Population genetics,diversity, forensic characteristics of four Chinese populations inferred from X-chromosomal short tandem repeats

We genotyped 19 X-STRs included in the AGCU X19 kit in 712 unrelated Chinese individuals from four populations (Li, Hui, Tibetan, and Han) and then merged with 4156 previously genotyped subjects from 19 populations to investigate genetic relationships and population structure, as well as the association between the genetic affinity and language classification. The combined mean paternity exclusion chances and the combined probabilities of discrimination based on single allele frequencies of 19-X-STRs and haplotype frequencies of seven linkage groups were high, indicating that this set of markers was very polymorphic in the four populations studied. Therefore, this panel can complement autosomal or uniparental markers in kinship analysis and complex deficient paternity testing. Subsequently, population differentiation analyses among 23 populations based on 19 STRs and 15 populations based on over 62 million single nucleotide polymorphisms consistently demonstrated that genetic stratifications exist between the different language-speaking populations, especially Tibeto-Burman-speaking, Tungusic and Turkic-speaking populations. Our newly studied populations are genetically close to ethnolinguistically adjacent populations. Our datasets can and should be used as an allele and haplotype frequencies reference database to facilitate the use of 19-X-STRs panel in routine forensic practice.     

Allele and haplotype frequencies of 12 X-STRs in Mexican population

The use of X-chromosome short tandem repeats (X-STRs) to solve complex kinship cases has been facilitated by commercial human identification kits, such as the Argus X-12 kit (Qiagen), and the free-access software FamlinkX. For this purpose, allele and haplotype frequencies are required in the populations to be employed. Therefore, we obtained Argus X-12 haplotypes in 933 unrelated males from seven different geographic regions from Mexico. Forensic parameters for individual X-STRs and for three-loci linkage groups are reported. The observed homogeneity between the studied population samples support to use a global Mexican population database (Fst p-value >0.05). In brief, forensic validation of the Argus X-12 kit was performed to facilitate incorporation of X-STRs in forensic casework in this country.     

Genetic diversity study on 12 X-STR loci of investigator® Argus X STR kit in Bangladeshi population

The X-chromosome short tandem repeat (STR) loci are of particular interest for solving complex kinship and paternity cases. Here, we report the genetic data from 209 unrelated Bangladeshi individuals (102 males and 107 females) that were genotyped using the 12 X-chromosomal STR markers included in the Investigator® Argus X-12 kit (Qiagen). The 12 X-STR markers are located in four linkage groups (linkage group I: DXS10135, DXS10148, and DXS8378; linkage group II: DXS7132, DXS10079, and DXS10074; linkage group III: DXS10103, HPRTB, and DXS10101; and linkage group IV: DXS10146, DXS10134, and DXS7423). Allelic frequencies of the 12 X-STR loci and haplotype frequencies of the four linkage groups were investigated. No significant difference was observed in the allele frequencies of males and females. Distributions of heterozygosity were observed from 64.5 to 92.5% among the studied 12 X STR loci. DXS10135 and DXS10101 loci were found to be most polymorphic. For all the four linkage groups, the haplotype diversity was found to be greater than 0.986. A total of 95, 73, 66, and 74 haplotypes were observed in linkage groups I, II, III, and IV, respectively. Hardy–Weinberg equilibrium tests showed no significant deviation from expected values for all 12 loci (p > 0.05). The exact test for pairwise linkage disequilibrium for the 12 loci in the male samples did not show any significant linkage disequilibrium except the DXS10103 and DXS10101 loci after the p values were corrected by Bonferroni’s correction for multiple testing (p > 0.05/66). A combined power of discrimination in male and female individuals were 0.999999998159791 and 0.999999999999993, respectively. The combined mean exclusion chance were 0.999997635 in deficiency cases, 0.999999996 in normal trio cases, and 0.999999178 in duo cases. The currently investigated Bangladeshi population showed significant differences when compared with previously reported X-STR data from other 12 populations. The results of the data analysis indicated that all the loci in the Investigator® Argus X 12 kit were fairly informative and might be useful in forensic application and kinship analysis in Bangladeshi population.

     

Use of X-linked markers for forensic purposes

In forensic science, X-chromosomal short tandem repeats (ChrX STRs) bear the potential to efficiently complement the analysis of other genetic markers (autosomal, Y-chromosomal or mitochondrial). We review the population genetic properties and forensic utility of selected ChrX markers, and discuss the problems and limitations arising with their practical use. Formulae required to assess the evidential power of individual markers in different contexts are summarised and applied to ChrX STRs of interest. Since linkage and linkage disequilibrium between markers affect the inferential interpretation of genotype data, practically relevant information regarding the co-localisation and haplotypic association of ChrX STRs is provided. Finally, two examples of complex kinship testing are presented which serve to highlight the particular importance of ChrX STRs for solving deficiency cases and cases involving blood relatives.     

