- Email: [email protected]
Haplotype diversity in mitochondrial DNA hypervariable regions I and II in Maracaibo population (Venezuela)
Forensic Science International: Genetics Supplement Series 2 (2009) 334–335
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Haplotype diversity in mitochondrial DNA hypervariable regions I and II in Maracaibo population (Venezuela) J.M. Quintero *, L. Borjas, T. Pardo, D. Bracho, K. Huerta, K. Me´ndez, Y. Sa´nchez, W. Zabala Laboratorio de Gene´tica Molecular, Unidad de Gene´tica Me´dica, Facultad de Medicina, Universidad del Zulia, Maracaibo, Estado Zulia 4001, Venezuela
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 August 2009 Accepted 26 August 2009
The control region of the human mitochondrial DNA (mtDNA) is highly polymorphic due to a rapid rate of evolution. The hypervariable segments HVI and HVII of the mitochondrial control region (D-loop) are the most variable part of human mitochondrial DNA. Blood samples from 50 unrelated individuals born from Maracaibo City (Venezuela) were obtained from routine paternity. Polymerase chain reaction amplification products were purified and fluorescent-based capillary electrophoresis sequencing method, the sequences were analysed and it is compared DNA sequences with the rCRS. The dominant haplogroups corresponded to Amerindians followed by African. The nucleotide positions most common for HVII was 263G (100%), 315.1C (100%), 73G (94%), and 309.1C (66%) and for HVI 16223T (74%), 16362C (56%), 16319A (40%) and 16111T (34%). ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Venezuelan population Hypervariable segments I Hypervariable segments II D-loop
1. Introduction
2.3. Amplification and sequencing of mtDNA
The analysis of the control region has been of great value in forensic science since this region was not involved in the gene coding for a product tends to accumulate mutations at a frequency of 5–10 times higher than the rest of the mitochondrial DNA (mtDNA) [1], and then in this region where the highest variability as interpersonal, mtDNA sequences usually differ between Caucasian Europeans on average at 8 nucleotide position [2], providing important support for the identification of human biological evidence.
Blood samples from 50 unrelated individuals born from Maracaibo City (Venezuela) were obtained from routine paternity at West region of Venezuela.
The following sets of PCR primers were used to amplified two DNA fragments corresponding to HVR I (476 bp) and HVR II (361 bp): L15926 50 -TCA AAG CTT ACA CCA GTC TTG TAA ACC-30 , H16401 50 -TGA TTT CAC GGA GGA TGG TG-30 , L048 50 -CTC ACG GGA GCT CTC CAT GC-30 , H408 50 -CTG TTA AAA GTC CAT ACC GCC A-30 . ‘L’ and ‘H’ designate the light and the heavy strands of the mtDNA molecule, respectively. Each PCR was performed using GoTaq (Promega) and 30 amplification cycles in a PTC-200 thermocycler device (MJ Research1). PCR products were purified using Wizard1 PCR Preps DNA Purification System (Promega) and both strands sequenced with BigDyeTM Terminator v. 3.1 kit (Applied Biosystems) using the same primers as for the amplification and purified using Wizard1 MagneSil1 Sequencing Reaction Clean-Up System (Promega), the sequencing was performed on an ABI PrismTM 310 Genetic Analyzer.
2.2. DNA extraction
2.4. Quality control
DNA was extracted from blood, collected on FTA1 cards and processed with the FTATM Purification Reagent from GIBCO-BRL according to the manufacturer’s protocol.
Proficiency testing of the GEP-ISFG WG (http://www.gepisfg.org).
2. Materials and methods 2.1. Population
* Corresponding author. Tel.: +58 261 6110736. E-mail address: [email protected] (J.M. Quintero). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.149
2.5. Analysis of data Sequences were aligned and compared to the revised Cambridge reference sequence (rCRS) [3] using the sequence analysis
J.M. Quintero et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 334–335 Table 1 Mutations found in Maracaibo population (including C stretch).
