Determining Mitochondrial 3243A>G Heteroplasmy Using an ARMS-ddPCR Strategy

Abstract

Abstract Objectives Determining mitochondrial DNA (mtDNA) A-to-G substitution at nucleotide 3243 (m.3243A>G) heteroplasmy is essential for both precision diagnosis of m.3243A>G–associated mitochondrial disease and genetic counseling. Precise determination of m.3243A>G heteroplasmy is challenging, however, without appropriate strategies to accommodate heteroplasmic levels ranging from 1% to 100% in samples carrying thousands to millions of mtDNA copies. Methods We used a combined strategy of amplification-refractory mutation system–quantitative polymerase chain reaction (ARMS-qPCR) and droplet digital PCR (ddPCR) to determine m.3243A>G heteroplasmy. Primers were specifically designed and screened for both ARMS-qPCR and ddPCR to determine m.3243A>G heteroplasmy. An optimized ARMS-qPCR–ddPCR–based strategy was established using artificial standards, with different mixtures of m.3243A-containing and m.3243G-containing plasmids and further tested using clinical samples containing the m.3243A>G mutation. Results One of 20 primer pairs designed in the study was omitted for ARMS-qPCR–ddPCR strategy application according to criteria of 85% to 110%, R2  > 0.98 amplification efficiency, melt curve with a single clear peak, and specificity for m.3243A and m.3243G artificial standards (|CtWt-CtMut|max). Using plasmid standards with various m.3243A>G heteroplasmy (1%-100%) at low, mid, and high copy numbers (3,000, 104, and 105-107, respectively) and DNA from the blood of 20 patients carrying m.3243A>G with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, we found that ARMS-qPCR was reliable for determining m.3243A>G at 3% to 100% for low copy number and 1% to 100% for mid to high copy number samples. Meanwhile, ddPCR was reliable for determining m.3243A>G at 1% to 100% at low to mid copy number samples. Conclusions An ARMS-qPCR–ddPCR–based strategy was successfully established for precise determination of m.3243A>G heteroplasmy in complex clinical samples.