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The association of HLA class II phenotype with the development of insulin-dependent (Type 1) diabetes mellitus (IDDM) is well established but the contribution of the various HLA-DR and -DQ alleles and haplotypes to disease predisposition is not fully understood. We have determined haplotype and genotype odds ratios, and further employed multivariate tree analysis to explore the contribution of individual HLA-DRDQ haplotypes to the genetic risk for developing IDDM in the Dutch population. Next to haplotype and genotype odds ratios, multivariate tree analysis techniques provide overall risk calculations for each modeled parameter, and offer insight in the interaction of the model parameters via tree-shaped reports, in which subsequent stratifications on the data can easily be followed. We compared 206 Dutch IDDM patients with 840 serologically typed random healthy unrelated Dutch Caucasoid controls. The multivariate tree analysis showed that the HLA-DR7DQ9 and DR15DQ6 haplotype were strongly associated with disease protection (OR = 0.04, P = 0.0003, and OR = 0.07, P < or = 0.0001, respectively). The highest ORs were found for the DR4DQ8/DR8DQ4 genotype (OR = 21.04, P = 0.001), followed by DR4DQ8/DR17DQ2 (OR = 12.45, P < 0.0001) and DR9DQ9/DR17DQ2 (OR = 10.87, P = 0.02). DR4DQ8 homozygous and DR17DQ2 homozygous individuals have a disease OR of 9.0 and 3.0 (P = 0.01 and 0.03), respectively. In conclusion, the results from haplotype, genotype, and tree analyses provide insight into the disease associations for combinations of HLA-DRDQ haplotypes. We confirm that the DR9DQ9/DR17DQ2 genotype is associated with susceptibility in the Dutch population, which has previously been noticed as a HLA risk genotypes in Asian populations only.

Background. Although HLA typing and matching have been used for 3 decades, that practice has been poorly implemented in corneal transplantation, mainly because of inconclusive or contradictory analytical results. Consequently, we studied the immune response of corneal transplant recipients to HLA histoincompatibilities in a large homogeneous study. Methods. All corneal transplantations performed by a single surgeon between 1976 and 1996 were studied. HLA-AB matching was used for recipient selection. All HLA typings were performed by a single experienced laboratory. Population genetic techniques were used to assess the validity of the HLA typings. Mono- and multivariate analyses were performed to identify the factors which significantly influence the survival of corneal allografts. Simulation studies were carried out to demonstrate the effects of mis-typed donor and recipient HLA-DR typings on analytical results. Results. Retransplantation, degree of vascularization, HLA-AB and DR matching, endothelial cell count, graft size, recipient gender, and storage method were identified as significant factors by our monovariate analyses. A Cox proportional hazards survival analysis model identified degree of vascularization and HLA-AB and DR matching as significant prognostic factors when all immunological rejection episodes were used, P=0.000001. When only irreversible immunological rejection episodes were used, panel reactive antibodies, retransplantation, and number of rejection events were also identified, P=0.000001. Simulation studies showed that the effects of HLA-DR matching are abrogated by poor HLA-DR typings. Conclusions. Corneal allograft recipients have a normal alloimmune response to histoincompatibilities. Demonstration of that fact requires accurate HLA typings.

HLA-DRB1, DRB3/4/5 and DQB1 polymorphism has been studied in a population of 80 unrelated healthy Macedonians using molecular methods. Twenty-five different DRB1 alleles were identified of which DRB1*1104, *1501, *1601, and *1101 were found most frequently. Among the 15 identified DQB1 alleles, two were predominant: DQB1*0301 and *0502. The most frequent three-locus haplotypes were DRB1*1104-DRB3*02-DQB1*0301 (18%/), DRB1*1101-DRB3*02-DQB1*0301 (9%) and DRB1*1601-DRB5*02-DQB1*0502 (10%). Polymorphism for DRB1*04, *13 and *15 haplotypes was extensive. Eleven different DR2-related haplotypes were found, some of which were unusual for European populations: DRB1*1501-DRB5*0102-DQB1*0502, DRB1*1501-DRB5*02-DQB1*0502, DRB1*1501-DRB5*0102-DQB1*0601.

Typing of human platelet alloantigens (HPA) is necessary in various clinical situations. The purpose of this study was to type a random sample of the Slovenian population for HPA alleles, in order to obtain genetic population data. A total of 152 unrelated Slovenian blood donors were genotyped for HPA-1, -2, -3, -4 and -5 alleles using a simple method that enables simultaneous and complete determination of HPA genotypes. Ten different polymerase chain reactions employing sequence-specific priming (PCR-SSP), which worked in identical cycling conditions, were used. The allele frequencies were 0.809 for HPA-1a, 0.191 for HPA-1b, 0.891 for HPA-2a, 0.109 for HPA-2b, 0.591 for HPA-3a, 0.407 for HPA-3b, 0.997 for HPA-4a, 0.00 for HPA-4b, 0.934 for HPA-5a and 0.066 for HPA-5b. When compared to results of studies of various other Caucasian populations, our population displayed a slightly but not significantly higher proportion of the HPA-1b and 2b alleles.

