Molecular characterization of the HLA-DR2LUM haplotype

Abstract: Molecular studies of HLA-DRB, -DRA and -DQB1 genes in the variant DR2 haplotype, DR2LUM, were performed using the homozygous lymphoblastoid cell line, CTS. The results of HLA Class II gene RFLP and PCR analyses suggest that DR2LUM was created by a homologous recombination event between HLA-DR1 and HLA-DR15 haplotypes. Evidence for the presence of a recombinational "hotspot" in haplotypes possessing a DRB6 pseudogene is presented. The results of this study have important implications for detection of HLA-DR2 alleles in DRB gene oligotyping strategies, and suggest that the CTS cell line will be a useful addition to cell panels for characterizing HLA antisera.     

Analysis of HLA-DRB and -DQB gene RFLPs in DR7 homozygous cell lines: associations with Dw11, Dw17 and DB1

The DR7-associated Dw specificities, Dw11, Dw17 and DB1 were investigated with regard to DRB- and DQB-gene polymorphism, as revealed by RFLP analysis using the restriction enzyme TaqI. In the 22 DR7 homozygous cell lines investigated, each of these Dw specificities was found to correlate to one specific RFLP defined DR-DQ haplotype. In addition, a clear linkage disequilibrium to a specific HLA-B locus allele for each Dw specificity was noted, indicating that the Dw subtypes of DR7 often are associated with a conserved HLA-B-DR-DQ haplotype. Only one genetically homozygous cell line, PLH, deviated from these correlations. This cell line, notably derived from an individual with a deletion of the 21-hydroxylase B-gene (21-OHB), caries the HLA haplotype Bw47, DR7, DQw2, DB1, but displayed a DRB RFLP otherwise found in association with Dw17.     

HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: An alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.     

Lack of HLA-DR2—associated DRB1 gene in a family

HLA-DR51 haplotypes co-express one DRB1 (B1*15 or B1*16) gene and one DRB5 gene. These haplotypes also carry two pseudogenes (DRB6 and DRB9). During routine HLA typing for transplantation, we observed an unexpected DR51 haplotype in two subjects (mother and daughter) in a family. Serological typing showed that these subjects are positive for DR51 and DQ6 but negative for DR15 and DR16. Investigations of genomic DNA by molecular techniques showed that these individuals carry DRB5*0101, DRB6*02 and DQB1*0602 genes, whereas the DRB1 gene associated with either DR15 (B1*1501 to B1*1504) or DR16 (B1*1601 to B1*1606) was not detected in the mother and daughter. It is possible that the new haplotype, DQB1*0602, DRB6*02, DRB5*0101, arose by deletion of DR2-associated DRB1 gene.     

Multi-locus analysis of HLA class II genes in DR2-positive IDDM haplotypes in Finland

Abstract: In this study we characterized the haplotypes found in IDDM patients that normally confer resistance to the disease in order to localize the polymorphisms relevant for the protection. We studied 15 DR2-positive subjects with IDDM for their DRB1, DRB5 and DQB1 genes using RFLP, polymerase chain reaction (PCR), oligonucleotide typing, and in some specific cases direct sequencing after allele-specific PCR. In addition we analyzed 39 DR2-positive, IDDM non-associated haplotypes representing those haplotypes that are not inherited to probands and hence are present only in healthy family members. The frequency of the DRB1*1501-DRB5*0101-DQB1*0602 haplotype was slightly decreased among diabetic patients (80% vs. 92%). In addition, two unconventional haplotypes DRB1*1501-DRB5*0101-DQB1*05031 and DRB1*1501-DRB5*0101-DQB1*0502 were found in patients with IDDM while all the control ones were conventional. The sequencing of the DQB1*0602 allele present in IDDM haplotypes showed no differences when compared to the controls. These results support the primary but not absolute role of DQ in the protection against IDDM. An additional role of factors centromeric to DQB1 gene was suggested by findings based on the biallelic TaqI RFLP polymorphism of the DQA2 gene. All DR2-DQB1*0602 IDDM haplotypes were associated with the 2.1-kb fragment while in the control group the 2.1-kb and 1.9-kb fragments were evenly distributed.     

DNA typing of HLA class II genes (HLA-DR, -DQ and -DP) in Japanese patients with histiocytic necrotizing lymphadenitis (Kikuchi''s disease)

The pathogenesis of histiocytic necrotizing lymphadenitis (HNL), which was reported first by Kikuchi et al. and Fujimoto et al. in 1972, is as yet unknown. HNL is frequently reported in Asian countries including Japan, however it is rare in Europe and North America. To elucidate whether the human leukocyte antigen (HLA) alleles and haplotypes are associated with HNL, we performed DNA typing of HLA class II genes (HLA-DR, -DQ, and -DP) in 86 patients with HNL and 525 unrelated healthy Japanese controls with polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP). In this study, we found DPA1*01 and DPB1*0202 allele frequencies in HLA class II genes are significantly higher in HNL patients than in normal controls. It is known that the frequency of DPB1*0202 alleles is extremely low or absent in Caucasians (e.g., French 0.4%, Italian 0.8%) and Negroid (e.g., South African 0%, Hottentot 0%), but relatively frequent in Asians (e.g., Korean 9.9%, Japanese 4.5%). Previous reports have said the incidence of HNL is frequent in Asians but rare in other races. In light of this background, HLA class II genes of HNL and the incidence of HNL in Asian countries, including Japan, might have a positive relationship to DPA1*01 and DPB1*0202 allele.     

