Determination of NA gene frequencies in the Spanish population by polymerase chain reaction with sequence‐specific primers
Authors: Torío A. , Marín L. , Muro M. , Álvarez‐López M. R. , García‐Alonso A. M. 
Published: March 2003

The granulocytic antigens NA1 and NA2 are two allelic gene products of the FcγRIIIB gene. These antigens are frequently targets of granulocytic‐specific allo‐antibodies and auto‐antibodies that cause neutropenias. Different distributions of these antigens have been found in population studies. In our study, the genomic DNA of 345 healthy, unrelated Spanish individuals was typed for NA1 and NA2 using polymerase chain reaction (PCR) with sequence‐specific primers (SSP). The gene frequencies found in the Spanish population were 0.29 for NA1 and 0.71 for NA2. Flow cytometry immunotyping was also carried out in 20 of these samples, and an exact correlation was observed among the results. The gene frequencies determined in our study are similar to those reported in other Caucasian populations. The null phenotype reported in other serological studies was not found. This typing system can be used in population studies instead of serological tests as a more rapid, simple and reproducible method.Since the 1980s, scientific research has been carried out into the molecular characterization of as yet enigmatic neutrophil allo‐antigens and as a result, the role of neutrophil‐specific allo‐antibodies in a number of clinically important situations is now well established. Allo‐immune neonatal neutropenia, chronic benign autoimmune neutropenia in infancy, and dramatic transfusion‐associated lung injury have all been linked with antibodies to neutrophil‐specific allo‐antigens such as NA1, NA2, NB1, ND1, NE1 and the recently described antigen SH (Huizinga et al., 1990; Fromont et al., 1992; Puig et al., 1995; Bux et al., 1997). Human Fcγ receptor III (FcγRIII; CD16) is encoded by two highly homologous genes. The FcγRIIIA gene, which is expressed on macrophages and natural killer (NK) cells, shows no polymorphism. NA1 and NA2 are isoforms of the FcγRIIIB gene expressed on neutrophils. The two alleles differ at five nucleotides predicting four amino acid substitutions (Ory et al., 1989).The NA antigens are usually identified by granulocyte immunofluorescence (GIFT), agglutination test (GAT) and monoclonal antibody immobilization of granulocyte antigens (MAIGA). However, serological tests sometimes prove ineffective in identifying the antigens and depend on the availability of NA‐specific sera and freshly isolated cells. The polymerase chain reaction (PCR) with sequence‐specific primers (SSP) provides a powerful technique for discrimination of alleles arising from single or multiple base substitutions without the use of post‐amplification procedures such as digestion with restriction enzymes (Stein et al., 1994), or hybridization with allo‐specific oligonucleotides (Stein et al., 1995). PCR‐SSP analysis has been used to determine NA gene frequencies in populations and differences in the distribution of these frequencies have been found (Bux et al., 1995; Hessner et al., 1996). In this paper, we report NA frequencies in a Spanish population from the Murcia region of Spain, using PCR‐SSP analysis, as well as the correlation with the results of immunophenotyping on blood samples from selected donors.Genomic DNA was extracted from whole blood using the Dynabeads DNA direct system (Dynal, Oslo, Norway), following the manufacturer’s instructions. The PCR reaction was performed in a 9600 Perkin‐Elmer thermal cycler (Perkin‐Elmer, Norwalk, CT), using the conditions previously described (Bux et al., 1995). In every assay a negative control without DNA was included to detect contamination, as well as a positive DNA control with a known genotype. Peripheral blood collected in Na2EDTA was typed using flow cytometry and anti‐NA1 (M 1390, CLB, Amsterdam, Netherlands) and anti‐NA2 (VTS‐591–5, Sera‐Lab, Sussex, UK) monoclonal antibodies following standard methods.NA genotypes of 345 healthy, unrelated Caucasian donors from the Murcia region of Spain were determined by PCR‐SSP. The percentage of homozygous DNA samples for was 6.7 (n = 23) for NA1 and 48.9 (n = 169) for NA2, while 44.3 (n = 153) were heterozygous. No samples were found to be the null genotype, with positive amplification for the growth hormone gene and negative amplification for both NA alleles. The gene frequency, determined by the counting method, was 0.29 for NA1 and 0.71 for NA2. The 20 samples also immunophenotyped by flow cytometry showed an exact concordance with the results obtained by genotyping (data not shown).Significant differences in NA gene frequency have been reported among population groups (Ohto & Matsuo, 1989; Fromont et al., 1992; Bux et al., 1995; Bierling et al., 1990; Muñiz‐Diaz et al., 1995; Hessner et al., 1996; data shown Table 1). The genomic frequencies that we found correlate well with those described in other studies of Caucasian populations using PCR‐SSP techniques (Bux et al., 1995; Hessner et al., 1996). As in those studies, we did not detect the presence of a null genotype, which was detected in population studies using serological techniques (Bierling et al., 1990; Fromont et al., 1992; Muñiz‐Diaz et al., 1995). This technique can identify homozygous individuals for a FcγRIIIB gene deletion (NA null), but it cannot identify heterozygous individuals for a FcγRIIIB gene deletion if they are typed as NA1 or NA2 homozygous. In some of the serological studies, the frequency of the null phenotype was lower than that originally described when carrying out new typing (NA null phenotype frequency = 0.0026 instead of 0.0045) (Bierling et al., 1990), or when there was an increase in the population typed (NA null genotype frequency = 0.06 instead of 0.08) (Muñiz‐Diaz et al., 1997). In a previous study of genomic frequencies in a Spanish population, where serological techniques were used (Muñiz‐Diaz et al., 1995), it was suggested that there was a greater frequency of null individuals in the Spanish population compared with other Caucasian groups. In our study we were unable to confirm this hypothesis. This could be due to the fact that the individuals typed came from different regions (although in HLA studies carried out in our laboratory we have not found genetic differences between the Murcia region population and the rest of the Spanish population). Other factors could be the different number of typed samples, or indeed the discrepancies caused by the different typing techniques employed.

. NA gene frequencies in different populations determined by serological test and PCR‐SSP
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In conclusion, the gene frequencies observed in a Spanish population from the Murcia region are similar to those reported in studies of other Caucasian populations using PCR‐SSP, even though the null genotype was not found. We consider this technique to be a useful typing method for population studies which can be used instead of a serological test.