- Email: [email protected]
Identification of Azores islands haplogroups by mitochondrial DNA analysis
Forensic Science International: Genetics Supplement Series 3 (2011) e131–e132
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Identification of Azores islands haplogroups by mitochondrial DNA analysis H. Afonso Costa a,b,*, M. Carvalho a,b, A.M. Bento a,b, F. Balsa a,b, M.J. Anjos a,b, F. Corte-Real b,c,d a
Forensic Genetics Service, Centre Branch, National Institute of Legal Medicine, I.P. Coimbra, Portugal CENCIFOR, Forensic Sciences Centre, Portugal c National Institute of Legal Medicine, I.P. Coimbra, Portugal d Faculty of Medicine, University of Coimbra, Portugal b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2011 Accepted 29 August 2011
Mitochondrial DNA (mtDNA) has enormous potential in population genetics allowing the correlation and identification of the origin of individuals in a population. In forensic genetics mtDNA enables the identification of the matrilineal lineage from degraded samples. Genetic testing can be performed using mtDNA noncoding region and/or mtDNA coding region. However, to apply this inclusive approach as evidence in legal practice is necessary to determine the genetic structure of populations. Traditional mtDNA sequencing and Single Nucleotide Polymorphisms (SNP) analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups. Forty-eight haplotypes were identified. All mtDNA haplotypes were included into specific haplogroups: forty-three haplotypes belonging to macrohaplogroup R; three haplotypes belonging to macrohaplogroup N; and two haplotypes belonging to macrohaplogroup M. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA typing Coding region SNPs West Eurasian Haplogroup Haplotype Azores
1. Introduction Mitochondrial DNA (mtDNA) haplotypes analysis found an important role in forensic genetics, especially when nuclear DNA analysis does not give a conclusive response; also mtDNA haplogroups assignment became noteworthy to clarify the history and demographic past of a population. Features that increase the vested interest of mitochondrial DNA are the high copy number per cell, maternal inheritance [1], absence of recombination [2] and high mutation rate. Due to higher overall mutation rate, control region is comparatively enriched in sequence variation and therefore its analysis is important to establish haplotypes and haplogroups. However, the information obtained from the mtDNA control region may be insufficient to discriminate between samples belonging to the same haplogroup. It is pertinent to corroborate predicted haplogroups based on control region sequences using informative Single Nucleotide Polymorphisms (SNPs) from the coding region. To achieve more information several protocols to analyse mtDNA SNPs from coding region were developed [3–6]. Western Eurasian may be classified into 9 major haplogroups and several sub-haplogroups: H, I, J, K, T, U, V, W and X [7,8].
* Corresponding author at: Largo da Se´ Nova, 3000-213 Coimbra, Portugal. Tel.: +351 239854230; fax: +351 239820549. E-mail address: [email protected] (H. Afonso Costa). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.066
Traditional mtDNA sequencing and SNP analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups.
2. Material and methods Blood samples were obtained from fifty, healthy and unrelated, individuals from Azores. DNA was extracted using Chelex1 100 method [9]. Hypervariable regions of the mtDNA control region, for fifty samples, were amplified using the primers L15997 and H16401 for hipervariable region I (HVI) and the primers L408 and H048 for hipervariable region II (HVII) [10]. Thermocycling conditions were performed in a GeneAmp1 PCR System 2700 (Applied Biosystems, Foster City, CA). Both light and heavy chains of each hypervariable region were sequenced using BigDye1 Terminator v1.1 Cycle Sequence Kit (Applied Biosystems, FosterCity, CA). Electrophoresis was performed on an ABI PRISM1 3130 Genetic Analyzer (Applied Biosystems, FosterCity, CA). Analysis was done with ABI DNA Sequencing Analysis V5.2 and SeqScape v2.5. Data, of HVI and HVII sequences, stored in EMPOP with accession number EMP00396. Sixteen mtSNPs were amplified in two multiplex reaction, for thirty-four samples, using primers proposed by Bransta¨tter et al. [5] and amplification protocol published by Parson et al. [11]. Multiplex I consist of the SNP target sites G709A, G1719A, G3010A, C7028T, A11251G, G12372A, T14798C and C15904. Multiplex II
e132
H. Afonso Costa et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e131–e132
includes the SNP target sites A1811G, T6365C, T6776C, G8251A, G8697A, G9055A, G13708A and C14766T. Previous to minisequencing reaction, PCR product was treated with ExoSAP-IT1 (USB corporation, Cleveland, Ohio) to remove unincorporated dNTPs and excess primers. Minisequencing reaction was performed with ABI PRISM1 SNaPshotTM Multiplex Kit (Applied Biosystems, FosterCity, CA). Electrophoresis was accomplished on an ABI PRISM1 3130 Genetic Analyzer using GeneScanTM 120 LIZ1 Size Standard (Applied Biosystems, FosterCity, CA). Analysis was done using GeneMapper1 ID v3.2 software.
