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

Authors
  • Xu, Pu1, 2
  • Jia, Manli2
  • Yan, Jimei2
  • Yuan, Xiangshu2
  • Yu, Weidong2
  • Zhou, Zhuohua2
  • Fang, Hezhi2
  • Gao, Feng3
  • Shen, Lijun2
  • 1 Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, China. , (China)
  • 2 School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China. , (China)
  • 3 Zhejiang Provincial People's Hospital, Affiliated People's Hospital of Hangzhou Medical College, Hangzhou, China. , (China)
Type
Published Article
Journal
American Journal of Clinical Pathology
Publisher
Oxford University Press
Publication Date
May 04, 2022
Volume
157
Issue
5
Pages
664–677
Identifiers
DOI: 10.1093/ajcp/aqab174
PMID: 34698344
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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. 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. 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. An ARMS-qPCR-ddPCR-based strategy was successfully established for precise determination of m.3243A>G heteroplasmy in complex clinical samples. © American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: [email protected]

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