Typing of HLA-DQA1 and DQB1 using DNA single-strand conformation polymorphism.

The technique of single-strand conformation polymorphism (SSCP), which is capable of distinguishing DNA sequence variability, was adapted to the identification of the HLA-DQA1 and DQB1 alleles. Eight DQA1 alleles and 12 DQB1 alleles were distinguished by amplifying the second exon of the genes in the presence of radioactive deoxynucleotide, denaturing the products with heat, and separating the single strands by electrophoresis in nondenaturing gels. For DQA1, it was possible to distinguish the eight alleles with standard bis-acrylamide or with a Hydrolink gel matrix. Twelve DQB1 alleles were identified by a protocol employing a combination of oligohybridization and SSCP using products amplified by specific DQB1 primers.     

Complete analysis of HLA-DQB1 polymorphism and DR-DQ linkage disequilibrium by oligonucleotide typing

HLA class II polymorphism is functionally important in the control of immune responses, in transplantation immunology, and in the suceptibility to autoimmune diseases. HLA-DQA1 and -DQB1 genes exhibit a larger degree of allelic polymorphism than usually recognized by routine serology. We have therefore performed an extensive analysis of DQB1 polymorphism by oligotyping. A set of 12 oligo probes was hybridized on polymerase chain reaction-amplified DNA, thus allowing the detection of 12 DQB1 alleles, as demonstrated in homozygous as well as in heterozygous individuals. This highly sensitive detection system is particularly relevant within the DQw1 specificity where the 7 allelic sequences can easily be identified. The DQ-DR linkage disequilibrium was analyzed by oligotyping of 80 Caucasoid heterozygous individuals (160 haplotypes), and very tight associations were observed between DRB1 and DQB1 alleles. Five DRB1 alleles, DR-BON, DR4/Dw4 or Dw14, DR7, DRw8.3, and DRw11, however, can be associated with different DQB1 alleles. Moreover the DRB1 and DQB1 oligotyping analysis performed on 20 randomly chosen DRw8 Caucasoid individuals showed a high prevalence of the DRB1*0801-DQB1*0402 haplotype. By combining the analysis of allelic variations at DRB1, DRB3, and DQB1 loci, we can detect 33 different DR-DQ combinations in our panel of Caucasoid individuals. We now apply DQB1 oligotyping on a routine basis for optimal matching of unrelated donors for bone marrow transplantation.     

The complexity of HLA class II (DRB1, DQB1, DM) associations with disseminated Mycobacterium avium complex infection among HIV-1-seropositive whites

Earlier associations of polymorphism in classic HLA class II (DRB1 and DQB1) genes have been extended to include the accessory genes DMA and DMB as determinants of disseminated Mycobacterium avium complex (DMAC) infection among HIV-1-seropositive whites. From the Multicenter AIDS Cohort study, 176 DMAC cases were matched with 176 controls in a nested case-control study. PCR-based HLA genotyping techniques were used to resolve variants of DRB1 and DQB1 to their four-digit or five-digit alleles, and single-strand conformation polymorphism was used to resolve sequences in exon 3 at each DM locus. The DMA*0102 allele occurred less frequently among DMAC cases than among controls (OR = 0.46, p =.02). Combinations of DRB1 alleles with or without specific DMA and DMB variants showed significant differences in distributions between the cases and controls, but both of the previously associated class II alleles (DRB1*1501 and DRB1*0701) showed stronger positive associations with DMAC in the absence than in the presence of DMA*0102. Apparent joint effects of DRB1 and DM allelic combinations on occurrence and timing of DMAC suggest that class II disease relationships may be better predicted by biologically plausible interactive combinations than by polymorphisms in individual genes.     