Analysis of proteolipid protein (PLP)-specific T cells in multiple sclerosis: identification of PLP 95 116 as an HLA-DR2,w15-associated determinant

Multiple sclerosis (MS) is a putative autoimmune disease thatis linked with HLA-DR2,w15. Proteollpid protein (PLP) is a candidateautoantigen in MS, but the disease-associated epitopes havenot been determined. Using overlapplng and non-overlapping PLPpeptides, we have studied the T cell response to the major hydrophllicdomain PLP 85–159 in the peripheral blood of MS and healthysubjects (HS). Short-term T cell lines (TCL) were selected againsteach peptide using microwell plates and the frequency of peptide-specificTCL was estimated. PLP 95–116-specific TCL were most efficientlygenerated and the frequency was significantly higher in MS comparedwith HS (P <0.05). When compared between DR2,w15⁺ and DR2,w15^–MS, TCL frequency to PLP 95–116 was significantly higherin DR2,w15⁺ MS (P<0.005) and TCL reactive to the overlappingpeptide 105–124 were also increased in DR2,w15⁺ MS (P<0.025). Using DR gene-transfected L cells, we could show thatthe DRB1^*1501 product of the DR2 haplotype presents PLP 95–116to TCL selected against the pepide. These results imply thatPLP 95–116 represents a major epitope for the DR2,w15⁺MS.     

Polymorphism and distribution of HLA-DR2 alleles and haplotypes in a Greek population

Abstract: HLA-DR2 serological subtyping has indicated that the DR16 serotype appears at a higher frequency relative to the DR15 serotype in the Greek population, differing from the distribution observed in most other Caucasian groups. In this study, we have analyzed by the PCR-SSP technique a DR2-positive group of unrelated Greek individuals selected from our normal control panel for the different DRB1, DRB5, DQB1 and DQA1 DR2-associated alleles present. Six of the 50 individuals analyzed were homo-zygous for DR2, contributing a total of 56 haplotypes for DR2. The observed frequencies of the DR2-related DRB1 alleles were as follows: 58.9% for the DRB1*1601, 7.1% for the DRB1*1602, 25.0% for the DRBl*1501 and 7.1% for the DRB1*1502 allele. The rare allele DRB1*1605 was detected in one heterozygous sample and its presence was definitively established by DNA sequencing. The alleles *1503, *1504, *1505, *1603 and *1604 were not detected. Three DRB5 alleles were identified: DRB5*0202 (67.8%), DRB5*0101 (25.0%) and DRB5*0102 (7.1%). Ten different DRB1/DQB1/ DQA1 DR2-associated haplotypes were denned. The most frequently observed haplotype was DRBl*1601-DQBl*0502-DQAl*0102 (relative frequency =57%) followed by DRB1*1501-DQB1*0602-DQA1*0102 (relative fre-quency=14.3%). In conclusion, the refined analysis of the DR2-associated DRB1 alleles in the Greek population revealed the prevalence of the DRB1*1601 allele. The rare allele DRB1*1605 was demonstrated once. A considerable variety of different DR2-related DR/DQ haplotypes was detected and the overall haplotypic frequencies in the Greek population are distributed differently compared to those reported for most other Caucasian populations.     

No effect of a BglI polymorphism at the renin (REN) locus on blood pressure level or variability

We have studied a normal restriction fragment length polymorphism at the renin locus, detected with the restriction enzyme BgII in healthy Norwegians. No association with blood pressure level or variability was found. Thus, the normal genes detected by examination of this restriction fragment length polymorphism at the renin locus have neither "level gene" nor "variability gene" effects on normal blood pressure.     

Diversity of HLA-DR2 in North Indians: the changed scenario after the discovery of DRB1*1506

Abstract: DRB1*1506, a new allele of DR2, differs from DRB1*1501 only at codon 50 in the second exon, where the nucleotide sequence has changed from GTG to GCG resulting in an amino acid substitution from valine to alanine in DRB1*1506. Since codon 50 was considered non-polymorphic until the discovery of this new allele by sequence-based typing, it became necessary to study what fraction of subjects thought to have DRB1*1501 actually had DRB1*1506. For this purpose, 116 DNA samples with DR2 coming from normal healthy individuals, leprosy patients and childhood tuberculosis patients were amplified using PCR and hybridized with ³²P-labeled probes specific for DRB1*1501, DRB1*1502, DRB1*1503, DRB1*1506, DRB1*1601 and DRB1*1602. The oligonucleotide probe for DRB1*1506 was designed to span codons 47–52 based on the published nucleotide sequence. DRB5, DQA1 and DQBl-specific amplifications and hybridizations were also carried out to study the diversity of DR2 haplotypes. It was found that 21 % of the samples identified previously as DRB1*1501 were actually DRB1*1506. DRB1*1506 was found to be associated with DQBl*0502 and DQB1*0601. Haplotypes of DRB1*1501, DRB1*1502, DRB1*1506 and DRB1*1602 showed a marked heterogeneity. Besides the rare haplotypes which have not yet been reported in any other populations, haplotypes characteristic of different ethnic groups, such as Croatians, South Chinese and Gypsies, were also found in the North Indians, suggesting the extent of racial admixture and migrations to and from India.