HLA Allele Frequencies in Sudanese Population: a Lab Report

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ABSTRACT

Background: Human Leukocyte Antigen (HLA) are cell surface glycoproteins encoded by the Major Histocompatibility Complex (MHC) genes of the human genome. They play a crucial role in solid organ transplantation, bone marrow transplantation, and autoimmune diseases. This study aimed to determine the distribution and frequencies of HLA-A, HLA-B, and HLA-DRB1 alleles among healthy unrelated renal and bone marrow donors. Additionally, it aimed to compare these findings with geographically African and Arab related populations. The data generated will provide valuable insights for anthropological and HLA-associated disease studies.

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Materials and Methods: A cross-sectional study was conducted on a cohort of 351 unrelated renal and bone marrow donors. They underwent HLA-A, HLA-B, and HLA-DRB1 typing using low-resolution Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) techniques at the Tissue Typing Laboratory of the Ibn Sina Center for Kidney Diseases and Renal Transplantation. The study was carried out between April and October 2017. Allele frequencies for HLA-A, HLA-B, and HLA-DRB1 were calculated as the percentage of the population possessing each specific antigen.

Samples containing only one allele were considered homozygous, with that allele being counted twice in the analysis.

Results: The study revealed significant allele diversity at each locus. The most prevalent HLA-A* alleles were A*02 (54.4%), A*30 (39.8%), A*68 (16.8%), A*03 (13.1%), A*01 (11.4%), and A*24 (11.4%). In the HLA-B* locus, the most frequent alleles were B*51 (23%), B*41 (12.8%), B*39 (12.0%), B*50 (12.0%), B*15:03 (11.0%), and B*35 (10.8%). All HLA-DRB1 alleles were observed, with HLA-DRB1*13 (49.0%) being the most frequent, followed by DRB1*301 (22.8%), DRB1*15 (21.0%), DRB1*07 (19.9%), DRB1*08 (19.9%), and DRB1*11 (19.4%).

Conclusions: This study corroborates previous observations in other Sudanese population studies [7], with slight differences but overall consistent allele frequency values.

These variations may be attributed to regional differences within the same country. The allele distribution and frequencies reported here demonstrate that the Sudanese population shares HLA patterns with Arab-related populations, particularly those from North Africa and neighboring African regions. This data has implications for solid organ transplantation, bone marrow transplantation, and the study of HLA antigen-associated diseases. Furthermore, it serves as a valuable resource for population genetics enthusiasts and contributes to anthropological research.

Key words: HLA-A, HLA-B, HLA-DRB1, Allele frequency, Population, Sudan, Transplant.

Introduction

The Human Major Histocompatibility Complex (MHC), commonly referred to as the human leukocyte antigen (HLA) complex, comprises a specific group of molecules expressed on the cell surface. These molecules play a critical role in the recognition of non-self-molecules by the acquired immune system¹. Human Leukocyte Antigens are categorized into three classes (I, II, and III), all of which are encoded by a gene complex situated on the short arm of chromosome 6 (6p21.3).

HLA class I molecules are found on the surface of nearly all nucleated cells, while MHC class II antigens are primarily expressed on antigen-presenting cells (APCs), including B lymphocytes, activated T lymphocytes, monocytes, macrophages, Langerhans cells, dendritic cells, endothelial cells, and epithelial cells. The class III region of the MHC complex contains genes encoding components of the complement system (C2, C4, factor B), tumor necrosis factors (TNF-α and TNF-β), and heat shock proteins². HLA genes are closely linked, and the entire MHC complex is inherited as an HLA haplotype in a Mendelian fashion from each parent, with one haplotype inherited from the father and one from the mother.

HLA molecules are the primary antigens that vary among individuals of the same species and are closely associated with rejection in solid organ transplantation and graft-versus-host disease in hematopoietic stem cell transplantation. The study of HLA allele frequencies holds significant importance in both related and unrelated bone marrow transplantation programs. It serves as the basis for HLA compatibility assessment to prevent graft rejection and to identify suitable donors³. Investigations into HLA genes and proteins have proven to be valuable tools in transplantation, anthropological research, and studies related to disease associations⁴.

