MOXEKymPHÂS EHOmma, 2010, m0M 44, № 6, c. 980-984
= TEHOMHKA. TPAHCKPHnTOMHKA =
UDC 577.2
HAPLOTYPES OF mtDNA -HV1/HV2 IN NON-RELATED INDIVIDUALS OF CAUCASIAN POPULATION LIVING IN THE SLOVAK REPUBLIC
© 2010 V. Repiska1*, I. Lehocky2, J. Galatova3, D. Bohmer1
1Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava,
811 08 Bratislava, Slovak Republic 2Department of Biology, Institute of Forensic Science of Police Corps, 812 72 Bratislava, Slovak Republic 3Institute of Foreign Languages, Faculty of Medicine, Comenius University in Bratislava, 811 08 Bratislava, Slovak Republic
Received February 09, 2010 Accepted for publication April 29, 2010
Evaluation of the frequency index for mtDNA particular sequences in the Caucasian population is crucial for forensic practice. There are two hypervariable regions in the mtDNA D-loop, HV1 and HV2. Both of them, 610 bp altogether, were sequenced, 342 bp from the hypervariable region HV1 (16024—16365) and 268 bp from the hypervariable region HV2 (73—340). We have analyzed 374 randomly selected non-related individuals of the Caucasian population and 192 individuals of the Roma subpopulation living in Slovakia. The main goals of the work were to introduce and standardize methods of analysis ofvariability of the HV1 and HV2 regions of mtDNA for forensic use; to characterize the variability of mtDNA in the Slovakian population taking into account the subpopulations; classification of mtDNA profiles into haplotype groups, and comparison with other haplotype groups in Europe in the frame of phylogenetic studies.
Keywords: haplotype groups, mitochondrial DNA analysis, PCR.
A database of mitochondrial DNA (mtDNA) haplo-types, which is an equivalent of a database of DNA profiles, plays unique role in the identification of cadavers or parts of human bodies of unrecognized identity. A database ofthis type is very important in case ofabsence ofrel-atives and during ethnicity identification. mtDNA typing is performed in samples wherein successful typing of nuclear DNA (ncDNA) is unlikely — bones, teeth, hair, material fixed in paraffin blocks, excrements and other samples which were resolved or exposed to unfavorable conditions of the environment [1, 2].
The largest variability among mtDNA haplotypes in the human population was detected in the region of the D-loop [3]. There are two hypervariable regions in that area labelled as HV1 and HV2. mtDNA haplogroup distribution correlates with the polymorphism ofHV1/HV2, as well as with polymorphism of the whole mitochondrial genome. Haplogroups A, B, C, D, E, F, G and M are typical for Asian populations, while the majority of native American populations have haplogroups A, B, C and D. Haplogroups L1, L2 and L3 are African, whereas haplogroups H, I, J, K, T U, V W and X are linked with the European populations [4, 5].
EXPERIMENTAL
DNA was obtained by standard phenol-chloroform extraction from 374 peripheral blood samples from randomly selected non-related individuals ofthe Slovak Cau-
* E-mail: vanda.repiska@fmed.uniba.sk
casian population and from 192 individuals of the Roma subpopulation living in Slovakia.
For PCR amplification of the HV1 region of mtDNA the following primers were used: H15806 (5'-GCATC-CGTACTATACTTCACAACAATCC- 3') and L16545 (5'-AACGTGTGGGCTATTTAGGC-3'). The algorithm of the PCR reaction was as follows:
94 ° C 1 min
94°C 20 s
52 ° C 15 s 38 cycles 72° C 1 min
72 ° C 5 min 4° C
For PCR amplification of the HV2 of mtDNA the next primers: H8 (5'-GGTCTATCACCCTATTAAC-CAC-3') and L429 (5'-CTGTTAAAA GTGCATAC-CGCC-3') — were used. The PCR reaction algorithm of was as follows:
95°C 2 min
94°C 45 s
66°C 1 min 30 cycles
72°C 1 min
72°C 7 min 4°C
HAPLOTYPES OF MTDNA -HV1/HV2 IN NON-RELATED INDIVIDUALS
981
Amplification was performed on thermal cycler GeneAmp® PCR System 2700 ("Applied Biosystems", USA). The PCR products were revised in the coloured by ethidium bromide agar gel with using an UV transilumi-nator. All PCR products were purified, and the DNA was sequenced. The algorithm for the DYE-PCR reaction of the HV1 region of mtDNA was the following:
94 ° C 20 s 50°C 15 s 60 ° C 1 min
34 cycles
The algorithm for the DYE-PCR reaction of the HV2 region of mtDNA was the following:
96°C 1 min
96°C 10 s 50°C 5 s 60 ° C 4 min
25 cycles
In the DYE-PCR reaction we used three primers (two forward primers and one reverse primer): H15909 (5'-ACACCAGTCTTGTAAACCGG-3'), H15975 (5'-CTCCACCATTAGCACCCAAAG-3'), and L16420 (5'-TGATTTCACGGAGGATGGTGG-3').
Amplification products of the mtDNA HV1 region were sequenced on a MegaBACE™ 1000 Sequencer ("Amersham Biosciences").
