Complex Kinship Cases Research Articles

Genetic Diversity and Forensic Utility of X-STR Loci in Punjabi and Kashmiri Populations: Insights into Population Structure and Ancestry

Background: X-chromosomal short tandem repeats (X-STRs) are crucial in forensic applications, particularly in complex kinship cases, and play an important role in population genetics. However, there is limited data on X-STR variation in Pakistani populations, especially among ethnic groups like Kashmiri and Punjabi. Methodology: This study investigates the forensic and genetic properties of 12 X-STRs from the Investigator Argus X-12 Kit (QIAGEN, Hilden, Germany) in 125 families (75 Kashmiri, 50 Punjabi) from Azad Jammu and Kashmir and Punjab, Pakistan. Results: In both populations, a total of 222 alleles were identified across the 12 X-STR loci (Punjabi 171 alleles, Kashmiri 161 alleles), with allele frequencies ranging from 0.0056 to 0.3033. DXS10148 was the most polymorphic locus with 28 alleles, while DXS7132 was the least polymorphic with 9 alleles. Most loci were in linkage equilibrium, except for the DXS10135/DXS10148 pair in males, with no loci exhibiting significant linkage disequilibrium in females. The combined power of discrimination was 0.999 999 9977 for Kashmiri males, 0.999 999 999 999 9746 for Kashmiri females, and 0.999 999 999 999 9781 for Punjabi females. In Kashmiri males, 34, 31, 28, and 32 haplotypes were observed across the four linkage groups (LG1, LG2, LG3, and LG4), though these groups did not form stable haplotypes, as indicated by Linkage Equilibrium within and significant Linkage Disequilibrium between groups. Conclusions: Genetic structure analysis using Principal Component Analysis and STRUCTURE revealed distinct clustering patterns for the Kashmiri and Punjabi populations, indicating unique genetic backgrounds and ancestry influences, particularly distinguishing them from East Asian populations. This study provides a comprehensive analysis of X-STR variation in Punjabi and Kashmiri populations, offering valuable insights for forensic and population genetic studies.

Population data for 12 X-STRs loci in Malaysian Malay and Chinese populations

The utilization of X-chromosome short tandem repeats (X-STRs) for human identification particularly in resolving complex kinship cases has been advocated. Since, forensic statistical parameters vary among different populations, and because the X-STRs population data representing the diverse population of Peninsular Malaysia remain unavailable, the specific attempt reported here for the Malays (n = 224) and Chinese (n = 201) populations appears forensically relevant to support the evidential value of the 12 X-STRs markers for human identification in Malaysia. Results derived from the Qiagen Investigator® Argus X-12 kit revealed that DXS10135 as the most polymorphic locus with high genetic diversity, polymorphic information content, heterozygosity as well as power of exclusion. Based on allele frequencies, the combined power of discrimination as well as the mean exclusion chance (MECKrüger, MECKishida, MECDesmarais and MECDesmaraisDuo) values for the Malays and Chinese were individually ≥0.999995532964908. As for the combined power of discrimination as well as the mean exclusion chance (MECKrüger, MECKishida, MECDesmarais and MECDesmaraisDuo) calculated based on haplotype frequencies, the values were ≥0.9999986410567 for the Malays and Chinese populations. In addition, results from the genetic distance, neighbor-joining phylogenetic tree and principal component analysis revealed close biogeographical distributions of the studied populations with other South East Asian populations. Hence, the utilization of the X-STRs data for identifying individuals among the Malays and Chinese populations in Peninsular Malaysia for forensic application appears as highly supported.

Determination of paternity on the basis of half-sibling testing.

This paper presents the results of genetic testing in two cases of shared paternity between half-siblings. In the first example, the research was based on the analysis of half-siblings and their mothers with respect to 21 autosomal markers. In the second, the research was based on both the analysis of 30 autosomal markers and X chromosome markers in putative sisters and the mother of one of them. Selected examples are presented to illustrate how the use of different research strategies affects the outcome of the likelihood ratio and the ability to make inferences in complex kinship cases.

Recombulator-X: A fast and user-friendly tool for estimating X chromosome recombination rates in forensic genetics.

