The authors have declared that no competing interests exist.
Conceived and designed the experiments: ABK KTM ARA. Performed the experiments: ABK ANI. Analyzed the data: ABK MNV KTM. Contributed reagents/materials/analysis tools: ABK KTM GZhA CAM. Wrote the paper: ABK MNV MNB KTM. Contributed to sample collection and preparation: ABK GZhA ARA ANI EVZ.
Kazakhstan has been inhabited by different populations, such as the Kazakh, Kyrgyz, Uzbek and others. Here we investigate allelic and haplotypic polymorphisms of human leukocyte antigen (HLA) genes at DRB1, DQA1 and DQB1 loci in the Kazakh ethnic group, and their genetic relationship between world populations.
A total of 157 unrelated Kazakh ethnic individuals from Astana were genotyped using sequence based typing (SBT-Method) for HLA-DRB1, -DQA1 and -DQB1 loci. Allele frequencies, neighbor-joining method, and multidimensional scaling analysis have been obtained for comparison with other world populations. Statistical analyses were performed using Arlequin v3.11. Applying the software PAST v. 2.17 the resulting genetic distance matrix was used for a multidimensional scaling analysis (MDS). Respectively 37, 17 and 19 alleles were observed at HLA-DRB1, -DQA1 and -DQB1 loci. The most frequent alleles were HLA-DRB1*07:01 (13.1%), HLA-DQA1*03:01 (13.1%) and HLA-DQB1*03:01 (17.6%). In the observed group of Kazakhs DRB1*07:01-DQA1*02:01-DQB1*02:01 (8.0%) was the most common three loci haplotype. DRB1*10:01-DQB1*05:01 showed the strongest linkage disequilibrium. The Kazakh population shows genetic kinship with the Kazakhs from China, Uyghurs, Mongolians, Todzhinians, Tuvinians and as well as with other Siberians and Asians.
The HLA-DRB1, -DQA1and -DQB1 loci are highly polymorphic in the Kazakh population, and this population has the closest relationship with other Asian and Siberian populations.
Kazakh Khanate (Kazakhskoye khanstvo) was established as the first Kazakh state in 1456 (1465/66) and was located in the territory of the present day Republic of Kazakhstan (
The targets of our study were: HLA-Typing of HLA-DRB1, DQA1 and DQB1 loci in the Kazakh population living in the new capital city of Kazakhstan; investigation of allele and haplotype frequencies in relation to HLA-DRB1 polymorphism; and comparisons with other world populations with different historical backgrounds in order to further understand the genetic background and the origin of the Kazakh population. The HLA class I and II are recognized as essential components of the immune response with a high polymorphism. More than 10,000 alleles are in the latest version 3.15. (2013-07) of the IMGT/HLA Database, which provides a specialised database for sequences of the HLA-complex and official sequences for the WHO nomenclature Committee for factors of the HLA system
Results of the HLA-study in populations with different ethnic backgrounds are the basis for development in several areas of clinical transplantation, diagnostics, forensics and can be considered as an anthropological guide. This is a prerequisite for research of HLA-diversity in the population of Kazakhstan. The distribution of specific HLA genes in representatives of a healthy group can be used as reference markers to search for genetic predispositions of various diseases in the Kazakh ethnic group. This could serve as a theoretical basis for clinical transplantation and to find donors of allogeneic bone marrow from the same ethnic group. In our study, we focused on the study of HLA-DRB1 alleles in the Kazakh population living in Astana. There were also other classic distributions of alleles in the HLA class II. The aim of this work was to investigate HLA-genetic heterogeneity among Kazakhs by studying allele- and haplotype frequencies in relation to the HLA-DRB1 locus based on its high polymorphism. We hypothesized that, relying on the use of HLA-distribution, the origin of the Kazakh population can be determined.
This project was approved by the Ethics Committee of the National Center for Biotechnology, Kazakhstan (№ 10, 14.02.2010). The ethics committee approved the informed consent for this study. The investigation was conducted in accordance with humane and ethical research principles of National Center for Biotechnology. All 314 study participants completed a questionnaire requiring them to be healthy, provided informed consent, and included information regarding family history, lineage, etc. We confirm in our consent statement that consent was provided by 314 healthy individuals.