A genetic overview of Atlantic coastal populations from Europe and North-West Africa based on a 17 X-STR panel

The forensic use of X-STRs requires the creation of allele and haplotype frequency databases in the populations where they are going to be used. Recently, an updated Spanish allele and haplotype frequency database for the new 17 X-STR panel has been created, being the only database available up to now for this new multiplex. In order to broaden the forensic applicability of the 17 X-STR panel, 513 individuals from four different populations located on the Atlantic Coast of Europe and North–West Africa have been studied, i.e. Brittany (France), Ireland, northern Portugal, and Casablanca (Morocco). Allele and haplotype frequency databases, as well as parameters of forensic interest for these populations are presented. The obtained results showed that the 17 X-STR panel constitutes a highly discriminative tool for forensic identification and kinship testing in the studied populations. Furthermore, we aimed to study if these populations located on the Atlantic coast actually share alike allele and haplotype frequency distributions since they have experienced genetic exchanges throughout history. This would allow creating larger forensic databases that include several genetically similar populations for its use in forensic casework. For this purpose, pairwise F_ST genetic distances between the analyzed populations and others from the Atlantic Coast previously studied with the 17 X-STR panel or the ten coincident markers included in the decaplex of the GHEP-ISFG were estimated. Our results suggest that certain nearby populations located on the European Atlantic coast could have underwent episodes of genetic interchange as they have not shown statistically significant differentiation between them. However, the population of Casablanca showed significant differentiation with the majority of the European populations. Likewise, the autochthonous Basque Country and Brittany populations have shown distinctive allele frequency distributions between them. Therefore, these findings seem to support that the use of independent allele and haplotype frequency databases for each population instead of a global database would be more appropriate for forensic purposes.     

Haplotyping of STR cluster DXS6801–DXS6809–DXS6789 on Xq21 provides a powerful tool for kinship testing

Short tandem repeat (STR) markers DXS6801 (GATA41B11), DXS6809 (GATA69B129) and DXS6789 (GATA31F01) are located in a 3-Mb region on human chromosome Xq21, spanning approximately 3–6 cM. Theoretically, this cluster could give rise to 1,144 different haplotypes in the German population. In fact, genotyping of 806 males revealed the presence of 207 different haplotypes. Since the three STRs have been shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of single locus allele frequencies alone, but have to be estimated directly instead. In this work, we present data on linkage, haplotype frequencies and LD in the German population. To highlight the potential of the STR cluster for forensic analysis, we also report two examples of its successful application in pedigree-based kinship testing.Electronic Supplementary Material Supplementary material is available for this article at .     

Forensic parameters of 19 X-STR polymorphisms in two Chinese populations

Application of X-STRs as complements of autosomal STR application in the forensic genetics has become a tendency for kinship testing, especially in deficiency paternity cases. Recently, a novel kit of 19 X-STR loci was developed, which permitted the analysis of 19 STR in the same PCR reaction, and these markers can be clustered into seven groups for the physical linkage. The objective of this study was to evaluate the allele and haplotype diversity of 19 X-STR loci in the Uygur (n = 220) and Tibetan nationality (n = 270) and to estimate the usefulness for complex kinship analysis. In the Tibetan and Uygur populations, a total of alleles of all loci were 188 and 212, with the allele frequencies ranged from 0.0037 to 0.5593 and from 0.0045 to 0.5409, respectively. Compared with previous studies, DXS10135 was the most polymorphic locus in the two population groups, whereas the least variant locus was DXS10164 in the Uygur population and DXS7423 in the Tibetan nationality. Haplotype diversity obtained in this investigation was greater than 0.9 across all LGs. This study indicated the new kit could be used as a supplementary tool in kinship testing in China. In addition, the data sets can be used as supplementary national X-STR references to enlarge the database.

     

X-chromosome data for 12 STRs: Towards an Argentinian database of forensic haplotype frequencies

The analysis of X-chromosome STRs is useful in certain kinship cases for which autosomal markers provide insufficient statistical power. Particularly, powerful results are achieved in paternity cases with a daughter, when the alleged father is not accessible for analysis, contrarily to his unquestioned mother or daughter. However, representative haplotype frequencies for this type of markers are not available for some populations, as is the case of Argentina, which prevents the quantification of the proof in routine forensic analyses. In this work we present haplotype frequencies for the 12 X-chromosome STRs included in the Investigator Argus X-12 kit, as well as segregation data, obtained from the analysis of the genetic profiles of 457 father-daughter duos, which gave us information on 914 (unrelated) haplotypes from residents of all Argentinian provinces.     