Genetic markers (especially short tandem repeats or STRs) located on the X chromosome are a valuable resource to solve complex kinship cases in forensic genetics in addition or alternatively to autosomal STRs. Groups of tightly linked markers are combined into haplotypes, thus increasing the discriminating power of tests. However, this approach requires precise knowledge of the recombination rates between adjacent markers. The International Society of Forensic Genetics recommends that recombination rate estimation on the X chromosome is performed from pedigree genetic data while taking into account the confounding effect of mutations. However, implementations that satisfy these requirements have several drawbacks: they were never publicly released, they are very slow and/or need cluster-level hardware and strong computational expertise to use. In order to address these key concerns we developed Recombulator-X, a new open-source Python tool. The most challenging issue, namely the running time, was addressed with dynamic programming techniques to greatly reduce the computational complexity of the algorithm. Compared to the previous methods, Recombulator-X reduces the estimation times from weeks or months to less than one hour for typical datasets. Moreover, the estimation process, including preprocessing, has been streamlined and packaged into a simple command-line tool that can be run on a normal PC. Where previous approaches were limited to small panels of STR markers (up to 15), our tool can handle greater numbers (up to 100) of mixed STR and non-STR markers. In conclusion, Recombulator-X makes the estimation process much simpler, faster and accessible to researchers without a computational background, hopefully spurring increased adoption of best practices.

Optimization and Development of an Efficient 13 X-STRs Multiplex PCR System for Paternity Testing

X-chromosomal short tandem repeats (X-STRs) markers complement autosomal STR identification systems and valuable tools in complex kinship cases. Objective: To develop a multiplex PCR system that consists of 13 X-chromosome STR markers, including GATA172D05, DXS8378, DXS6801, DXS6793, DXS6810, DXS7132, GATA31E08, DXS9902, HPRTB, DXS6789, DXS7423, DXS8377, DXS981 and sex-determining locus Amelogenin. Methods: Primer sequences of all X-STR markers were acquired from the Genome databases, and the original sequences for HPRTB, DXS6789, DXS7423, DXS8377 and DXS981 were modified to eliminate primer-dimer formation and optimize melting temperatures to increase annealing efficiency. All primer pairs were labelled with fluorescent dyes to support amplification in a multiplex PCR, and the cycling conditions for multiplex PCR were optimized. Alleles for each locus were bi-directionally sequenced to determine the exact repeat size, and alleles generated in multiplex reactions were undistinguishable from alleles produced in a single marker PCR reaction. Results: The combined power of discrimination of 13 X-STRs was 2.96 x 10-13 and 2.58 x10-8 in females and males, respectively. Conclusions: In conclusion, we have developed a 14-plex PCR system that can potentially be used for parentage testing and forensic casework studies.

Three mutations at a Y-STR haplotype defy a paternal half-brothers kinship case analysis.

This work presents the results of a DNA test aimed to determine a possible biological link of paternal half brotherhood of two males. The combined use of biparentally inherited markers (autosomal STRs) and a panel of 27 Y-STRs allowed us to determine the existence of a biological relationship of kinship, even after detecting three mutations at their Y-STR haplotypes along the analyses, constituting an infrequent multiple mutation situation. This case is an example illustrating the importance of having different analytical markers sets and strategies for clarifying complex kinship cases where mutations occur.

Resolving the recombination pattern of 38 X-STRs from Chinese Han three-generation pedigrees

X-chromosomal markers have been proved as a useful tool for solving complex kinship cases due to its sex-linked inheriting feature. Among these markers, tightly linked X-STR clusters forming haplotypes are highly informative. The analysis of the haplotypes requires determination of linkage disequilibrium. In this study, genetic linkage, recombination fractions and mutation rates of 38 X-STR loci in 177 three-generation pedigrees were investigated. Genetic linkage analysis and calculation of recombination fractions were performed within each pair of markers and clusters. Then mutation rates were calculated. The results showed that, a) 22 recombination events happened within the tightly linked X-STR clusters, which span<1.0 Mb; b) significantly linked marker pairs were observed with the LOD (logarithm of the odds) scores > 2.0 (2.0104 to 54.8316); c) the average mutation rate of the 38 X-STR loci was 1.32 × 10−3 per meiosis in the Chinese Han population, with DXS10135 and DXS8377 presenting notably high mutation rate (6.5 × 10−3). Our results confirmed that meiotic recombination was not a simple function of physical distance, so that whether recombination occurred at the closely clustered X-STRs or not should be assumed cautiously considering the stability of haplotypes in inheritance process for kinship analysis. This study supplemented the existing database and laid an experimental foundation for the future study on genetic characteristics, recombination, and mutation of the X-STRs.

