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Evaluation of rapidly mutating Y-STRs in Pakistani population

Published:September 27, 2019DOI:https://doi.org/10.1016/j.fsigss.2019.09.094

      Abstract

      Y-chromosomal Short Tandem Repeats have been widely used in forensic investigations, identification of males for criminal justice purpose and population genetics. Commercially available Y-STRs kits allow the identification of male pedigrees and has a limited application in forensic genetics because of its limitation in differentiating closely related male individuals. Recent research with the Rapidly Mutating Y-STRs (RM Y-STRs) have revealed that these loci deliver significantly higher discrimination capacity and haplotype diversity in worldwide populations when compared with the conventional Y-STRs. Although a number of RM Y-STRs have found their way in most updated commercial kits, there are still some loci that are not yet used in such kits. The aim of this study is to develop RM Y-STR haplotypes frequency database for the Pakistani population, in order to appraise the resolution power of these loci. A total of 212 unrelated males from the Pakistani population were typed with 13 RM Y-STRs which comprise DYF399S1, DYF387S1, DYS570, DYS576, DYS518, DYS526a + b, DYS626, DYS627, DYF403S1a + b, DYF404S1, DYS449, DYS547 and DYS612. 211 unique haplotypes were identified, out of which 1 haplotype was shared between two individuals, accounting for 0.9952 discrimination capacity (DC). Haplotype diversity was found to be 0.999925. Gene diversity (GD) values of all the loci were higher than 0.5, where the highest GD values were observed at DYF399S1, DYF403S1a and DYF404S1; with values of 0.99419, 0.98252 and 0.93061 respectively. Results of our study revealed that these 13 RM Y-STRs produced significantly stronger discriminatory power in Pakistani populations.

      Keywords

      1. Introduction

      Y chromosomal short tandem repeats (Y-STRs) are extensively used in forensic genetics, specifically in cases of sexual assault, paternity testing and missing person investigation. Y-STRs show paternal inheritance due to the non-recombining nature of the Y chromosome which allows to distinguish lineages of human populations but have less abilities to differentiate between related males who belong to the same paternal lineage and conclusive identification cannot be drawn on the individual level [
      • Budowle B.
      • et al.
      Utility of Y-chromosome short tandem repeat haplotypes in forensic applications.
      ]. A number of studies have revealed that Y-STR haplotype diversity, as measured with current Y-STR sets, can be enriched and male differentiation can be enhanced by including carefully chosen additional Y-STRs. A panel of rapidly mutating Y-STRs, composed of 13 markers including DYF399S1, DYF387S1, DYS570, DYS576, DYS518, DYS526a + b, DYS626, DYS627, DYF403S1a + b, DYF404S1, DYS449, DYS547 and DYS612, with mutation rates above 1 × 10−2 was reported [
      • Kayser M.
      Uni-parental markers in human identity testing including forensic DNA analysis.
      ], whereas most Y-STRs, including all of those currently used in forensics, have mutation rates in the order of 1 × 10-3 or lower.

      2. Materials and methods

      2.1 Sample collection and extraction

      For the purpose of this study, blood samples were randomly collected on FTA® paper from 212 unrelated male individuals that belong to different areas of Pakistan.

      2.2 Multiplex development

      PCR amplification of 13 RM Y-STRs was performed using a Veriti™ Thermal Cycler in a 15 μl reaction volume comprising 7 μl of Platinum® PCR Multiplex Master Mix, 1.5 μl of the 13 RM Y-STRs primer mix [
      • Alghafri R.
      • et al.
      A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs.
      ], 6.5 μl of PCR grade water and a 0.5 mm disk of FTA® stained with blood. The following cycling conditions were used: 95 °C for 10 min; followed by 12 cycles of 94 °C for 30 s, 58 °C for 45 s, 72 °C for 60 s; then 20 cycles of 94 °C for 30 s, 55 °C for 45 s, 72 °C for 60 s; and a final cycle of 72 °C for 45 min. Three male controls which include TaqMan, 007 and 9948 positive controls, were used in this study, where 1 μl of each was used as DNA template. Prior to electrophoresis, 1 μl of the amplified products was added to 0.4 μl of GeneScan™ 600 LIZ® Size Standard v. 2.0 and 9.6 μl of Hi-Di™ formamide. Samples were then electrophoresed on an ABI 3500 Genetic Analyzer using 36 cm capillaries and POP-4 polymer, and the fragment analysis was performed with GeneMapper® IDX v.1.4.

