International Journal of Legal Medicine

, Volume 119, Issue 1, pp 50–53

Mitochondrial DNA CA dinucleotide repeats in Koreans: the presence of length heteroplasmy

Authors

  • Ukhee Chung
    • Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceCollege of Medicine Yonsei University
    • Biometrics Engineering Research CenterYonsei University
  • Hwan Young Lee
    • Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceCollege of Medicine Yonsei University
  • Ji-Eun Yoo
    • Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceCollege of Medicine Yonsei University
  • Myung Jin Park
    • Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceCollege of Medicine Yonsei University
    • Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceCollege of Medicine Yonsei University
    • Biometrics Engineering Research CenterYonsei University
    • Human Identification Research InstituteYonsei University
Short Communication

DOI: 10.1007/s00414-004-0487-7

Cite this article as:
Chung, U., Lee, H.Y., Yoo, J. et al. Int J Legal Med (2005) 119: 50. doi:10.1007/s00414-004-0487-7

Abstract

The mitochondrial DNA HV3 region contains CA dinucleotide repeats which display length polymorphism. To analyze this for forensic purposes, we designed a fluorescence-labelled PCR primer set for short amplification products and carried out genotyping by using capillary electrophoresis. A total of 4 alleles with 4–7 repeat units were observed and the genetic diversity was estimated to be 0.5120 in 500 unrelated Koreans. Interestingly, three individuals showed two or three length variants, i.e. length heteroplasmy.

Keywords

Mitochondrial DNACA dinucleotide repeatsLength polymorphismLength heteroplasmyKoreans

Introduction

The analysis of human mitochondrial DNA (mtDNA) is of central importance for forensic identity testing. The properties of mtDNA that make it valuable for human identification include a high copy number, maternal inheritance and a rapid rate of evolution [1, 2]. Especially, the two non-coding HV1 (first hypervariable region) and HV2 (second hypervariable region) fragments of the control region, which are the most polymorphic regions in mtDNA, generally have been targeted for forensic purposes and analyzed in several kinds of biological evidence [3, 4, 5]. Although HV1/HV2 haplotypes have a high evidential value, other regions within the control region and variations within the mtDNA coding region with forensic utility were sometimes targeted for better differentiation of mtDNA [6, 7, 8, 9, 10].

It is noteworthy that CA dinucleotide repeats between nucleotide position 514 and 523 in HV3 (third hypervariable region) show length variability like STRs (short tandem repeats) in nuclear DNA [11, 12, 13], and the genetic diversity is relatively high. As this length polymorphism was expected to provide additional information for forensic purposes with simple genotyping methods as for STR analysis, i.e. PCR and capillary electrophoresis, we analyzed the CA dinucleotide repeats using a fluorescence-labelled PCR primer set for short amplification products in 500 unrelated Koreans. Also, we found three heteroplasmic samples during the analyses and confirmed the presence of length heteroplasmy by identifying each length variant in an individual.

Materials and methods

DNA samples

Buccal swabs or blood samples were obtained from 500 unrelated Koreans for the present study. DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cell line sample 9948 DNA (Promega, Madison, WI) was used for control DNA to calibrate the allelic ladder.

PCR amplification and genotyping

Primers were designed to amplify CA dinucleotide repeats in the HV3 region using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR amplification was carried out in a 10 µl reaction volume containing 10–20 pg of template DNA and the following set of primers: 0.2 µM of F447 (5’-HEX-CATTATTTTCCCCTCCCACTCC) and 0.2 µM of R569 (5’-GGTGTCTTTGGGGTTTGGTTG). Thermal cycling was conducted using a PTC-200 DNA engine (MJ Research, Waltham, MA) under the following conditions: 95°C for 11 min, 25 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min and a final extension at 60°C for 60 min. The PCR products were analyzed by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) using GeneScan analysis software version 3.1 (Applied Biosystems, Foster City, CA). Allele typing was carried out based on the sequenced allelic ladder using Genotyper 2.5 software (Applied Biosystems, Foster City, CA).

Sequence analysis

To analyze sequences including CA dinucleotide repeat units in the HV3 region, mtDNA was amplified in a total volume of 25 µl using an unlabelled primer set. PCR was performed under the same conditions of GeneScan PCR except for 35 thermal cycles. The PCR products were purified using a QIAquick purification kit (Qiagen, Hilden, Germany) and sequenced from both ends with a BigDye Terminator Cycle Sequencing v2.0 Ready Reaction kit (Applied Biosystems, Foster City, CA). The resulting sequences were analyzed using Sequence Analysis Software Version 3.4 and Sequence Navigator 1.01 (Applied Biosystems, Foster City, CA). The genetic diversity of CA dinucleotide repeats was calculated according to Tajima [14].

Confirmation of length heteroplasmy by cloning

The PCR products of heteroplasmic samples which showed consecutive multiple peaks with two base differences in the GeneScan electropherogram were cloned into the pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturer’s instructions. Each clone was sequenced as previously described. In addition, clones with various repeat unit lengths were subjected to GeneScan analysis to investigate whether stutter occurs due to insertion or deletion during the PCR amplification of CA dinucleotide repeats.

