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2POLYMORPHISM OF HLA-DQA1, HLA-DQB1, AND HLA-DRB1 GENES AND THEIR CLINICAL SIGNIFICANCE
1'2Kurganov S.K., 1Akhmedova D.Sh., 1Normatov A.E., 1Tosheva D.M., 1Akhmedov B.B.,
1Saitova N.S.
Republican Centre for Forensic Expertise, Tashkent, Uzbekistan 2Institute of Biophysics and Biochemistry at the National University of Uzbekistan named after
Mirzo Ulughbek, Tashkent. https://doi.org/10.5281/zenodo.14015640
Abstract. This article investigates the polymorphism of HLA-DQA1, HLA-DQB1, and HLA-DRB1 genes and their clinical significance. The study involved 220 blood samples from healthy individuals, with molecular genetic analysis used to identify the alleles of HLA class II genes. The results demonstrated an asymmetric distribution of these alleles. HLA-DQA15*01, HLA-DQB1*02, and HLA-DRB1*07 were identified as the most prevalent alleles. The findings highlight the significance of these results in evaluating immunological compatibility in autoimmune diseases, allergic conditions, and organ transplantation.
Keywords: HLA-DQA1, HLA-DQB1, HLA-DRB1, genetic polymorphism, immunological compatibility, autoimmune diseases, allergic reactions, organ transplantation.
Introduction
The HLA (Human Leukocyte Antigen) system plays a crucial role in immune response processes [5]. These antigens are recognized as molecules of the Major Histocompatibility Complex (MHC), enabling the immune system to identify pathogens that pose a threat to the organism and to initiate appropriate defense mechanisms [3]. Testing for HLA genes is essential for assessing the human immune system, determining genetic susceptibility to autoimmune diseases, enhancing the effectiveness of organ transplantation, personalizing drug therapies, evaluating predisposition to allergic reactions, and elucidating the pathogenesis of diseases in clinical research [1, 15].
HLA genes are primarily divided into HLA class I (HLA-A, HLA-B, HLA-C) and HLA class II (HLA-DP, HLA-DQ, HLA-DR) genes. HLA class I genes are recognized by CD8+ T-lymphocytes and stimulate immune responses against viruses, bacteria, and malignant cells [3]. HLA class II genes, on the other hand, are recognized by CD4+ T-lymphocytes and are associated with autoimmune diseases and allergic reactions. HLA genes, particularly the HLA class II group (HLA-DP, HLA-DQ, HLA-DR), regulate the activity of the immune response, and their polymorphisms across different populations and individuals can influence susceptibility to various diseases by either increasing or decreasing the risk [8, 11, 12].
Studying HLA class II genes, investigating their polymorphisms, and understanding genetic susceptibility to diseases is crucial.
Based on the above, the objective is to identify population-specific genetic polymorphisms of HLA class II (HLA-DQ, HLA-DR) genes in a group of conditionally healthy individuals and to study their distribution. The results of this research may provide a foundation for the future identification and prevention of genetic diseases, as well as improving the effectiveness of organ transplantation.
Research Objective: Blood samples were collected from 220 conditionally healthy individuals for the study. The samples were taken from veins using vacuum tubes containing 3% EDTA (ethylenediaminetetraacetic acid), which are designed for blood hemostasis studies, and were used for DNA extraction.
Materials and Methods
DNA extraction was carried out using the PROBA-GS-GENETIKA kits (DNA-Technology, Russia) and the QIAamp DNA Blood Kits 250 (QIAGEN Inc., Valencia, CA, USA). The concentration of the extracted DNA was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, USA) by comparing it to the fluorescence standard curve of X phage DNA. The DNA samples were diluted to a working concentration of 50-60 ng/^L and stored frozen at -20°C. In the analysis, all DNA preparations contained more than 25 ng/^L of genomic DNA, ensuring that the DNA samples were adequately diluted for subsequent studies.