haplotypes in specific haplogroups. MtDNA control region typing in combination with SNP analysis are extremely useful in population genetics and due to their characteristics can be used as a technique to differentiate among degraded samples frequently found in forensic genetics and establish its global frequency when having knowledge of the genetic structure of populations.
Conflict of interest None.
3. Results All fifty samples were successfully typed for HVI and HVII and forty-eight haplotypes were identified. From the fifty samples, thirty-two of them were successfully typed with the two multiplexes. All the mtDNA haplotypes were included into specific macrohaplogroups: forty-three haplotypes belong to macrohaplogroup R (86%); three haplotypes belong to macrohaplogroup N (6%); two haplotypes belong to macrohaplogroup M (4%). Distribution in macrohaplogroup R was as follows: Hg H: 24%, Hg R: 14%, Hg J: 12%, Hg T: 12%, Hg K: 10%, Hg U: 10%, Hg HV0: 6%, Hg V: 2%. 4. Discussion From the 9 major West Eurasian Caucasian haplogroups only 6 were observed, since none of the analysed individuals belong to Hg I, W or X. The most observed haplogroup was haplogroup H and the minor haplogroup observed was haplogroup V. The haplogroups determined from Azores population are in accordance with other European populations. 5. Conclusion Information obtained by combining the sequences of control region and coding region SNPs allowed the inclusion of all
References [1] R.E. Giles, H. Blanc, H.M. Cann, et al., Maternal inheritance of human mitochondrial DNA, Proc. Natl. Acad. Sci. 77 (1980) 6715–6719. [2] J.W. Ballard, M.O. Whitlock, The incomplete natural history of mitochondria, Mol. Ecol. 13 (4) (2004) 729–744. [3] B. Quinta´ns, V. A´lvarez-Iglesias, A. Salas, et al., Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SnaPshot minisequencing, Forensic Sci. Int. 140 (2004) 251–257. [4] H. Young Lee, J.E. Yoo, M.J. Park, et al., East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogrouplevel control region sequence analysis, Electrophoresis 27 (2006) 4408–4418. [5] A. Bransta¨tter, T. Parson, W. Parson, Rapid Screening of mtDNA coding region SNPs for the identification of West European Caucasian haplogroups, Int. J. Legal Med. 117 (2003) 291–298. [6] P.M. Vallone, R.S. Just, M.D. Coble, et al., 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] A. Torroni, K. Huoponen, P. Francalacci, et al., Classification of European mtDNAs from an analysis of three European populations, Genetics 144 (1996) 1835–1850. [8] V. Macaulay, M. Richards, E. Hickey, et al., The emerging three of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLP, Am. J. Hum. Genet. 64 (1999) 232–249. [9] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (1991) 506–513. [10] M.R. Wilson, J.A. DiZinno, D. Polanksey, et al., Validation of mitochondrial DNA sequencing for forensic casework analysis, Int. J. Legal Med. 108 (1999) 68–74. [11] W. Parson, L. Fendt, D. Ballard, et al., Identification of west Eurasian mitochondrial haplogroups by mtDNA screening: results of the 2006–2007 collaborative exercise, Forensic Sci. Int.: Genet. 2 (2008) 61–68.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Identification of Azores islands haplogroups by mitochondrial DNA analysis H. Afonso Costa a,b,*, M. Carvalho a,b, A.M. Bento a,b, F. Balsa a,b, M.J. Anjos a,b, F. Corte-Real b,c,d a
Forensic Genetics Service, Centre Branch, National Institute of Legal Medicine, I.P. Coimbra, Portugal CENCIFOR, Forensic Sciences Centre, Portugal c National Institute of Legal Medicine, I.P. Coimbra, Portugal d Faculty of Medicine, University of Coimbra, Portugal b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2011 Accepted 29 August 2011