Analysis of 12 X-chromosomal markers in the population of central Croatia

Investigator® Argus X-12 Kit is a commercially available set that allows simultaneous PCR amplification of 12 X-STR markers belonging to four linkage groups (LG). To assess the forensic efficiency of these markers for the population of central Croatia and consequent applicability in routine forensic casework, DNA from 200 blood samples of unrelated donors (100 female and 100 male) was amplified by Investigator® Argus X-12 Kit and analyzed by capillary electrophoresis. Statistical computations based on allele and haplotype frequencies for LG1 – LG4 were performed using Arlequin 3.5 software and on-line tool available at ChrX-STR.org. In female samples, all X-STR markers were in Hardy-Weinberg equilibrium (HWE). The most informative marker for central Croatia population was DXS10135 with polymorphism information content (PIC) 0.9296. The least polymorphic locus was DXS8378 (PIC = 0.6363). Power of discrimination (PD) varied from 0.6968 to 0.9336 in male and from 0.8476 to 0.9916 in female samples. Combined PD exceeded 0.999999999 in both men and women. In male samples, linkage disequilibrium (LD) test revealed significant association (P = 0.0000) of one marker pair in LG4 and two marker pairs in LG3. Portion of observed haplotypes in the number of possible haplotypes varied from 2.86% to 7.47% across all LGs. LG1 was the most informative with haplotype diversity (H) 0.9972. High PD of all analyzed markers exhibited for central Croatia population confirms suitability of Investigator® Argus X-12 for forensic pertinence. Moreover, results of this study will be included in establishing a national reference X-STR database based on 12 X-STR loci, which is necessary for the correct interpretation of the forensic casework results.     

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135–DXS8378, DXS7132–DXS10074, HPRTB-DXS10101 and DXS7423–DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1–4. The genetic distance within the STR pairs is assumed to be <1 cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.     

Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both “rural” and “urban” samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes.     

Genetic polymorphisms of twelve X-STRs of the investigator Argus X-12 kit and additional six X-STR centromere region loci in an Egyptian population sample

Recently, many researchers have focused on analysis of different X-chromosomal STRs as they bear the potential to efficiently complement the analysis of autosomal and Y-chromosomal STRs in solving special complex kinship deficiency cases. In the current study we examined a sample of 250 unrelated Egyptian males with the Investigator Argus X-12 kit (Qiagen GmbH, Hilden, Germany) which detects 12 X-STR markers distributed over the entire X-chromosome as four closely linked clusters. Microvariant off ladder alleles as well as null alleles have been detected in some loci. Furthermore, discordant results were observed between the Investigator Argus X-12 and the Mentype^® Argus X-8 kits (Biotype AG, Dresden, Germany). New primers were designed for loci DXS10101, DXS10146 and DXS10148 to correct the allele drop outs observed in these loci with the Investigator Argus X-12 kit. Additionally, DNA sequence analysis revealed the polymorphisms responsible for the allele drop outs. Furthermore, six additional X-STRs (DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165) located in the centromere region at Xp11.21–Xq11.1 were examined in a single multiplex reaction. Allele and haplotype frequencies as well as different forensic statistical parameters of the 18 X-STR loci tested indicated that they are highly informative in different forensic applications in the Egyptian population. However, some modifications still need to be performed on the Investigator Argus X-12 kit before its use in forensic casework is validated.     

Potentials and limits of pairwise kinship analysis using autosomal short tandem repeat loci

At least in principle, most instances of complex kinship testing can be reduced to pairwise kinship cases involving two critical family members that either link or separate presumed sub-branches of a family. In the European population, the 34 short tandem repeats (STRs) currently used in forensic genetics are sufficiently powerful to allow assessment of disputed first and second but not lower degrees of pairwise blood relatedness. We provide estimates of the means and variances of marker-specific log-likelihood ratios, using large-sample approximation and assuming different scenarios of pairwise kinship analysis. These estimates allow power calculations to be performed for any combination of the available STRs. Since some of the markers considered are physically linked, chromosome-wide likelihood calculations in kinship cases other than parent–child duos (and trios) have to take the reduced rates of meiotic inter-marker recombination into account. We show by simulation that this requirement may be ignored when discriminating distant hypotheses about kinship, but that linkage may play an important role in the biostatistical analysis of more intricate cases.     

