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Evaluation of 13 rapidly mutating Y-STRs on a Dravidian pedigree

Published:September 26, 2019DOI:https://doi.org/10.1016/j.fsigss.2019.09.083

      Abstract

      The effectiveness of a rapidly mutating Y-STRs multiplex assay (RM Y-STRs) containing 13 RM Y-STR markers was compared with the AmpFℓSTR™ Yfiler™ PCR Amplification Kit (Yfiler) with conventional Y-STR markers for their efficiency in differentiating males within the same paternal lineage. Samples from 4 generations comprising 16 Dravidian males (15 father-son pairs) were analysed with both assays. Mutations were observed in 3 father-son pairs in the RM Y-STRs profiles while only 2 mutations were observed in the Y-filer profiles. Even though not many mutations were observed as anticipated, this study still emphasised the importance of RM Y-STRs when differentiation between males within the same paternal lineage is required and also indicated the need for mutation rates for different populations.

      Keywords

      1. Introduction

      Y-chromosome short tandem repeats (Y-STRs) profiling is a useful tool for forensic analysis to process challenging cases such as sexual assault when the recovered male DNA is in a small amount compared to the female DNA. However, in cases where the suspects have the same paternal lineage, conventional Y-STR markers would not be very robust to differentiate them. Thus, Y-STR markers with more discriminative power are required to resolve such cases [
      • Ballantyne K.N.
      • Goedbloed M.
      • Fang R.
      • et al.
      Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.
      ].

      2. Materials and methods

      2.1 Sample collection and DNA extraction

      Buccal swabs from 4 generations comprising 16 Dravidian males (15 father-son pairs) within the same paternal lineage were collected with informed consent. DNA from buccal swabs was extracted using the QIAamp® DNA Mini Kit (Qiagen®). DNA quantification was carried out using the Quantifiler™ Human DNA Quantification Kit (Applied Biosystems™) on a 7500 Real-Time System (Applied Biosystems™) following the manufacturer’s protocol.

      2.2 DNA amplification

      Extracted DNA samples were amplified with the AmpFℓSTR™ Yfiler™ PCR Amplification Kit (Applied Biosystems™) and the rapidly mutating Y-STRs multiplex assay. Yfiler amplification was carried out following the manufacturer’s recommendation, while a previously published method was used for the RM Y-STRs [
      • Alghafri R.
      • Goodwin W.
      • Ralf A.
      • et al.
      A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs.
      ].

      2.3 Electrophoresis and typing

      Capillary electrophoresis was performed on the ABI Prism® 3500xL Genetic Analyzer (Applied Biosystems™) and alleles were determined using the GeneMapper® ID-X software (Applied Biosystems™).

      2.4 Quality controls

      Manufacturer provided AmpFℓSTR™ Control DNA 007 and AmpFℓSTR™ Control DNA 9947A were used as the positive and negative controls respectively in Yfiler amplification while previously genotyped controls were used in the RM Y-STRs amplification. The paternity of all father-son pairs were confirmed using the VeriFiler™ Express PCR Amplification Kit (Applied Biosystems™) with a threshold of paternity probability set at 99.99%.

      3. Results

      In this study, RM Y-STRs amplified 20 alleles compared to 17 in Yfiler. There is no overlapping marker between both assays. The core DNA profiles for this Dravidian pedigree which were developed using both assays are shown in Table 1.
      Table 1Table below shows the core DNA profiles of this Dravidian pedigree for RM Y-STRs and Y-filer loci.
      RM Y-STRs locusCore profileY-filer locusCore profile
      DYS526a13DYS45615
      DYS61239DYS389I12
      DYF399S120,21,24.1DYS39024
      DYS526b33DYS389II30
      DYS54744DYS45820
      DYF404S113,14DYS1915
      DYS62629DYS38511,14
      DYF403S1a12,13,14DYS39314
      DYF403S1b47DYS39110
      DYS57619DYS43913
      DYS51838DYS63522
      DYS62719DYS39211
      DYS57019Y_GATA_H412
      DYF387S138DYS43714
      DYS44929DYS43811
      DYS44819
      From the total of 15 father-son pairs, mutations were observed in 3 father-son pairs in the RM Y-STRs profiles while only 2 mutations were observed in the Y-filer profiles. All the observed mutations were identified at different loci and were 1-step mutations. Mutations were also observed in the same father-son pair on both the assays and coincidently, both the mutations in this father-son pair were with repeat losses while the remaining 3 mutations were with repeat gains (Fig. 1).
      Fig. 1
      Fig. 1Pedigrees with observed mutations from RM Y-STRs (a) and Yfiler (b) in 16 Dravidian male samples comprising 15 father-son pairs.

      4. Discussion and conclusion

      The geographical location of Dravidians is mainly in South India and they belong to the Dravidian linguistic group [
      • Basu A.
      • Sarkar-Roy N.
      • Majumder P.P.
      Genomic reconstruction of the history of extant populations of India reveals five distinct ancestral components and a complex structure.
      ]. The YHRD website (Y-STR Haplotype Reference Database, www.yhrd.org/) comprises 1327 haplotypes (at least minimal) and 13 unique population samples for the Dravidian Metapopulation. The core Yfiler DNA profile from the Dravidian pedigree in this study was searched on this website. However, no match was found even with the minimal haplotype [
      • Marino M.
      • Sala A.
      • Corach D.
      Genetic attributes of the YHRD minimal haplotype in 10 provinces of Argentina.
      ] search (accessed on 13th September 2019).
      Also based on the YHRD website, the highest combined mutation rate (μ) for the Yfiler loci is at locus DYS458 (μ: 0.00617), followed by locus DYS439 (μ: 0.00546) and locus DYS456 (μ: 0.00441). However, no mutations were identified at these loci. The observed mutations were at locus DYS385 (μ: 0.00251) and locus DYS390 (μ: 0.00208).
      According to Ballantyne et al. [
      • Ballantyne K.N.
      • Goedbloed M.
      • Fang R.
      • et al.
      Mutability of Y-chromosomal microsatellites: rates, characteristics, molecular bases, and forensic implications.
      ], the highest mutation rate in the RM Y-STRs is at locus DYF399S1 (μ: 0.0773), followed by locus DYF403S1a (μ: 0.031). Mutations at both these loci were also observed in this Dravidian pedigree. Another mutation was observed at locus DYS570 (μ: 0.0144). In a study by Chen et al. [
      • Chen Y.
      • Zhou W.
      • Li M.
      • et al.
      Mutation rates of 13 RM Y-STRs in a han population from Shandong province, China.
      ], the mutation rate at locus DYS570 (μ: 0.0278) was found to be much higher than the mutation rate reported by Ballantyne et al. The samples used in both these studies were from different populations; Chen et al. used samples of Han population from Shandong province, China, while samples used by Ballantyne et al. were obtained from the Berlin, Leipzig, and Cologne areas of Germany and the Warsaw and Wroclaw areas of Poland. Also, only 3 mutations were observed among 15 father-son pairs in this Dravidian pedigree while 17 mutations were identified in 28 father-son pairs in a study of UAE Arabs [
      • Alghafri R.
      • Goodwin W.
      • Ralf A.
      • et al.
      A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs.
      ]. These findings indicate that the mutation rates for RM Y-STRs could be different among different populations.
      Conclusively, incorporation of the RM Y-STRs based on the mutation rates in different populations could improve the differentiation between males within the same paternal lineage.

      Declaration of Competing Interest

      None.

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