Roles of HLA-B, HLA-C and HLA-DPA1 incompatibilities in the outcome of unrelated stem-cell transplantation.

In unrelated stem-cell transplantation, the value of matching at the HLA-A, -B and -DR loci between donor and recipient is well documented. The effect of HLA-C, DPB1 and DPA1 mismatches on transplantation outcome is unclear. In this study, 104 donor recipient-pairs, transplanted at Huddinge University Hospital between 1988 and 1999, were retrospectively HLA class I- and class II-typed by PCR-SSP. The samples were typed for HLA-A, -B and -C and HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPB1 and -DPA1 with allele level resolution. Isolated HLA-B allele level mismatches were associated with an increased incidence of acute graft versus host disease grades II-IV and grades III-IV. HLA-C-mismatched, but killer cell immunoglobulin-like receptor (KIR) ligand motif-matched stem-cell grafts were significantly associated with improved survival rates and relapse-free survival (RFS). In patients receiving HLA-DPA1-mismatched stem cell grafts, reduced survival and shorter RFS were seen. These patients also had an increased frequency of relapses (64%vs 26%). We conclude that genomic HLA class I- and class II-typing may improve the outcome after unrelated stem-cell transplantation. The awareness of HLA class I- and II-mismatches in a recipient-donor pair makes it possible to give appropriate pre- and post-transplantation treatment.

HLA-DPB1 typing by polymerase chain reaction amplification with sequence-specific primers.

DPB1 is the second most polymorphic class II locus with currently 84 recognized alleles, i.e. DPB1*0101 to DPB1*8101. Most of the alleles have been described during the last few years using oligonucleotide and sequencing techniques and relatively little is known about the role and importance of the polymorphic residues as regards to the function of DP molecules. In the present study, polymerase chain reaction (PCR) primers were designed for identification of all the phenotypically different DPB1 alleles by PCR amplification with sequence-specific primers. Forty-eight standard genomic PCR reactions per sample were performed in order to achieve this resolution. Unique amplification patterns were obtained in 2983 of 3160 (94.4%) possible genotypes. The primers were combined so that only very rare genotypes gave rise to ambiguous patterns. Sixty-four Histocompatibility Workshop cell lines and 150 DNAs provided by the UCLA DNA exchange were investigated by the DPB1 primer set. All typing results were conclusive. Analysis of the distribution of DPB1 alleles was performed in 200 Caucasian samples, 100 African samples and 40 Oriental samples. The population study by the DPB1 PCR-SSP method showed a characteristic distribution of HLA-DPB1 alleles. Each ethnic group had one, or two, frequent DPB1 allele(s) and the frequency of homozygotes was high, suggesting that balancing selection does not appear to be affecting the evolution of the DPB1 locus.

DQB1*0202 and the new DQB1*0203 allele: a fourth pair of DQB1 alleles differing only at codon 57.

Amino acid 57 of DQ beta chains is of functional importance as it influences peptide binding, is part of B and T cell epitopes, and is associated with susceptibility and resistance to insulin-dependent diabetes mellitus and humoral immunodeficiencies. Polymorphism of codon 57 is conserved in primates and in HLA class II B genes implying that balancing selection operates on this residue. Previously, three DQB1 allele pairs have been described, that only differ at residue 57. In an African-American Black individual with the HLA phenotype A23.30;B58,63;Cw6;DR18,12;DR52;DQ5,2, we found a fourth example of this dimorphism: the new DQB1*0203 allele, that was identical to DQB1*0202 except for codon 57, which encodes aspartic acid and alanine respectively in the two alleles. The class II haplotype carrying the new allele was deduced to be DRB1*0302,DRB3*0101,DQA1*05011,DQB1*0203.

HLA-DPA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) and distribution of DPA1 alleles in Caucasian, African and Oriental populations.

In the present study PCR primers were designed for detecting all known DPA1 variability, i.e., the presently recognized six DPA1 alleles 0103 to 0401, and also for separation of the four DPA1*02 alleles, by PCR amplification with sequence-specific primers (PCR-SSP). For each sample seven different PCR reactions were performed which allowed the identification of all DPA1 alleles and the resolution of all DPA1 genotypes. Forty-eight cell lines and 100 donor spleen cells were investigated by the DPA1 PCR-SSP technique. In the forty-eight known workshop cell-lines no false positive or false negative results were obtained. The 100 donor spleen cells were only typed by the PCR-SSP technique and in their DNAs only one or two DPA1 alleles were found. Twenty cell lines and twenty donor spleen cells were typed on two separate occasions and interpreted blindly. The reproducibility between the repeated typings was 100%. The length of the specific products ranged from 103 to 258 base pairs and the amplification patterns obtained were easy to interpret. In conclusion, DPA1 typing by the PCR-SSP method is an accurate typing technique with high sensitivity, specificity and reproducibility. Analysis of the distribution of DPA1 alleles was performed in 100 Caucasian samples, 100 African samples and 80 Oriental samples, including separation of the four of HLA-DPA1 alleles. Each ethnic group appeared to have one (Caucasians), or two (Africans and Orientals), frequent DPA1 allele(s) and a high frequency of DPA1 homozygotes, suggesting that, like for the DPB1 locus, balancing selection does not appear to be affecting the evolution of the DPA1 locus.

HLA-DOB1 "low-resolution' typing by PCR amplification with sequence-specific primers (PCR-SSP).

In the study described here primers were designed for DQB1 'low-resolution', i.e. generic, typing by PCR amplification with sequence-specific primers (PCR-SSP) considering all the currently recognized DQB1 alleles, i.e. 0501-0504, 0601-0609, 0201, 0301-0305 and 0401-0402. This resolution was achieved by performing eight PCR reactions per individual. The DQB1 alleles corresponding to the serological specificities DQ4, DQ5, and DQ6 were uniquely identified, whereas the DQ2, DQ7, DQ8 and DQ9 specificities were amplified by two primer mixes. All homozygous and heterozygous combinations of the serological series DQ1 to DQ9 could be distinguished. The yield of amplified products were increased compared to our previously described DQB1 'high-resolution' typing technique by lengthening many of the primers, modifying the PCR cycling parameters and by including glycerol in the PCR reaction mixtures. Thirty-one cell lines and 90 donor spleen cells were investigated by the DQB1 'low-resolution' PCR-SSP technique as well as by TaqI DRB-DQA-DQB RFLP analysis. The concordance between PCR-SSP typing and RFLP analysis was 100%. The cell lines and 20 of the spleen cells were typed twice with complete reproducibility. No false positive or false negative typing results were obtained. DQB1 'low-resolution' PCR-SSP typing, including DNA extraction, PCR amplification, gel detection, documentation and interpretation, were performed in 2 h which renders the PCR-SSP technique suitable also for the genotyping of cadaveric organ donors.