Mitochondrial DNA control region database in Banco Nacional de Datos Genéticos, Argentina

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
• 3. Results and discussion
• Conflict of interest statement
• References
• Copyright

Abstract 

To analyze the distribution of different haplotypes of mitochondrial control region DNA in a large sample of argentine population who assisted to the Banco Nacional de Datos Genéticos (BNDG) looking for their identity in cases of civil state suppression during the dictatorial government (1976–1983).

A total of 1168 unrelated argentine individuals who assisted to the BNDG were sampled for the analysis.

Mitochondrial DNA Control Region sequences were determined for both hypervariable regions 1 (HV1) and 2 (HV2).

Blood samples were used as the DNA source.

The amplification for both hypervariable regions was performed in a Perkin Elmer 9700 thermal cycler.

The cycle sequencing was performed using the BigDye^® Terminator v 1.1 Cycle Sequencing Kit (Applied Biosystems) and automated sequencing was performed in an ABIPRISM^® 3100 Genetic Analyzer (Applied Biosystems).

Analysis of mitochondrial DNA sequencing data was performed by manual method.

The hypervariable region 1 (HV1) was analyzed between positions 16023 and 16428 and the hypervariable region 2 (HV2) between 50 and 480 according to the Anderson Sequence.

To analyze these results a database was created and statistical parameters were calculated.

From 1168 haplotypes analyzed, 794 were unique. Haplotype frequencies were estimated by haplotype counting. Values of Genetic Identity (P) and Genetic Diversity (h) were calculated, being 3.3939×10⁻³ and 0.9974 respectively.

This Mitochondrial Control Region DNA database provides useful information for genetic profiles comparison and maternal lineage determination in our lab.

Keywords: Mitochondrial DNA, Forensics, Population, Polymorphisms

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1. Introduction 

Analysis of mitochondrial DNA (mt DNA) has become a useful tool for individualisation in forensic casework, being in some cases, the more convenient and successful method to be considered, specially, with low DNA or degraded samples due to its high copy number and hence its relative abundance. That is the reason why, actually, great efforts are made to build up mitochondrial databases for forensic purposes. Sequencing of mitochondrial DNA D-Loop region [1], [2] has been incorporated since 1993 in our laboratory to study maternal lineage. Since then, it has become the tool of choice for forensic casework.

Automated DNA sequencing of PCR products has made mitochondrial DNA analysis easier and faster.

The aim of our study is to analyze the distribution of different haplotypes of Mitochondrial Control Region DNA (HV1 and HV2 segments) and the sequence polymorphisms in a large sample or argentine population who assisted to the Banco Nacional de Datos Genéticos (BNDG) looking for the identity in cases of civil state suppression during the dictatorial government (1976–1983).

We analyzed the sequence polymorphisms of Mitochondrial Control Region DNA [3] in our population, and for that purpose, we studied the D-Loop hypervariable regions 1 and 2 (HV1 and HV2). Statistical interpretation of the results let us create a database with sequences of our population conformed by a large sample of unrelated argentine who assisted to the Banco Nacional de Datos Genéticos (BNDG) looking for their identity in cases of civil state suppression during the dictatorial government (1976–1983).

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2. Materials and methods 

EDTA plasma samples were collected from a total of 1168 unrelated argentine individuals who assisted to the BNDG looking for their identity as we described before.

Total DNA was extracted from blood samples, according to the Miller's method (salting out) and, from forensic samples, according to the chloroform/phenol/isoamyl alcohol extraction method. Mitochondrial DNA Control Region sequences were determined for both hypervariable regions 1 (HV1) and 2 (HV2).

PCR amplification of mt DNA Control Region of approximately 1300bp was performed using the following set of primers: L-15926 and H-00580.

For forensic samples, we amplified three fragments of HV1 segment (259, 157 and 193bp) and three fragments of HV2 segments (233, 227 and 172bp) using the following primers: L-15997/H-16255, L-16099/H-16255, L-16209/H-16401, L-00030/H-00262, L-00140/H-00366 and L-00241/H-00412, respectively.

PCR was performed in a Gene Amp PCR System 9700^® (Perkin Elmer). PCR products were purified using Microspin S-300 HRColumns (Amersham/Pharmacia) and then sequenced by cycle sequencing using fluorescent dideoxynucleotides (Big Dye Terminator Cycle Sequencing Kit v. 1.1 – Applied Biosystems), followed by capillary electrophoresis in an ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems).

Analysis of mitochondrial DNA sequencing data was performed by manual method.

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3. Results and discussion 

We analyzed the sequence polymorphisms of Mitochondrial Control Region DNA in 1168 unrelated individuals. We studied both regions (HV1 and HV2) in all the samples. For HV1 region, we analyzed 406bp (between positions 16023 and 16428), and for HV2 region, 431bp (between positions 50 and 480) according to the Cambridge Reference Sequence (CRS).

From 1168 haplotypes analyzed we found that 794 (68%) were unique.

For both regions the haplotypes frequencies were estimated by haplotype counting. In Table 1, we described the most frequent combined haplotypes we had in our population considering the complete analysis of HV1 and HV2 regions. The HV1 haplotypes 16223-T, 16298-C, 16325-C, 16327-T were as frequent in our population as that described in Amerindians [4].

Table 1. mt DNA control region. Most frequent complete haplotypes found in the BNDG.

In the combined results of 1168 individuals, 223 variable sites were detected in HV1 region and 167 variable sites in HV2 region. Transition types of substitution in both regions were more predominant than transversions. In our population HV1 region did not present either insertions or deletions. In contrast, HV2 region presented both.

The nucleotide substitution at the position 16223 (nucleotide transition from C to T) was the most frequent transition we found in HV1 region (61.7%). This mutational event is present in Amerindian, Asian and African haplotypes [5].

The nucleotide sequence from 16184 to 16193 in HV1 region contained serially repeated stretches of C, when the nucleotide transition from T to C at the position 16189 was present. In the present study this nucleotide transition from T to C was observed in 302 (26%) individuals, showing the polycytosine tract.

In HV2 region the insertion of an additional cytosine at the cytosine tract 311–315 was found in all individuals.

We observed that the HV1 haplotype: 16223-T, 16298-C, 16325-C, 16327-T was associated with an HV2 haplotype sharing three deletion positions (249, 290 and 291), which are described in Mapuches [6].

In four cases we observed a 6-bp deletion (from nucleotide 106 to nucleotide 111 also found in Chibcha population (http://www.mitomap.org).

Other observation of association of HV1 region with HV2 region was that when the HV1 haplotype was CRS (common in Europeans) [7], the HV2 haplotype did not present the point mutation 73-G.

Genetic Identity testing using P=Σx² (where x is the frequency of mt DNA genotype) for both regions, gave the value of P=3.3939×10⁻³ for the two regions considered together.

The Genetic Diversity utilizing h=(1−Σx²)×n/(n−1) (n: sample size) [8], of argentine mt DNA Control Region was estimated as 0.9974 for both regions. Similar values were also obtained from other populations analyzed for the both regions combined. For example: Caucasians [9].

Complete sequence analysis showed a high level of diversity (99.74%), which let us conclude that sequencing of Mitochondrial DNA D-Loop region is an essential marker to be considered in human identification.

In summary, the Mitochondrial Control Region DNA database created proved to be useful for the purpose of genetic profiles comparison and parental lineage determination in the Banco Nacional de Datos Genéticos (BNDG).

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Conflict of interest statement 

None

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References 

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