The Association of Human Leukocyte Antigen Genotyping Among Sudanese Patients With Rheumatoid Arthritis: Reference to Ethnicity

Introduction: Human leukocyte antigens (HLA) account for up to one-half of the total genetic contribution to rheumatoid arthritis (RA) risk. The study investigated the association of HLA class II genotyping with RA susceptibility in Sudanese ethnic groups. Methods: The DRB1 and DQB1 alleles and haplotypes were determined in 122 RA patients (i.e., Gaalia = 54, Johayna = 24, Baggara = 17, Nile Nubian = 12, and others = 15) and 120 healthy controls of ethnic groups (i.e., Gaalia = 44, Johayna = 11, Baggara = 15, Nile Nubian = 9, and others = 21) using a polymerase chain reaction with sequence-specific primers method. Results: Susceptibility to RA was associated with a high frequency of DRB1*04 (P = 0.04), DRB1*10 (P = 0.04), and DQB1*03 (P = 2.2 x 10-8/Pc = 6.6 x 10-8) between study ethnic groups, while protective effects were shown with DRB1*07 (P = 0.01), DQB1*02 (P = 0.02), and DQB1*06 (P = 2.2 x 10-6/Pc = 6.6 x 10-6), with an inconsistent frequency between study ethnic groups. The HLA haplotypes that were high in frequency among RA ethnic groups and showed susceptibility associations were DRB1*03-DQB1*03, DRB1*04-DQB1*03, DRB1*08-DQB1*03, DRB1*13-DQB1*02, and DRB1*13-DQB1*03 (P = 0.00003/Pc = 0.0003, P = 0.0001/Pc = 0.0001, P = 0.03, P = 0.004/Pc = 0.03, and P = 3.79x10-8/Pc = 3.3x10-9, respectively). On the contrary, DRB1*03-DQB1*02, DRB1*07-DQB1*02, and DRB1*13-DQB1*06 were lower in frequency in the ethnic groups with RA and may confer protection (P = 0.004/Pc = 0.032, P = 0.002/Pc = 0.02, and P = 0.01, respectively). Conclusions: Our findings indicate an association between HLA-DRB1 and DQB1 genotypes and the susceptibility to RA in the Sudanese population, with a moderate frequency between our ethnic groups.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease with features of chronic inflammation that affects synovial joints, leading to the destruction of joint cartilage [1]. RA pathogenesis is multifactorial, and disease susceptibility is associated with genetic and environmental factors [2].
The anti-citrullinated peptide antibodies (ACPA), which are more frequent than the rheumatoid factor (RF), present in 70-80% of RA patients, were considered predictive markers of bone destruction since they could be detected years before the disease and be stable after, and they were associated with disease erosion and aggressiveness [3,4]. Likewise, ACPA specificity is higher in RA than RF and was found to be strongly associated with human leukocyte antigen (HLA)-DRB1 shared epitope (SE) alleles such as DRB1*03 and *04, particularly in ACPA seropositive RA patients [2,5].
The HLA genes are situated on chromosome 6 (6p21. 3), and their genomic map spans about 7.6 Mb and contains approximately 421 gene loci in a contiguous region on chromosome 6 [6]. Among the HLA genes, HLA-DRB1 SE alleles that encode for a common amino acid sequence are the strongest genetic risk factors for RA [7,8].
The expression of HLA class II, particularly the HLA-DRB1/DQB1 genotypes in RA, varies between racial groups and is geographically distributed from one population to another [9]. It has been reported that patients who carry HLA-DRB1 alleles are at high risk for developing a more progressive type of disease in many populations [10][11][12], including Sub-Saharan African populations [13,14]. Therefore, the genetic risk of reclassified HLA-DRB1/DQB1 alleles may vary among different ancestries, including our population, and raises the possibility that the specific HLA-DRB1/DQB1 alleles responsible for disease risk may differ in African and European ancestral populations [8].
Studies linking HLA-DRB1/DQB1 haplotypes and their association with RA susceptibility in the variable ethnic groups in our population are inadequately studied. Thus, investigating the allele frequency patterns of HLA genes that contribute to the development of RA in our population can provide insight into the disease's nature, vulnerability, and prognosis. This study relates HLA class II genotypes to RA vulnerability among our different ethnic populations.

