Gallimore Jamie M, McElhoe Jennifer A, Holland Mitchell M
Forensic Science Program, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 014 Thomas Building, University Park, PA 16802, United States.
Forensic Science Program, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 014 Thomas Building, University Park, PA 16802, United States.
Forensic Sci Int Genet. 2018 Jan;32:7-17. doi: 10.1016/j.fsigen.2017.09.013. Epub 2017 Oct 2.
Resolution of mitochondrial (mt) DNA heteroplasmy is possible when applying a massively parallel sequencing (MPS) approach. However, interpretation criteria for matching heteroplasmic sequences will need to be established that address a number of important topics, including the drift of variants in sample types such as human hair shafts. Prior to MPS analysis, we compared three different DNA extraction methods for hair using a custom mtDNA quantitative PCR (mtqPCR) assay, and found that a method involving bead capture significantly outperformed methods currently in place in forensic laboratories. The findings were similar for both fine (head) and coarse (pubic) hairs. Using the favored DNA extraction approach, hair shaft extracts were subjected to MPS analysis to assess heteroplasmic drift and the potential impact of the observations on interpretation of mtDNA MPS data. Hairs from different regions of the head were evaluated in individuals with varying percentages of heteroplasmy (low-level, high-level, and no detectable heteroplasmy), as measured in buccal and blood cells. The range of variant ratios was broad and was not significantly different between individuals in the low and high-level groups. While the range was also broad for the group of individuals with no heteroplasmy, the vast majority of hairs from these donors still exhibited a lack of heteroplasmy. A model was developed to predict the amount of heteroplasmy expected in hair samples when knowledge of the percentage of heteroplasmy in buccal cells is available. While significant, the model was best applied when levels of heteroplasmy in buccal cells was high. No correlation was observed between rates of heteroplasmy in blood cells and the predicted amount of heteroplasmy in hairs. Of particular interest, unexpected sites of mixed mtDNA sequence that could be interpreted as heteroplasmy were observed for 13% of the 75 hairs tested. These sites can be explained as heteroplasmy not observed in buccal or blood cells, or sites of DNA damage, with inherent heteroplasmy a likely cause, possibly due to de novo mutation events. Overall, when applying an MPS approach to hair analysis, heteroplasmic variant ratios may be quite different than those observed in blood cells, may be correlated to rates in buccal cells, and may include unexpected mixed sites. The results of this study directly impact MPS analysis of minor sequence variants from hair samples, and are particularly relevant to clinical and forensic investigations.
应用大规模平行测序（MPS）方法时，线粒体（mt）DNA异质性的解析是可行的。然而，需要建立匹配异质序列的解释标准，以解决一些重要问题，包括人类毛发等样本类型中变异的漂移。在进行MPS分析之前，我们使用定制的线粒体DNA定量PCR（mtqPCR）检测方法，比较了三种不同的毛发DNA提取方法，发现一种涉及磁珠捕获的方法明显优于法医实验室目前使用的方法。对于细（头部）毛发和粗（阴部）毛发，结果相似。使用最受青睐的DNA提取方法，对毛干提取物进行MPS分析，以评估异质性漂移以及这些观察结果对mtDNA MPS数据解释的潜在影响。对不同头部区域的毛发进行评估，这些个体的异质性百分比各不相同（低水平、高水平和未检测到异质性），异质性百分比通过颊细胞和血细胞进行测量。变异比率范围很广，低水平组和高水平组个体之间没有显著差异。虽然在没有异质性的个体组中变异比率范围也很广，但这些供体的绝大多数毛发仍然没有表现出异质性。当已知颊细胞中的异质性百分比时，开发了一个模型来预测毛发样本中预期的异质性量。虽然该模型具有显著意义，但在颊细胞中异质性水平较高时应用效果最佳。血细胞中的异质性比率与毛发中预测的异质性量之间没有相关性。特别值得注意的是，在测试的75根毛发中，有13%观察到了意想不到的混合mtDNA序列位点，这些位点可被解释为异质性，在颊细胞或血细胞中未观察到，或者是DNA损伤位点，固有异质性可能是一个原因，可能是由于新生突变事件。总体而言，在将MPS方法应用于毛发分析时，异质变异比率可能与血细胞中观察到的变异比率有很大不同，可能与颊细胞中的比率相关，并且可能包括意想不到的混合位点。本研究结果直接影响毛发样本中微小序列变异的MPS分析，对临床和法医调查尤为重要。