HLA typing and population studies were performed on 157 Kazakh individuals, 69 male and 88 female, living in Astana during 2010–2011. All individuals included in the present study were unrelated, without any sign of clinically diagnosed diseases, and randomly selected from different regions of Kazakhstan. The study participants consisted of representatives of Kazakh nationality only, and were classified as mono-ethnic according to their phenotype characteristics and family origin
For HLA-DRB1, -DQB1 and -DQA1 loci, allele polymorphisms were typed using the sequence-based typing (SBT) method. Genomic DNA from whole blood samples was extracted using a DNA Purification Kit (PROMEGA, Madison, WI) according to the manufacturer's protocol. The concentration of DNA was 50–100 ng/ml, with the purity of the extracted DNA ranging from a 1.5 to a 1.8 OD value. PCR and sequencing were performed for exon 2 of the HLA-DRB1, -DQA1 and -DQB1 genes using the SBT-method and locus, group, and sequence-specific primers according to multiple sources
Allelic frequencies of HLA-DRB1, -DQB1 and -DQA loci were estimated by the direct counting method. Allele frequencies, haplotype frequencies, neighbor-joining dendrograms and multidimensional scaling analysis were obtained for comparing Kazakhs and worldwide populations. Statistical analyses were performed using Arlequin v3.11. The resulting genetic distance matrix was used for a multidimensional scaling analysis (MDS), for two dimensions. MDS for pairwise populations was computed using allele frequencies, based on the Euclidean distance matrix
This first HLA-study was preferred to compare HLA-DRB1 frequencies in Kazakh population with Mediterraneans, Europeans, Scandinavians, Asians and Siberians. The HLA-DRB1 neighbor-joining dendogramm shows the Kazakh population together with Asian and Siberian populations, and separated from the European, Scandinavian and Mediterranean populations. The multidimensional scaling analysis (MDS) based on variances of the mean genetic distances were perfomed, and it was also observed here that Kazakhs clustered together with Asian and Siberian populations, however were separated from the Mediterranean, Scandinavian, European, and American populations (
Dendroram constructed by the neighbor-joining method showing the relationship between Kazakh populations with other populations based on the frequencies of HLA-DRB1 loc.
Number of alleles per locus, observed and expected heterozygosity | ||||
Locus | Number of Alelles | Hetobs | Hetexp | P-Values |
HLA-DRB1 | 37 | 0.93631 | 0.94593 | ns |
HLA-DQB1 | 19 | 0.91083 | 0.91758 | ns |
HLA-DQA1 | 17 | 0.87898 | 0.91144 | ns |
DRB1 | Allelic frequency, % | DQA1 | Allelic frequency, % | DQB1 | Allelic frequency,% |
*01:01 | 4.2 | *01:01 | 3.5 | *02:01 | 13.6 |
*01:02 | 0.3 | *01:02 | 10.2 | *02:02 | 6.3 |
*03:01 | 10.0 | *01:03 | 9.6 | *02:03 | 1.3 |
*03:02 | 0.3 | *01:04 | 9.0 | *02:04 | 2.6 |
*03:07 | 0.6 | *02:01 | 9.6 | *03:01 | 17.6 |
