Application of dual-genome oligonucleotidearray-based comparative genomic hybridization to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes

Abstract

Purpose: Mitochondrial disorders constitute a group of clinically and genetically heterogeneous diseases for which molecular diagnosis has been a challenge. The current procedures for diagnosis of mitochondrial DNA deletion and depletion syndromes based on Southern analysis and quantitative polymerase chain reaction are particularly inefficient for determining important parameters of deletion endpoints and percent heteroplasmy. We have developed an improved approach for routine analyses of these disorders in a clinical laboratory. Methods: A customdesigned oligonucleotide array-based comparative genomic hybridization platform was developed to provide both tiled coverage of the entire 16.6-kb mitochondrial genome and high-density coverage of nuclear genes involved in mitochondrial biogenesis and function, for quick evaluation of mitochondrial DNA deletion and depletion. Results: For initial validation, the performance of this array was characterized in 20 samples with known mitochondrial DNA deletions and 12 with apparent depletions. All previously known deletions were clearly detected and the break points were correctly identified by the oligonucleotide array-based comparative genomic hybridization, within the limits of resolution of the array. The extent of mitochondrial DNA depletion and the percentage of deletion heteroplasmy were estimated using an automated computational approach that gave results comparable to previous methods. Conclusions from subsequent application of this approach with Ͼ300 new clinical samples have been in 100% concordance with those from standard methods. Finally, for one sample, we were able to identify an intragenic deletion in a nuclear gene that was responsible for the observed mitochondrial DNA depletion. Conclusion: We conclude that this custom array is capable of reliably detecting mitochondrial DNA deletion with elucidation of the deletion break points and the percentage of heteroplasmy. In addition, simultaneous detection of the copy number changes in both nuclear and mitochondrial genomes makes this dual genome array of tremendous value in the diagnoses of mitochondrial DNA depletion syndromes. Genet Med 2009:11(7): 518 -526.

Capture-based high-coverage NGS: a powerful tool to uncover a wide spectrum of mutation types

“…Targeted array comparative genomic hybridization or multiplex ligation-dependent probe amplification is commonly used for copy-number changes involving one or more exons. [27][28][29] However, these methods cannot detect point mutations. This report demonstrates the power of target capture deep NGS methods in the simultaneous detection of point mutations, exonic deletions, and genomic rearrangement.…”

Comprehensive One-Step Molecular Analyses of Mitochondrial Genome by Massively Parallel Sequencing

“…Although the gold standard, Sanger sequencing, reliably detects most nucleotide changes, it does not provide quantitative information and is not adequately sensitive for detecting mutant heteroplasmy below 15% (11 ). Large mtDNA deletions are analyzed separately by Southern blotting (12 ) or by custom-designed array comparative genome hybridization (aCGH) (13,14 ). Nevertheless, neither method by itself can provide deletion breakpoints and heteroplasmy.…”

Detection of uniparental isodisomy in autosomal recessive mitochondrial DNA depletion syndrome by high-density SNP array analysis

“…The array-comparative genomic hybridization (aCGH) experiment and analysis of results were performed as previously published. [11][12][13][14] Real-time quantitative PCR was used to measure the mtDNA content as previously described. 15 The Human610-Quad single-nucleotide polymorphism (SNP) array was used as per the manufacturer's protocol (Illumina Inc., San Diego, CA, USA).…”