Department of Genetic Identification, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.
Division of Biological Traces, Netherlands Forensic Institute, The Hague, the Netherlands.
Forensic Sci Int Genet. 2021 Nov;55:102595. doi: 10.1016/j.fsigen.2021.102595. Epub 2021 Sep 11.
Y-chromosomal short tandem repeats (Y-STRs) with high mutation rates are recognized as valuable genetic markers for differentiating paternally related men, who typically cannot be separated with standard Y-STRs, and were shown to provide paternal lineage differentiation on a higher resolution level than standard Y-STRs. Both features make Y-STRs with high mutation rates relevant in criminal casework, particularly in sexual assault cases involving highly unbalanced male-female DNA mixtures that often fail autosomal forensic STR profiling for the male donor. Previously, the number of known Y-STRs with mutation rates higher than 10 per locus per generation termed rapidly mutating Y-STRs (RM Y-STRs) was limited to 13, which has recently been overcome by the discovery and characterization of 12 additional RM Y-STRs. Here, we present the development and validation of RMplex, an efficient genotyping system for analyzing 30 Y-STRs with high mutation rates, including all currently known RM Y-STRs, using multiplex PCR with capillary electrophoresis (CE) or massively parallel sequencing (MPS), overall targeting a total of 44 male-specific loci. If previously unavailable, repeat number assignations were provided based on newly generated MPS data. Validation tests based on the CE method demonstrated that the results were both repeatable and reproducible, full profiles were achieved with minimal input DNA of 250 pg for RMplex 1 and 100 pg for RMplex 2, and in the presence of inhibitors, or with a surplus of female DNA, the assays performed reasonably well. Application of RMplex to differentiate between paternally related men was exemplified in 32 males belonging to five different paternal pedigrees. Given further successful forensic validation testing, we envision the future application of RMplex in criminal cases where it is suspected, or cannot be excluded, that the crime scene trace originated from a male relatives of the suspect who is highlighted with standard Y-STR matching. Other applications of RMplex are in criminal cases without known suspects to differentiate between male relatives highlighted in familial searching based on standard Y-STR matching.
Y 染色体短串联重复序列（Y-STRs）具有较高的突变率，被认为是区分亲缘关系男性的有价值的遗传标记，这些男性通常无法通过标准 Y-STRs 区分，并且在提供父系血统分化方面的分辨率高于标准 Y-STRs。这些特征使得高突变率 Y-STRs 在刑事案件中具有相关性，特别是在涉及高度不平衡的男女 DNA 混合物的性侵犯案件中，这些混合物通常无法通过男性供体的常染色体法医 STR 分析进行分析。此前，具有高于每代每座点 10 个突变率的已知 Y-STR 数量被限制在 13 个，这一限制最近通过发现和描述 12 个额外的 RM Y-STR 而得到突破。在这里，我们介绍了 RMplex 的开发和验证，这是一种用于分析 30 个高突变率 Y-STR 的高效基因分型系统，包括所有当前已知的 RM Y-STR，使用带有毛细管电泳（CE）或大规模平行测序（MPS）的多重 PCR，总体目标是总共 44 个男性特异性位点。如果以前不可用，则根据新生成的 MPS 数据提供重复数赋值。基于 CE 方法的验证测试表明，结果具有可重复性和可再现性，使用 RMplex1 的最小输入 DNA 为 250pg，使用 RMplex2 的最小输入 DNA 为 100pg 即可获得完整的图谱，并且在存在抑制剂或女性 DNA 过剩的情况下，该测定也能很好地进行。将 RMplex 应用于区分亲缘关系男性的示例是在属于五个不同父系血统的 32 名男性中进行的。在进一步成功的法医验证测试之后，我们设想在犯罪案件中应用 RMplex，在这些案件中，怀疑或不能排除犯罪现场痕迹来源于嫌疑人的男性亲属，而这些亲属的标准 Y-STR 匹配突出显示。RMplex 的其他应用是在没有已知嫌疑人的刑事案件中，基于标准 Y-STR 匹配的家族搜索来区分突出显示的男性亲属。
