标题：Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers
中文标题：新型经济、准确、灵敏的单细胞分析方法用于线粒体 DNA 突变携带者的线粒体脱氧核糖核酸定量
引用信息：Zou W, Zong K, Zhang Z, Shen L, Wang X, Su X, Wang X, Yin T, Liang C, Liu Y, Liang D, Hu C, Cao Y, Ji D. Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers. J Assist Reprod Genet. 2023 Sep;40(9):2197-2209. doi: 10.1007/s10815-023-02878-w. Epub 2023 Jul 18. PMID: 37462790; PMCID: PMC10440311.
摘要：
Purpose: Although a variety of analytical methods have been developed to detect mitochondrial DNA (mtDNA) heteroplasmy, there are special requirements of mtDNA heteroplasmy quantification for women carrying mtDNA mutations receiving the preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PD) in clinic. These special requirements include various sample types, large sample number, long-term follow-up, and the need for detection of single-cell from biopsied embryos. Therefore, developing an economical, accurate, high-sensitive, and single-cell analytical method for mtDNA heteroplasmy is necessary.
Methods: In this study, we developed the Sanger sequencing combined droplet digital polymerase chain reaction (ddPCR) method for mtDNA quantification and compared the results to next-generation sequencing (NGS). A total of seventeen families with twelve mtDNA mutations were recruited in this study.
Results: The results showed that both Sanger sequencing and ddPCR could be used to analyze the mtDNA heteroplasmy in single-cell samples. There was no statistically significant difference in heteroplasmy levels in common samples with high heteroplasmy (≥ 5%), low heteroplasmy (< 5%), and single-cell samples, either between Sanger sequencing and NGS methods, or between ddPCR and NGS methods (P > 0.05). However, Sanger sequencing was unable to detect extremely low heteroplasmy accurately. But even in samples with extremely low heteroplasmy (0.40% and 0.92%), ddPCR was always able to quantify them. Compared to NGS, Sanger sequencing combined ddPCR analytical methods greatly reduced the cost of sequencing.
Conclusions: In conclusion, this study successfully established an economical, accurate, sensitive, single-cell analytical method based on the Sanger sequencing combined ddPCR methods for mtDNA heteroplasmy quantification in a clinical setting.
中文摘要：
目的: 虽然已有多种分析方法用于检测线粒体线粒体脱氧核糖核酸异质性，但对于在临床上接受胚胎植入前遗传筛选(PGD)和产前诊断(PD)检测的携带线粒体 DNA 突变的妇女，对线粒体 DNA 异质性定量有特殊要求。这些特殊要求包括各种样本类型、大样本数量、长期随访以及需要从活检胚胎中检测单细胞。因此，开发一种经济、准确、高灵敏度、单细胞的线粒体 DNA 异质性分析方法是十分必要的。
方法: 在这项研究中，我们发展了桑格测序联合液滴数字聚合酶链式反应(ddPCR)方法用于线粒体 DNA 定量，并将结果与下一代测序(NGS)进行比较。在这项研究中共招募了十七个具有十二个 mtDNA 突变的家庭。
结果: 结果显示，桑格测序和 ddPCR 均可用于分析单细胞样本的线粒体 DNA 异质性。高度异质性(≥5%)、低度异质性(< 5%)和单细胞样本的异质性水平在桑格测序和 NGS 方法之间或 ddPCR 和 NGS 方法之间没有统计学差异(P > 0.05)。然而，桑格测序无法准确地检测出极低的异质性。但即使在异质性极低(0.40% 和0.92%)的样本中，ddPCR 总是能够对它们进行定量。与 NGS 相比，桑格测序联合 ddPCR 分析方法大大降低了测序成本。
结论: 总之，本研究成功地建立了一种经济、准确、灵敏的单细胞分析方法，该方法基于桑格测序联合 ddPCR 方法，用于临床定量 mtDNA 异质性。
原文链接：
https://link.springer.com/article/10.1007/s10815-023-02878-w