*03:12 | 0.3 | *03:01 | 13.1 | *03:02 | 7.5 |
*03:16 | 1.9 | *03:02 | 8.3 | *03:03 | 2.6 |
*03:29-57 | 0.6 | *03:03 | 5.1 | *03:04-22 | 11.7 |
*04:01 | 3.8 | *04:01 | 2.6 | *04:01 | 0.6 |
*04:02 | 0.6 | *04:02-04 | 0.3 | *04:02 | 2.6 |
*04:03 | 0.9 | *05:01 | 9.6 | *05:01 | 7.9 |
*04:04-32 | 2.8 | *05:02 | 0.6 | *05:02 | 4.8 |
*07:01 | 13.1 | *05:03 | 1.0 | *05:03 | 5.1 |
*08:01 | 0.9 | *05:05 | 9.0 | *05:04 | 1.0 |
*08:02 | 0.6 | *05:06-10 | 5.7 | *05:05 | 0.3 |
*08:03 | 1.6 | *06:01 | 2.6 | *06:01 | 2.6 |
*08:04-24 | 0.6 | *06:02 | 0.3 | *06:02 | 5.1 |
*09:01 | 4.5 | *06:03 | 5.1 | ||
*09:02 | 0.3 | *06:04-39 | 1.6 | ||
*10:01 | 1.2 | ||||
*11:01 | 2.2 | ||||
*11:02 | 0.6 | ||||
*11:03 | 2.8 | ||||
*11:04 | 3.8 | ||||
*11:06-62 | 2.5 | ||||
*12:01 | 1.9 | ||||
*12:02 | 0.6 | ||||
*13:01 | 8.6 | ||||
*13:02 | 2.9 | ||||
*13:03-103 | 5.7 | ||||
*14:01 | 4.9 | ||||
*14:03 | 1.9 | ||||
*14:04 | 1.9 | ||||
*14:05-72 | 4.2 | ||||
*15:01 | 5.1 | ||||
*15:02 | 1.2 | ||||
*16:01 | 0.6 |
DRB1 | Allelic frequency, % | DRB1 | Allelic frequency, % | DRB1 | Allelic frequency,% |
*01:01 | 5.4 | *07:01 | 1.9 | *12:02-08 | 4.1 |
*01:02 | 1.3 | *08:01 | 1.6 | *13:01 | 8.0 |
*03:01 | 7.6 | *08:02 | 1.9 | *13:02 | 3.8 |
*03:02 | 0.6 | *08:03 | 1.9 | *13:03-103 | 4.8 |
*03:07 | 1.6 | *08:04-24 | 3.5 | *14:01 | 1.9 |
*03:12 | 0.6 | *09:01 | 4.8 | *14:03 | 1.6 |
*03:16 | 0.3 | *09:02 | 1.3 | *14:04 | 0.3 |
*03:29-57 | 2.5 | *10:01 | 1.3 | *14:05-72 | 7.1 |
*04:01 | 3.0 | *11:01 | 3.5 | *15:01 | 7.6 |
*04:02 | 1.0 | *11:04 | 4.1 | *15:02-05 | 3.0 |
*04:03 | 0.6 | *11:06-62 | 2.5 | *16:01 | 0.3 |
*04:04-32 | 2.2 | *12:01 | 2.5 |
Three-loci haplotypes MHC Class II (DRB1-DQB1-DQA1
Haplotypes | HF, % | HF in the World | Possible Origin |
DRB1*07:01-DQA1*02:01-DQB1*02:01/02:02 |
8.0 | very common | European/Asian |
DRB1*03:01-DQA1*05:01-DQB1*02:01 |
3.4 | very common | European/Asian |
DRB1*13:01-DQA1*01:03-DQB1*06:03 |
2.9 | very common | European/Asian |
DRB1*14:01-DQA1*01:04-DQB1*05:02 |
2.9 | common | Asian |
DRB1*13:01-DQA1*03:01-DQB1*03:01 |
1.6 | not found | Kazakhs |
DRB1*15:01-DQA1*01:02-DQB1*06:02 |
1.3 | very common | European/Asian |
DRB1*07:01-DQA1*03:01-DQB1*02:02 |
1.3 | not found | Kazakhs |
DRB1*15:01-DQA1*01:02-DQB1*06:02 |
1.3 | very common | European |
DRB1*07:01-DQA1*03:02-DQB1*02:02 |
1.0 | not found | Kazakhs |
DRB1*15:01-DQA1*01:02-DQB1*06:03 |
1.0 | rare | European/Asian |
DRB1*15:02-DQA1*01:03-DQB1*06:01 |
1.0 | very common | European/Asian |
DRB1*03:01-DQA1*03:01-DQB1*03:02 |
1.0 | not found | Kazakhs |
DRB1*09:01-DQA1*03:03-DQB1*02:02 |
1.0 | not found | Kazakhs |
DRB1*14:03-DQA1*05:01-DQB1*03:01 |
1.0 | common | Asian |
DRB1*13:01-DQA1*01:03-DQB1*06:04 |
1.0 | very rare | African |
DRB1*13:02-DQA1*01:02-DQB1*06:04 |
1.0 | common | European/Asian |
DRB1*04:01-DQA1*03:02-DQB1*03:01 |
1.0 | not found | Kazakhs |
DRB1*13:01-DQA1*01:03-DQB1*05:01 |
1.0 | not found | Kazakhs |
Found in Buryats (22.0%); Khanty-Mansi (16.9%) Kazakhs (China) (8.3%).