The association between cervical neoplasia and certain HLA phenotypes observed in different studies has not been consistent. By serological typing, the association between HLA antigens, cervical carcinoma and cervical intraepithelial neoplasia (CIN) was studied in a group of 172 and 116 patients, respectively. We demonstrated an increased frequency of B63 in patients with HPV types other than HPV 16 or 18, and B55 in patients that were negative for all HPV types. The association between cervical carcinoma and DQ3, described in various populations, was not observed in the present study. However, we confirmed other previously observed associations between cervical cancer and class II antigens, i.e., a positive correlation with DR15 irrespective of the HPV status, with DR3 in patients harboring HPV types other than HPV 16 or 18, and with DR11 among HPV 16 positive patients. In contrast, a negative correlation between DR13 and HPV positive cervical cancer was observed which suggests protection of this antigen against HPV-associated cervical cancer. A slight increase of DR15 and DQ4 antigens was observed in CIN patients, suggesting that these specific HLA antigens may be important in determining the risk of CIN.

Vaccination with peptides that induce a specific immune response is a potential prophylactic or therapeutic strategy against viral infections and tumors. Because of the extensive polymorphism of the HLA loci, synthetic peptide vaccines must consist of a cocktail of peptides that bind specifically to different HLA molecules. Such cocktails should be optimized for the target population as each population has its specific HLA gene frequencies. To achieve maximum population coverage with a minimum number of peptides, information is needed on the ranking of the most frequent HLA phenotypes. We introduce the minimal phenotype panel, which is the smallest combination of HLA antigens selected so that the proportion of individuals in a population that express at least one of the antigens in the panel exceeds a desired minimum value. We developed a method for assembling minimal phenotype panels based on known HLA class I gene frequencies. We give an example based on a set of 2446 well-defined HLA-typed, random, healthy, unrelated, Dutch Caucasoid individuals. In addition, we discuss the possibility of assembling minimal phenotype panels based on two-locus haplotypes, which enables the assembly of phenotype panels from the antigens of both loci.

We analyzed the HLA-A, -B, -C, -DR and -DQ phenotypes of 2,440 healthy, unrelated, Dutch Caucasoid blood donors and of 20,814 Dutch blood donors who were registered as volunteer bone marrow or platelet donors. Phenotype and gene frequencies, Hardy-Weinberg equilibrium fit and homozygosity were calculated as well as 2- and 3-locus haplotype frequencies, deltas, relative deltas and significance levels of the deltas. The population appears to be in Hardy-Weinberg equilibrium. Many haplotypes are in strong positive linkage disequilibrium. A phylogenetic tree, based on the HLA-A, -B and -DR gene frequencies of blood donors in different Dutch regions, reflects the limited but manifest heterogeneity of the Dutch population. Additionally we introduce a stepwise test for Hardy-Weinberg equilibrium and discuss the applicability of this test and of the single test for Hardy-Weinberg equilibrium for tissue typing quality control and for selection of split antigens prior to gene and haplotype frequency analyses.

In bone marrow transplantation, the advantages of family donors over unrelated donors are threefold. (1) Family donors are better matched because they share complete haplotypes. (2) The time between the start of the search and the actual transplantation can be much shorter than for unrelated donors. (3) Related bone marrow transplantation is cheaper. We developed a procedure for calculating the probability of finding a suitable donor among cousins and blood-related aunts and uncles (the extended family). The procedure calculates the following probabilities (P): (1) P that any blood-related uncle or aunt is a suitable donor, (2) P that a suitable donor exists in all blood-related uncles/aunts (from [1]), (3) P that any cousin is a suitable donor (as in [1]), (4) P that a suitable donor exists in all cousins (from [3]), (5) P that a suitable donor exists in the entire extended family (from [2] and [4]). Additionally, we discuss the suitability of this procedure in the daily practice of donor procurement. The procedure is also suitable to search for family donors who have a single antigen mismatch with the patient. We also discuss the differences between our method and the one recently described by Kaufman (Bone Marrow Transplant 15:279, 1995).

Vaccination with peptides recognized by antigen-specific CTLs can prevent lethal virus infections and tumor growth. In order to avoid the synthesis and testing of the numerous overlapping peptide of long AA sequences of proteins of interest, we developed a computer program which utilizes the rules, "motifs" which govern how peptides bind to HLA class I molecules, to derive a predicted binding score for each overlapping peptide. Correlations between the predicted and actual binding results to HLA-A*0201 for 100 peptides selected from six early and two late protein sequences of human papillomavirus type 1a revealed an acceptable level (61%) of concordance. The program is very flexible with regard to the input of protein sequences and motif definitions and is able to handle various motif and peptide lengths.

