Polymorphism of HLA-DRB4 gene in Croatia

Abstract

U ovom istraživanju analiziran je polimorfizam gena HLA-DRB4 i haplotipova HLA-DRB1-DRB4 u Hrvatskoj među kadaveričnim davateljima organa (N=144). Svi uzorci bili su odabrani na temelju prisustva jednog od alela skupine HLA-DRB53 (aleli DRB1*04, DRB1*07 i DRB1*09). Određivanje alela lokusa HLA-DRB4 provedeno je metodom lančane reakcije polimerazom i početnicama specifičnim za jedan ili više alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Primers, PCR-SSP). Aleli lokusa HLA-DRB1 i -DQB1 određeni su metodom lančane reakcije polimerazom i probama specifičnim za jedan ili grupu alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Oligos, PCR-SSO). Na lokusu HLA-DRB4 otkriveno je sedam različitih alela, 5 funkcionalnih alela (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05 i DRB4*01:08) i dva nul alela (DRB4*01:03:01:02N i HLA-DRB4*03:01N). Među ispitanicim pozitivnim za jedan od alela skupine HLA-DRB1*04 nije otkrivena statistički značajna razlika u raspodjeli alela DRB4 s obzirom na prisustvo alela DQB1. U haplotipovima HLA-DRB1*04-DQB1 najčešći prisutni alel je bio DRB4*01:03 (86,7%). Među HLA-DRB1*07:01 pozitivnim haplotipovima uočena je statistički značajna razlika (p<0,05) u raspodjeli alela HLA-DRB4 s obziroma na prisustvo alela HLA-DQB1. Haplotipovi: HLA-DRB1*07:01-DQB1*03:03 na lokusu HLA-DRB4 u 98,0% slučajeva imali su nul alel DRB4*01:03:01:02N, dok su haplotipovi DRB1*07:01-DQB1*02:02 na lokusu DRB4 u 65,5% slučajeva imali prisutan alel DRB4*01:03. Unutar skupine HLA-DRB1*04 pozitivnih haplotipova nije uočena statistički značajna razlika u raspodjeli alela HLA-DRB4 s obzirom na prisustvo alela HLA-DQB1. Alel HLA-DRB1*09:01 je kod svih ispitanika uočen u kombinaciji s alelima HLA-DRB4*01:03-DQB1*03:03.In this study the polymorphism of HLA-DRB4 alleles and HLA-DRB1-DRB4 haplotypes among Croatian cadaveric organ donors (N = 144) was analyzed. All samples were selected based on the presence of one of the alleles of HLA-DRB53 group (DRB1*04, DRB1*07, and DRB1*09 alleles). Typing of HLA-DRB4 alleles was carried out by polymerase chain reaction sequence specific primers for one or more HLA alleles (method: PCR-SSP). HLA-DRB1 and -DQB1 alleles were determined by polymerase chain reaction sequence specific oligos for one allele or a group of HLA alleles (method: PCR-SSO). On the HLA-DRB4 locus seven different alleles were discovered; 5 functional alleles (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05, and DRB4*01:08) and two null alleles (DRB4*01:03:01:02N and HLA-DRB4*03:01N). Among individuals positive for one of the HLA-DRB1*04 alleles no statistically significant difference was detected in the distribution of the HLA-DRB4 alleles given the presence of the DQB1 alleles. Among HLA-DRB1*04-DQB1 haplotypes the most present was DRB4*01:03 allele (86.7%). Among HLA-DRB1*07:01 positive haplotypes a statistically significant difference (p<0.05) in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles was observed. The HLA-DRB1*07:01-DQB1*03:03 haplotype in 98.0% of cases had the DRB4*01:03:01:02N allele on the HLA-DRB4 locus, while DRB1*07:01-DQB1*02:02 haplotype on the HLA-DRB4 locus in 65.5% of cases had the DRB4*01:03 allele (65.5%). Within the group of the HLA-DRB1*04 positive haplotype, there was no statistically significant difference in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles. The HLA-DRB1*09:01 allele was observed in combination: HLA-DRB4*01:03-DQB1*03:03 in all the cases