Mitochondrial DNA CA dinucleotide repeats in Koreans: the presence of length heteroplasmy
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- Chung, U., Lee, H.Y., Yoo, J. et al. Int J Legal Med (2005) 119: 50. doi:10.1007/s00414-004-0487-7
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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.
KeywordsMitochondrial DNACA dinucleotide repeatsLength polymorphismLength heteroplasmyKoreans
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
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).
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 .
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 frequencies, sequence structures and genetic diversities of the CA dinucleotide repeats in the mtDNA HV3 region
Number of CA repeats
CCAG (CA)4 CCGC
CCAA (CA)4 CCGC
CCAG (CA)3CC CCGCb
CCAG (CA)5 CCGC
CCAA (CA)5 CCGC
CCAG TA (CA)4 CCGCc
CCAG (CA)6 CCGC
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 . 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 .
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 . 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.
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.