Expanding the Swiss autosomal marker set to 32 STRs

By genotyping 1198 individuals with the Qiagen Investigator® HDplex Kit, we expand the Swiss autosomal STR dataset to 32 loci, providing additional resources for complex kinship cases. We present the first high-quality allele frequency dataset for loci D2S1360, D5S2500, D7S1517, and D10S2325 that will be accessible through the ENFSI reference database STRidER. For loci D3S1744, D4S2366, D6S474, D8S1132, and D21S2055, we provide a first European STRidER dataset.

German population data for 18 X-STRs: a hexaplex PCR adding two clusters of X-STRs to the Argus X-12 set and expanding the German haplotype databases.

In kinship analysis, large data sets with estimated haplotype frequencies for marker clusters are very important for the likelihood calculation. Practical use of the X-STRs demonstrated that in some complex kinship cases, the marker set of the Investigator Argus X-12 kit can be insufficient. This study aimed to extend the German data base of the Argus X-12 kit (1037 haplotypes) and for a cluster in Xq21 (806 haplotypes) with additional 700 male haplotypes and to include a further cluster in Xp22.3 to complete the X-STR marker set for complex kinship cases.

Application of CPI cutoff value based on parentage testing of duos and trios typed by four autosomal kits.

In this study, we analyzed the application of four autosomal kits and the sensitivity of the combined paternity index (CPI) cutoff value (CPI≥10000) in parentage testing. First, 1442 real trios and 803 real duos were tested using the Goldeneye 25A kit. The Goldeneye 25A kit covers the autosomal short tandem repeat (STR) loci of the other three kits, so we calculated the CPI value of every case for the four kits. Second, three complex close relative kinship cases were also analyzed to evaluate the application of the CPI cutoff value. The CPI values of all trio cases were higher than 10000 using the four kits; the CPI values of all duo cases were higher than 10000 using the Goldeneye 25A kit; and the CPI values of a portion of the duo cases were lower than 10000 using the other three kits. In the three complex close relative cases, the alleged father or mother was not excluded using 40 autosomal STRs. Adding X chromosome short tandem repeats (X-STR) and samples of biological fathers or mothers, the conclusions were confirmed. The four kits were adequate to draw conclusions in the trio cases; the Goldeneye 25A Kit was adequate to draw conclusions in the duo cases; and the other three kits were not sufficient for a portion of the duo cases. The CPI cutoff value was sensitive for real trio and duo cases. In complex close relative kinship cases, high CPI values may result in false conclusions.

Genetic characterization of 12 STRs (Investigator® HDplex kit) in the Ecuadorian population

Allele frequencies, and genetic data for 12 autosomal Short Tandem Repeat (STRs) loci (D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D21S2055, and SE33 [ACTBP2]) using the Investigator® HDplex kit (QIAGEN) were obtained from 200 non-related individuals from the Ecuadorian mestizo population. Subjects included 100 men and 100 women, from the 24 provinces of Ecuador. Heterozygosity (Ho), power of discrimination (PD), power of exclusion (PE) and polymorphism information content (PIC) were estimated for the population sample. Locus SE33 had the highest values for power of discrimination (0.9372), polymorphism information content (0.9338), and power of exclusion (0.7852). The most informative STRs for the Ecuadorian population were SE33, D7S1517, D10S2325 and the least informative were D2S1360, D3S1744 and D6S474. The Investigator® HDplex makers have shown to be highly informative in the Ecuadorian population and are suitable as a powerful complementary tool in forensic analysis and complex kinship cases.

Genetic polymorphisms and forensic efficiency of 19 X-chromosomal STR loci for Xinjiang Mongolian population.

AimX-chromosomal short tandem repeat (X-STR) loci are playing an increasingly important role in some complex kinship cases in recent years. To investigate the forensic efficiency of X-STRs of Mongolian minority group from Xinjiang Uygur Autonomous Region, China, and further depict the genetic relationship among Xinjiang Mongolians and other populations, 267 blood samples from unrelated healthy Xinjiang Mongolians were amplified by an AGCU X-19 STR kit.ResultsNo deviations for all 19 X-STR loci were observed from the Hardy–Weinberg equilibrium after Bonferroni correction (p > 0.0026) in female samples. The most frequent allele was allele 10 at locus DXS10164 with the frequency 0.5663. The polymorphism information content values of the 19 X-STR loci were more than 0.5 with the highest polymorphism at the locus DXS10135. The cumulative power of discrimination were 0.99999999999999999999988761005481 in females and 0.999999999999903 in males, respectively; and the cumulative mean exclusion chances were 0.9999999969738068321121 in duos and 0.999999999998952 in trios, respectively. The seven linkage groups were extremely informative, with all the haplotype diversities greater than 0.9487. No linkage disequilibrium was observed for a significance level of 0.00029 (p = 0.05/171) after Bonferroni correction. The DA distances, multidimensional scaling plot and phylogenetic tree based on the 11 overlapping X-STR loci all presented that the Xinjiang Mongolian population was genetically different from other Asian populations, including the Mongolian population from Inner Mongolia Autonomous Region, China.ConclusionThis study indicated that the 19 X-STR multiplex PCR system was of high utility value for both forensic practices and population genetic research in Xinjiang Mongolian group.

