansion of massively parallel sequencing has revolutionized mitochondrial genetics. [24][25][26] Sanger sequencing has long been considered the gold standard for molecular diagnosis of mtDNA-based disorders. The vast majority of deleterious mtDNA mutations are heteroplasmic, and it is challenging to identify all mutations in the mitochondrial genome and simultaneously quantify the mtDNA heteroplasmy levels.…”

“…In comparison, massively parallel sequencing has been reported to detect mtDNA heteroplasmies of ≥5% with virtually no false-positive results. 16,26,28 The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has significantly improved the molecular diagnosis of mtDNA disorders. 24 Large mtDNA deletions have been reported in mitochondrial diseases, and the ability of massively parallel sequencing to detect mtDNA deletions has not been firmly established.…”

“…Massively parallel sequencing is a cost-effective approach with the high sensitivity, specificity, and accuracy necessary to detect a wide spectrum of mtDNA mutations while also quantifying levels of heteroplasmy. 16,24,26,28 Without functional correlates, distinguishing mildly pathogenic mtDNA mutations from benign variants is challenging and can depend on interpretation. Future studies should correlate the mtDNA mutational spectrum with functional assays to more fully explore the role mitochondrial mutations and dysfunction play in the pathogenesis of glaucoma.…”

Challenges of Bringing Next Generation Sequencing Technologies to Clinical Molecular Diagnostic Laboratories

“…In our laboratory, we use one pair of immediate backto-back LR-PCR primers to generate a single amplicon of the entire mitochondrial genome (Fig. 1) [29,30,45]. Enrichment by multiplex PCR or by mixing PCR products usually does not provide even coverage upon sequencing [30,45].…”

“…1) [29,30,45]. Enrichment by multiplex PCR or by mixing PCR products usually does not provide even coverage upon sequencing [30,45]. Coverage within the same PCR fragment may be uniform, but there is large variation in coverage among different PCR fragments, that renders the inability to detect large deletions.…”