BACKGROUND HLA typing and matching have been poorly implemented in corneal transplantation, mainly because of inconclusive or contradictory analytical results. Consequently, we studied the immune response of corneal transplant recipients to HLA histoincompatibilities in a large homogeneous study. METHODS All corneal transplantations were performed by a single surgeon in a single center between 1976 and 1996. Population genetic and other statistical analyses were performed. Simulation studies assessed the effects of HLA-DR mistypings on analytical results. RESULTS Mono- and multivariate analyses identified retransplantation, degree of vascularization, HLA-AB and -DR match grades, endothelial cell count, graft size, recipient gender, storage method and panel-reactive antibodies as significantly influencing the survival of corneal transplants. Simulation studies showed that the beneficial effect of HLA-DR matching is abrogated by HLA-DR mistypings. CONCLUSIONS Corneal transplant recipients have a normal immune response to HLA incompatibilities. Demonstration of that fact requires accurate HLA typings.

We analyzed the HLA-A, -B, -DR and -DQ phenotypes and 12 microsatellite locus genotypes within and close to the major histocompatibility complex in a panel of 98 randomly selected, healthy, unrelated Dutch Caucasoid individuals. Allele frequencies and Hardy-Weinberg equilibrium (HWE) were calculated. Also, the linkage disequilibrium patterns between HLA and microsatellite loci were studied. The HLA-A, -B, -DR, -DQ and six microsatellite loci centromeric of the HLA-A showed HWE. In contrast, all microsatellites telomeric of the HLA-A showed deviation from HWE due to excess of homozygosity. Linkage disequilibrium analyses provided strong evidence that among the tested microsatellite loci only the alleles of the D6STNFa locus are in linkage disequilibrium with both HLA-B and -DR. Our results suggest that selection acting on the HLA genes includes the D6STNFa locus and linked genes.

In order to investigate the performance of haplotype frequency estimation methods using unrelated individuals, we compared the results of three estimation methods with those from the haplotypes deduced from family pedigrees. To that end we used the HLA phenotypes of the parents of 1040 families as data for the estimation methods and the full pedigree information as data for the deductive method. We evaluated the results of the following estimation methods: the method using two by two tables described by Mattiuz et al ., the maximum likelihood method described by Yasuda and Tsuji and a crude method that uses the information on homozygosity in the phenotypes. All estimation methods generate reliable haplotype frequencies for the more frequent haplotypes, but are unreliable for the less frequent haplotypes. The maximum likelihood estimation method shows the best overall correlation with the results of the deductive method.

A recent case-control study in Indonesia suggested that the course of Brugian filariasis and in particular resistance to the development of elephantiasis was associated with certain HLA class II alleles. In order to see whether these data could be confirmed we conducted a similar study in another Indonesian population from South Sulawesi. We could not confirm our earlier results and therefore concluded that HLA-DR and -DQ alleles are at least not strongly associated with progression to elephantiasis in Brugian filariasis. The complete data are presented also for anthropological reference purposes.

To calculate reliable HLA gene and haplotype frequencies of bone marrow donors in various regions in the world, we have analyzed the HLA-A, -B, and -DR phenotype frequencies of 18 bone marrow donor registries with a total of more than 300,000 HLA-A, -B-typed donors. These registries were included in the 22nd edition of Bone Marrow Donors Worldwide. Maximum likelihood gene frequencies, Hardy-Weinberg equilibrium fit, and 2- and 3-locus haplotype frequencies were calculated as well as deltas, relative deltas, and their significance. Remarkable gene and haplotype frequency differences exist between the registries. The genetic distances between the different registries were used to draw phylogenetic trees that clearly show that the degree of similarity between registries is related to their geographic locations. The resulting frequencies can be used for the estimation of the probability of finding a hyplotypically identical related or unrelated bone marrow donor for an individual patient. Phylogenetic trees are useful representations of the similarity between donor pools and can also aid in the selection of donors.

Large data sets like the Bone Marrow Donors Worldwide (BMDW) data set can be used for population genetic analyses. The qualities of such data sets are unique. To be able to use the BMDW data for analyses, several problems, like limited size and selective DR typing, of the data have to be solved and the quality of the registry data subsets has to be examined. We describe these problems and methods to overcome them. Also, we give an overview of the qualities of the different registry subsets. Sixteen of the twenty-nine examined subsets contain data that can be used for population genetic analysis. We will deal with these analyses in the future. Additionally, we present a method to calculate the minimum number of individuals required for reliable haplotype frequency estimation.

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.

The use of specific cDNA probes and selected combinations of restriction endonu-cleases makes it possible to characterize specific antigens by the molecular weights of the hybridized restriction enzyme digests of DNA. We have developed a simple program suitable for personal computers which utilizes such results to deduce HLA-DR phenotypes of any nucleated cell from which DNA may be extracted.