Frequency of HLA-DQB*06 in Caucasian,African American, and Mexican American patients with a positive direct antiglobulin test.

A reduced frequency of HLA-DQ6 in patients with a positive direct antiglobulin test (DAT) was previously reported but race was undisclosed. Therefore, we investigated a total of 275 patients (80 Caucasian, 113 African American, and 82 Mexican American) and 518 normal controls (205 Caucasian, 208 African American, and 105 Mexican American). These were typed for class II HLA antigens using molecular techniques. A DAT was performed on each patient's red cells drawn into EDTA using both mouse and rabbit polyspecific reagents. Of 275 patients tested, 73 (27%) had a positive DAT (12 Caucasians, 35 African Americans, and 26 Mexican Americans). We found that 5 (42%) Caucasian patients and 103 (50%) Caucasian controls possessed the DQB*06 allele (p =.56). In the African American group, 15 (43%) patients and 91 controls (44%) were DQB*06 positive (p =.92). Six Mexican American patients (23%) and 21 controls (20%) had the DQB*06 allele (p =.72). This article underscores the need to use race-matched controls when genetic disease associations are sought.

DNA analysis of Duffy genes in American blacks.

BACKGROUND: Because limited studies have shown that the Duffy genotype is not accurately reflected by the erythrocyte phenotype, we used PCR-RFLP to determine the true FY1 and FY2 gene frequencies in blacks. METHODS: Genomic DNA was extracted from 81 randomly selected black individuals, amplified using PCR, digested with BanI and electrophoresed in 1.8% gels. Direct DNA sequencing of the FY regulatory region was done on a sample which had weak Fyb antigens. Standard serological techniques were used to type the erythrocytes for Fya and Fyb. RESULTS: The most common genotype (83%) in blacks was Fy(a-b+) but almost all of the samples serologically typed as Fy(a-b-). The new gene frequencies are 0.08 for FY1 and 0.92 for FY2. Two donors whose erythrocytes typed weakly positive for Fyb and appeared to be Fyx were homozygous for FY2 by DNA testing. DNA sequencing of the regulatory region for Duffy found a mutation in an Sp1 binding site in the Fyx donor. CONCLUSIONS: We conclude that (1) the FY2 gene occurs in a much higher frequency in blacks than previously reported which may explain the lack of anti-Fyb in this ethnic group and (2) Fyx appears to be a the product of a mutant FY2 gene giving a quantitative variation in expression.

Evaluation of column technology for direct antiglobulin testing.

Preliminary reports have suggested that microcolumn technology might be too sensitive for direct antiglobulin testing (DAT). We studied 228 samples from patients with autoimmune diseases and 30 samples from healthy controls to determine the sensitivity of column techniques. Both Sephadex gel and protein A/G columns were compared with manual methods using rabbit or murine polyspecific reagents. Of the 187 samples that were negative by both manual methods, an additional 29 (15%) and 42 (22%) samples gave weakly positive reactions with the Sephadex and protein A/G bead columns, respectively. Subsequently, there was poor correlation between manual and column techniques (r = 0.40-0.61). Acid eluates from these samples were negative. We concluded that the column technology may detect too many weakly positive DATs that are clinically insignificant.

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression.

Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH+ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 - 1106, S at position 1157, and VDLL at positions 1188 - 1191. A second donor typed as C4A2, A4, B1 and was also WH+. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 - 1106, S at position 1157, and ADLR at positions 1188 - 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene.