Aportaciones y avances de la genética forense en los sucesos con víctimas múltiples / Contributions and advances of forensic genetics in mass fatality incidents

La identificación de los afectados por un suceso con víctimas múltiples es una prioridad por razones humanitarias y legales. La genética forense juega un importante papel en estas situaciones que, por su complejidad, a menudo se convierten en un reto para los distintos profesionales implicados. El establecimiento de guías y recomendaciones facilita el seguimiento de protocolos estandarizados que permiten garantizar la fiabilidad del resultado final de la identificación. Así mismo, los avances en la genética forense contribuyen a agilizar la respuesta, aportando nuevas estrategias de análisis y herramientas de tipo bioinformático. Con este artículo, se pretende ofrecer una visión general de cómo la genética forense y sus avances pueden contribuir en estas situaciones, así como algunas claves para entender la labor de los laboratorios de genética forense en la identificación de cadáveres en sucesos con víctimas múltiples. (AU)

Disaster victim identification is crucial for humanitarian and legal reasons. Forensic genetics plays an important role in these situations which often become a challenge for the different professionals involved due to their complexity. The establishment of guidelines and recommendations makes it easier to follow standardized protocols that make it possible to guarantee the reliability of the identification final result. Likewise, advances in forensic genetics contribute to speeding up the response, providing new analysis strategies and bioinformatic tools. This article aims to provide an overview of how forensic genetics and its advances can contribute in these situations, as well as some keys to understanding the work of forensic genetics laboratories in the identification of corpses in events with multiple victims. (AU)

Age Prediction of Human Based on DNA Methylation by Blood Tissues.

In recent years, scientists have found a close correlation between DNA methylation and aging in epigenetics. With the in-depth research in the field of DNA methylation, researchers have established a quantitative statistical relationship to predict the individual ages. This work used human blood tissue samples to study the association between age and DNA methylation. We built two predictors based on healthy and disease data, respectively. For the health data, we retrieved a total of 1191 samples from four previous reports. By calculating the Pearson correlation coefficient between age and DNA methylation values, 111 age-related CpG sites were selected. Gradient boosting regression was utilized to build the predictive model and obtained the R2 value of 0.86 and MAD of 3.90 years on testing dataset, which were better than other four regression methods as well as Horvath's results. For the disease data, 354 rheumatoid arthritis samples were retrieved from a previous study. Then, 45 CpG sites were selected to build the predictor and the corresponded MAD and R2 were 3.11 years and 0.89 on the testing dataset respectively, which showed the robustness of our predictor. Our results were better than the ones from other four regression methods. Finally, we also analyzed the twenty-four common CpG sites in both healthy and disease datasets which illustrated the functional relevance of the selected CpG sites.

Variants in linkage status at D5S818 detected by multiple STR kits comparison and Sanger sequencing.

BACKGROUND: D5S818 discrepancies have been reported in forensic parental testing due to null alleles. However, more cases may be ignored since proportional null alleles were missed without detection of heredity discrepancy between parents and offspring. RESULTS: In this study, null allele 12 at D5S818 was detected by the PowerPlex® 21 System with a higher occurrence rate on the basis of review on 2824 samples from the 1282 routine cases in Chinese Han population. Sequencing results revealed novel variant of guanine (G) into adenine (A) in the 7th [AGAT] repeats in the core repeat region accompanied by rs1187948322 in the samples with null allele 12. CONCLUSIONS: Forensic STR typing may benefit from this discovery: (1) primer design of CE profiling system could be improved for sensitive population and (2) polymorphic information could be enriched for the accuracy and precision of NGS genotyping system. Peak area of D5S818 was also analyzed through different commercial STR kits. It is suggested that more attention should be paid on observed homozygosity with reduced peak area, especially for the samples from Chinese Han population.

Prediction of Eye Colour in Scandinavians Using the EyeColour 11 (EC11) SNP Set.

Description of a perpetrator's eye colour can be an important investigative lead in a forensic case with no apparent suspects. Herein, we present 11 SNPs (Eye Colour 11-EC11) that are important for eye colour prediction and eye colour prediction models for a two-category reporting system (blue and brown) and a three-category system (blue, intermediate, and brown). The EC11 SNPs were carefully selected from 44 pigmentary variants in seven genes previously found to be associated with eye colours in 757 Europeans (Danes, Swedes, and Italians). Mathematical models using three different reporting systems: a quantitative system (PIE-score), a two-category system (blue and brown), and a three-category system (blue, intermediate, brown) were used to rank the variants. SNPs with a sufficient mean variable importance (above 0.3%) were selected for EC11. Eye colour prediction models using the EC11 SNPs were developed using leave-one-out cross-validation (LOOCV) in an independent data set of 523 Norwegian individuals. Performance of the EC11 models for the two- and three-category system was compared with models based on the IrisPlex SNPs and the most important eye colour locus, rs12913832. We also compared model performances with the IrisPlex online tool (IrisPlex Web). The EC11 eye colour prediction models performed slightly better than the IrisPlex and rs12913832 models in all reporting systems and better than the IrisPlex Web in the three-category system. Three important points to consider prior to the implementation of eye colour prediction in a forensic genetic setting are discussed: (1) the reference population, (2) the SNP set, and (3) the reporting strategy.

Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

The first GHEP-ISFG collaborative exercise on forensic applications of massively parallel sequencing.

One of the main goals of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. The GHEP-ISFG supports several Working Commissions which develop different scientific activities. One of them, the Working Commission on "Massively Parallel Sequencing (MPS): Forensic Applications", organized its first collaborative exercise on forensic applications of MPS technology in 2019. The aim of this exercise was to assess the concordance between the MPS results and those obtained with conventional technologies (capillary electrophoresis and Sanger sequencing), as well as to compare the results obtained within the different MPS platforms and/or the different kits/panels and analysis software packages (commercial and open-access) available on the market. The seven participating laboratories analyzed some samples of the annual GHEP-ISFG proficiency test (EIADN No. 27 (2019)), using Ion Torrent™ or MiSeq FGx® platforms. Six of them sent autosomal STR sequence data, five laboratories performed MPS analysis of individual identification SNPs, four laboratories reported MPS data of Y-chromosomal STRs, and X-chromosomal STRs, three laboratories performed MPS analysis of ancestry informative SNPs and phenotype informative SNPs, two labs performed MPS analysis of the mitochondrial DNA control region, and only one lab produced MPS data of lineage informative SNPs. Autosomal STR sequencing results were highly concordant to the consensus obtained by capillary electrophoresis in the EIADN No. 27 (2019) exercise. Furthermore, in general, a high level of concordance was observed between the results of the participating laboratories, regardless of the platform used. The main discordances were due to errors during the analysis process or from sequence data obtained with low depth of coverage. In this paper we highlight some issues that still arise, such as standardization of the nomenclature for STRs analyzed by sequencing with MPS, the universal uptake of a nomenclature framework by the analysis software, and well established validation and accreditation of the new MPS platforms for use in routine forensic case-work.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics on forensic Y chromosome typing.

Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.

The STRidER Report on Two Years of Quality Control of Autosomal STR Population Datasets.

STRidER, the STRs for Identity ENFSI Reference Database, is a curated, freely publicly available online allele frequency database, quality control (QC) and software platform for autosomal Short Tandem Repeats (STRs) developed under the endorsement of the International Society for Forensic Genetics. Continuous updates comprise additional STR loci and populations in the frequency database and many further STR-related aspects. One significant innovation is the autosomal STR data QC provided prior to publication of datasets. Such scrutiny was lacking previously, leaving QC to authors, reviewers and editors, which led to an unacceptably high error rate in scientific papers. The results from scrutinizing 184 STR datasets containing >177,000 individual genotypes submitted in the first two years of STRidER QC since 2017 revealed that about two-thirds of the STR datasets were either being withdrawn by the authors after initial feedback or rejected based on a conservative error rate. Almost no error-free submissions were received, which clearly shows that centralized QC and data curation are essential to maintain the high-quality standard required in forensic genetics. While many errors had minor impact on the resulting allele frequencies, multiple error categories were commonly found within single datasets. Several datasets contained serious flaws. We discuss the factors that caused the errors to draw the attention to redundant pitfalls and thus contribute to better quality of autosomal STR datasets and allele frequency reports.

Evaluation of a six-dye multiplex composed of 27 markers for forensic analysis and databasing.

BACKGROUND: Short tandem repeat (STR) markers play a significant role in genetic applications and have proved to be effective for the personal identification in forensic medicine. In this study, a six-dye multiplex composed of 23 autosomal STR loci (TH01, D3S1358, Penta D, D6S1043, D21S11, TPOX, D1S1656, D12S391, Penta E, D10S1248, D22S1045, D19S433, D8S1179, D2S1338, D2S441, D18S51, vWA, FGA, D16S539, CSF1PO, D13S317, D5S818, D7S820), one Y chromosome STR (DYS391), two internal quality control markers (Quality Sensor QS1 and QS2), and Amelogenin was evaluated. METHODS: Evaluation studies, including PCR-based studies, sensitivity studies, species specificity studies, stability studies, DNA mixture studies, concordance studies, and precision evaluations were performed according to the guidelines of "Validation Guidelines for Forensic DNA Analysis Methods (2016)" by the Scientific Working Group on DNA Analysis Methods (SWGDAM). In addition, the forensic characteristics of 357 unrelated male samples from Han and Hui populations in China were investigated using 27 markers. RESULTS: Full STR profiles were obtained from different reaction volumes (5 ~ 25 µl), cycle numbers (28 ~ 34 cycles) and annealing temperatures (58 ~ 62°C). All STR profiles were obtained at humic acid concentration of up to 200 ng/µl and hematin concentration of up to 500 µM. No peaks were observed in most common animal samples except two innovative internal PCR controls (Quality Sensor QS1 and QS2). The six-dye multiplex showed a notably high value for the combined probability of exclusion (CPE), exhibiting values of with 0.99999999977688 in the Han population and 0.999999999583875 in the Hui population. The values of combined probability of discrimination (CPD) were 0.999999999999999999999999999997453 in the Han population and 0. 999999999999999999999999999994398 in the Hui population. In addition, concordance studies showed that there was no difference with the AGCU Express Marker 22 Kit. CONCLUSION: The results indicated that the Investigator® 26plex QS Kit is a robust, reliable, and suitable tool for forensic analysis and databasing.

DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis.

Forensic genetic laboratories perform a large amount of STR analyses of the Y chromosome, in particular to analyze the male part of complex DNA mixtures. However, the statistical interpretation of evidence retrieved from Y-STR haplotypes is challenging. Due to the uni-parental inheritance mode, Y-STR loci are connected to each other and thus haplotypes show patterns of relationship on the familial and population level. This precludes the treatment of Y-STR loci as independently inherited variables and the application of the product rule. Instead, the dependency structure of Y-STRs needs to be included in the haplotype frequency estimation process affecting also the current paradigm of a random match probability that is in the autosomal case approximated by the population frequency assuming unrelatedness of sampled individuals. Information on the degree of paternal relatedness in the suspect population as well as on the familial network is however needed to interpret Y-chromosomal results in the best possible way. The previous recommendations of the DNA commission of the ISFG on the use of Y-STRs in forensic analysis published more than a decade ago [1] cover the interpretation issue only marginally. The current recommendations address a number of topics (frequency estimators, databases, metapopulations, LR formulation, triage, rapidly mutating Y-STRs) with relevance for the Y-STR statistics and recommend a decision-based procedure, which takes into account legal requirements as well as availability of population data and statistical methods.

Population genetic data from 23 autosomal STR loci of Huaxia Platinum system in the Jining Han population.

BACKGROUND: Genetic polymorphisms at 23 short tandem repeat (STR) loci were investigated in 1,215 Jining Han individuals from Jining city, Shandong province, eastern China. METHODS: We used population genetic data of 23 autosomal STR loci included in the Huaxia Platinum system to evaluate 1,215 unrelated Chinese Han individuals in the Jining Han population. Allele frequencies and forensic parameters of the STR loci were determined and genetic relationships among the Jining Han and other Chinese populations were evaluated. RESULTS: In total, we observed 321 alleles, with frequencies ranging from 0.00041 to 0.52222. The combined discrimination power and probability of excluding paternity were 0.99999999999999999999999999919 and 0.99999999962, respectively. No deviations from HWE were observed at any loci. Population comparisons showed that the Xinjiang groups (Uyghur and Kazakh) and the Mongolian and Tibetan groups were isolated, while the Jining Han population clustered together with other populations, except the Guizhou Han population. CONCLUSION: This study demonstrated that 23 autosomal STR loci included in the Huaxia Platinum system are highly polymorphic and suitable for personal forensic identification and paternity testing in this population.

The yield of postmortem genetic testing in sudden death cases with structural findings at autopsy.

Sudden cardiac death (SCD) is often associated with structural abnormalities of the heart during autopsy. This study sought to compare the diagnostic yield of postmortem genetic testing in (1) cases with structural findings of uncertain significance at autopsy to (2) cases with autopsy findings diagnostic of cardiomyopathy. We evaluated 57 SCD cases with structural findings at cardiac autopsy. Next-generation sequencing using a panel of 77 primary electrical disorder and cardiomyopathy genes was performed. Pathogenic and likely pathogenic variants were classified using American College of Medical Genetics (ACMG) consensus guidelines. In 29 cases (51%) autopsy findings of uncertain significance were identified whereas in 28 cases (49%) a diagnosis of cardiomyopathy was established. We identified a pathogenic or likely pathogenic variant in 10 cases (18%); in 1 (3%) case with non-specific autopsy findings compared with 9 (32%) cases with autopsy findings diagnostic of cardiomyopathy (p = 0.0054). The yield of genetic testing in SCD cases with autopsy findings consistent with cardiomyopathy is comparable with the yield in cardiomyopathy patients that are alive. Genetic testing in cases with findings of uncertain significance offers lower clinical utility than in cardiomyopathy, with lower yields than detected previously. This highlights the need for stringent evaluation of variant pathogenicity.

Genetic polymorphism and phylogenetic analyses of 21 non-CODIS STR loci in a Chinese Han population from Shanghai.

BACKGROUND: Short tandem repeats (STRs) are essential genetic markers for forensic applications and population estimations; thus the population genetics of STR loci have been extensively studied and discussed. METHODS: In the present study, we detected 21 autosomal noncombined DNA index system (non-CODIS) STR loci in a Chinese Han population from Shanghai, calculated their forensic parameters and analyzed their genetic relationships with reported reference populations in mainland China. RESULTS: A total of 173 alleles were observed, with corresponding allele frequencies from 0.0020 to 0.5512. The cumulative power of discrimination (CPD) and the cumulative probability of exclusion (CPE) values of the 21 STR loci were 0.999999999999999999997337058271 and 0.99999953732495, respectively. The results of interpopulation differentiation, phylogenetic, multidimensional scaling, and structure analyses indicated a closer genetic relationship of the studied population with Han populations from other regions of China than with other populations. CONCLUSIONS: The 21 STR loci exhibited high genetic polymorphism in the studied Shanghai_Han population and could be used for forensic applications and population genetic studies.

Investigation of the forensic GlobalFiler loci in the genetically isolated Circassian subpopulation in Jordan.

Circassians are a Caucasian ethnic group who make up a significant minority in Jordan. Although other ethnic groups have been the subject of forensic genetic analysis, no published study has investigated the forensic genetic efficiency of short tandem repeats (STRs) in Circassians, neither in Jordan nor in any other country. The main objective of the current study is to determine the allelic frequencies and evaluate the forensic efficiency parameters of 21 highly polymorphic autosomal STR loci among the Circassian subpopulation in Jordan. The GlobalFiler loci were amplified using DNA extracted from the whole blood samples of 150 Jordanian Circassians. The SE33 locus was found to be the most informative and polymorphic STR marker while TPOX was the least informative. However, allele 8 of TPOX was the most common across all of the investigated 21 loci in Jordanian Circassians. The combined matching probability (CMP) and combined power of discrimination (CPD) were 5.02E-24 and 0.9999999, respectively.

An innovative panel containing a set of insertion/deletion loci for individual identification and its forensic efficiency evaluations in Chinese Hui ethnic minority.