Identifying the frequencies of HLA alleles within nations is of paramount importance. Such data eases the burden of searching for compatible donors, reduces transplant waiting times, and consequently enhances the chances of survival for transplant recipients⁵.

Sudan, until 2011, was the largest country in Africa. With the separation of South Sudan into an independent nation known as the Republic of South Sudan, Sudan's diverse population remains a complex mosaic, comprising approximately 19 distinct ethnic groups and nearly 600 subgroups⁵. This study aims to investigate the distribution and frequencies of HLA-A, HLA-B, and HLA-DRB1 alleles within the Sudanese population. Additionally, it seeks to compare these findings with those of geographically related African and Arab populations. The data generated in this study will contribute to further investigations in anthropological research and studies related to HLA-associated diseases. It is noteworthy that there have been relatively few studies of this nature conducted in Sudan, and this research endeavor aims to address this significant gap in knowledge.

Materials and Methods

The study was conducted at Al-Neelain University, specifically within the Faculty of Medical Laboratory Sciences, in collaboration with the Ibn Sina Center for Kidney Diseases and Transplantation, specifically the Tissue Typing Laboratory. The research spanned from April to October 2017. Buffy coats, which had been separated from blood samples collected in Acid Citrate Dextrose (ACD), were employed for the study. A total of 351 randomly selected unrelated donors for renal and bone marrow transplants were included in the study. These samples were completely anonymized, containing no donor-specific information or names. The age range of donors varied, with a minimum age of 3 years for bone marrow donors and an age requirement of over 18 years for both bone marrow and kidney donors. The study included individuals of both genders and represented various states within Sudan. It followed a descriptive cross-sectional design.

DNA Extraction and HLA Typing: Buffy coat samples were collected and stored at -20°C in accordance with the procedures and guidelines of the Tissue Typing Laboratory. DNA extraction was carried out from the buffy coat samples using the Qiagen Blood Mini Kit, a product of QIAgene Blood Mini Kit, Germany. The extraction process followed the manufacturer's instructions and was subsequently quantified through standard UV spectrophotometric analysis.

The extracted DNA was then subjected to amplification for HLA-A, HLA-B, and HLA-DRB1 using the HISTOTYPE SSP kit as outlined in the manufacturer's manual (BAG Health Care GmbH, 35423 Lich / Germany). Following the PCR-SSP process, the amplified DNA fragments underwent separation via agarose gel electrophoresis. The gel was visualized and documented using a UV transilluminator integrated into the photo gel system with an ethidium bromide filter, connected to a computer. An amplification reaction was considered successful when bright bands were visible.

The results were subsequently analyzed with the aid of HLA software provided by the manufacturer, specifically BAG Health Care GmbH (2017).

Statistical Analysis: Allele frequencies for HLA-A, HLA-B, and HLA-DRB1 were determined as a percentage of the population possessing a specific antigen. This calculation was achieved by dividing the total number of occurrences of a particular allele by the total number of individuals (n/N) × 100, where 'n' represents the number of occurrences of a particular allele, and 'N' denotes the total number of individuals in the study cohort. In cases where samples contained only one allele, they were considered homozygous, and that allele was counted twice in the analysis.

Results

In this study, the frequencies and distribution of HLA-A, HLA-B, and HLA-DRB1 alleles were examined among 351 unrelated Sudanese renal and bone marrow donors, comprising 142 (40.5%) females and 209 (59.25%) males. A total of 18 HLA-A alleles, 33 HLA-B alleles, and 14 HLA-DRB1 alleles were identified.

