An unusual occurrence of repeated single allele variation on Y-STR locus DYS458

Egyptian Journal of Forensic Sciences (2015) xxx, xxx–xxx

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Egyptian Journal of Forensic Sciences journal homepage: http://www.journals.elsevier.com/egyptian-journal-of-forensic-sciences

CASE REPORT

An unusual occurrence of repeated single allele variation on Y-STR locus DYS458 Pankaj Shrivastava *, Veena Ben Trivedi, Toshi Jain, Mehmood Ali DNA fingerprinting Unit, State Forensic Science Laboratory, Department of Home (Police), Govt. Of MP, Sagar 470001, India Received 12 January 2015; revised 19 April 2015; accepted 5 May 2015

KEYWORDS Forensic science; DNA typing; Y-STR; Mutation; DYS458; Allele variation

Abstract Six brothers were accused of gagging and raping a woman. A single male Y-STR profile was obtained from vaginal smear swab and clothes of the victim, which did not match with the DNA profile of the accused brothers. As a reference point, the blood sample of their father (aged 87 years) was also analyzed with the same kit. The Y-STR haplotype of all six brothers was found to be the same as that of their father except at locus DYS458. At this locus, while the eldest, second and fourth siblings share allele 18 with their father, a loss of one repeat (allele 17 instead of 18) is observed in the third son while fifth and sixth siblings have allele 19 representing a gain of one repeat. Thus, two changes viz. a gain (twice) and loss of one repeat at this locus in one generation is both interesting and unusual. ª 2015 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences (IALFS).

1. Introduction Y-Chromosome short tandem repeats (Y-STR’s) have proven to be of immense value in anthropo-sociological, genealogical and other scientific applications including determination of paternity.1–3 Forensic DNA laboratories are initializing Y-STR analysis in their routine case work, especially in cases of sexual assault. With the increasing use of Y STR markers in forensics it is important to estimate mutation rates using larger studies. In most of the forensic laboratories 17 loci Y STR kit is being utilized for accessing the presence of male DNA. Reliable estimation of mutation rates for these loci is a valuable asset in the interpretation of Y STR test results.4 There * Corresponding author. Tel.: +91 7049154272. E-mail address: [email protected] (P. Shrivastava). Peer review under responsibility of The International Association of Law and Forensic Sciences (IALFS).

are a large number of articles reporting mutation rates for the minimal haplotype loci, but only a few5,6 have dealt with 17 Y STR loci. The present study describes a family with three mutational events in a single generation at Y-STR DYS458 loci. We report here the findings in a case of sexual assault in which six brothers were accused of raping a woman. Referral blood samples of all six brothers were received along with the vaginal smear swab and clothes of the victim. Though the findings in the case proved this to be a case of exclusion, the Y STR analysis yielded interesting and unusual results. 2. Materials and methods DNA was isolated from all the referral blood samples using the Magtration 12 GC (Precision System Science Co., Ltd., Japan) DNA extraction system. DNA from vaginal smear swabs and clothes of the victim was isolated by a phenol chloroform differential extraction method.7

http://dx.doi.org/10.1016/j.ejfs.2015.05.003 2090-536X ª 2015 Hosting by Elsevier B.V. on behalf of The International Association of Law and Forensic Sciences (IALFS). Please cite this article in press as: Shrivastava P et al. An unusual occurrence of repeated single allele variation on Y-STR locus DYS458, Egypt J Forensic Sci (2015), http://dx.doi.org/10.1016/j.ejfs.2015.05.003

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P. Shrivastava et al. Table 1

AmpFlSTR Identifiler DNA profile of the blood samples.

