In the present study PCR primers were designed for detecting all phenotypically expressed DQB1 and DQA1 allelic variability, 19 and 10 alleles, respectively, by PCR amplification with sequence-specific primers (PCR-SSP). For DQB1 typing, each sample was amplified by a first set of 14 PCR primer pairs, followed in some cases by two to six additional PCR reactions. The first 14 primer pairs allowed the identification/separation of all but a few of the recently described DQB1 alleles: DQB1*0504, DQB1*0605, DQB1*0606 and DQB1*0607 would not be identified; DQB1*0603 and DQB1*0608; and DQB1*0301 and DQB1*0304, respectively, would not be distinguished. Therefore an additional set of eight DQB1 primer pairs was used for a complete DQB1 typing, including all homozygous and heterozygous combinations. For DQA1 typing, 12 PCR reactions were performed per sample, 10 for detecting variability within the second exon and two for identifying first exon polymorphism. All homozygous and heterzoygous combinations of DQA1 alleles could be resolved by these primer pairs. In addition, four primer mixes were designed for determining codon 57 of the HLA-DQB1 gene. Thirty cell lines and 120 individuals were investigated by the DQB1 and DQA1 PCR-SSP technique, as well as with the HLA-DQ beta 57 primers. The concordance between PCR-SSP typing and assigning DQB1 and DQA1 alleles from TaqI DRB-DQA-DQB RFLP analysis was 100%. The reproducibility was 100% in 30 samples investigated on two separate occasions. Amplification patterns, investigated in 15 nuclear families, segregated according to dominant Mendelian inheritance. DQB1 and DQA1 PCR-SSP typing can be performed in 2 hours, including DNA extraction, PCR amplification and post-amplification processing. The method is technically simple and the typings are easy to interpret. The cost for typing one individual is low and is independent of the number of samples analyzed simultaneously, i.e. the technique is well-suited for routine clinical use.

Multiple sclerosis (MS) has, since the 1970s, been known to be associated with the HLA-Dw2 and -DR2 specificities in Caucasian Europeans and North Americans. By the use of genomic typing techniques, the association has been specified to be with the DRw15,DQw6,Dw2, i.e. the DRB1*1501-DQA1*0102-DQB1*0602 haplotype. A significant DPw4 association in Scandinavian MS patients has been described in one report. However, this association has not been confirmed in several subsequent studies with patients from the same and other ethnic groups. During the last few years several reports, based on serological, RFLP and PCR-SSO data, have suggested that the HLA class II-associated MS susceptibility gene(s) may be more closely associated with the DQ than with the DR subregion. The observations that the HLA-DQB1 genes of MS patients share long stretches of sequence motifs and also carry DQA1 alleles encoding glutamine at position 34 of the DQ alpha chain have received considerable attention. It has been suggested that the susceptibility to develop MS might be determined by the corresponding DQ alpha-beta heterodimers either encoded in cis or in trans. We have investigated these issues in a large group of Swedish MS patients (n = 179). We found that the associations with the suggested DQB1 sequences and position 34 of the DQ alpha chain were due to linkage disequilibrium and secondary to the association with the DRw15,DQw6,Dw2 haplotype (p less than 10(-9) and p less than 10(-8), respectively). No overrepresentation of the implicated DQ alpha-beta heterodimers was observed in DRw15,DQw6,Dw2-negative patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Although most patients with sarcoidosis have a good prognosis, a significant proportion runs a more severe and prolonged disease course. There is no marker to distinguish these subpopulations of patients, however. To investigate the relationship between HLA haplotype and clinical course, 122 Scandinavian patients with sarcoidosis were genomically typed for HLA-DR, -DQA1 and -DQB1 alleles using PCR amplification with sequence-specific primers. Control subjects were 250 healthy Swedish volunteers. Patients were carefully clinically monitored for up to 10 yr. We found that HLA-DR17(3) was overrepresented among sarcoidosis patients (33%) compared with control subjects (17%, p < 0.001). Ninety-one patients were followed for more than 2 yr and classified into chronic or nonchronic patients, according to disease outcome. Among the 34 patients with a nonchronic form of sarcoidosis, 65% were DR17(3)-positive (p < 10(-5) versus control subjects). On the other hand, DR14(6) and DR15(2) were significantly associated with chronic disease. Even in patients with clinical manifestations that are normally associated with good prognosis, HLA typing enabled a subgrouping into two categories with significantly different clinical courses. Therefore, HLA class II typing is a valuable tool in predicting the outcome of the disease in Scandinavian sarcoidosis patients.

Multiple sclerosis (MS) is a presumed autoimmune disease of the central nervous system, shown to be associated with the HLA class II haplotype DRB1*15,DQB1*06. Carrying the HLA class II haplotype DRB1*15,DQB1*06 increases the risk of MS by 3.6. By adopting a polymerase chain reaction (PCR)-based typing technique for HLA class I and class II genes, 200 Swedish MS patients and 210 Swedish healthy controls were analysed for their HLA alleles. Additional HLA class I alleles that increase and decrease the genetic susceptibility to MS were identified. The HLA-A*0301 allele increases the risk of MS (odds ratio=2.1) independently of DRB1*15,DQB1*06. HLA-A*0201 decreases the overall risk (odds ratio= 0.52) and the presence of A*0201 reduces the risk of MS for DRB1*15,DQB1*06 carriers from 3.6 to 1.5. Our findings are the first to identify a major modulating effect of HLA class I alleles on the susceptibility to a human autoimmune disease; a phenomenon that has previously only been observed in animal models.

The association of primary sclerosing cholangitis (PSC) to HLA class II genes was studied by comparing patients from five different European populations. Deduced HLA-DRB1, DQA1, DQB1 haplotypes of 256 PSC patients from England, Italy, Norway, Spain and Sweden were compared to those observed in 764 ethnically-matched controls. Increased frequencies of the DRB1*03, DQA1*0501, DQB1*02 (RR=3.0, P<0.00001) and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes (RR=2.4, P<0.0001) were observed in all five patient groups. A total of 16% of the PSC patients were homozygous for the DRB1*03, DQA1*0501, DQB1*02 haplotype compared to 1% of the controls (RR=20, P<0.0001). The DRB1*04, DQA1*03, DQB1*0302 haplotype was significantly reduced in frequency(RR=0.4, P<0.00001). Among Norwegian, Swedish and British patients that did not carry neither the DRB1*03, DQA1*0501, DQB1*02 nor the DRB1*13, DQA1*0103, DQB1*0603 haplotype, an increased frequency of the DRB1*15, DQA1*0102, DQB1*0602 haplotype was observed (RR=2.0, P<0.0001). Thus, PSC was found to be positively associated to three different HLA class II haplotypes (i.e. the DRB1*03, DQA1*0501, DQB1*02, the DRB1*15, DQA1*0102, DQB1*0602 and the DRB1*13, DQA1*0103, DQB1*0603 haplotypes) and negatively associated to one HLA class II haplotype (i.e. the DRB1*04, DQB1*0302 haplotype).