Tian Meihui, Liu Xiangnian, Wang Danyang, Wang Yuxi, Wang Siwen, Wei Jiayi, Guan Dawei, Yao Jun
Department of Forensic Genetic and Biology, China Medical University School of Forensic Medicine, No.77 Puhe Road, Shenyang North New Area, Shenyang 110122, PR China; China Medical University Center of Forensic Investigation, No.77 Puhe Road, Shenyang North New Area, Shenyang 110122, PR China; Liaoning Province Key Laboratory of Forensic Bio-evidence Science, No.77 Puhe Road, Shenyang North New Area, Shenyang 110122, PR China.
Department of Clinical Medicine, Second Clinical College of China Medical University, Shenyang 110122, PR China.
Forensic Sci Int Genet. 2025 Apr;77:103242. doi: 10.1016/j.fsigen.2025.103242. Epub 2025 Feb 21.
Monozygotic twins (MZTs) share nearly identical genomic DNA sequences, making traditional forensic short tandem repeats (STR) genotyping methods ineffective for distinguishing between them. In recent years, the use of epigenetic factors in forensic applications has gained traction. The dynamic epigenetic factors can be influenced by inherited traits or acquired environmental factors. This study analyzed the expression profiles of transfer RNA-derived small RNAs (tsRNAs) in peripheral blood from four pairs of adult MZTs using Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing (PANDORA-seq). Differentially expressed tsRNAs (DEtsRNAs) were identified and validated using the reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (ddPCR) in both adult and newborn MZTs. The study also evaluated the longitudinal temporal stability, resistance to degradation, and suitability of DEtsRNAs for aged bloodstains. A total of 8795 expressed tsRNAs were identified in the four pairs of adult MZTs by PANDORA-seq. After screening with a normalized | log (fold change) | > 1 and an adjusted p-value < 0.05, 10, 187, and 1520 DEtsRNAs were shared by 4, 3, and 2 pairs of MZTs. RT-qPCR and ddPCR confirmed the expression of the 10 DEtsRNAs identified by PANDORA-seq. Six candidate tsRNAs (tRNA-Gly-GCC, tRNA-Leu-TAA, tRNA-Lys-CTT, tRNA-Val-AAC_5_end, tRNA-iMet-CAT_5_end, and tsRNA-3023a/b-PheGAA) were identified as effective discrimination markers, even in neonatal MZTs which are largely unaffected by environment factors. Forensic applicability assessment revealed that tRNA-Gly-GCC and tRNA-Leu-TAA remained detectable in the 180-day-series bloodstains, while tRNA-Lys-CTT, tRNA-Val-AAC_5_end, and tRNA-iMet-CAT_5_end were relatively stable after 15 times of freeze-thaw cycles. Additionally, tRNA-Gly-GCC and tRNA-Lys-CTT exhibited long-term stability, with consistent expression over six months. In conclusion, this study demonstrates that differential tsRNAs expression can serve as a novel biomarker for MZT identification in forensic medicine.
同卵双胞胎（MZTs）共享几乎相同的基因组DNA序列，这使得传统的法医短串联重复序列（STR）基因分型方法无法有效区分他们。近年来，表观遗传因素在法医应用中的使用越来越受到关注。动态表观遗传因素可能受到遗传特征或后天环境因素的影响。本研究使用克服RNA修饰中止测序的全景RNA显示技术（PANDORA-seq）分析了四对成年MZTs外周血中转移RNA衍生的小RNA（tsRNAs）的表达谱。在成年和新生儿MZTs中，使用逆转录定量聚合酶链反应（RT-qPCR）和液滴数字PCR（ddPCR）对差异表达的tsRNAs（DEtsRNAs）进行了鉴定和验证。该研究还评估了DEtsRNAs的纵向时间稳定性、抗降解性以及对陈旧血迹的适用性。通过PANDORA-seq在四对成年MZTs中共鉴定出8795个表达的tsRNAs。经过标准化的|log（倍数变化）|>1且校正后的p值<0.05筛选后，4对、3对和2对MZTs分别共享10个、187个和1520个DEtsRNAs。RT-qPCR和ddPCR证实了PANDORA-seq鉴定出的10个DEtsRNAs的表达。六种候选tsRNAs（tRNA-Gly-GCC、tRNA-Leu-TAA、tRNA-Lys-CTT、tRNA-Val-AAC_5_end、tRNA-iMet-CAT_5_end和tsRNA-3023a/b-PheGAA）被确定为有效的鉴别标记，即使在很大程度上不受环境因素影响的新生儿MZTs中也是如此。法医适用性评估显示，tRNA-Gly-GCC和tRNA-Leu-TAA在180天系列的血迹中仍可检测到，而tRNA-Lys-CTT、tRNA-Val-AAC_5_end和tRNA-iMet-CAT_5_end在经过15次冻融循环后相对稳定。此外，tRNA-Gly-GCC和tRNA-Lys-CTT表现出长期稳定性，在六个月内表达一致。总之，本研究表明，差异tsRNAs表达可作为法医学中鉴定MZTs的一种新型生物标志物。