Patients and controls
This is a hospital-based case-control study that retrospectively encompassed 122 RA patients (16 males with a mean age of 44.81 ± 14.65 years and 106 females with a mean age of 44.97 ± 14.01 years) and 120 healthy controls (11 males with a mean age of 38.55 ± 14.37 years and 89 females with a mean age of 43.62.55 ± 9.89 years).
The study was conducted in two different rheumatology outpatient clinics at the Academy Teaching Hospital and Ibrahim Malik Hospital in Khartoum, Sudan.
Sudan is a large country located in the northeast (i.e., the heart) of the African continent and shares borders with seven countries. Sudan's population structure is rooted in many diverse cultural, social, ethnic, verbal, and religious characteristics.
The patients were selected directly or indirectly from referrals to the clinics based on the clinical and laboratory examinations conducted under the supervision of two rheumatologists from each clinic. The RA diagnosis was assessed based on the criteria established in 2010 by the American College of Rheumatology [15].
The laboratory examinations were conducted accordingly and recorded together with the demographic and clinical findings on a well-structured questionnaire.
The healthy controls were selected based on the fact that they are apparently healthy, have no family history of RA, have a current rheumatic picture, or have any other autoimmune disease, and are recruited from the same hospitals and geographical areas. We considered the self-reports for the determination of age, sex, and ethnicity. Study subjects were categorized into 11 ethnic groups extracted from the Sudanese official documents of tribes and family histories as follows: Gaalia (44.3% vs. 44.0%), Johayna (19.7% vs. 11.0%), Baggara (13.9% vs. 15.0%), Nile Nubian (9.8% vs. 9.0%), Nubian (1.6% vs. 8.0%), Darfurian (0.8% vs. 5.0%), and Moroccan and Egyptian (3.3%). The Nubian, Darfurian, Moroccan, Egyptian, Nigerian, Beja, Fonj, and Chadian tribes were collectively named "others" because of their low frequency among study subjects.
Verbal and written informed consent was obtained from all the study participants. The study was approved by the Institutional Ethics Committee of the University of Gezira (approval number: UGIREC/2020).

HLA genotyping
DNA was extracted from the peripheral blood according to the manufacturer's instructions provided by the QIA amp® DNA mini kit (Qiagen, Germantown, MD). Low-resolution kits (HLA-SSP Typing Kits, R.O.S.E. Europe GmbH, Frankfurt, Germany) for HLA class II typing were used with sequence-specific primers for a polymerase chain reaction (PCR-SSP). DNA amplification was done using an Applied Biosystem 9700 thermocycler (Thermo Fisher Scientific, Waltham, MA), and the protocol was applied consistently with the instructions provided by the company. The amplified DNA fragments were separated using 2% agarose gel electrophoresis and visualized under ultraviolet light. The typing of HLA genotypes was achieved via HLA software (R.O.S.E. Europe GmbH, Frankfurt, Germany).

Autoantibody analysis
Peripheral blood samples were collected from each study participant in vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) and processed to obtain serum under good laboratory practice standards. Serum ACPA was detected using anti-cyclic citrullinated peptides (anti-CCP) IgG antibodies and was quantitatively and semiquantitatively detected as per the instructions provided in the Immunoscan CCPlus® ELISA kits (Euro Diagnostica AB, Malmö, Sweden). Positive results for ACPA were defined as concentrations >25 U/ml. A rapid latex agglutination test (NS Biotec, Alexandria, Egypt) and an automated chemistry analyzer (Cobas Mira Plus, Roche, Basel, Switzerland) were used to measure serum C-reactive proteins (CRP) and IgM RF, respectively. Erythrocyte sedimentation rate (ESR) measurement was performed

Patients and healthy controls
The study included 122 RA patients, and their demographic and clinical findings are presented in Table 1.
The mean levels of ACPA, CRP, and ESR were remarkably higher in RA patients compared with healthy controls. Gaalia was the most frequent tribe, accounting for 54 (44.3%) patients, followed by Johayna (24; 19.7%), Baggara (17; 13.9%), and Nile Nubian (12; 9.8%). Other tribes were collected together only because their numbers were too small for meaningful comparisons.
HLA-DQB1*03 allele was high between Gaalia and Johayna tribes in patients, while HLA-DQB1*02 and *06 were higher in frequency in ethical groups of healthy controls ( Table 4).
Out of the 13 haplotypes observed in the ethnic groups, nine were found in patients and healthy controls ( Table 6). The shared haplotypes *03/03, 04/03*, *08/03, and *13/03 were more frequent in patients' ethnic groups, while *03/02, *07/02, and *13/06 were more frequent among the ethnic groups in healthy controls, which were found consistent with their association with RA susceptibility and protection, respectively ( Table 6). The RA-susceptible *03/03 and *13/03 haplotypes were absent in the healthy ethnic groups, while the protective *13/06 haplotype was absent in the patient ethnic groups.

