Transition to next generation analysis of the whole mitochondrial genome: a summary of molecular defects.

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed "Next-Generation Sequencing" (NGS) technology offers a robust high-throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS-NGS cohort of 391 patients, 21 mutation-positive cases (5.4%) have been identified. The mtWGS-NGS provides a one-step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

Tang S ，Wang J ，Zhang VW ，Li FY ，Landsverk M ，Cui H ，Truong CK ，Wang G ，Chen LC ，Graham B ，Scaglia F ，Schmitt ES ，Craigen WJ ，Wong LJ ... -  《-》

Comprehensive Mitochondrial Genome Analysis by Massively Parallel Sequencing.

Palculict ME ，Zhang VW ，Wong LJ ，Wang J ... -  《-》

The development of next-generation sequencing assays for the mitochondrial genome and 108 nuclear genes associated with mitochondrial disorders.

Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.

Dames S ，Chou LS ，Xiao Y ，Wayman T ，Stocks J ，Singleton M ，Eilbeck K ，Mao R ... -  《-》

Next generation molecular diagnosis of mitochondrial disorders.

Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6k b mitochondrial genome and ~1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders.

Wong LJ 《-》

Targeted array CGH as a valuable molecular diagnostic approach: experience in the diagnosis of mitochondrial and metabolic disorders.

Oligonucleotide array-based comparative genomic hybridization (aCGH) targeted to coding exons of genes of interest has been proven to be a valuable diagnostic tool to complement with Sanger sequencing for the detection of large deletions/duplications. We have developed a custom designed oligonucleotide aCGH platform for this purpose. This array platform provides tiled coverage of the entire mitochondrial genome and high-density coverage of a set of nuclear genes involving mitochondrial and metabolic disorders and can be used to evaluate large deletions in targeted genes. A total of 1280 DNA samples from patients suspected of having mitochondrial or metabolic disorders were evaluated using this targeted aCGH. We detected 40 (3%) pathogenic large deletions in unrelated individuals, including 6 in genes responsible for mitochondrial DNA (mtDNA) depletion syndromes, 23 in urea cycle genes, 11 in metabolic and related genes. Deletion breakpoints have been confirmed in 31 cases by PCR and sequencing. The possible deletion mechanism has been discussed. These results illustrate the successful utilization of targeted aCGH to detect large deletions in nuclear and mitochondrial genomes. This technology is particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing. For female carriers of X-linked disorders, if sequencing analysis does not detect point mutations, targeted aCGH should be considered for the detection of large heterozygous deletions.

Wang J ，Zhan H ，Li FY ，Pursley AN ，Schmitt ES ，Wong LJ ... -  《-》