The application of mtDNA SNPs to a forensic case

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Forensic Science International: Genetics Supplement Series 1 (2008) 295–297 www.elsevier.com/locate/FSIGSS

Research article

The application of mtDNA SNPs to a forensic case Kimberly A. Sturk, Michael D. Coble, Suzanne M. Barritt, Thomas J. Parsons 1, Rebecca S. Just * Armed Forces DNA Identification Laboratory, 1413 Research Boulevard, Building 101, Rockville, MD 20850, United States Received 18 August 2007; accepted 9 October 2007

Abstract A multiplex allele specific primer extension (ASPE) assay was optimized to type 11 single nucleotide polymorphisms (SNPs) in the control and coding regions of the mitochondrial genome. The SNPs provide additional discrimination when one of the most common W. European Caucasian hypervariable region types (HV types) is encountered. The SNP assay was applied in a nearly 40-year-old missing persons case involving highly degraded human remains in which four potential reference families matched the case sample across hypervariable regions 1 and 2 (HV1/HV2). The SNP assay identified two coding region positions (5250 and 12438) at which the case sample differed from one reference family. The exclusion eliminated the need for any further testing for comparison to those reference samples, and narrowed the focus to the remaining families. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Mitochondrial DNA; Single nucleotide polymorphism; Allele specific primer extension; SNaPshot

1. Introduction

2. Materials and methods

Mitochondrial DNA (mtDNA) is frequently used in cases of highly degraded remains, when the case samples lack sufficient nuclear DNA (e.g. shed hairs), or when only maternal references are available [1]. The maternal inheritance and lack of recombination with mtDNA, however, can result in identical HV1/HV2 profiles from unrelated individuals. In such cases, additional genetic testing of select targets in the coding region and control region outside of HV1/HV2 of the mitochondrial genome (mtGenome) can sometimes distinguish individuals [2–5]. This paper presents a forensic casework application of an mtDNA SNP assay. ‘‘Multiplex C’’ targets 11 control and coding region SNPs chosen specifically to identify additional variation in samples of two of the most common W. European Caucasian HV types, V:1 (16298C, 263G, 315.1C; frequency 1.0%) and H:5 (263G, 315.1C, 16304C; frequency 0.9%) [2]. We describe a missing persons case in which four families matched HV type V:1, and the exclusion of one family through the typing of Multiplex C mtDNA SNPs.

We have previously described the selection of the 11 SNPs typed by Multiplex C (72, 513, 4580, 5250, 11719, 12438, 12810, 14770, 15833, 15884, and 16519) [2]. Multiplex amplification and extension primers were designed and quantified as described in [6]. Multiplex primer concentrations were optimized to provide balanced signal strength for each SNP [7]. The multiplexed assay was tested to confirm sensitivity to at least 3 pg genomic DNA, and identification of the minor component of a mixture to at least 10% [7]. Genomic DNA was extracted from dried bloodstains on filter paper with Chelex1 100 beads (Bio-Rad Laboratories, Hercules, CA) and from skeletal material using a standard phenol/chloroform method, both as described in [8]. MtDNA HV1/HV2 sequencing of the reference samples and skeletal remains was performed as described in [8]. SNP typing was performed as described in [6] with the modifications described here. Reference sample amplifications were performed in a 15 ml total volume reaction, using 2.5 U AmpliTaq Gold1 DNA polymerase (Applied Biosystems, Foster City, CA) and 2 ml sample extract. Skeletal extract amplifications were performed using 36 cycles in a 25 ml total volume reaction, using 6.25 U AmpliTaq Gold DNA polymerase and 1 ml sample extract. PCR reaction cleanup was performed using exonuclease I (EXO) and shrimp alkaline phosphatase (SAP; USB Corporation, Cleveland, OH) in a ratio of 2.5 units EXO and

* Corresponding author. Tel.: +1 301 319 0224; fax: +1 301 295 5932. E-mail address: [email protected] (R.S. Just). 1 Present address: International Commission on Missing Persons, Alipasˇina 45A, 71000 Sarajevo, Bosnia and Herzegovina. 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.148

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Table 1 HV1/HV2 and Multiplex C SNP profiles for four reference families and case sample Sample HV1/HV2 Reference family 1 1a

C

G

C

G

C

A

T

C

A

C

G

C

G

C

G

C

A

T

C

A

C

G

Reference family 2 2a

16298C, 263G, C 309.1C, 309.2N, 315.1C

G

T

G

T

A

T

C

A

C

G

Reference family 3 3a

16298C, 263G, 309.1C, 315.1C 16298C, 263G, 309.1C, 315.1C

C

G

T

G

T

A

T

C

A

C

G

C

G

T

G

T

A

T

C

A

C

G

C

G

T

G

T

A

T

C

A

C

G

C

G

T

G

T

A

T

C

A

C

G

C

G

T

G

T

A

T

C

A

C

G

Reference family 1 1b

Reference family 3 3b Reference family 4 4a Reference family 4 4b Case sample

A

16298C, 263G, 309.1C, 315.1C 16298C, 263G, 309.1C, 315.1C

72 513 5250 11719 12438 12810 16519 14770 4580 15833 15884 (113 bp) (154 bp) (110 bp) (117 bp) (104 bp) (101 bp) (183 bp) (149 bp) (130 bp) (152 bp) (152 bp)

