Gene frequencies of the HNA‐4a and ‐5a neutrophil antigens in Brazilian persons and a new polymerase chain reaction‐restriction fragment length polymorphism method for HNA‐5a genotyping

Abstract

The HNA-4a (Mart) and HNA-5a (Ond) antigens are polymorphic variants of alpha(M) (CD11b) and alpha(L) (CD11a) subunits of the beta(2)-integrin, and are associated with single nucleotide polymorphisms (SNP) leading to amino acid dimorphisms. HNA-4a has been linked to alloimmune neonatal neutropenia, but the HNA-5a clinical significance is unclear. Using a polymerase chain reaction (PCR) with sequence-specific primers, the frequency of HNA-4a among 121 Brazilian blood donors and 114 Amazon Indians was determined. A PCR-restriction fragment length polymorphism (RFLP) method for HNA-5a genotyping was developed, and the gene frequencies of this antigen were determined among 123 blood donors and 114 Indians. To validate the genotyping method, the amplified DNA from six previously obtained samples (two of each genotype) was sequenced. The HNA-4a (+/+), HNA-4a (+/-), and HNA-4a (-/-) genotype frequencies of blood donors (0.686, 0.273, 0.041) and Indians (1.000, 0.000, 0.000) were different (p < 0.01). The frequencies of HNA-5a (+/+), HNA-5a (+/-), and HNA-5a (-/-) genotypes among blood donors (0.512, 0.399, 0.089) and Indians (0.746, 0.219, 0.035) also differed (p < 0.01). Sequencing demonstrated concordance with PCR-RFLP genotyping in all six evaluated samples. Comparing to another populations, Brazilians present a higher frequency of HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization. Moreover, the distribution of the HNA-4 alleles observed in Amazon Indians is quite similar to that reported among Koreans. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.