3.1. Other remarks
HVI
HVII
HVI + HVII
126 90 14 36
11 52 141 11
137 142 155 47
266
215
481
3 13 1 0 0 0
0 7 0 1 1 3
3 20 1 1 1 3
Total
17
12
29
Insertions +C
12
96
108
Deletions A
0
11
11
295
334
629
Polymorphic sites (C stretch) [5] Group I 38 Group II 0 Group III 1 Group IV 11
27 12 11 0
Transitions C>T T>C A>G G>A Total Transversions C>G A>C A>T G>C G>T C>A
Total mutations
Total
335
The most frequent mutations found were 263G and 315.1C present in a 100% of the sample, followed by 73G (94%), 16223T (74%) 309.1C (66%), and 16362C (56%). A mutation previously reported by N. Lander et al., [6], the deletion at nucleotide position 115, was confirmed by forward and reverse sequencing in HVII. The predominant haplogroups were essentially Amerindian A2 (30%), B4 (18%), C1 (4%), and African confirming hypothesis that European Caucasian migratory groups made little contribution to the mitochondrial DNA pool. This paper follows the guidelines for mitochondrial DNA typing and for publication of population data [7]. 4. Conflict of interest None. References
50
50
and alignment software Staden Package v.2.0, following international guidelines for mtDNA typing [4]. 3. Results Table 1 presents an overview of the different types of mutations found.
[1] C.F. Aquadro, B.D. Greenberg, Human mitochondrial DNA variation and evolution: analysis of nucleotide sequences for seven individuals, Genetics 103 (February (2)) (1983) 287–312. [2] B. Budowle, M.R. Wilson, J.A. DiZinno, C. Stauffer, M.A. Fasano, M.M. Holland, K.L. Monson, Mitochondrial DNA regions HVI and HVII population data, Forensic Sci. Int. 12 (1999) 23–35. [3] R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 147. [4] A. Carracedo, W. Bar, P. Lincoln, W. Mayr, N. Morling, B. Olaisen, P. Schneider, B. Budowle, B. Brinkmann, P. Gill, M. Holland, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [5] S. Lutz-Bonengel, T. Sanger, S. Pollak, R. Szibor, Different methods to determine length heteroplasmy within the mitochondrial control region, Int. J. Legal Med. 118 (2004) 274–281. [6] N. Lander, M.G. Rojas, M.A. Chiurillo, J.L. Ramı´rez, Haplotype diversity in human mitochondrial DNA hypervariable regions I–III in the city of Caracas (Venezuela), Forensic Sci. Int. Genet. 2 (4) (2008) e61–e64. [7] P. Lincoln, A. Carracedo, Publication of population of human polymorphisms, Forensic Sci. Int. 110 (2000) 3–5.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Haplotype diversity in mitochondrial DNA hypervariable regions I and II in Maracaibo population (Venezuela) J.M. Quintero *, L. Borjas, T. Pardo, D. Bracho, K. Huerta, K. Me´ndez, Y. Sa´nchez, W. Zabala Laboratorio de Gene´tica Molecular, Unidad de Gene´tica Me´dica, Facultad de Medicina, Universidad del Zulia, Maracaibo, Estado Zulia 4001, Venezuela
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 August 2009 Accepted 26 August 2009
The control region of the human mitochondrial DNA (mtDNA) is highly polymorphic due to a rapid rate of evolution. The hypervariable segments HVI and HVII of the mitochondrial control region (D-loop) are the most variable part of human mitochondrial DNA. Blood samples from 50 unrelated individuals born from Maracaibo City (Venezuela) were obtained from routine paternity. Polymerase chain reaction amplification products were purified and fluorescent-based capillary electrophoresis sequencing method, the sequences were analysed and it is compared DNA sequences with the rCRS. The dominant haplogroups corresponded to Amerindians followed by African. The nucleotide positions most common for HVII was 263G (100%), 315.1C (100%), 73G (94%), and 309.1C (66%) and for HVI 16223T (74%), 16362C (56%), 16319A (40%) and 16111T (34%). ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Mitochondrial DNA Venezuelan population Hypervariable segments I Hypervariable segments II D-loop
1. Introduction
2.3. Amplification and sequencing of mtDNA
The analysis of the control region has been of great value in forensic science since this region was not involved in the gene coding for a product tends to accumulate mutations at a frequency of 5–10 times higher than the rest of the mitochondrial DNA (mtDNA) [1], and then in this region where the highest variability as interpersonal, mtDNA sequences usually differ between Caucasian Europeans on average at 8 nucleotide position [2], providing important support for the identification of human biological evidence.
Blood samples from 50 unrelated individuals born from Maracaibo City (Venezuela) were obtained from routine paternity at West region of Venezuela.
