Publication Date:
2009-11-01
Description:
Abstract LBA-4 The HNA-3a/b (5b/a) leukocyte alloantigens were identified in 1964 by van Leeuwen et al. as a biallelic system with gene frequencies of 0.82 (HNA-3a) and 0.18 (HNA-3b), respectively. Despite the passage of more than 40 years, the presumptive protein carrier for HNA-3a/b and the molecular composition of the antigens themselves is unknown. The importance of defining the HNA-3a/b antigens more fully has been heightened by the recognition that HNA-3a-specific antibodies are especially prone to cause severe, often fatal, transfusion-related lung acute injury (TRALI), now the single most important cause of transfusion-related mortality. About 4-5% of the general population is HNA-3a-negative and is at risk to produce HNA-3a-specific antibodies. We performed genome-wide single nucleotide polymorphism (SNP) scanning of DNA from 8 HNA-3a-negative (HNA-3b/b) and 107 unrelated normal individuals and found that each of the HNA-3a-negatives, but only 5 of the normal subjects was homozygous (AA) for a SNP (G〉A) located at nt542 of the SLC44A2 gene encoding the choline transporter-like protein CTL2 (pA542) in the gene SLC44A2 encoding CTL2.HNA-3-a negative subjects (n = 8)Normal subjects (N = 107)GGGAAAGGGAAA008*64385*p = 2.4 × 10−12 This SNP (rs2288904) is the only one known in SLC44A2 that correlates with the HNA-3a-negative phenotype and predicts an extracellular amino acid substitution (R〉Q154) in the mature protein. Gene frequencies of the high and low frequency alleles of SNP rs2288904 are 0.82 and 0.18, respectively, identical to the gene frequencies of HNA-3a and HNA-3b determined by serologic typing. Although HNA-3a is widely considered to be a neutrophil antigen, we found that lymphocytes carry slightly more HNA-3a than neutrophils, and that persons heterozygous for HNA-3a have 50% as much antigen as homozygotes, allowing heterozygotes to be identified by flow cytometry. To validate the SNP analyses, we directly sequenced DNA encoding CTL2 from 4 HNA-3a/a, 4 HNA-3a/b and 8 HNA-3b/b subjects and found that, without exception, nt542G and nt542A correlated with the phenotypes HNA-3a and HNA-3b (determined serologically), respectively. To obtain direct evidence that HNA-3a is carried on CTL2, we sensitized leukocytes with anti-HNA-3a, immunoprecipitated antibody-bound proteins, performed SDS electrophoresis and subjected candidate bands to mass spectrometric analysis. In three independent assays, peptides derived from CTL2 spanning the entire length of the protein were identified (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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