Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle.
Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle.
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DOI：
10.1007/s40520-020-01698-7
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发表时间：
2021-07
影响因子：
4
通讯作者：
Wanagat J
中科院分区：
文献类型：
--
作者：
Herbst A;Lee CC;Vandiver AR;Aiken JM;McKenzie D;Hoang A;Allison D;Liu N;Wanagat J
线粒体 DNA (mtDNA) 缺失突变会导致多种组织和哺乳动物物种中电子传递链缺陷细胞和年龄诱导的细胞损失。体细胞 mtDNA 缺失突变的准确定量可以作为年龄引起的细胞损失的指标。 mtDNA 缺失分子的定量因其在组织匀浆中的低丰度、缺失断点的多样性、单细胞中的随机积累以及细胞间的马赛克分布而受到干扰。通过技术和生物学验证，转化临床前测定以定量用于人类 DNA 样本中的 mtDNA 缺失，并在不同年龄的人类受试者上测试该测定。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 14 名男性受试者的人类股四头肌样本的分析表明，mtDNA 缺失频率随着年龄的增长呈指数增加——从 20 岁到 80 岁，平均增加了 98 倍。扩增产物的序列分析证实了人类 mtDNA 缺失断点测定的特异性。合成突变混合物的滴定发现检测下限至少为百万分之 0.6。使用六个月大 mtDNA 突变小鼠的肌肉 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），从生物学上验证了该方法。线粒体 DNA 缺失频率呈指数增加，伴随着已知的肌纤维损失和随年龄增长而加速的死亡率。改进的检测方法可以准确、灵敏地定量 DNA 样本中的缺失突变，并且足以测量健康个体整个生命周期中 mtDNA 缺失突变频率的变化，因此也可以测量疑似线粒体疾病患者的线粒体 DNA 缺失突变频率的变化。
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 six-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. 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.
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影响因子：
4.6
作者：
Belmonte FR;Martin JL;Frescura K;Damas J;Pereira F;Tarnopolsky MA;Kaufman BA
通讯作者：
Kaufman BA
影响因子：
4.8
作者：
Hepple RT
通讯作者：
Hepple RT
影响因子：
14.9
作者：
CORTOPASSI, GA;ARNHEIM, N
通讯作者：
ARNHEIM, N
DOI：
10.1152/ajprenal.00307.2006
发表时间：
2007-06-01
影响因子：
4.2
作者：
McKiernan, Susan H.;Tuen, Victoria C.;Aiken, Judd M.
通讯作者：
Aiken, Judd M.
DOI：
10.1093/geronj/48.6.b201
发表时间：
1993-11-01
期刊：
JOURNALS OF GERONTOLOGY
影响因子：
--
作者：
LEE, CM;CHUNG, SS;AIKEN, JM
通讯作者：
AIKEN, JM