Genetic markers

The father (87 years)

Son I (40 years)

Son II (38 years)

Son III (36 years)

Son IV (35 years)

Son V (24 years)

Son VI (22 years)

D8S1179 D21S11 D7S820 CSF1PO D3S1358 THO1 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 D5S818 FGA AMELOGENIN

11,13 29,33.2 8,12 8,14 17,17 9,9.3 8,11 11,14 22,23 15,15 17,19 8,8 14,14 12,12 19,25 XY

11,13 30,33.2 8,11 8,11 17,18 9,9 8,8 11,12 23,24 14,15 17,18 8,10 14,14 12,12 19,26 XY

11,12 29,30 8,11 8,11 17,18 6,9.3 8,11 12,14 22,24 14,15 17,18 8,10 14,17 12,12 19,26 XY

11,12 29,29 11,12 8,11 15,17 9,9 8,11 11,12 23,24 14,15 18,19 8,10 14,14 12,12 25,25 XY

12,13 29,33.2 8,11 11,14 17,18 9,9 8,11 12,14 18,22 14,15 18,19 8,10 14,17 12,12 25,26 XY

12,13 29,30 8,11 11,14 17,18 6,9 8,8 11,12 23,24 14,15 17,18 8,11 14,14 12,12 25,26 XY

11,12 30,33.2 11,12 11,14 15,17 9,9.3 8,11 12,14 18,22 14,15 18,19 8,10 14,17 12,12 25,25 XY

Table 2

AmpFlSTR Y DNA profile of the blood samples.

Genetic markers

The father (87 years)

Son I (40 years)

Son II (38 years)

Son III (36 years)

Son IV (35 years)

Son V (24 years)

Son VI (22 years)

DYS456 DYS389I DYS390 DYS389II DYS458 DYS19 DYS385 DYS393 DYS391 DYS439 DYS635 DYS392 YGATA H4 DYS437 DYS438 DYS448

13 12 23 28 18 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 18 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 18 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 17 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 18 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 19 15 13,17 14 11 12 20 11 11 15 9 19

13 12 23 28 19 15 13,17 14 11 12 20 11 11 15 9 19

Real Time PCR ABI 7000 (Applied Biosystems, Foster City, CA, USA) was used for quantification of the isolated DNA using Quantifiler Duo DNA Quantification Kit as per the recommended protocol by the manufacturer. All samples were amplified with the AmpFlSTR Yfiler and AmpFlSTR Identifiler PCR Amplification Kits (Applied Biosystems, Foster City, CA, USA) to check for autosomal allele sharing that would establish the father–son relationship. PCR was performed by taking the ½ reaction volume of the manufacturer’s recommended protocol and using an AmpFlSTR multiplex kit and a Gene Amp 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). The PCR mix comprised of Reaction Buffer – 5.0 lL, Primers – 2.5 lL, MQ water – 4.0 lL, Amplitaq gold – 0.3 lL, DNA – 1.0 lL to make final volume 12.8 lL. The samples were run on Genetic Analyzer (ABI 3100) on G5 matrix set using matrix standards DS-33 (Applied Biosystems, Foster City, CA, USA). The samples were prepared using 12 lL of Hi-Di formamide, 0.2 lL of Gene

Scan 500 LIZ size standard (both from Applied Biosystems, Foster City, CA, USA), and 1 lL of the PCR product. The samples were denatured for 3 min at 95 C and snap cooled on ice before loading onto the instrument. The analyzer is equipped with 36 cm arrays, with separation of fragments using POP-4 polymer (Applied Biosystems, Foster City, CA, USA) and the data were analyzed with GeneMapper Analysis Software v 3.5 (Applied Biosystems, Foster City, CA, USA). A peak detection threshold of 50 RFUs was used for allele designation. 3. Result and discussion A single male Y-STR profile was obtained from the vaginal smear swab and clothes of the victim, which failed to match with the DNA profile of the accused brothers. An unusual finding in the Y STR DNA profile was observed on DYS458 loci which asked for further analysis requiring blood sample of father of the six accused brothers to get an answer of this

Please cite this article in press as: Shrivastava P et al. An unusual occurrence of repeated single allele variation on Y-STR locus DYS458, Egypt J Forensic Sci (2015), http://dx.doi.org/10.1016/j.ejfs.2015.05.003

An unusual occurrence of repeated single allele variation on Y-STR locus DYS458 Table 3

Loss or gain of DYS458 repeats in offspring.

