Gene Frequencies of the HNA-4a and -5a Neutrophil Antigens in Brazilians and a New Polymerase Chain Reaction-Restriction Fragment Length Polymorfism Method for HNA-5a Genotyping.
Abstract The HNA-4 and HNA-5 systems are located on the b2-integrin, that share a common b subunit (b 2 or CD18) noncovalently associated with four different a subunits. The HNA-4a (Mart) and HNA-5a (Ond) antigens are polymorfic variants of a M (CD11b) and a L (CD11a) subunits, respectively, and both are associated with single nucleotide polymorphisms (SNP) leading to amino-acid dimorphisms. HNA-4a has been recently linked to alloimmune neonatal neutropenia while the HNA-5a clinical significance is not clear yet. Using a PCR with sequence-specific primers (SSP) the frequency of allelic polymorphism of HNA-4a among 121 Brazilian blood donors and 114 Amazon Indians was determined. A novel PCR-restriction fragment length polymorphism (RFLP) method digesting the PCR product with the enzyme Bsp1286 I for HNA-5a genotyping was developed, and the gene frequencies of this antigen were determined among 123 Brazilian blood donors and 114 Amazon Indians. In order to validate this proposed new genotyping method, the amplified genomic DNA from 6 previously genotyped samples (2 of each genotype) was purified using a commercial kit (GFX PCR DNA and Gel Band Purification Kit, Amersham Bioscience, Piscataway, NJ, USA), sequenced using a cycle-sequencing kit (BigDye Terminator, PE Applied Biosystems, Foster City, CA, USA) and analyzed on a genetic analyzer (ABI Prism 3100, PE Applied Biosystems). The calculated HNA-4a (+/+), HNA-4a (+/−) and HNA-4a (−/−) genotype frequencies found in blood donors (0.686, 0.273 and 0.041) and Amazon Indians (1.000, 0.000 and 0.000) presented statistically significant differences (p<0.01). The frequencies of HNA-5a (+/+), HNA-5a (+/−) and HNA-5a (−/−) genotypes among blood donors (0.512, 0.399 and 0.089) and Amazon Indians (0.746, 0.219 and 0.035) also differed significantly (p<0.01). Sequencing studies demonstrated absolute concordance with PCR-RFLP genotyping results in all 6 analyzed samples. Overall, the present data indicate that comparing to other population studies, the Brazilian population presents a higher frequency of the HNA-4a-negative allele, suggesting that Brazilians would be more susceptible to HNA-4a alloimmunization by pregnancy or blood transfusion. Moreover, the distribution of the HNA-4 system alleles observed in Amazon Indians is quite similar to that already reported among Korean individuals. Besides that, a new effective and efficient HNA-5a genotyping technique is now available for population studies.