Genetic risk assessment of HLA DRB1 alleles (01, 04, 12 and 15) with Rheumatoid arthritis susceptibility among the Pashtun population of Khyber Pakhtunkhwa, Pakistan

Abstract Background: Rheumatoid arthritis (RA) is a chronic autoimmune condition influenced by a complex interplay of genetic and environmental factors. Specific genetic variations, particularly in the human leukocyte antigen (HLA)-DRB1 gene, have been strongly linked to RA susceptibility. HLA-DRB1 alleles, part of the major histocompatibility complex (MHC) class II gene family, play a significant role in the immune system. Aims: This study aimed to investigate the association between HLA-DRB1*01, *04, *12, and *15 alleles and RA risk within the Pashtun population of Pakistan. Methods: A cohort of 100 RA patients and 80 healthy controls was recruited for this study. DNA was isolated from blood samples using the salting-out method, followed by genotyping using sequence-specific primer polymerase chain reaction (SSP-PCR). The association of the selected alleles with clinicopathological and demographic parameters of RA was assessed using the Medcalc odds ratio calculator and Chi-square test. Results: Allelic and genotypic analysis revealed that HLA-DRB1 *01 and *04 variants exhibited non-significant associations with RA risk (P=0.1421 and 0.8495, respectively). Similarly, mutant genotypes of HLA-DRB1 *01 and *04 showed non-significant associations with RA (P=0.2922 and P=0.8263, respectively). However, the heterozygous CA genotype of HLA-DRB1 *01 demonstrated a significant association with RA (P=0.0004), whereas HLA-DRB1 *04 did not (P=0.9120). Furthermore, HLA-DRB1 *12 showed a significant association with increased RA risk (P=0.0001), while HLA-DRB1 *15 did not (P=0.5519). Additionally, HLA-DRB1*01, *04, and *12 did not show significant associations with age group (P=0.08, P=0.11, P=0.16) and rheumatoid factor (RF) status (P=0.34, P=0.65, and P=0.74), whereas HLA-DRB1 *15 exhibited non-significant associations with age group and RF (P=0.01 each). Moreover, associations of HLA-DRB1 *01, *04, *12, and *15 with gender (P=0.81, P=0.52, P=0.27, and P=0.09, respectively) and anti-citrullinated protein antibody (ACPA) status (P=0.56, P=0.40, P=0.65, and P=0.008, respectively) were not significant, except for HLA-DRB1 *04, which displayed a significant association with ACPA. Conclusion: HLA-DRB1 *12 and heterozygous genotypes of HLA-DRB1 *01 were significantly associated with RA risk in the Pashtun population. However, further analysis utilizing whole exome sequencing with larger datasets is warranted for more precise results.