The HLA-DNA-TECH kit (DNA-Technology, Russia) was selected to identify the alleles of the DQA1, DQB1, and DRB1 genes from the DNA samples. The amplification of the DNA samples was performed using a Dtlite4 Real-Time PCR system with 48-well automated amplifiers according to the following program: initial denaturation at 80°C for 120 seconds and 94°C for 300 seconds, followed by five cycles of denaturation at 94°C for 30 seconds and annealing/extension at 64°C for 15 seconds. This was followed by 45 cycles of denaturation at 94°C for 10 seconds and annealing/extension at 64°C for 15 seconds, with a melt curve step at 25°C for 30 seconds. The melt curve analysis was repeated 50 times at 25°C for 15 seconds. (Fluorescence measurement was taken at each step.)
In addition, to verify the results, SeCore™ DQA1 Locus Seq Kit, SeCore™ DQB1 Locus Seq Kit, and SeCore™ DRB1 Locus Exon 2, 3 Seq Kit amplification sets, produced by Thermo Fisher Scientific, were used to identify alleles of HLA class II (HLA-DQ, HLA-DR) genes. Sanger sequencing analyses were performed using an automated system, the 3500 Genetic Analyzer (Applied Biosystems, USA). The identification of HLA gene alleles was carried out using the HLA SBT uTYPE CE-IVD 7.3 and uTYPE™ HLA Sequence Analysis Software.
A table (Table 1) was created based on the results of molecular-genetic analysis to present the absolute number of DQA1, DQB1, and DRB1 gene alleles and their occurrence frequency in the group of 220 conditionally healthy individuals examined in the study.
Table 1. Absolute number of DQA1, DQB1, and DRB1 gene alleles and their occurrence
frequency
DQA1 gene alleles (n) (n%) DQB1 gene alleles (n) (n%) DRB1 gene alleles (n) (n%)
101* 33 0,15 2* 55 0,25 1* 17 0,07
102* 30 0,14 301* 41 0,19 3* 25 0,13
103* 22 0,1 302* 29 0,14 4* 28 0,13
201* 32 0,14 303* 4 0,02 7* 32 0,15
301* 37 0,17 304* 1 0,005 8* 5 0,02
401* 4 0,019 305* 2 0,015 9* 9 0,04
501* 60 0,28 0401/0402* 5 0,02 10* 8 0,03
601* 2 0,001 501* 24 0,11 11* 27 0,13
0502/0504* 6 0,02 12* 7 0,03
503* 10 0,04 13* 23 0,1
601* 15 0,06 14* 9 0,04
0602-8* 27 0,13 15* 26 0,12
16* 3 0,01
Research Results and Analysis
It was found that the alleles of HLA-DQA1, HLA-DQB1, and HLA-DRB1 genes exhibit asymmetric distribution in the examined group. The significance of this study lies in its important role in understanding genetic diversity, the potential pathways to genetic diseases, and the complex mechanisms that affect the immune system of the population [13].
Among the alleles of the HLA-DQA1 gene, the DQA1501 allele was observed in 60 cases (28%) and was recorded as the most prevalent allele, confirming its genetic significance in the population. The least common allele was DQA1601, which was identified in only 2 cases (1%). The distribution of the remaining alleles is as follows: DQA1101 (33 cases, 15%), DQA1102 (30 cases, 14%), DQA1103 (22 cases, 10%), DQA1201 (32 cases, 14%), DQA1301 (37 cases, 17%), and DQA1401 (4 cases, 2%).
Among the alleles of the HLA-DQB1 gene, the most prevalent allele is DQB102, identified in 55 cases (25%). Following this, the DQB1301 allele was observed in 41 cases (19%). The distribution of the less common alleles is as follows: DQB1302 (29 cases, 14%), DQB10602-8 (27 cases, 13%), DQB1601 (15 cases, 6%), DQB10401/0402 (5 cases, 2%), and DQB1304 (5 cases, 1%). According to these results, the DQB102 allele is the most widely distributed in the population, indicating its significant role among the population, while the least common allele is DQB1*304.
