采用 ARMS-ddPCR 策略测定线粒体 3243A>G 异质性。

Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, China.

School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.

Am J Clin Pathol. 2022 May 4;157(5):664-677. doi: 10.1093/ajcp/aqab174.

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.

目的

确定核苷酸 3243（m.3243A>G）处线粒体 DNA（mtDNA）A 到 G 替换的异质性对于 m.3243A>G 相关线粒体疾病的精确诊断和遗传咨询至关重要。然而，如果没有适当的策略来适应携带数千至数百万 mtDNA 拷贝的样本中 1%至 100%的异质性水平，那么精确确定 m.3243A>G 异质性是具有挑战性的。

方法

我们使用扩增受阻突变系统-定量聚合酶链反应（ARMS-qPCR）和液滴数字 PCR（ddPCR）的组合策略来确定 m.3243A>G 异质性。专门设计并筛选了用于 ARMS-qPCR 和 ddPCR 的引物，以确定 m.3243A>G 异质性。使用不同混合物的 m.3243A 包含和 m.3243G 包含质粒的人工标准建立了优化的基于 ARMS-qPCR-ddPCR 的策略，并进一步使用含有 m.3243A>G 突变的临床样本进行了测试。

结果

根据 85%至 110%、R2>0.98 扩增效率、具有单个清晰峰的熔解曲线以及 m.3243A 和 m.3243G 人工标准的特异性标准（|CtWt-CtMut|max），研究中设计的 20 对引物中的一对被排除在 ARMS-qPCR-ddPCR 策略应用之外。使用具有不同 m.3243A>G 异质性（1%-100%）的质粒标准品，低、中和高拷贝数（分别为 3000、104 和 105-107），以及携带 m.3243A>G 的 20 名线粒体肌病、脑病、乳酸酸中毒和中风样发作患者的血液 DNA，我们发现 ARMS-qPCR 可用于可靠地确定低拷贝数为 3%至 100%和中至高拷贝数样本为 1%至 100%的 m.3243A>G。同时，ddPCR 可用于可靠地确定低至中拷贝数样本的 m.3243A>G 为 1%至 100%。