In-Depth Analysis of the Whole Human Mitochondrial Genome for Pathogenic Mutations And Variants By DHPLC, SURVEYOR® Nuclease and DNA Sequencing: A Cost-Effective Multi-Pronged Approach for Clinical Labs

Mitochondrial disorders are a group of heterogeneous disorders associated with organs that are high energy dependent. Alterations of the mitochondrial DNA (mt DNA) are implicated in various pathological conditions. Limited success has been obtained with mutation scanning techniques such as SSCP, DGGE, and TDGE for the screening the entire mitochondrial genome. Issues of sensitivity, specificity and cost of these techniques have been overcome by complementary heteroduplex-based scanning methodologies, denaturing high performance chromatography (DHPLC) and SURVEYOR Nuclease, combined with selective DNA sequencing. Clinical samples have been analyzed with our multi-pronged approach in which a total of 40 amplicons covering the entire mitochondrial genome are PCR-amplified with a high-fidelity DNA polymerase and analyzed by DHPLC under fully optimized conditions for mtDNA screening. Scanning results are further confirmed with SURVEYOR Nuclease and DNA sequencing. This strategy efficiently detected point mutations, insertions and deletions in the entire mitochondrial genome, with straightforward data analysis and a detection limit of 0.5% heteroplasmy. Furthermore, collection and re-amplification of heteroduplex peak-fractions allowed sequence analysis of low level mtDNA mutations. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples. With 100% sensitivity, the costs were greatly reduced with an initial mutation scanning by DHPLC using the WAVE® HS 4500HT platform followed by SURVEYOR-cleavage fragment analysis and double-stranded DNA sequencing of the positive variants. This is an efficient and cost-effective approach with a rapid turnaround time for clinical diagnostic labs. ABSTRACT Mitochondrial DNA (mtDNA) is a 16kb circle of maternally inherited, extra-nuclear, double-stranded DNA. 37 genes encoding two ribosomal RNAs, 22 transfer RNAs and 13 subunits of the respiratory chain. Metabolically active tissues such as the central nervous system, skeletal muscle and gastrointestinal system are largely affected by mitochondrial disorders. The presence of only a single allele at any given mtDNA locus is described as homoplasmy while a mixture of wild-type and mutant mtDNA alleles is described as heteroplasmy. The severity of phenotype is in proportion to the mutant load. Molecular diagnostic analysis of mitochondrial mutations and gene deletions has mainly used traditional methods such as restriction fragment length polymorphism (RFLP) analysis, allele specific oligonucleotide (ASO) hybridizations, allele specific PCR, direct sequencing and Southern blotting. In an effort to circumvent the time consuming and labor intensive techniques of mutation scanning that include SSCP and DGGE, an easier and more cost-effective approach was developed. This consisted of amplifying 40 amplicons spanning the entire mitochondrial genome, with universal PCR conditions, combined with automated and tiered mutation scanning. Rapid and sensitive mutation detection was achieved using DHPLC combined with heteroduplex cleavage fragment analysis both using the WAVE HS System. Using this approach, we present whole mitochondrial genome mutation scanning data from the analysis of 25 clinical samples, some of which demonstrate known pathogenic mutations, along with data demonstrating the sensitivity of the testing methodology for the detection of heteroplasmy. Validation of this mutation screening approach to regions that harbor the most prevalent pathogenic mutations was confirmed by the successful detection of all reference mutations in test samples. Amplicons were designed as overlapping fragments of 450-675 bp to optimize for both accuracy and efficiency in the mutation screening process. PCR primers and conditions were designed and optimized to facilitate the use a single thermal cycling condition, while improving specificity and yield of all products for analysis. DHPLC analysis conditions were optimized for mutation detection in each amplicon with use of a minimal range of different temperatures. Analytical sensitivity was determined using reference samples with known mtDNA mutations mixed with wild-type mtDNA at ratios varying from 0.5 to 50%. DHPLC and SURVEYOR Nuclease were able to detect levels of heteroplasmy as low as 0.5-1.0% versus 20% which was the limit of detection for direct sequencing. The entire mitochondrial genomes of 25 patients and control samples were screened using this multi-pronged approach with independent screening technologies. A mixture of two populations of mtDNA molecules does not always indicate a causative mutation is present. A large number of polymorphisms, usually homoplasmic in nature, were found in the cohort analyzed. We have developed and validated an in-depth screening methodology for mutational analysis of the entire mitochondrial genome.