Determining Mitochondrial 3243A>G Heteroplasmy Using an ARMS-ddPCR Strategy. Issue 5 (26th October 2021)

• Record Type: Journal Article
• Authors: Xu, Pu; Jia, Manli; Yan, Jimei; Yuan, Xiangshu; Yu, Weidong; Zhou, Zhuohua; Fang, Hezhi; Gao, Feng; Shen, Lijun
• Is Part Of: American journal of clinical pathology. Volume 157:Issue 5(2022)
• Page Start: 664
• Page End: 677
• Publication Date: 2021-10-26
• Subjects: ARMS-qPCR -- ddPCR -- Heteroplasmy -- mtDNA copy number -- Mitochondrial disease; Diagnosis, Laboratory -- Periodicals; Pathology -- Periodicals; 616.07
• DOI: 10.1093/ajcp/aqab174
• Languages: English
• ISSNs: 0002-9173

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%, R 2 > 0.98 amplification efficiency, melt curve with a single clear peak, and specificity for m.3243A and m.3243G artificial standards (|Ct Wt -Ct Mut | max ). Using plasmid standards with various m.3243A>G heteroplasmy (1%-100%) at low, mid, and high copy numbers (3, 000, 10 4, and 10 5 -10 7, 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.