Results and discussion

The constructed allelic ladder for CA dinucleotide repeats in the HV3 region and an example of genotyping results are shown in Fig. 1A. The 9948 control DNA displayed the genotype 5. Allele frequencies, genetic diversities and sequence structures of CA dinucleotide repeats are shown in Table 1. There are some variant allele sequence structures. The A to C transversion and C to T transition in CA dinucleotide repeat units were observed in alleles 4 and 5, respectively. Also, a G to A transition in the flanking region was observed in alleles 4 and 5. The genetic diversity calculated with consideration of the flanking region sequence structure is 0.5120, which is higher than 0.4951 when considering only the amplification product size. According to previous reports, the genetic diversities of Germans, Russians, Hungarians and from the Cameroons were 0.3600, 0.3400, 0.4000 and 0.5200, respectively. The genetic diversity of the Korean population (0.4951) is relatively higher than those of Germans, Russians and Hungarians, while closer to the Cameroons [11]. The most abundant allele in Koreans (allele 5) is the same as in Germans, Russians and Hungarians, but not in the Cameroons (allele 4). Although there are some distinctions in genetic diversities and allele distributions among geographically different populations, it can be easily inferred that the combined use of length polymorphism in CA dinucleotide repeats in the HV3 region with sequence polymorphism in HV1 and HV2 regions will increase the forensic utility of mitochondrial DNA to some extent. Moreover, the measuring method for CA dinucleotide repeats designed in this study is simpler and faster than sequencing analysis, and the PCR product size is small enough (122–128 bp) to analyze degraded or low copy DNA or hair shaft DNA without nuclear DNA with ease.
Fig. 1A–D

The electropherograms showing genotype results of normal and heteroplasmic mtDNA. Constructed allelic ladder is shown at the top. A 9948 male standard DNA of which sequence is CCAG (CA)5 CCGC in CA dinucleotide repeat area. B Heteroplasmic mtDNA which contains (CA)4 and (CA)5. C Heteroplasmic mtDNA which contains (CA)5 and (CA)6. D Heteroplasmic mtDNA which contains (CA)4, (CA)5 and (CA)6

Table 1

The frequencies, sequence structures and genetic diversities of the CA dinucleotide repeats in the mtDNA HV3 region

Number of CA repeats

Na

Frequency

Sequence structure

Na

Frequency

4

186

0.3720

CCAG (CA)4 CCGC

181

0.3620

CCAA (CA)4 CCGC

4

0.0080

CCAG (CA)3CC CCGCb

1

0.0020

5

303

0.6060

CCAG (CA)5 CCGC

299

0.5980

CCAA (CA)5 CCGC

1

0.0020

CCAG TA (CA)4 CCGCc

3

0.0060

6

7

0.0140

CCAG (CA)6 CCGC

7

0.0140

7

4

0.0080

CCAG (CA)7CCGC

4

0.0080

Total

500

500

Genetic diversity

0.4951

0.5120

aThe number of individuals.

b, cClassified as allele 4 and allele 5 as the lengths of PCR products are identical to those of (CA)4and (CA)5, respectively.

On the other hand, we found three heteroplasmic samples during the course of analysis. One had length variants with 4 and 5 CA repeat units, another had 5 and 6 CA repeat units and the other had 4, 5 and 6 CA repeat units (Fig. 1B–D). The presence of length heteroplasmy was established if multiple peaks with two base differences were found in the GeneScan electropherograms. Also, we analyzed the presence of length heteroplasmy by cloning, and the results coincided with those of the GeneScan analysis. According to previous reports [15, 16, 17], direct sequencing, fluorescence-labelled restriction fragment analysis and cloning analysis have been used to detect and identify length heteroplasmy in mitochondrial HV1 and HV2 regions. In comparing the detection limit of the three methods, the level of detection was found to increase in the order from direct sequencing of the mtDNA to restriction fragment analysis, to the cloning assay [17]. However, restriction fragment analysis was thought to be a sufficiently accurate alternative for most applications involving heteroplasmy analysis. In the present study, our previous method that is similar to the fluorescence-labelled restriction fragment analysis with some modifications, was employed [18].

As for the mechanism of heteroplasmy formation in the CA dinucleotide repeats, their propensity for insertion-deletion mutation of multiples of repeating unit during replication could explain the phenomena along with the low fidelity of mitochondrial DNA polymerase. The high slippage rate of dinucleotide repeats can be observed in STRs, and resultant stutter production is correlated to the length of repeat stretches consisting of uniform repeats [19]. In this study, it was also demonstrated that the PCR amplification of CA dinucleotide repeats did not produce stutters for up to 6 CA repeat units, while clones with 7 CA repeats showed traces of stutter in the electropherograms. Taken together, polymerase slippage is the driving force not only in stutter artifact genesis during PCR but also causing new mutations (plus/minus one repeat) in dinucleotide repeats.

In summary, the analysis of length variability of CA dinucleotide repeats in the HV3 region will help increase the power of discrimination of mitochondrial DNA sequence analyses. Also, the length heteroplasmy of CA dinucleotide repeats in the HV3 region will increase the chance of forensic identification and help to understand the mechanism of mitochondrial evolution and microsatellite formation.

Acknowledgements

This work was supported by a grant from the Korea Science and Engineering Foundation (KOSEF) through the Biometrics Engineering Research Center (BERC) at Yonsei University.

Copyright information

© Springer-Verlag 2004