Investigation of 12 X-STR loci in Mongolian and Eastern Han populations of China with comparison to other populations

Due to the unique inheritance pattern, X-chromosomal short tandem repeats (X-STRs) have several advantages in complex kinship cases, such as deficiency cases or grandparent-grandchild and half-sisters testing. In our study, 541 unrelated individuals gathered from Mongolian and Eastern Chinese Han populations were successfully genotyped using the Investigator Argus X-12 kit. We calculated allele/haplotype frequencies and other forensic parameters of the two populations and further explored their genetic distance with already published Chinese populations and six global populations. Our results showed that the 12 X-STR markers were highly informative in the two populations when compared with nine other Chinese populations: significant differences were found at several loci. Geographically neighboring populations or different ethnic groups within the same area appeared to have closer evolutionary relationships. We also analyzed population genetic structure by performing clustering with the STRUCTURE program and Principal Coordinate Analysis (PCoA), and we found that the Chinese and other populations enrolled in this study could be distinguished. Furthermore, Mongolian males were distinguishable from the other studied males by a moderate genetic distance. Our study also expanded the X-STR database, which could facilitate the appropriate application of the 12 X-STR markers in the forensic field in China.

Allele and haplotype diversity of 12 X-STRs in Sardinia

The analysis of clusters of tightly linked X-chromosome short tandem repeat (STR) markers can assist the interpretation of complex kinship cases. However, when linkage disequilibrium (LD) is present in the population of origin of tested individuals, haplotype rather than allele frequencies should be used in likelihood calculations. The diversity of twelve X-STRs arranged in four linkage groups (I: DXS10148-DXS10135-DXS8378; II: DXS7132-DXS10079-DXS10074; III: DXS10103-HPRTB-DXS10101; IV: DXS10146-DXS10134-DXS7423) was tested in a Sardinian population sample (n=516) including three open populations from the Northern, Central and Southern part of the island, and three isolates (Benetutti, Desulo, Carloforte). Evidence of LD was detected in Sardinia within each linkage group. Significant differences in haplotype and allele frequency distribution of X-STR markers was seen between isolates and open populations, which on the contrary appeared highly homogeneous.The percentage of Sardinian haplotypes previously unobserved in a similar dataset compiled for the Italian population was: 76.3% (linkage group I), 61.3% (linkage group II), 54.1% (linkage group III), 58.9% (linkage group IV). Significant pairwise genetic differences were seen between mainland Italy, the three Sardinian isolates, and the open population of Southern Sardinia.The study confirms the presence of high levels and complex patterns of LD along the X chromosome in Sardinia, and provides population-specific haplotype data for biostatistical evaluation in kinship testing.

Analysis of four novel X-chromosomal short tandem repeats within 71kb of the Xp22.3 region.

DNA testing using X-chromosomal short tandem repeat (X-STR) polymorphisms has been used in maternity/paternity and complex kinship cases. Analyses of repeat sequences, surveys on racial statistics, and development of practical applications for DNA testing continue to be reported. In this study, we identified four novel tetranucleotide STR loci located in the X chromosome, which is the basis of X-STR research. These four tetranucleotide STRs were located within 71kb of the chromosome Xp22.3 region. Using sequence analysis of the structure of repeat sequences, we identified simple repeat sequences of TAAA, CTTT, TATC, and GATA with rare insertions. We then calculated forensic statistical parameters using base length analysis. In the Japanese population, the polymorphism information content was 0.597-0.687, power of discrimination in females was 0.829-0.884, and power of discrimination in males was 0.635-0.729. As these tetranucleotide STRs are closely linked, we conducted haplotype analysis and detected that three loci (LC149476, LC149479, and LC149480) were in linkage disequilibrium. We demonstrated that the simultaneous analysis of these loci may be useful in complex kinship cases. Because these four loci can be detected by multiplex PCR, the detection of alleles at these loci can be rapidly and easily achieved. We conclude that the X-STR loci detected in this study may be useful tools in complex kinship cases and may increase the reliability of genetic testing.