      2.3 Statistical analysis

      Sequencing information was subjected to various forensic statistical analyses to obtain information about gene diversity, match probability, haplotype diversity, power of discrimination and allele frequencies. STRAF (STR Analysis for Forensics) was used for the computation of all these relevant parameters based on population studies [
      • Gouy A.
      • Zieger M.
      STRAF—a convenient online tool for STR data evaluation in forensic genetics.
      ].

      3. Results and discussion

      Various forensic statistical analysis parameters were studied, the results of which have been summarized in Table 1. Gene diversity (GD) values, which estimates the probability that two alleles drawn at random from the population will be different, were higher than 0.5 at all loci. The highest GD values have been observed at DYF399S1, DYF403S1a and DYF404S1 with values of 0.99419, 0.98252 and 0.93061 respectively. The lowest GD value was observed at DYS526a with the value of 0.7475. All of the 13 RM Y-STR loci were found to be highly polymorphic, indicating their great potential for forensic individual identification.
      Table 1Gene Diversity (GD), Haplotype Diversity (HD) and Discrimination Capacity (DC) values calculated for the set of 13 RM Y-STRs.
      LocusGD
      DYF387S10.92524
      DYF399S10.99419
      DYF403S1a0.98252
      DYF403S1b0.81691
      DYF404S10.93061
      DYS4490.84480
      DYS5180.84574
      DYS526a0.74725
      DYS526b0.85321
      DYS5470.83426
      DYS5700.78739
      DYS5760.81865
      DYS6120.79540
      DYS6260.84803
      DYS6270.81825
      Average GD0.85616
      Haplotype Diversity (HD)0.99995
      Discrimination Capacity (DC)0.99520
      Compared with previously published results, 5 samples manifested up to 6 alleles at the DYF399S1 locus, which was found to be an aberrant result. These samples additionally exhibited deletions at five different single allelic markers including DYF403S1b, DYS576, DYS627, DYS570 and DYS449. Although a large number of deletions were present in these samples, all of them were differentiated by only two markers (DYF399S1 and DYF403S1a), signifying the power of RM Y-STRs in forensic applications, especially when it comes to closely related individuals.

      4. Conclusion

      RM Y-STRs show high discrimination capacity (0.9952) which significantly increases the ability to differentiate between related males in forensic caseworks. 212 haplotypes of 13 RM Y-STRs were identified, out of which 1 haplotype was shared by two individuals, accounting for 99.52% unique haplotypes. Haplotype diversity was found to be 0.999955. All of the 13 RM Y-STR loci were found to be highly polymorphic which indicated their great potential for forensic individual identification. Gene diversity (GD) values of all the loci were higher than 0.5, with the lowest being 0.7475 at DYS526a. On the other hand, the highest GD values were observed at DYF399S1, DYF403S1a and DYF404S1 with values of 0.99419, 0.98252 and 0.93061 respectively. In comparison with previously published results, five samples manifested up to 6 alleles at locus DYF399S an example shown in Fig. 1. These samples also exhibited deletions at five different single allelic markers including DYF403S1b, DYS576, DYS627, DYS570 and DYS449; yet two markers, which include DYF399S1and DYF403S1a, were able to differentiate all of those samples. The results of our study revealed that the 13 RM Y-STRs produced significantly stronger discriminatory power in Pakistani populations.
      Fig. 1
      Fig. 1RM –Yplex haplotype showing the atypical number of alleles detected at DYF399S1 along with deletion detected at DYF403S1b, DYS570, DYS627, DYS576 and DYS449.

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