A134T, G156A and G1333A SNPs in the CD177 Gene Are Associated with Atypical Expression of Human Neutrophil Antigen 2a (HNA-2a)

Introduction: The HNA-2a has gained clinical importance for being involved in immune neutropenias, febrile transfusion reactions and transfusion-related acute lung injury (TRALI). The HNA-2a shows the unique feature of being expressed only on a neutrophil subpopulation. The gene encoding the HNA-2a, CD177, is located on chromosome 19 at 19q13.2 and the cDNA consists of 2364 bp (RefSeq: NM 020406) coding for 437 amino acids including a signal peptide of 21 amino acids. In this study we investigated the presence of SNPs in the CD177 and its association with atypical expressions of HNA-2a.

Methods: After evaluating the expression of HNA-2a in 135 randomly selected Brazilians by flow cytometry (FACSCalibur, BD) using two pure monoclonal antibodies (TAG4 and 7D8), we observed that some subjects revealed an atypical expression of the antigen (a non reactive cell population plus two distinct reactive cell populations). Subjects were grouped according to cytometric results as having high (≥80%), low (≤50%) or atypical expression of HNA-2a. We isolated granulocyte RNA from the selected individuals and reverse transcription was performed according to kit protocol (SuperScript III First-Strand Synthesis, Invitrogen). Full-length cDNA was amplified and then sequenced according the manufacturer’s protocol (Mega BACE, GE). Data were analyzed using the Bioedit program.

Results: The flow cytometry analysis revealed that 131 (97%) subjects were HNA-2a positive, 4 (3%) HNA-2a negative, while 8 subjects (5.9%) showed an atypical expression of HNA-2a with levels ranging from 49 to 80%. Sequencing analysis of subjects with high (n=14), low (n=22) or atypical (n=8) HNA-2a expression identified six polimorfic sites (C49G, A134T, G156A, A793C, G1084A and G1333A), five of them predicting amino acid exchanges (except the SNP G156A). Comparing the cDNA sequences between individuals with typical (n=36) or atypical expression (n=8), we observed three polimorphisms (A134T, G156A and G1333A) that showed a significantly higher frequency in the group with atypical expression comparing to the group of individuals with typical expression (P= 0.004; 0.006; and <0.0001, respectively; Fisher exact test).

Conclusions: The analysis of individuals with two distinct positive cell populations revealed three SNPs strongly related to this atypical HNA-2a expression, but do not seem to be related to the size of the HNA-2a-expressing neutrophil subpopulations.