BACKGROUND: Individual identification is one of the most important tasks in the field of forensic genetics. Insertion/Deletion (InDel) polymorphism marker has been a promising marker for individual identification. However, a part of InDel loci in commonly used commercial kit show low polymorphisms in Chinese populations. METHODS: We evaluated a panel of 35 InDel loci constructed previously for individual identifications in Hui group. Subsequently, population data of three Chinese populations from 1,000 Genomes Project database were used to evaluate individual identification performance of these 35 InDels. Forensic parameters, such as heterozygosity, power of exclusion, match probability and power of discrimination, were calculated to evaluate the forensic efficiency of these loci in Hui group. The heatmap of insertion allelic frequencies, Nei's genetic distances, pairwise fixation index values, principal component analyses and admixture analyses were used to analyze the genetic differentiations and structure between Hui group and other populations. RESULTS: In studied Hui group, besides rs3054057, polymorphism information content values of the remaining loci were greater than 0.3. Values of expected heterozygosity of these loci were close to 0.5. The combined power of discrimination and power of exclusion values were 0.99999999999999659609 and 0.998682, respectively. Analyses of population genetics revealed that Chinese Hui group had closer genetic relationships with East Asian populations than other intercontinental populations. CONCLUSION: The forensic statistical analyses revealed these loci showed relatively high genetic polymorphisms in Chinese Hui group, and could be served as a useful tool for individual identifications in Hui group. Population genetic evaluations indicated that Chinese Hui group had close genetic relationships with East Asian populations.

Prediction of Y haplogroup by polymerase chain reaction-reverse blot hybridization assay.

BACKGROUND: The analysis of Y-SNPs from crime scene samples is helpful for investigators in narrowing down suspects by predicting biogeographical ancestry. OBJECTIVE: In this study, a PCR-reverse blot hybridization assay (REBA) for predicting Y-chromosome haplogroups was employed to determine the major haplogroups worldwide, including AB, DE, C, C3, F, K, NO, O, O2, and O3 and evaluated. METHODS: The REBA detects nine biallelic Y chromosome markers (M9, M89, M122, M145, M175, M214, M217, P31, and RPS4Y711) simultaneously using multiple probes. RESULTS: The REBA for Y-single nucleotide polymorphisms (SNP) genotyping was performed using 40 DNA samples from Asians-14 Koreans, 10 Indonesians, six Chineses, six Thais, and four Mongolians. 40 Asian samples were identified as haplogroup O2 (40%), O3 (32.5%), C3 (17.5%), O (7.5%) and K (2.5%). These cases were confirmed by DNA sequence analysis (κ = 1.00; P < 0.001). CONCLUSION: PCR-REBA is a rapid and reliable method that complements other SNP detection methods. Therefore, implementing REBA for Y-SNP testing may be a useful tool in predicting Y-chromosome haplogroups.

Technical note: developmental validation of a novel 6-dye typing system with 36 Y-STR loci.

Y-chromosomal short tandem repeats (Y-STRs) have proven to be very useful in investigating sexual assault cases and in paternity lineage differentiation. However, currently available commercial Y-STR multiplex amplification systems bear the limitations in the identification of related males from the same paternal lineage due to there being an insufficient number of loci in any single amplification kit. The aim of this study was to establish and validate a novel 6-dye, 36-plex Y-STR multiplex amplification system that incorporated all of the loci present in the Yfiler™ Plus kit (DYS19, DYS385a/b, DYF387S1, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, DYS635, Y_GATA_H4) as well as a further nine highly polymorphic Y-STR loci (DYS388, DYS444, DYS447, DYS522, DYS527a/b, DYS549, DYS596, DYS643). The novel system was optimized and validated by a series of studies that tested the effect of different PCR-based conditions as well as the species specificity, sensitivity, stability, stutter precision, suitability for use on DNA mixtures, reproducibility, and parallel testing of the system, as well as its performance on casework samples and population analysis, according to the SWGDAM developmental validation guidelines. A total of 246 haplotypes were found for the 36 Y-STRs among 247 Guangdong Han unrelated males. Collectively, the results demonstrate that the developed 36-plex Y-STR system is sensitive, robust, reliable, and highly informative for use in forensic genetics.