Discussion
The HLA genes consist of three main subregions (DP, DQ, and DR), which code HLA class II molecules important in antigen presentation, particularly to immune cells that are involved in adaptive immune activation via T cell activation and differentiation. The polymorphisms in the HLA class II molecules account for approximately 30-40% of the genetic risk for RA [18]. Among the HLA-linked genes, many studies have reported that polymorphisms at the HLA-DRB1 locus confer more risk for RA and other autoimmune diseases [19]. Thus, particular HLA-DRB1 alleles like HLA-DRB1*01, HLA-DRB1*04, and HLA-DRB1*10 were found to be associated with the development of RA in ethnic groups from Europe and Asia [20][21][22][23]. On the other hand, certain HLA-QB1 alleles, such as HLA-QB1*05, *04, and *602, were also found to be associated with RA risk in populations from Asia [22,24] and Africa [25].
Another study from the USA showed the HLA-DRB1*04 frequency was higher among non-Hispanic white and Hispanic ethnic groups compared with African Americans [33]. African Americans were characterized by a low prevalence of RA due to the low frequency of the high-risk alleles HLA-DRB1*04:01 and *04:04 [8].
Collectively, these studies indicate that the HLA antigens are associated with RA susceptibility among ethnic groups.
We found that HLA-DRB1*07 had a higher frequency in healthy control ethnic groups than in RA patients and may confer protection. This protective effect has been observed in different populations [42][43][44][45][46][47]. While other studies found an equal distribution of the allele between the patients and controls [21,27].
Our data showed that HLA-DQB1*03 frequency was significantly higher in patients compared with the healthy controls, with an inconsistent frequency between ethnicities. This result agreed with an ethnic group from Iran [28]. On the contrary, HLA-DRQB1*02 and HLA-DRQB1*06 were significantly higher in healthy controls.
Interestingly, the aforementioned two alleles showed protective associations in populations from Albania [48], susceptibility associations in populations from Iraq [49], and no associations in populations from Turkey [47].
Collectively, the association between HLA genotyping and inheritable makeup is still controversial due to the differences in race between populations.
We demonstrated a protective association of HLA-DRB1*03/DRB1*02, HLA-DRB1*13/DRB1*06, and HLA-DRB1*07/DRB1*02 haplotypes with RA. The HLA-DRB1*07/DRB1*02 haplotype protective association has been previously stated in a population from Iraq [49]. In resemblance, a study conducted among the Moroccan population states that DRB1*13-DQB1*06 and DRB1*07-DQB1*02 haplotypes are more frequent in RA patients positive for RF as compared with healthy controls [51]. Likewise, the HLA-DRB1*03/DRB1*02 protective association has been reported in patients with type 1 diabetes [52] and celiac disease [53]. The differences in RA risk and protective alleles between our data and others could be explained by the differences in the prevalence of RA between populations, while the effects of genetic and environmental factors cannot be excluded.
The present study has some limitations. The small sample size, the expensive cost of HLA typing tests, and the wide discrepancy in Sudanese ethnic groups lead to a lack of association of allele frequencies between the tribal stock. More hands-on studies with large sample sizes and further studies using high-resolution genotyping are needed to verify our findings.

Conclusions
This study highlighted the relevance between the ethical background (i.e., tribal stock) of the Sudanese population, the HLA class II molecules, and RA risk that can be used as a foundation for future populationbased assessments. Our data identified nine HLA-DRB1 and four DQB1 alleles among 11 ethnic groups and their modest consistency with RA susceptibility, despite their modest differences in frequency. Further studies with a large sample size are needed to prove these associations.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. The University of Gezira Ethical Committee issued approval UGIREC/2020. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.