Genetic study of 12 X-STRs in Malay population living in and around Kuala Lumpur using Investigator Argus X-12 kit

We investigated 12 X-chromosomal short tandem repeat (STR) polymorphisms in 283 unrelated Malay individuals (160 males and 123 females) living in and around Kuala Lumpur using the Investigator Argus X-12 kit. Heterozygosity among the present 12 X-STRs showed a distribution of from 55.3 to 93.5 %. The diversity values of the haplotypes constructed using four closely linked groups were all higher than 0.9865. A comparison of allelic frequency in each system and haplotype variation indicated that the nature of these X-STRs in the Malay population differed from that in East Asian, European, or African populations. Several microvariant alleles found in the Malay population were characterized and compared with known sequence data. The present data may be helpful in forensic casework such as personal identification and kinship testing in the Malay population in Malaysia.     

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.     

POSTER PRESENTATIONS

X-chromosome markers have become a useful set of markers of choice when certain complex kinship cases need to be unravelled. The Argus X-12 kit allows the co-amplification in a single PCR reaction of 12 X-chromosome short tandem repeats located in four linkage groups. A number of 507 unrelated individuals from Greenland, Denmark and Somalia together with two generation families were typed using the Argus X-12 kit. Silent alleles for the DXS10148 and DXS10146 systems were observed in males, mostly from Somalia. High levels of intrapopulation variability and therefore high forensic parameter values were calculated for the three studied populations. The population in Greenland showed a significantly lower intrapopulation variability and a high genetic differentiation compared with 13 other populations. Significant levels of linkage disequilibrium were observed between markers belonging to the same linkage group, mainly in the populations in Greenland and Somalia. Family studies allowed the calculation of mutation and recombination frequencies. A higher male versus female mutation rate was obtained, with an average value of 3.3 × 10⁻³. Recombination fraction calculations performed on two generation families showed, as previously described, a not complete independence between X-chromosome linkage groups 3 and 4.     

DXS10079, DXS10074 and DXS10075 are STRs located within a 280-kb region of Xq12 and provide stable haplotypes useful for complex kinship cases

The evaluation of the short tandem repeat (STR) markers DXS10079, DXS10074 and DXS10075 was amended to establish a STR cluster spanning a genetic distance<1 cM. These three STRs are located within a 280-kb region at Xq12 and provide stable haplotypes useful for solving complex kinship cases. Theoretically, this cluster could give rise to 2,548 different haplotypes in the German population and the genotyping of 781 men revealed the presence of 172 haplotypes. Since the three STRs were shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of a single locus allele frequency alone but have to be estimated directly. Here, we present data on linkage, haplotype frequencies and LD in a German population. Further clusters from other regions of the X chromosome will be published in the future to cover the chromosome with a well-structured network of highly informative sites.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Haplotype analysis of two X-chromosome STR clusters in the Pakistani population

Haplotype analysis of closely associated markers has proven to be a powerful tool in kinship analysis especially when X-chromosome short tandem repeats fail to resolve uncertainty in relationship analysis. Microsatellites located on the X chromosome show stronger linkage disequilibrium compared with autosomal microsatellites; hence, it is necessary to estimate the haplotype frequencies directly from population studies as linkage disequilibrium is population-specific. Here, we describe five markers residing in two clusters; cluster I harboring three STR markers DXS6801–DXS6809–DXS6789 and cluster II harboring two STR markers DXS7424–DXS101. A total of 302 male DNA samples of Pakistani descent were analyzed. Theoretically, 847 and 160 different combinations of haplotypes are possible in clusters I and II, but genotyping identified only 129 and 75 haplotypes, respectively. No evidence of linkage disequilibrium was detected, except for the pair (DXS6801–DXS6789), consistent with results obtained with the cluster I in a German population. Our results demonstrate that 83% haplotypes of cluster I and 65% haplotypes of cluster II show <1% frequency in the Pakistani population. This strongly suggests that haplotypes of these two clusters provide a powerful tool for kinship testing and relationship investigations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported in part by Higher Education Commission and Ministry of Science and Technology Islamabad, Pakistan.     

NGMSElect™ and Investigator® Argus X-12 analysis in population samples from Albania,Iraq, Lithuania,Slovenia, and Turkey

The analysis of STRs is the main tool when studying genetic diversity in populations or when addressing individual identification in forensic casework. Population data are needed to establish reference databases that can be used in the forensic context. To that end, this work investigated five population samples from Albania, Iraq, Lithuania, Slovenia, and Turkey. Individuals were typed for 16 autosomal STRs and 12 X-chromosomal STRs using the NGMSElect™ and Investigator^® Argus X-12 kits, respectively. The aim of the study was to characterize the diversity of both STR kits in these population samples and to expand our forensic database.The results showed that all markers were polymorphic in the five populations studied. No haplotype was shared between the males analysed for X-STRs. No statistically significant deviations from Hardy–Weinberg equilibrium were observed for any of the genetic markers included in both the kits. Pairwise LD was only detected in X-STRs between markers located in the same linkage group. Power of discrimination values for males and females and the probability of exclusion in duos and trios were high for the populations in this study.