Xiaojiang Gao

Alleles at four HLA class II loci determined by oligonucleotide hybridization and their associations in five ethnic groups

The use of polymerase chain reaction (PCR) and oligonucleotide hybridization offers a new approach for the definition of HLA class II alleles. It has been possible to determine 43 alleles of DRB1, four of DRB3, two of DRB4, four of DRB5, eight of DQA1, and 14 of DQB1. These alleles are inherited together in members of families and form closely associated groups which are found repeatedly and in characteristics patterns in different populations. We have determined the HLA class II alleles and analyzed their association in 431 healthy unrelated subjects including 161 North American Caucasians, 53 Latin Americans, 61 Blacks, 88 Chinese, and 68 Israeli Jews. For-locus haplotypes (DRB1; DRB3/4/5; DQA1; DQB1) were derived from 79 B cell lines and the analysis of segregation in 34 nuclear families. The B-cell lines yielded 37 and the families showed the same, and 20 other, haplotypic combinations. In addition to these 57 haplotypes, associated alleles were assigned in the unrelated panels following certain rules. The resulting haplotypes were assigned to groups known to share associated alleles. The groups were: (1) DR1, DR2, and DRw10 (13 haplotypes); (2) DR3 and DRw6 (26 haplotypes); (3) DR5 and DRw8 (24 haplotypes); (4) DR4, DR7, and DR9 (24 haplotypes). Their distribution in populations with different ethnic backgrounds was analyzed. The expressed DRB4 allele and its null mutant were determined by PCR and oligonucleotide hybridization. The different DR7 haplotypes resulting from these determinations were analyzed in a panel of 130 North American Caucasoids. This comprehensive analysis of class II HLA haplotypes in human populations should be useful in understanding the role of these genes and in various applications including anthropolgy, disease susceptibility, and transplantation of allogeneic organs and tissues.

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. III, Typing for 24 alleles of HLA-DP

The second exon of HLA-DPB includes five polymorphic segments with extensive sharing of sequences between alleles. In order to facilitate assignment of specificities in heterozygous individuals, we have used group-specific amplification of two nonoverlapping sets of DPB alleles (here called group A and group B) with especially designed primers. Group A and group B polymerase chain reaction products were hybridized with sequence-specific oligonucleotide probes generating easily recognizable patterns which defined 24 distinct HLA-DPB alleles. We also established a routine procedure for distinguishing HLA-DP homozygosity from failed amplification in one of the alleles. Our results showed that when only one allele was detected, failure of amplification had occurred in less than 4% of the cases. DNA typing with this method correlated well with primed-lymphocyte typing for HLA-DP in the Tenth Workshop, as determined by us in assays performed on the workshop B-cell lines. Two normal panels of unrelated subjects were tested to obtain population frequencies. We conclude that this method is simple, relatively quick, and accurate. It is the method of choice for studies to determine the role of HLA-DP alleles in T cell reactions, in various diseases, and in transplantation.

Class II human leukocyte antigen genes and T cell receptor polymorphisms in patients with rheumatoid arthritis

Human leukocyte antigen (HLA) typing was performed in 174 patients with rheumatoid arthritis and 222 white control subjects. Increases in HLA-DR4 and HLA-DR1 were observed as in previous studies. Each of these appeared to be inherited as dominant risk factors. Southern blotting with a DR-beta probe after digestion of genomic DNA with the restriction enzyme Bam HI showed seven bands. Three of them correlated with DR4 and were increased in rheumatoid arthritis patients. Subsets of DR4 were determined by polymerase chain reaction amplification followed by hybridization with oligonucleotide probes. Dw4 was increased in rheumatoid arthritis patients, and the frequency of the other subsets appeared to be similar in rheumatoid arthritis patients and control subjects. A polymorphism associated with the T cell receptor V-beta-8 gene family was significantly increased in rheumatoid arthritis patients.