Population genetic data for 12 X-STR loci in the Northern Han Chinese and StatsX package as tools for population statistics on X-STR

The X-chromosomal short tandem repeat (X-STR) has the advantage in female traces identification against male contamination and in complex kinship cases. In this study, a total of 516 Northern Han Chinese were genotyped using the Investigator® Argus X-12 QS Kit and pairwise genetic distances (FST) were measured between Northern Han Chinese and 35 published populations using the same 12 X-STR loci in four presumed linkage groups (LG). Meanwhile, the StatsX package was compiled to aid the analysis of population statistics on X-STR. Off-ladder alleles were investigated by Sanger sequencing or next generation sequencing (NGS). The results showed that high combined PDM, PDF, MECKrüger, MECKishida, MECDesmarais and MECDesmarais Duo based on allele frequencies were achieved as 0.999999998, >0.999999999, 0.999996425, 0.999999993, 0.999999993 and 0.999998732, respectively, so did they based on haplotype frequencies. Averaged FST and multidimensional scaling (MDS) plot generally mirrored with the biogeography distribution of the studied populations and their historical relationships. A total of 16 unique off-ladder alleles were observed in this experiment, four of which have not been reported yet. The StatsX package could obtain full concordance with established software. Overall, the Investigator® Argus X-12 QS Kit may provide high polymorphic information for forensic identification and kinship analysis in the Northern Han Chinese population, and the StatsX package can make the workflow smoother for researchers to do population statistical analysis on X-STR.

Detection of a novel X-chromosomal short tandem repeat marker in Xq28 in four ethnic groups.

DNA testing of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several studies, mainly due to its applicability in the investigation of complex kinship cases. Studies of X-STR in analyses of DNA sequences, population studies and DNA testing applications have been reported. We performed detection and population genetic study of a novel tetranucleotide X-STR locus in the present study. We identified a unique X-STR locus consisting of two tetranucleotides in Xq28. Although the STR is a simple tetranucleotide, its polymorphism was comparatively high [polymorphism information content (PIC)=0.7140] in Japanese subjects. In addition, the STR varied in structure among ethnic groups. We conclude that this locus will be useful for forensic DNA testing and anthropological studies.

A new 17 X-STR multiplex for forensic purposes

The use of STRs located in the X chromosome (X-STRs) is very efficient for determining kinship between fathers and daughters as well as other complex kinship cases. Currently, X-STR markers are analyzed by using two different multiplexes, the decaplex of the GHEP-ISFG and the Investigator Argus X-12 Kit. Here, we present a new multiplex whose advantage is the analysis of 17 X-STRs in a single reaction. Population samples from four different continents were analyzed. A sensitivity test showed the efficacy of this new multiplex for samples with DNA quantities as low as 100pg.

Belarusian experience of the use of FamLinkX for solving complex kinship cases involving X-STR markers

Use of X-STR markers extends and gives additional possibilities to a forensic DNA expert for kinship analysis. FamLinkX is software for the calculation of likelihood ratios (LRs) for kinship cases based on X-STR DNA profiles that takes linkage and linkage disequilibrium as well as mutations of X-STR markers into account. Belarusian haplotype frequencies database of 360 unrelated Belarusian males has been uploaded to FamLinkX. LR values for 6 deficient paternity cases and 2 forensic cases involving two putative full siblings, females, were calculated with different values of lambda (λ=1, 10, 100) in order to account for uncertainties in the haplotype frequency estimation. To demonstrate the ethnic sensitivity of X-STR markers and importance of the choice of the appropriate X-STR population database for LR calculation we calculated LR values for the same kinship cases using Belarusian and Swedish X-STR databases. LR values for the same kinship case differed from each other up to 6000 times depending on the chosen database. X-STRs genotyping lets us to determine relatedness successfully in all cases whereas the results of autosomal STR profiling and mitochondrial sequencing (for the cases where only the samples of putative full-siblings were available) were not sufficient to resolve family relationships issues. LR values were considered to be high enough in all cases.

Genetic diversity of 12 X-chromosomal short tandem repeats in Jewish populations

Haplotype and allele frequencies of 12 X-STRs included in the Investigator Argus X-12 kit were studied for 313 individuals, representing five populations with known Jewish ancestry. The high efficiency in forensic parameters in all the populations studied demonstrates that this set of markers provides a powerful tool for solving complex kinship cases in Jewish populations.