Additionally, among the alleles of the HLA-DRB1 gene, the most frequent allele is DRB107, which was identified in 32 cases (15%). This is followed by the DRB104 (28 cases, 13%) and DRB103 (25 cases, 13%) alleles. The less common alleles include DRB111 (27 cases, 13%), DRB115 (26 cases, 12%), and the rarest allele, DRB116 (3 cases, 1%). These results reflect the genetic diversity of HLA-DRB1 gene alleles within the population.
Among the list of alleles identified through various studies and their associated developed diseases, the HLA-DQA1, HLA-DQB1, and HLA-DRB1 genes are key complex genes that regulate the immune response in the human body, playing a significant role in the development of various autoimmune diseases and induced immune reactions.
The presence of HLA-DQB1 alleles 02 and 08 in individuals may significantly increase the risk of developing celiac disease. However, the presence of these gene alleles is not the sole cause of the disease's development, and their presence does not guarantee the specific onset of the disease. HLA-DQ2 and HLA-DQ8 genes play a key role in the pathogenesis of autoimmune diseases characterized by damage to the intestinal mucosa due to excessive sensitivity to gluten. The allele combination HLA-DQ2 (DQA105/DQB102) is found in approximately 90-95% of patients with celiac disease. This gene complex consists of the alleles DQA105 andDQB102, and individuals who are homozygous for HLA-DQ2 (with two copies of DQA105 andDQB102) have an even higher risk of developing the disease [10].
Type 1 diabetes is an autoimmune disease in which the immune system attacks and destroys the insulin-producing beta cells of the body. This condition leads to insulin deficiency and an increase in blood sugar levels. Certain alleles of the HLA-DQA1 and HLA-DQB1 genes, primarily HLA-DQ2 (DQA105/DQB102) and HLA-DQ8 (DQA103/DQB103:02), play a crucial role in the
development of Type 1 diabetes. The presence of HLA-DQ2 and HLA-DQ8 alleles significantly increases the risk of developing Type 1 diabetes, as these genes trigger autoimmune reactions that damage beta cells. However, the presence of these alleles does not guarantee the development of the disease, as environmental factors (such as infections, stress, and diet) also play an important role in the disease's onset [9].
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by damage to nerve fibers due to the immune system attacking the myelin sheath. Genetic factors play a significant role in the development of MS, with the HLA-DQB106 allele substantially increasing the risk of the disease. This allele can trigger an autoimmune response, leading to damage to nerve cells. Additionally, environmental factors (such as viral infections and vitamin D deficiency) also influence the development of the disease. Although individuals with the HLA-DQB106 allele have a higher risk of MS, the absence of this allele does not guarantee complete protection from the disease [4].
Rheumatoid arthritis (RA) is an autoimmune disease primarily characterized by inflammation in the joints and damage to bone tissues. The HLA-DRB104 allele is considered a significant genetic factor in the development of this disease. This allele triggers an autoimmune response of the immune system against the bones and joints, leading to inflammation and damage. While genetic predisposition increases the risk of RA, environmental factors (such as smoking and infections) also play a role in the disease's progression. The presence of the HLA-DRB104 allele raises the likelihood of disease development, but its absence does not guarantee complete protection from RA [7].
Multiple sclerosis is an inflammatory autoimmune disease of the central nervous system, closely associated with a high risk linked to the HLA-DRB115 allele. This allele triggers the immune system to attack the myelin sheath, resulting in damage to nerve fibers. The presence of the HLA-DRB115:01 allele significantly increases the risk of MS development [4].
Systemic lupus erythematosus (SLE) is an autoimmune disease associated with increased risk linked to the HLA-DRB103 and HLA-DRB115 alleles. These alleles may disrupt the communication between the immune system and body functions, leading to inflammation and attacks on various body tissues [6].