Offspring

Age of father when the child was born (years)

Allele call at locus DYS458

Loss/gain of repeat

1st 2nd 3rd 4th 5th 6th

47 49 51 52 63 65

18 18 17 18 19 19

No loss or gain No loss or gain Loss of 1 repeat No loss or gain Gain of 1 repeat Gain of 1 repeat

unusual finding. As a reference point, the blood sample of their father was also analyzed with the same kit. Identifiler profiles of all six brothers showed that they all share paternal alleles with their father, establishing them to be the offspring of the same father. A comparative study of the father’s Identifiler profile with the profiles of each brother confirmed that they are all offspring of the same father (Table 1). The Y-STR haplotype of all six brothers was found to be the same as that of their father except at locus DYS458 where unusual results were observed (Table 2). This may be due to mutation during spermatogenesis. At this locus, while the eldest, second and the fourth siblings share allele 18 with their father, a loss of one repeat (allele 17 instead of 18) is seen in the third son at the same locus (Table 3). Of even more interest is the profile of the last two brothers which showed allele 19 (a gain of one repeat on the same marker). Thus, two changes a gain (twice) and loss of one repeat at this locus in one generation is both interesting and unusual (Table 3). The father was 47 years old when he fathered his first son and was 65 years old when his youngest son was born (Table 3). Mulero et al. (2006) have identified the two cases where the DYS458 allele was not in agreement with the other paternal lineage family members. A discordant DYS458-18 allele was identified instead of DYS458-17 in the sibling (the first case) and the discordant DYS458-16 allele instead of DYS458-17 in the sibling (the second case).6 The present study describes a family with the three mutational events in a single generation at Y-STR DYS458.

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Funding None. Conflict of interest None declared. Acknowledgement The authors are thankful to The Director, State Forensic Science Laboratory Sagar for support. References 1. Gusmao L, Sanchez-Diz P, Calafell F, Martin P, Alonso CA, Alvarez- Fernandez F, et al. Mutation rates at Y chromosome specific microsatellites. Hum Mutat 2005;26:520–8. 2. Bianchi NO, Catanesi CI, Bailliet G, Martinez-Marignac VL, Bravi LB, Vidal-Rioja LB, et al. Characterization of ancestral and derived Y-chromosome haplotypes of New World native populations. Am J Hum Genet 1998;63(6):1862–71. 3. Kayser M, Knijff P, Dieltjes P, Krawczak M, Nagy M, Zerjal T, et al. Applications of microsatellite-based Y-chromosome haplotyping. Electrophoresis 1997;18:1602–7. 4. Kayser M, Roewer L, Hedman M, Henke L, Brauer S, et al. Characteristics and frequency of germline mutations at microsatellite loci from the human Y chromosome, as revealed by direct observation in father/son pairs. Am J Hum Genet 2005;66(5):1580–8. 5. Berger B, Lindinger A, Niederstatter H, Grubwieser P, Parson W. Y-STR typing of an Austrian population sample using a 17-loci multiplex PCR assay. Int J Legal Med 2005;119:241–6. 6. Mulero JJ, Chang CW, Calandro LM, Green RL, Li Y, Johnson CL, et al. Development and validation of the AmpFlSTR Yfiler PCR amplification kit: a male specific, single amplification 17 YSTR multiplex system. J Forensic Sci 2006;51(1):64–75. 7. Yoshida K, Sekiguchi K, Mizuno N, Kasai K, Sakai I, Sato H, et al. The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen. Forensic Sci Int 1995;72:25–33.

3.1. Quality control Author(s) have passed proficiency testing of the GITAD, Spain http://gitad.ugr.es/principal.htm), and quality control exercise of the YHRD, Germany (www.yhrd.org).

Please cite this article in press as: Shrivastava P et al. An unusual occurrence of repeated single allele variation on Y-STR locus DYS458, Egypt J Forensic Sci (2015), http://dx.doi.org/10.1016/j.ejfs.2015.05.003