Sequences ofhypervariable region HV1 were analyzed using SeqLab (GCG Wisconsin Package 10, Genetics Computer Group). Sequence analysis of the hypervariable region HV2 was performed using DNA Sequencing Analysis Software version 5.2 ("Applied Biosystems"). The reference sequence of the complete human mitochondrial genome is available in the Genbank index access code NC_001807. In order to compare the obtained sequences with the reference one we used the ABI PRISM® SeqScape® Software version 2.5 ("Applied Biosystems").
Genetic diversity was calculated according to Tajima [6], random match probability was detected according to Stoneking et al. [7], and the average number ofnucleotide differences was calculated using the DnaSP version 3 programm [8]. Particular haplotypes were classified into the corresponding haplogroups and respectively labelled.
RESULTS AND DISCUSSION
For better differentiation of haplotypes some new terms were used. The region between positions 16024 and 16465, overlapping HV1, was called the "HV1 over-passing region" (HV1ex); the region between positions 72 and 340, overlapping HV2, was called the "HV2 over-passing region" (HV2ex). All calculations were performed for the regions HV1, HV1ex, HV2 and HV2ex, and also for HV1HV2 and HV1exHV2ex.
The most polymorphic region was HV1, which involved 342 bp with 97 variable positions. The extended
HV1 region (HV1ex), being significantly longer the HV1, had only 10 polymorphic places more (107 over 442 bp). Mutation at the 16399 position was observed in 22 individuals (5.9%). The HV2 region was 268 bp in length and had only 56 mutating positions. The extended HV2ex region being 269 bp in length carried an additional, 57th, polymorphic place. The HV2 region was extended by the 72 position (HV2ex), mutation at which was observed in 10 individuals (2.7%).
Comparison of the detected sequences with the Cambridge reference [9] allowed us to identify 284 mtDNA lines according to the 164 variable positions, 43 of which characterized only one of the examined mtDNA (26%), while the other 121 positions were not unique (the each nucleotide difference appeared in at least 2 individuals). These results obtained do not agree with Lutz's data [10] according to which the unique changes were at 92 variable positions (46% of all detected ones)Most of the polymorphic places are related to only one type of mutation, and only in 8 out of164 variable positions (4.9%) two different types of mutation were reported.
By comparing sequences of the whole HV1exHV2ex segment 284 haplotypes were detected. At that, 240 sequences (64.2%) were unique, 27 haplotypes (14.4%) occurred twice, 9 (7.2%) were observed 3 times, 4 (4.3 %) were found 4 times, and also more 4 different haplotypes every of which occurred 5 (1.3%), 9 (2.4%), 10 (2.7%) and 13 (3.5%) times were identified.
The most prevailing haplotype in the population investigated is the 263G, 315.1C one (Table 1) that was found in 3.5% of individuals. It is a characteristic haplo-type observed in the Caucasian population of other European countries, namely: 3% of all individuals in Austria [11], 2% in Germany [12], 2.6% in Switzerland [13], 4.3% in the Czech [14], in 4.5% the United Kingdom [15] and 4% in France [16] of all individuals have this haplo-type. It would be very interesting to expand a scale of our investigation to the Russian population which, unfortunately, such data are available only for the HV1 region for [17, 18].
Basing on the monitored haplotype frequency occurrence in our population, calculation of the genetic diversity [6], random match probability [7] and average number of nucleotide differences [8] were performed for hypervariable regions HV1, HV1ex, HV2, HV2ex, HV1 and HV2 together, and HV1ex and HV2ex together (Table 2). Genetic diversity of374 individuals of Caucasian population of Slovakia in the whole region sequenced is 0.997; probability that two randomly selected individuals from the population will have an identical haplotype of mtDNA is 0.62%. The average number of nucleotide differences was fixed to 8.03 for the two hypervariable regions together.
Most substitutions were distributed randomly. In hypervariable region HV1 position 16126C had a mutation in 17.4%, position 16311C was mutated in 16.6%, the 16270T position was different in 11.5% of samples. In HV2 region position 263G was mutated in 99.5%, posi-
982 REPISKA et al.
Table 1. Most frequent occurrence of haplotypes in the Caucasian population of the Slovak Republic
Times Haplotypes in the database Frequency, %
13 263G 315.1C 3.48
10 263G 309.1C 315.1C 2.67
9 263G 309.1C 309.2C 315.1C 2.41
5 16298C 72C 263G 309.1C 315.1C 1.34
4 16311C 263G 315.1C 1.07
4 16129A 16223T 16291T 16298C 73G 263G 309.1C 315.1C 1.07
4 16069T 16126C 16145A 16172C 16222T 16261T 73G 242T 263G 295T 315.1C 1.07
4 16126C 16294T 16296T 73G 263G 309.1C 315.1C 1.07
Table 2. Genetic diversity (GD), random match probability (RMP) and average number of nucleotide differences (ANND) in HV1, HV1ex, HV2, HV2ex, HV1HV2 and HV1exHV2ex regions of mtDNA from 374 individuals of the Caucasian population of Slovakia
Parametr HV1 16024-16365 HV1ex 16024-16465 HV2 73-340 HV2ex 72-340 HV1HV2 H
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.