Polymorphism in the upstream regulatory region of DQA1 gene in the Italian population

Abstract: Polymorphism in the 5'-upstream regulatory region of the DQA1 gene has been recently described. Using PCR-SSO method and SSCP analysis we have investigated this polymorphism in a group of 111 Italian blood donors which had been oligotyped for DRB1, DQA1 and DQB1 genes. Eight allelic variants were detected. Looking at the relationships among QAP sequences and DQA1 and DRB1 genes, three alternative situations were found: 1. a one-to-one relation between QAP and DQA1 alleles, independently of the other class II genes; 2. the same QAP allele in association with different DQA1-DRB1 haplotypes; 3. the same DQA1 allele with different QAP sequences according to the DRB1 specificity. No unexpected associations with DQB1 gene were found. These results must be interpreted considering that DQA1 and DRB1 genes are transcribed in opposite directions so that the promoter region of DQA1 gene lies between DQA1 and DRB1, close to the former but several hundreds kb away from the latter.     

Differential presentation of group A streptococcal superantigens by HLA class II DQ and DR alleles

Superantigens have been implicated as pivotal mediators of severe invasive group A streptococcal (GAS) infections, by virtue of their potent immunostimulatory activity. HLA polymorphism has been suggested to influence the susceptibility to severe invasive GAS infection. Here we studied the influence of allelic and isotypic variation of HLA class II molecules on GAS superantigen-induced immune responses using cells derived from patients with bare lymphocyte syndrome, untransfected or transfected with various HLA class II alleles. Significantly higher proliferative responses were detected when streptococcal pyrogenic exotoxin (Spe) A was presented by cells expressing DQA1*0101/DQB1*0302 (DQ3.2), as compared to cells expressing DR1, DR4, or DR5 alleles (p=0.0002-0.01). In contrast to SpeA, SpeC was preferentially presented by DR4 as compared to DQB1*03 (p=0.04). In agreement with the proliferation results, a significantly higher frequency of IL-2-, TNF-alpha-, TNF-beta-, and IFN-gamma-producing cells was detected when SpeA was presented by HLA class II DQB1*03 alleles as compared to DR4 (p=0.0002-0.04). Binding experiments showed a high affinitybinding of SpeA to both class II DR4 and DQB1*0302, and there was no significant difference in SpeA binding affinity between the two alleles. The data confirm the effect of allelic polymorphism in superantigen responses and show that different superantigens are preferentially presented by distinct class II alleles.     

High resolution HLA-DQB1 typing by combination of PCR-RFLP and PCR-SSCP.

A reliable method for high-resolution HLA-DQB1 typing using a combination of PCR- restriction fragment length polymorphism (RFLP) and PCR-single strand conformation polymorphism (SSCP) analysis is described. The second exon of the DQB1 gene was subjected to PCR using generic primers and digested with two restriction enzymes, MspA1I and HaeIII, and the DQB1 alleles were divided into seven groups. According to the RFLP patterns, appropriate group specific primers for DQ5, 6 and DQ2, 3, 4 groups were used to selectively amplify the alleles and the SSCP technique was used to distinguish the individual alleles. A total of 88 quality control samples of various ethnic groups distributed in the International Cell Exchange and HLA DNA Exchange programs and the ASHI/CAP Proficiency Tests were investigated by the PCR-RFLP/SSCP method. The concordance between our typing results and the consensus results of the surveys were 100%, and a total of 14 DQB1 alleles in 49 homozygous and heterozygous combinations were all correctly identified by the method described. This method is accurate, economical and relatively easy to interpret and well suited for routine clinical and research uses.     

Rapid allelic diversification and intensified selection at antigen recognition sites of the Mhc class II DPB1 locus during hominoid evolution