16298C, 263G, 309.1C, 315.1C 16298C, 263G, 309.1C, 315.1C 16298C, 263G, 309.1C, 315.1C

0.1 units SAP for each 1 ml PCR product. Unincorporated dideoxynucleotide removal was performed using 2 units SAP per reaction. Extension products were prepared for electrophoresis using 14 ml of Hi-Di formamide, 0.5 ml of LIZ-120 internal sizing standard (Applied Biosystems) and 1.0 ml of the extension product. Separation and detection was performed on an ABI PRISM1 3130xl Genetic Analyzer using POP-6TM polymer (Applied Biosystems). Data were analyzed using GeneMapper v3.2 (Applied Biosystems) and alleles were assigned automatically using a customized panel and bin set.

3. Results and discussion The mtDNA sequence of skeletal remains found at the site of a 1968 U.S. military plane crash was compared to maternal references representing several families of U.S. servicemen believed to have been lost in the region from which the remains were recovered. Comparison of the sequences from 60 reference families identified just 4 that matched the bone specimen exactly across HV1/HV2. These samples shared a common Caucasian HV type, V:1 (16298C, 263G, 315.1C), and

Fig. 1. MtDNA SNP profiles from reference family 1 (top panel) and case sample A (bottom panel) identified sequence differences at positions 5250 and 12438.

K.A. Sturk et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 295–297

therefore could not be resolved. Multiplex C typing of the references identified two positions (5250 and 12438) at which the mtDNA sequence for reference family 1 differed from the other three reference families (Table 1). Multiplex C typing of the skeletal element produced a full profile that excluded reference family 1 based on the sequence differences at the same two nucleotide positions (Fig. 1). The case described here represents a typical application of an mtDNA SNP assay; the carefully-selected discriminatory SNPs were typed to provide additional resolution in a case in which multiple reference families and a highly degraded skeletal element matched across the mtDNA hypervariable regions. The typing of the 11 Multiplex C mtDNA SNPs did not completely resolve all matching samples in this case; however, the two differences identified confidently excluded one potential reference family. Further resolution in this case was ultimately obtained through the use of reduced amplicon size short tandem repeats (STRs) and new low copy number STR typing protocols. Disclaimer The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or as views of the U.S. Department of Defense or the U.S. Department of the Army. Acknowledgements The authors would like to thank Colin R. Steven for assistance with the family reference samples; Brion Smith, James Canik, Louis Finelli, James Ross, and Richard Coughlin (AFDIL) for administrative, logistical, and computer support;

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Vinh Lam, Jon Norris and others at Future Technologies Inc. for database support; Alec Christensen, Tom Holland, and others at JPAC CIL for case material and historical context; and Peter Vallone at the National Institute of Standards and Technology for discussion. Conflict of interest None. References [1] M.M. Holland, T.J. Parsons, Mitochondrial DNA sequence analysis— validation and use forensic casework, Forensic Sci. Rev. 11 (1999) 21–50. [2] M.D. Coble, R.S. Just, J.E. O’Callaghan, I.H. Letmanyi, C.T. Peterson, J.A. Irwin, T.J. Parsons, Single nucleotide polymorphisms over the mtDNA genome that increase the power of forensic testing in Caucasians, Int. J. Legal Med. 118 (2004) 137–146. [3] R.S. Just, J.A. Irwin, J.E. O’Callaghan, J.L. Saunier, M.D. Coble, P.M. Vallone, J.M. Butler, S.M. Barritt, T.J. Parsons, Toward increased utility of mtDNA in forensic identifications, Forensic Sci. Int. 146S (2004) S147– S149. [4] M.D. Coble, P.M. Vallone, R.S. Just, T.M. Diegoli, B.C. Smith, T.J. Parsons, Effective strategies for forensic analysis in the mitochondrial DNA coding region, Int. J. Legal Med. 120 (2006) 27–32. [5] S. Sigurdsson, M. Hedman, P. Sistonen, A. Sajantila, A.C. Syva¨nen, A microarray system for genotyping 150 single nucleotide polymorphisms in the coding region of human mitochondrial DNA, Genomics 82 (2006) 534– 542. [6] P.M. Vallone, R.S. Just, M.D. Coble, J.M. Butler, T.J. Parsons, A multiplex allele specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome, Int. J. Legal Med. 118 (2004) 147–157. [7] T.J. Parsons, AFDIL, http://www.ncjrs.gov/pdffiles1/nij/grants/213502.pdf. [8] S.M. Edson, J.P. Ross, M.D. Coble, T.J. Parsons, S.M. Barritt, Naming the dead—confronting the realities of rapid identification of degraded skeletal remains, Forensic Sci. Rev. 16 (2004) 64–89.