Nakanishi Hiroaki, Shojo Hideki, Ohmori Takeshi, Hara Masaaki, Takada Aya, Adachi Noboru, Saito Kazuyuki
Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan,
Int J Legal Med. 2015 Jan;129(1):23-9. doi: 10.1007/s00414-014-1065-2. Epub 2014 Aug 27.
A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.
建立了一种基于检测X染色体数量的新型性别鉴定方法。目前基于检测Y染色体的方法，可以直接将未知样本鉴定为男性，但女性性别是通过未检测到Y染色体来间接确定的。因此，直接确定女性性别很重要，因为Y染色体DNA的质量（如片段化和牙釉蛋白-Y无效等位基因）可能导致错误结果。因此，我们开发了一种新型性别鉴定方法，通过使用拷贝数变异（CNV）检测技术（比较Ct法）分析X染色体数量。在本研究中，我们设计了一组引物，以不含CNV区域的牙釉蛋白-X基因为靶点来确定X染色体拷贝数，以排除CNV区域对比较Ct值的影响。使用实时PCR，通过CopyCaller软件对X染色体数量进行统计学测定。使用该方法，以1 ng模板DNA对所有参与者（20名男性，20名女性）的DNA样本进行了正确评估。发现使用该方法进行准确性别鉴定至少需要0.2 ng模板DNA。当使用紫外线照射的模板DNA作为模拟法医样本时，无法通过短串联重复序列（STR）分析确定样本性别，但使用我们的方法可以正确确定。因此，我们成功开发了一种基于X染色体数量的性别鉴定方法。我们的新方法将在法医实践中用于性别鉴定。