Distribution of HLA-A* Alleles (Table 1)

A total of 18 HLA-A alleles were observed in this study, with an estimated percentage of heterozygosity at 79.8%. The most prevalent HLA-A alleles were A*02 (54.4%) and A*30 (39.8%), followed by A*68 (16.8%), A*03 (13.1%), A*24 (11.4%), and A*01 (11.4%). HLA-A*34, HLA-A*36, HLA-A*66, and HLA-A*69 were present at lower frequencies. In the case of HLA-B antigens, a total of 33 alleles were detected, with an estimated percentage of heterozygosity at 88.3%. The highest frequency was observed for B*51 (23%), followed by B*41 (12.8%), B*50 (12.0%), B*39 (12.0%), B*15:03 (11.0%), and B*35 (10.8%). The least frequent alleles were B*55 (0.28%) and B*73 (0.28%). Several homozygous alleles, including B51 (3.1%), B41 (1.7%), and B07 (1.4%), were also detected.

Distribution of HLA-DRB1* Alleles (Table 3)

For HLA-DRB1, all alleles were observed, with a heterozygous percentage of 94.2%. The most frequent allele was HLA-DRB1*13 (49.0%), followed by DRB1*301 (22.8%), DRB1*15 (21.0%), DRB1*07 (19.9%), DRB1*08 (19.9%), and DRB1*11 (19.4%). The alleles with minimal frequencies were DRB1*302 (1.4%) and DRB1*09 (0.8%). The highest frequencies of homozygosity for the DRB1 locus were 31%, 19%, 16.7%, and 11.9% for DRB1*13, DRB1*07, DRB1*15, and DRB1*301, respectively.

Discussion

Understanding the distribution of HLA antigens is crucial for assessing the likelihood of finding matched donors for individuals in need of transplants. Previous studies have shown that the Sudanese population is highly diverse, consisting of approximately 19 different ethnic groups and nearly 600 subgroups⁵. Sudanese individuals share genetic similarities with both sub-Saharan Africans (such as Nigerians, Congolese, and Senegalese) and North Africans, particularly Egyptians. This suggests that the genetic profile of the Sudanese population reflects a historical admixture between North Africans, especially Egyptians, and sub-Saharan Africans⁶.

In this study, our objective was to determine the predominant frequencies of HLA-A, HLA-B, and HLA-DRB1 alleles among unrelated Sudanese renal and bone marrow donors. We also compared our HLA antigen data with previous studies conducted on various populations.

In our findings, HLA-A*02 emerged as the most prevalent allele, with a frequency of 54.4%, followed by HLA-A*30 (39.8%), HLA-A*68 (16.8%), HLA-A*03 (13.1%), and HLA-A*24 (11.4%). Several HLA-A alleles, such as HLA-A*34, HLA-A*36, HLA-A*66, and HLA-A*69, were present at lower frequencies. In the case of HLA-B antigens, the highest frequency (23%) was observed for B*51, followed by B*41 (12.8%), B*50 (12.0%), B*39 (12.0%), B*15:03 (11.0%), and B*35 (10.8%). Rare alleles like B*55 (0.28%) and B*73 (0.28%) were observed, while several homozygous alleles were also identified, including B51 (3.1%), B41 (1.7%), and B07 (1.4%).

For HLA-DRB1, all alleles were present, with a heterozygous percentage of 94.2%. The most frequent allele was HLA-DRB1*13 (49.0%), followed by DRB1*301 (22.8%), DRB1*15 (21.0%), DRB1*07 (19.9%), DRB1*08 (19.9%), and DRB1*11 (19.4%). Less common alleles included DRB1*302 (1.4%) and DRB1*09 (0.8%). Homozygosity was observed at varying frequencies, with the highest rates for DRB1*13, DRB1*07, DRB1*15, and DRB1*301.

Conclusion

The Sudanese population exhibits a notable diversity in the frequencies and distribution of HLA alleles, reflecting the complex history of population movements and dynamics. HLA antigen frequencies within Sudanese ethnic groups do not appear to be unique to any particular group but instead reflect the admixture of the Sudanese community over time. Our study demonstrates that the Sudanese population shares HLA patterns with Arab-related countries, North African populations, and some neighboring African populations.

Furthermore, the documentation of HLA phenotype frequencies and distribution among the Sudanese population serves multiple important purposes. It facilitates solid organ transplantation, bone marrow transplantation, and the study of HLA antigen-associated diseases. Additionally, it provides a valuable resource for individuals interested in population genetics and contributes to anthropological research.