Aggarwal Kangana, Sharma Nidhi, Mohapatra Braja Kishore, Chauhan Kamal, Singh Harpreet, Behera Chittaranjan
Department of Forensic Medicine and Toxicology, All India Institute of Medical Sciences, Room No.303, Second Floor, New Delhi, New Delhi, 110029, India.
Principal Scientific Officer and Head of Department, Central Forensic Science Laboratory, DFSS, New Delhi, 110003, India.
Forensic Sci Med Pathol. 2025 Aug 8. doi: 10.1007/s12024-025-01051-2.
Formalin fixation is a common practice for preserving forensic samples in developing countries like India, but it can adversely affect DNA quality and hinder downstream molecular analyses. This study investigated the effectiveness of mitochondrial DNA (mtDNA) typing in formalin-fixed, non-paraffin-embedded tissues when conventional Short Tandem Repeat (STR) profiling fails due to DNA degradation. Tissue samples from 30 deceased individuals were fixed in 10% unbuffered formalin and divided into four groups based on fixation duration (1-4 weeks). DNA was extracted, quantified, and subjected to STR profiling. Samples with incomplete STR profiles underwent mtDNA sequencing targeting the hypervariable regions to assess maternal lineage and haplogroup distribution. DNA quantification revealed significant degradation across all groups, with decreasing DNA concentrations over time. STR profiling showed limited success, with only 13 out of 27 loci amplified in group 1 and 7 loci in group 2, while groups 3 and 4 yielded no STR profiles. In contrast, mtDNA typing was successful in all samples, revealing distinct haplogroups and haplotypes. Haplogroup M was the most prevalent (50%), with 12 distinct subhaplogroups identified. The presence of East Asian (A11, G1b) and West Eurasian (H10g, T2H2, HV2a, U, R) haplogroups reflects historical migrations and admixture in the Indian population. The study highlights the limitations of STR profiling in formalin-fixed tissues and demonstrate the robustness of mtDNA typing. Incorporating mtDNA analysis into forensic protocols can enhance the reliability and comprehensiveness of DNA profiling results, particularly when dealing with challenging samples. Further research is needed to refine tissue preservation methods and optimize DNA recovery from formalin-fixed tissues to advance molecular analyses in forensic and retrospective studies.
在印度这样的发展中国家，福尔马林固定是保存法医样本的常见做法，但它会对DNA质量产生不利影响，并阻碍下游的分子分析。本研究调查了在传统短串联重复序列（STR）分析因DNA降解而失败时，福尔马林固定、非石蜡包埋组织中线粒体DNA（mtDNA）分型的有效性。从30名死者身上采集的组织样本用10%无缓冲福尔马林固定，并根据固定时间（1 - 4周）分为四组。提取DNA、进行定量，并进行STR分析。STR图谱不完整的样本进行针对高变区的mtDNA测序，以评估母系血统和单倍群分布。DNA定量显示所有组均有显著降解，DNA浓度随时间降低。STR分析成功率有限，第1组27个位点中仅13个被扩增，第2组为7个位点，而第3组和第4组未得到STR图谱。相比之下，mtDNA分型在所有样本中均成功，揭示了不同的单倍群和单倍型。单倍群M最为普遍（50%），共鉴定出12个不同的亚单倍群。东亚（A11、G1b）和西欧亚（H10g、T2H2、HV2a、U、R）单倍群的存在反映了印度人群历史上的迁徙和混合情况。该研究突出了福尔马林固定组织中STR分析的局限性，并证明了mtDNA分型的稳健性。将mtDNA分析纳入法医检测流程可以提高DNA图谱分析结果的可靠性和全面性，特别是在处理具有挑战性的样本时。需要进一步研究来改进组织保存方法，并优化从福尔马林固定组织中回收DNA的方法，以推进法医和回顾性研究中的分子分析。