Mitochondrial DNA (mtDNA) has enormous potential in population genetics allowing the correlation and identification of the origin of individuals in a population. In forensic genetics mtDNA enables the identification of the matrilineal lineage from degraded samples. Genetic testing can be performed using mtDNA noncoding region and/or mtDNA coding region. However, to apply this inclusive approach as evidence in legal practice is necessary to determine the genetic structure of populations. Traditional mtDNA sequencing and Single Nucleotide Polymorphisms (SNP) analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups. Forty-eight haplotypes were identified. All mtDNA haplotypes were included into specific haplogroups: forty-three haplotypes belonging to macrohaplogroup R; three haplotypes belonging to macrohaplogroup N; and two haplotypes belonging to macrohaplogroup M. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA typing Coding region SNPs West Eurasian Haplogroup Haplotype Azores
1. Introduction Mitochondrial DNA (mtDNA) haplotypes analysis found an important role in forensic genetics, especially when nuclear DNA analysis does not give a conclusive response; also mtDNA haplogroups assignment became noteworthy to clarify the history and demographic past of a population. Features that increase the vested interest of mitochondrial DNA are the high copy number per cell, maternal inheritance [1], absence of recombination [2] and high mutation rate. Due to higher overall mutation rate, control region is comparatively enriched in sequence variation and therefore its analysis is important to establish haplotypes and haplogroups. However, the information obtained from the mtDNA control region may be insufficient to discriminate between samples belonging to the same haplogroup. It is pertinent to corroborate predicted haplogroups based on control region sequences using informative Single Nucleotide Polymorphisms (SNPs) from the coding region. To achieve more information several protocols to analyse mtDNA SNPs from coding region were developed [3–6]. Western Eurasian may be classified into 9 major haplogroups and several sub-haplogroups: H, I, J, K, T, U, V, W and X [7,8].
* Corresponding author at: Largo da Se´ Nova, 3000-213 Coimbra, Portugal. Tel.: +351 239854230; fax: +351 239820549. E-mail address: [email protected] (H. Afonso Costa). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.066
Traditional mtDNA sequencing and SNP analysis techniques were combined to establish the mtDNA variability of the Azorean population and classify the haplotypes into haplogroups.
2. Material and methods Blood samples were obtained from fifty, healthy and unrelated, individuals from Azores. DNA was extracted using Chelex1 100 method [9]. Hypervariable regions of the mtDNA control region, for fifty samples, were amplified using the primers L15997 and H16401 for hipervariable region I (HVI) and the primers L408 and H048 for hipervariable region II (HVII) [10]. Thermocycling conditions were performed in a GeneAmp1 PCR System 2700 (Applied Biosystems, Foster City, CA). Both light and heavy chains of each hypervariable region were sequenced using BigDye1 Terminator v1.1 Cycle Sequence Kit (Applied Biosystems, FosterCity, CA). Electrophoresis was performed on an ABI PRISM1 3130 Genetic Analyzer (Applied Biosystems, FosterCity, CA). Analysis was done with ABI DNA Sequencing Analysis V5.2 and SeqScape v2.5. Data, of HVI and HVII sequences, stored in EMPOP with accession number EMP00396. Sixteen mtSNPs were amplified in two multiplex reaction, for thirty-four samples, using primers proposed by Bransta¨tter et al. [5] and amplification protocol published by Parson et al. [11]. Multiplex I consist of the SNP target sites G709A, G1719A, G3010A, C7028T, A11251G, G12372A, T14798C and C15904. Multiplex II
e132
H. Afonso Costa et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e131–e132
includes the SNP target sites A1811G, T6365C, T6776C, G8251A, G8697A, G9055A, G13708A and C14766T. Previous to minisequencing reaction, PCR product was treated with ExoSAP-IT1 (USB corporation, Cleveland, Ohio) to remove unincorporated dNTPs and excess primers. Minisequencing reaction was performed with ABI PRISM1 SNaPshotTM Multiplex Kit (Applied Biosystems, FosterCity, CA). Electrophoresis was accomplished on an ABI PRISM1 3130 Genetic Analyzer using GeneScanTM 120 LIZ1 Size Standard (Applied Biosystems, FosterCity, CA). Analysis was done using GeneMapper1 ID v3.2 software.
haplotypes in specific haplogroups. MtDNA control region typing in combination with SNP analysis are extremely useful in population genetics and due to their characteristics can be used as a technique to differentiate among degraded samples frequently found in forensic genetics and establish its global frequency when having knowledge of the genetic structure of populations.
Conflict of interest None.
3. Results All fifty samples were successfully typed for HVI and HVII and forty-eight haplotypes were identified. From the fifty samples, thirty-two of them were successfully typed with the two multiplexes. All the mtDNA haplotypes were included into specific macrohaplogroups: forty-three haplotypes belong to macrohaplogroup R (86%); three haplotypes belong to macrohaplogroup N (6%); two haplotypes belong to macrohaplogroup M (4%). Distribution in macrohaplogroup R was as follows: Hg H: 24%, Hg R: 14%, Hg J: 12%, Hg T: 12%, Hg K: 10%, Hg U: 10%, Hg HV0: 6%, Hg V: 2%. 4. Discussion From the 9 major West Eurasian Caucasian haplogroups only 6 were observed, since none of the analysed individuals belong to Hg I, W or X. The most observed haplogroup was haplogroup H and the minor haplogroup observed was haplogroup V. The haplogroups determined from Azores population are in accordance with other European populations. 5. Conclusion Information obtained by combining the sequences of control region and coding region SNPs allowed the inclusion of all
References [1] R.E. Giles, H. Blanc, H.M. Cann, et al., Maternal inheritance of human mitochondrial DNA, Proc. Natl. Acad. Sci. 77 (1980) 6715–6719. [2] J.W. Ballard, M.O. Whitlock, The incomplete natural history of mitochondria, Mol. Ecol. 13 (4) (2004) 729–744. [3] B. Quinta´ns, V. A´lvarez-Iglesias, A. Salas, et al., Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SnaPshot minisequencing, Forensic Sci. Int. 140 (2004) 251–257. [4] H. Young Lee, J.E. Yoo, M.J. Park, et al., East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogrouplevel control region sequence analysis, Electrophoresis 27 (2006) 4408–4418. [5] A. Bransta¨tter, T. Parson, W. Parson, Rapid Screening of mtDNA coding region SNPs for the identification of West European Caucasian haplogroups, Int. J. Legal Med. 117 (2003) 291–298. [6] P.M. Vallone, R.S. Just, M.D. Coble, et al., 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] A. Torroni, K. Huoponen, P. Francalacci, et al., Classification of European mtDNAs from an analysis of three European populations, Genetics 144 (1996) 1835–1850. [8] V. Macaulay, M. Richards, E. Hickey, et al., The emerging three of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLP, Am. J. Hum. Genet. 64 (1999) 232–249. [9] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques 10 (1991) 506–513. [10] M.R. Wilson, J.A. DiZinno, D. Polanksey, et al., Validation of mitochondrial DNA sequencing for forensic casework analysis, Int. J. Legal Med. 108 (1999) 68–74. [11] W. Parson, L. Fendt, D. Ballard, et al., Identification of west Eurasian mitochondrial haplogroups by mtDNA screening: results of the 2006–2007 collaborative exercise, Forensic Sci. Int.: Genet. 2 (2008) 61–68.