Found in Italians (Sardinia) (25.3%); Russia (North-west) (9.0%); Kazakhs (China) (13.1%).
Found in Khanty-Mansi (8.1%); Italians (7.6%), Todzhinians (6.8%); Russia (North-west) (5.5%). Kazakhs (China) (4.8%);
Found in South Korea (2.9%).
Not found in any other population.
Found in Todzhinians (22.5%); English (14.1%), Australia Aborigine (10.0%); Russia (North-west) (9.0%); Kazakhs (China) (2.4%).
Found in Italians (14.1%); Slovenes (11.4%).
Found in Australia Aborigine (7.0%).
Found in Japan (8.2%); Mongolians (6.5%); Kazakhs (China) (2.4%).
Found in Khanty-Mansi (8.1%); Italians (7.6%); Kazakhs (China) (4.8%).
Found in Cameroon Yaounde (1.2%).
Found in Italians (1.9%); Tuva (1.1%).
DRB1-DQB1 | n |
HF |
D |
t |
DQB1-DQA1 | n |
HF |
D |
t |
DRB1*07:01-DQB1*02:01 | 23 | 0.0732 | 0.4650 | 5.48 | DQB1*02:01-DQA1*02:01 | 23 | 0.0732 | 0.7296 | 4.11 |
DRB1*03:01-DQB1*02:01 | 11 | 0.0350 | 0.2552 | 4.15 | DQB1*03:02-DQA1*03:01 | 15 | 0.0478 | 0.5687 | 3.13 |
DRB1*07:01-DQB1*02:02 | 10 | 0.0318 | 0.4249 | 2.61 | DQB1*03:01-DQA1*05:01 | 15 | 0.0478 | 0.3914 | 5.35 |
DRB1*13:03-103-DQB1*03:01 | 7 | 0.0223 | 0.2562 | 3.21 | DQB1*03:01-DQA1*05:05 | 12 | 0.0382 | 0.3045 | 4.99 |
DRB1*11:04-DQB1*03:01 | 6 | 0.0191 | 0.3915 | 2.14 | DQB1*02:01-DQA1*05:01 | 10 | 0.0318 | 0.2275 | 4.11 |
DRB1*14:05-72-DQB1*03:01 | 6 | 0.0191 | 0.3446 | 2.32 | DQB1*03:04-22-DQA1*03:02 | 8 | 0.0254 | 0.2123 | 3.15 |
DRB1*03:01-DQB1*03:02 | 4 | 0.0127 | 0.0754 | 2.37 | DQB1*05:01-DQA1*01:04 | 7 | 0.0223 | 0.2095 | 2.23 |
DRB1*13:01-DQB1*03:01 | 4 | 0.0127 | -0.1693 | 4.82 | DQB1*03:01-DQA1*05:06-10 | 7 | 0.0223 | 0.2562 | 3.21 |
DQB1*03:04-22-DQA1*05:05 | 6 | 0.0191 | 0.1061 | 3.39 | |||||
DQB1*03:04-22-DQA1*03:01 | 6 | 0.0191 | 0.0314 | 4.96 | |||||
DQB1*03:01-DQA1*03:01 | 6 | 0.0191 | -0.1794 | 7.31 | |||||
DQB1*03:04-22-DQA1*05:06-10 | 5 | 0.0159 | 0.1783 | 2.18 | |||||
DQB1*05:01-DQA1*01:02 | 4 | 0.0127 | 0.0647 | 2.55 | |||||
DQB1*03:01-DQA1*03:02 | 4 | 0.0127 | -0.1373 | 4.64 |
Number of times;
Haplotype frequency;
D Linkage disequilibrium;
Only t values ≥ 2.0 were considered significant.
