Institute of Legal Medicine, University of Münster, Röntgenstraße 23, 48149 Münster, Germany; Institute of Legal Medicine, University of Munich, Nußbaumstraße 26, 80336 Munich, Germany.
Institute of Legal Medicine, University of Münster, Röntgenstraße 23, 48149 Münster, Germany.
Forensic Sci Int Genet. 2021 Jul;53:102521. doi: 10.1016/j.fsigen.2021.102521. Epub 2021 Apr 25.
The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites.
分析特定 CpG 位点的 DNA 甲基化水平是估计个体年龄最有前途的分子技术之一。最近发表了许多研究报告，提出了基于血液样本 DNA 甲基化模式的年龄估计模型，只有少数研究使用唾液或口腔拭子。本研究旨在确定 88 个 CpG 位点在 8 个不同标记区域（PDE4C、ELOVL2、ITGA2B、ASPA、EDARADD、SST、KLF14 和 SLC12A5）中在口腔拭子样本中的年龄相关性甲基化。总共从年龄在 21 至 69 岁之间的个体中采集了 141 个口腔拭子，将其分为训练集（n=95）和验证集（n=46）。训练集样本通过焦磷酸测序进行分析，并确定了与年龄相关性最佳的标记物。进行逐步线性回归分析，得到一个包含三个经检查 CpG 位点的年龄估计模型，该模型显示估计年龄与实际年龄的平均绝对偏差为 5.11 岁。为了允许在没有焦磷酸测序设备的情况下，将其轻松应用于法医实验室，开发了一种多重 mini 测序反应，其中包括通过焦磷酸测序先前鉴定的相同 CpG 位点。使用焦磷酸测序模型和 mini 测序模型对调整后的年龄估计模型进行了评估，其估计年龄与实际年龄的平均绝对偏差分别为 5.16 岁和 5.33 岁。使用 46 个口腔拭子的独立验证集测试模型性能。焦磷酸测序模型和 mini 测序模型的估计年龄与实际年龄的平均绝对偏差分别为 5.33 岁和 6.44 岁。两种方法的比较结果显示，在定性和定量方面，结果具有高度一致性。总之，口腔拭子为分子年龄估计提供了一种替代血液样本的合适选择，并且具有非侵入性采集的额外优势。此外，我们还表明，对于单个 CpG 位点甲基化状态的分析，mini 测序提供了一种经济高效且易于集成的替代焦磷酸测序的方法。