The evolution of polymorphism at the Mhc class II DPB1 locus was studied by comparison of chimpanzee ( Pan troglodytes ), pygmy chimpanzee ( Pan paniscus ), gorilla ( Gorilla gorilla ) and human DPB1 alleles. Extensive polymorphism was found in all hominoids. The clustering of sequences in the phylogenetic tree is consistent with rapid generation of the DPB1 polymorphism. Analysis of the substitution pattern for human alleles shows an excess of nonsynonymous changes to synonymous changes at antigen recognition sites, indicating that the amino acid polymorphism at these sites is being maintained by selection. By contrast, no excess of nonsynonymous changes was found at the antigen recognition sites of nonhuman hominoid species. Thus, it appears that diversifying selection on the DPB1 polymorphism has intensified in the lineage leading to humans. No evidence was found for the existence of ancient allelic lineages predating the divergence of the hominoid species. The number of synonymous differences among DPB1 alleles is lower than among DQB1 and DRB1 alleles, indicative of a more recent origin for the DPB1 polymorphism and consistent with the more rapid evolution suggested by the phylogenetic tree.     

Molecular analysis and polymorphism of the DLA-DQB genes

Abstract: Partial-length cDNA clones and full-length genomic clones corresponding to a complete canine DQB class It gene were isolated. Southern analyses suggested the presence of two DQB genes - one of which appeared to be a pseudogene lacking exon 2 called DQB2. The other DQB gene, called DQB1, was isolated from a genomic phage clone and contained six exons. The DQB1 clone was restriction mapped, and exon 2 was sequenced from 70 dogs. Twenty alleles were found. Most of the amino acid substitutions occurred at putative positions in the peptide binding site. Inheritance of these sequences showed Mendelian segregation with one or two alleles per dog. Cluster analysis of the nucleotide and predicted amino acid sequences subdivided the canine DQB1 alleles into four major allelic groups. The number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites suggestive of positive selection.     

Polymerase chain reaction--single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing.

A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.     

上海地区汉族人HLA-DQB1启动子多态性以及QBP-DQB1连锁分析

为分析上海地区汉族人HLA DQB1启动子 (QBP )多态性 ,以及QBP与DQB1的连锁不平衡 ,我们选用 2 4个序列特异性寡核苷酸探针 (SSO )检测QBP多态性。在目前已知的 12种QBP等位基因中 ,我们仅检测到 10种 (未发现新的QBP类型 ) ,并且发现QBP与DQB1的某些等位基因之间存在一定的连锁格局。我们的结果与高加索人群研究结果比较发现 ,无论是QBP基因频率还是QBP DQB1单元型频率 ,在两种不同人群中都存在明显差异。结果提示 ,由于不同的QBP等位基因功能不同 ,因此QBP DQB1之间不同的单倍型组合可能是HLA与疾病关联的另一个因素。     

Locus and population specific evolution in HLA class II genes

The population genetics of the HLA class II loci was studied with reference to variation in the frequency of (a) alleles at a locus and (b) amino acids at specific sites. Variation was surveyed at 4 loci (DRB1, DQA1, DQB1, and DPB1) in 22 populations from the Twelfth International Histocompatibility Workshop (Saint-Malo, 1996). Allele and amino acid variation was measured by computing heterozygosity and the effective number of alleles. Substantial variations in polymorphism were observed among the various populations and loci studied. In the majority of the populations, DRB1 has the highest heterozygosity and effective number of alleles. As previously shown, the Amerindian populations have lower levels of allelic diversity when compared to other populations. At the amino acid level, DRB1 antigen recognition sites (ARS) have the highest heterozygosities and effective number of alleles. For the other loci (DPB1, DQA1, and DQB1) for which there is no crystal structure and for which ARS sites were inferred from DRB1, non-ARS sites were often among the sites with highest levels of variation. It is possible that these putative non-ARS sites do play a role in antigen presentation.
The homozygosity test for neutrality was applied to allele and amino acid data. Of the four HLA class II loci studied, only DPB1 failed to show evidence of balancing selection. DQB1 and DQA1 depart significantly from neutrality in the largest number of populations. Genetic distances between populations were computed based on frequency of alleles and amino acids at ARS sites.     