The following sets of PCR primers were used to amplified two DNA fragments corresponding to HVR I (476 bp) and HVR II (361 bp): L15926 50 -TCA AAG CTT ACA CCA GTC TTG TAA ACC-30 , H16401 50 -TGA TTT CAC GGA GGA TGG TG-30 , L048 50 -CTC ACG GGA GCT CTC CAT GC-30 , H408 50 -CTG TTA AAA GTC CAT ACC GCC A-30 . ‘L’ and ‘H’ designate the light and the heavy strands of the mtDNA molecule, respectively. Each PCR was performed using GoTaq (Promega) and 30 amplification cycles in a PTC-200 thermocycler device (MJ Research1). PCR products were purified using Wizard1 PCR Preps DNA Purification System (Promega) and both strands sequenced with BigDyeTM Terminator v. 3.1 kit (Applied Biosystems) using the same primers as for the amplification and purified using Wizard1 MagneSil1 Sequencing Reaction Clean-Up System (Promega), the sequencing was performed on an ABI PrismTM 310 Genetic Analyzer.
2.2. DNA extraction
2.4. Quality control
DNA was extracted from blood, collected on FTA1 cards and processed with the FTATM Purification Reagent from GIBCO-BRL according to the manufacturer’s protocol.
Proficiency testing of the GEP-ISFG WG (http://www.gepisfg.org).
2. Materials and methods 2.1. Population
* Corresponding author. Tel.: +58 261 6110736. E-mail address: [email protected] (J.M. Quintero). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.149
2.5. Analysis of data Sequences were aligned and compared to the revised Cambridge reference sequence (rCRS) [3] using the sequence analysis
J.M. Quintero et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 334–335 Table 1 Mutations found in Maracaibo population (including C stretch).
3.1. Other remarks
HVI
HVII
HVI + HVII
126 90 14 36
11 52 141 11
137 142 155 47
266
215
481
3 13 1 0 0 0
0 7 0 1 1 3
3 20 1 1 1 3
Total
17
12
29
Insertions +C
12
96
108
Deletions A
0
11
11
295
334
629
Polymorphic sites (C stretch) [5] Group I 38 Group II 0 Group III 1 Group IV 11
27 12 11 0
Transitions C>T T>C A>G G>A Total Transversions C>G A>C A>T G>C G>T C>A
Total mutations
Total
335
The most frequent mutations found were 263G and 315.1C present in a 100% of the sample, followed by 73G (94%), 16223T (74%) 309.1C (66%), and 16362C (56%). A mutation previously reported by N. Lander et al., [6], the deletion at nucleotide position 115, was confirmed by forward and reverse sequencing in HVII. The predominant haplogroups were essentially Amerindian A2 (30%), B4 (18%), C1 (4%), and African confirming hypothesis that European Caucasian migratory groups made little contribution to the mitochondrial DNA pool. This paper follows the guidelines for mitochondrial DNA typing and for publication of population data [7]. 4. Conflict of interest None. References
50
50
and alignment software Staden Package v.2.0, following international guidelines for mtDNA typing [4]. 3. Results Table 1 presents an overview of the different types of mutations found.
[1] C.F. Aquadro, B.D. Greenberg, Human mitochondrial DNA variation and evolution: analysis of nucleotide sequences for seven individuals, Genetics 103 (February (2)) (1983) 287–312. [2] B. Budowle, M.R. Wilson, J.A. DiZinno, C. Stauffer, M.A. Fasano, M.M. Holland, K.L. Monson, Mitochondrial DNA regions HVI and HVII population data, Forensic Sci. Int. 12 (1999) 23–35. [3] R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 147. [4] A. Carracedo, W. Bar, P. Lincoln, W. Mayr, N. Morling, B. Olaisen, P. Schneider, B. Budowle, B. Brinkmann, P. Gill, M. Holland, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [5] S. Lutz-Bonengel, T. Sanger, S. Pollak, R. Szibor, Different methods to determine length heteroplasmy within the mitochondrial control region, Int. J. Legal Med. 118 (2004) 274–281. [6] N. Lander, M.G. Rojas, M.A. Chiurillo, J.L. Ramı´rez, Haplotype diversity in human mitochondrial DNA hypervariable regions I–III in the city of Caracas (Venezuela), Forensic Sci. Int. Genet. 2 (4) (2008) e61–e64. [7] P. Lincoln, A. Carracedo, Publication of population of human polymorphisms, Forensic Sci. Int. 110 (2000) 3–5.