The Neighbor-joining dendrogramm was created using the allelic frequencies at the HLA-DRB1 locus of various populations including the Kazakh group (
Because of the multiethnic background of the Kazakh population, multidimensional scaling analysis for the Kazakhs with different worldwide populations was performed. Multidimensional scaling analysis of the 74 ethnic groups were based on the allelic frequencies of the HLA-DRB1 locus shown in
Each point represents a population and its symbol its geographic region: ▪ - Asia 1-13 (1 Kazakhs (Astana), 2 Kazakhs (Tarbagatay), 3 Mongolian, 4 Han Chinese, 5 Japanese, 6 Kazakhs (China), 7 Koreans, 8 Taiwanese, 9 Uyghurs, 10 Malay, 11 Thai, 12 Vietnamese, 13 Turkish); Δ - Siberia 14–30 (14 Aleuts, 15 Chukchi, 16 Evenks, 17 Кеt, 18 Koryaks, 19 Buryats, 20 Nedigal, 21 Nentsy, 22 Nivkhs, 23 Nganasan, 24 Evenki (Okhotsk), 25 Todzhinians, 26 Tofalar, 27 Tuvinians-1, 28 Tuvinians-2, 29 Udegeys, 30 Ulchi); ♦ - America 31–36 (31 Argetine, 32 Mazatecans, 33 Ache, 34 Eskimos-1, 35 Eskimos-2, 36 Ypika_Alaska); ○ - Europe 37–74 (37 Austrians, 38 English, 39 German, 40, French_West, 41 Italians (North Italy), 42 Netherlands, 43 Portuguese, 44 Spanish (Granada), 45 Spanish (Madrid), 46 Albanians, 47 Bashkirs, 48 Belarussian, 49 Bulgarian, 50 Chuvashians, 51 Polish, 52 Russian (North-West), 53 Russian (Ural), 54 Serbs, 55 Slovaks, 56 Ukranian, 57 Armenians, 58 Cretans, 59 Georgians, 60 Greece, 61 Italians (South Italy), 62 Arabs, 63 Jews_Ashkenazi, 64 Kurds, 65 Lebanese, 66 Macedonians, 67 Palestinians, 68 Finnish, 69 Khanty-Mansi, 70 Komis, 71 Norway, 72 Pomors, 73 Saami, 74 Swedish). Stress value = 0.10.
This study aimed to determine the HLA class II (DRB1, DQA1 and DQB1) highly specific Kazakh alleles and specific HLA haplotypes, which have a low frequency in other world populations. The Kazakh's HLA alleles have been used for calculations of two-dimensional genetic distances, neighbor-joining dendrogramms, multidimensional scaling analysis (MDS), and the generation of extended HLA haplotypes (see
The study polymorphism of mitochondrial DNAdata (Berezina G, 2011) shows that Western Europe (55%) and Eastern Europe (41%) mtDNA linkages are present in the Kazakh population. It has been indicated that a high degree of intensity of gene exchange has occurred between the Kazakh population and populations of Russia on the North-West, North, North-East and East of Kazakhstan (Berezina G, 2011). It was also supported, that Kazakh Y-chromosome markers belong largely to the C3*, C3c and O3 haplogroups, which were obtained from people of southern Siberian or Mongolian lineage
In 1991, when the study of HLA allelic diversity was conducted few Caucasoid, Mongolian and mixed ethnic groups living in the territory of the former USSR were chosen. Based on these results, several authors concluded that the data on HLA-markers was broadly consistent with the anthropological information
The date of the HLA class II neighbor-joining tree shows the relatedness of world populations with the Kazakh population (
It should be noted that the relatively high degree of heterogeneity in Asian and Siberian populations compared to European populations may be associated with a wider habitat residence. Asian, especially the Siberian peoples, are relatively isolated from each other, whereas European populations are living in a more compact and limited area, providing more intense interactions. Previous studies and current results support a unique genetic origin of the Kazakhs, and this population could be genetically an admixture of three ethnic groups: Europeans, Siberians and Asians. Our results suggest that HLA loci and haplotypes in the Kazakh population are significant genetic polymorphisms, that will allow a future use of our results to find an HLA-matched donor, specifically for bone marrow transplantation, which in turn suggests the clinical relevance of ours and future research in the Kazakh population. Such studies are in high demand, as the data in this region is very limited. These data can be used for any research into HLA and disease, specifically relevant is data that has already been used in the study of tuberculosis in the Kazakh population
We would like to thank all donors enrolled in the current study. We thank Zhannur M. Nurkina, Pavel V. Tarlykov for technical assistance on this project. We also thank Mrs. Megan Rathbun for quickly and carefully editing our manuscript.