Structural characterization of the caprine major histocompatibility complex class II DQB1 (Cahi-DQB1) gene

The major histocompatibility class II genes have been extensively characterized in sheep and cattle, whereas in goats the only class II genes that have been completely sequenced are DRA and DRB. Herewith, we report the complete coding sequence of the goat DQB1 gene. This gene has a single open reading frame of 786bp, being organized in five exons and displaying 95-97% nucleotide identity with its bovine and ovine cDNA orthologous sequences. The structural features of the goat DQB1 molecule are well conserved with regard to its mammalian orthologues. Conserved glycosilation sites (beta19) and cysteine residues (beta15, beta79, beta117, beta173) forming disulfide bridges have been identified in the goat DQB1 molecule. The alignment of several Cahi-DQB1 exon 2 sequences has allowed to identify five different allelic variants Neighbor-joining phylogenetic analysis of caprine, ovine and bovine DQB sequences has allowed to ascertain that the five Cahi-DQB1 alleles we have found correspond to three different allelic lineages. We have identified fifteen polymorphic positions in the Cahi-DQB1 molecule, but only six of them are located in the peptide binding region. The high degree of conservation of these polymorphic sites located outside the peptide binding region in cattle and sheep suggests that they might play a functional role in antigen-presentation to CD4+ T cells.     

Sequence-specific priming and exonuclease-released fluorescence detection of HLA-DQB1 alleles

Molecular typing of HLA DQB1 alleles, employing sequence-specific primers (SSP) for PCR amplification, was used to test a novel method that eliminates the requirement for subsequent gel electrophoresis or additional hybridization steps by directly detecting positive reactions. We have evaluated the performance of this fluorescence-based oligonucleotide probe assay to assign the most common DQB1 alleles on DNA from 14 homozygous cell lines and in a blind study of 50 diabetic patient samples that had been previously typed at the DQB1 locus using SSOP and conventional SSP-based approaches. We used a panel of 14 DQB1 SSP primer pairs, internal control primers, and a combination of 4 fluorescent oligonucleotide probes to detect 14 alleles or groups of alleles and controls. We can reliably detect single-base allelic differences, observe 100% concordance with the results obtained using both of the standard methods, and are able to further subtype several alleles that are not easily distinguished using SSOP (e.g. DQB1 *0401/0402 and DQB1 *0302/ 0303). Sequence-specific priming and exonuclease-released fluorescence (SSPERF) detection is technically simple and can be performed in less than 2 hours, including DNA extraction, PCR amplification, data analysis and allele identification. This method is particularly useful for the analysis of large numbers of samples, for which high throughput is critical and for which gel-based approaches are difficult to perform. This technique may also be useful for small-scale class I and class II molecular typing in clinically oriented laboratories.     

Absence of DRwl5/3 and of DRwl5/7 heterozygotes in Caucasian patients with systemic lupus erythematosus

Polymorphic MHC class II molecules determine immune responsiveness towards pathogens and also contribute to susceptibility or resistance to a number of different autoimmune diseases, including systemic lupus erythematosus (SLE). The HLA-DR and -DQ alleles of 52 patients with SLE were analyzed by serology and, for 42 patients, HLA-DRB1, -B3 and DQB1 allelic polymorphism was determined by oligotyping on PCR-amplified DNA. While we confirm the increase of DR3 (44.2% versus 16% in controls; p less than 0.001) reported by others, we observed a complete absence of DRw15(2)/DR3 and DRw15(2)/DR7 heterozygotes among Caucasian patients. Moreover HLA-DQB1 oligotyping revealed the absence of DQB1*0602/0201 heterozygotes in our panel of Caucasoid SLE patients. Since both DR3 and DR7 haplotypes share the same DQB1*0201-encoded DQ beta chain, and since DRw15 is known to be in linkage disequilibrium with DQA1*0102, it can be predicted that DQA1*0102/DQB1*0201 combinations are absent in Caucasian patients. We therefore propose that a DQA1*0201/DQB1*0201-encoded HLA-DQ trans-dimer formed in these heterozygotes might function as a suppressor-inducer molecule that confers resistance against SLE.