Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada.
Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, USA.
Aging Clin Exp Res. 2021 Jul;33(7):1811-1820. doi: 10.1007/s40520-020-01698-7. Epub 2020 Sep 23.
Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells.
Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages.
We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency.
Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach.
DISCUSSION/CONCLUSIONS: The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.
线粒体 DNA（mtDNA）缺失突变导致电子传递链缺陷细胞和年龄诱导的多种组织和哺乳动物物种的细胞丢失。体细胞 mtDNA 缺失突变的准确定量可作为年龄诱导的细胞丢失的指标。组织匀浆中 mtDNA 缺失分子的丰度低、缺失断点的多样性、单细胞中的随机积累以及细胞间的镶嵌分布，使得对 mtDNA 缺失分子的定量变得复杂。
将一种用于量化 mtDNA 缺失的临床前检测方法转化为可用于人类 DNA 样本的方法，并进行技术和生物学验证，然后在不同年龄的人类受试者上测试该检测方法。
我们开发并验证了一种高通量液滴数字 PCR 检测方法，可定量人类 mtDNA 缺失频率。
对来自 14 名男性受试者的股四头肌样本进行分析表明，mtDNA 缺失频率随年龄呈指数增长——从 20 岁到 80 岁平均增加了 98 倍。对扩增产物的序列分析证实了该检测方法对人类 mtDNA 缺失断点的特异性。合成突变混合物的滴定发现，检测下限至少为 0.6 百万分率。使用 6 个月大的 mtDNA 突变体小鼠的肌肉 DNA，我们测量到 mtDNA 缺失频率增加了 6.4 倍（即与野生型小鼠相比），这在生物学上验证了该方法。
讨论/结论：mtDNA 缺失频率的指数增长与已知的肌肉纤维丢失和随着年龄的增长而加速的死亡率同时发生。改进后的检测方法可从 DNA 样本中准确而敏感地定量缺失突变，足以测量健康个体一生中 mtDNA 缺失突变频率的变化，因此也适用于疑似线粒体疾病的患者。