Graves' disease is an autoimmune disease of the thyroid gland, with the presence of the HLA-DRB 1*03:01 allele marking an increased risk factor. The presence of this allele is associated with the development of new complications related to the disease [14].
Autoimmune hepatitis is associated with the HLA-DRB103 and HLA-DRB104 alleles according to new international assessments. These alleles play a significant role in increasing the risk of disease development [5].
Ankylosing spondylitis is characterized by inflammation of bones and joints and is more associated with the HLA-B27 allele; however, HLA-DRB1 also serves as a risk factor. The risk associated with HLA-DRB1 alleles is notably higher in individuals with a family history of the disease [2].
In developed countries, checking HLA (Human Leukocyte Antigen) genes is crucial for ensuring immunological compatibility in organ transplantation. This testing helps reduce the risk of rejection by the recipient's immune system. Thus, compatibility based on HLA genes increases the success rate of transplants and guarantees the long-term functionality of the transplanted organ.
However, there are serious problems and negative consequences associated with this practice. One such issue is the development of organ trafficking and criminal activities that treat humans as living commodities.
While HLA genes are used to ensure immunological compatibility in transplantation, they also play a significant role as identification tools in criminal activities. HLA genes define the biological characteristics of tissues in the human body and are unique to each individual.
When the legality of a transplanted organ is questioned, comparing the HLA profiles of the donor and recipient can help determine whether the organ was obtained from a legal donor or through illicit means. If the HLA profiles do not match those of a legal donor, this indicates that the organs were obtained illegally, aiding in the resolution of crimes.
International databases store information about the HLA genes of donors and recipients. Utilizing this database is crucial in identifying crimes and preventing organ trafficking. When the HLA genes of donors and recipients are compared with global data, it becomes possible to ascertain whether the organs were acquired legally or illegally.
In criminal activities, vulnerable individuals—particularly those in economically difficult situations or migrant workers—often become victims. When they are coerced into illegally donating their organs, their HLA profiles can be used to identify and expose these crimes.
In Uzbekistan, organ transplantation is an emerging field in medicine, regulated by legislation. The law "On the Transplantation of Human Organs and Tissues," adopted in 2022, creates conditions for the legal transplantation of organs. However, if these laws are not sufficiently robust or if monitoring mechanisms do not function well, the risk of crime increases [16].
In countries where there is a high demand for organ transplantation, shortages of organs can lead to criminal activities. In Uzbekistan, there is also a significant need for healthcare and medical services, which may lead to shortages of organs for transplantation. This can result in the illegal sale of organs and exploitation of individuals.
While the practice of organ transplantation is developing in Uzbekistan, there are risks associated with the illegal trade of organs and the exploitation of people. Economic difficulties, issues within the healthcare system, and a lack of monitoring mechanisms facilitate the growth of these crimes. Strengthening legislation, enhancing international cooperation, and increasing oversight are crucial steps in addressing these issues.
In forensic genetic testing, checking HLA (Human Leukocyte Antigen) genes plays an important role in human identification and the resolution of criminal activities. This method can be effectively applied not only in medical transplantation processes but also in the fight against crimes such as organ trafficking.
Conclusions:
This study investigated the polymorphism of the HLA-DQA1, HLA-DQB1, and HLA-DRB1 genes in a population of conditionally healthy individuals. The distribution of identified alleles was asymmetric, with varying frequencies observed.
The most prevalent alleles were HLA-DQA1501 (28%), HLA-DQB102 (25%), and HLA-DRB1*07 (15%), indicating the significance of these alleles for the studied group.
Investigating the polymorphism of HLA-DQ and HLA-DR genes is crucial for assessing immunological compatibility in autoimmune diseases, allergic reactions, and organ transplantation.
The results of this study provide a foundation for future research aimed at identifying
genetic diseases, implementing preventive measures, and enhancing the success of organ
transplantation.
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21. https://lex.uz/ru/docs/6001286
