Identification of gene conversion events in horse IGHV suggests preferential hotspots for diversification.

The humoral immune response relies on a diverse antibody repertoire, which is expanded through processes such as somatic hypermutation, class-switch recombination and gene conversion. These processes are primarily mediated by activation-induced cytidine deaminase (AID). Gene conversion generates diversity in immunoglobulin heavy and light chains (IGHVs) in species such as chickens and rabbits, though it has not been widely studied. Since 80% of the equine IGHV repertoire originates from only three functional gene segments, we examined gene conversion events in horses to assess their role in antibody diversification. Using a modified version of BrepConvert, which optimized analysis time, we identified gene conversion events in 6.9% of immunoglobulin sequences. The results showed a local preference, with most events occurring at the beginning of framework region 1 (FR1) and within complementarity-determining region 2 (CDR2). Pseudogenes IGHV4-35, IGHV4-53, and IGHV4-38 were utilized most frequently, while functional genes IGHV4-21, IGHV4-22, and IGHV4-29 exhibited the highest event frequencies. Interestingly, while most mismatched regions were only three nucleotides long, 91% of these events are flanked by specific sequences (six nucleotides at the 5' end and one nucleotide at the 3' end). Furthermore, functional pseudogene pairs often share identical leader regions of 5-26 nucleotides, suggesting expanded events. We also identified a potential association between these events and local non-B DNA conformations, as well as with the zinc finger protein ZNF691, which supports the involvement of DNA-binding factors. Together, these findings demonstrate that gene conversion significantly contributes to equine antibody diversity by targeting specific IGHV regions.