Histocompatibility and corneal transplantation.

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.

HLA class II associations with Type 1 diabetes mellitus: a multivariate approach.

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.

Beneficial effect of HLA-DR matching on the survival of corneal allografts.

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.

MHC class I and class II phenotype, gene, and haplotype frequencies in Greeks using molecular typing data.

In the present study, DNA typing for HLA-A, C, B, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, and DPB1 was performed for 246 healthy, unrelated Greek volunteers of 20-59 years of age. Phenotype, genotype frequencies, Hardy-Weinberg equilibrium fit, and 3-locus haplotype frequencies for HLA-A, C, B, HLA-A, B, DRB1, HLA-DRB1, DQA1, DQB1, and HLA-DRB1, DQB1, DPB1 were calculated. Furthermore, linkage disequilibrium, deltas, relative deltas and p-values for significance of the deltas were defined. The population studied is in Hardy-Weinberg equilibrium, and many MHC haplotypes are in linkage disequilibrium. The most frequent specificities were HLA-A*02 (phenotype frequency = 44.3%) followed by HLA-A*24 (27.2%), HLA-B*51 (28.5%), HLA-B*18 (26.8%) and HLA-B*35 (26.4%) and HLA-Cw*04 (30.1%) and HLA-Cw*12 (26.8%). The most frequent MHC class II alleles were HLA-DRB1*1104 (34.1%), HLA-DQB1*0301 (54.5%) and HLA-DPB1*0401 with a phenotype frequency of 59.8%. The most prominent HLA-A, C, B haplotypes were HLA-A*24, Cw*04, B*35, and HLA-A*02, Cw*04, B*35, each of them observed in 21/246 individuals. The most frequent HLA-A, B, DRB1 haplotype was HLA-A*02, B*18, DRB1*1104 seen in 20/246 individuals, while the haplotype HLA-DRB1*1104, DQB1*0301, DPB1*0401 was found in 49/246 individuals. Finally, the haplotype DRB1*1104, DQA1*0501, DQB1*0301 was observed in 83/246 individuals. These results can be used for the estimation of the probability of finding a suitable haplotypically identical related or unrelated stem cell donor for patients of Greek ancestry. In addition, they can be used for HLA and disease association studies, genetic distance studies in the Balkan and Mediterranean area, paternity cases, and matching probability calculations for the optimal allocation of kidneys in Greece.

HLA-DR4 is associated with a diminished risk of the development of chronic myeloid leukemia (CML). Chronic Leukemia Working Party of the European Blood and Marrow Transplant Registry.

CML is characterized by the chromosomal translocation t(9;22) (q34;q11) resulting in the chimeric bcr-abl oncogene that encodes P210 fusion proteins with novel amino acid sequences in the breakpoint region. If these peptides derived from P210 are presented by HLA molecules on the cell membrane of leukemic cells an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region revealed that some peptides are capable of binding to the class I molecules HLA-A2,-A3,-A11 and -B8 and the class II molecules HLA-DR1, -DR2, -DR3, -DR4 and -DR11. Moreover T cell responses have been induced against bcr-abl-derived synthetic peptides bound to some of these HLA molecules. For HLA class I, we have previously shown that individuals expressing HLA-A3 and -B8 have a diminished risk of development of CML. To assess a similar protective effect of class II molecules we performed a large multi-center study. This study compared the frequencies of HLA-DR1, -DR2, -DR3, -DR4 and -DR11 of patients with CML from the database of the EBMT (n = 1462) with unaffected individuals from the registry of Bone Marrow Donors Worldwide (n = 500 596). Patients and controls were matched per country. This analysis yielded significantly lower odds ratios (ORs) of 0.86 (95% CI 0.75-0.98) for HLA-DR3 and of 0.80 (95% CI 0.71-0.89) for HLA-DR4. The OR was 0.91 (95% CI 0.80-1.04) for HLA-DR1, 1.05 (95% CI 0.94-1.18) for HLA-DR2 and 0.87 (95% CI 0.74-1.01) for HLA-DR11. To assess a possible effect of the linkage disequilibrium between HLA-B8 and HLA-DR3 we found that coexpression of HLA-B8 and HLA-DR3 gave an OR of 0.84 (95% CI 0.72-0.98), whereas HLA-DR3 positive/HLA-B8 negative individuals showed an OR of 1.02 (95% CI 0.84-1.24). This means that the protective effect of HLA-DR3 of the development of CML was probably caused by its linkage disequilibrium with HLA-B8. In contrast, as there is no linkage disequilibrium of HLA-DR4 with HLA-A3 or HLA-B8, the results indicate that HLA-DR4 expression itself is associated with a diminished incidence of CML possibly by the presentation of bcr-abl breakpoint peptides in these HLA molecules on the membrane of the leukemic cells.

HLA-DRB and -DQB1 polymorphism in the Macedonian population.

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.

Genotyping for human platelet-specific antigens HPA-1, -2, -3, -4 and -5 in the Slovenian population reveals a slightly increased frequency of HPA-1b and HPA-2b as compared to other European populations.

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.

HLA and susceptibility to cervical neoplasia.

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.

HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukemia. The Chronic Leukemia Working Party of the EBMT.

Chronic myeloid leukemia (CML) is characterized by the chromosomal translocation t(9;22) resulting in the chimeric bcr-abl oncogene that encodes the P210 fusion protein, which contains a unique amino acid sequence. If peptides derived from the leukemia-specific part of P210 are expressed in HLA molecules on the cell membrane of leukemic cells, an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region showed that some peptides are capable of binding to HLA-A3, -A11, and -B8 molecules. Cytotoxic T-cell responses have been induced against bcr-abl-derived synthetic peptides bound to HLA-A3 and -B8. We hypothesized that if antigen processing of the P210 fusion protein leads to presentation of peptides from the fusion region by major histocompatibility complex (MHC) molecules in vivo, this may be reflected in a diminished incidence of CML in individuals expressing HLA-A3, -A11, or -B8. Consequently, lower frequencies of these antigens would be expected in patients with CML compared with unaffected individuals. A case-control study and a meta-analysis were performed to test this hypothesis. The multicenter case-control study compared patients with CML from the data base of the European Group for Blood and Marrow Transplantation (EBMT) with unaffected individuals from the registry of Bone Marrow Donors Worldwide. Patients and controls were matched per country. The meta-analysis consisted of five studies reported in the literature. The multicenter case-control study consisting of 1,899 patients and 512, 363 bone marrow donors as controls yielded odds ratios (ORs) of 0.90 (95% confidence interval [CI], 0.80 to 1.00) for HLA-A3, 1.16 (95% CI, 1.02 to 1.33) for HLA-A11, and an OR of 0.73 (95% CI, 0.65 to 0. 82) for HLA-B8. Coexpression of HLA-A3 and HLA-B8 gave an OR of 0.51 (95% CI, 0.40 to 0.67). This can be translated in a protective effect of 27% for HLA-B8, 10% for HLA-A3, and 49% protection for the combination of HLA-A3 and HLA-B8. The meta-analysis comprising 463 CML patients and 4,912 controls showed a 29% risk reduction for individuals expressing HLA-B8 (OR of 0.71; 95% CI, 0.52 to 0.97), but an OR of 1.19 (95% CI, 0.90 to 1.56) for HLA-A3 and an OR of 1. 09 (95% CI, 0.80 to 1.50) for HLA-A11. In conclusion, these results indicate that HLA-B8 expression, in particular when HLA-A3 is coexpressed, is associated with a diminished incidence of CML. A biological mechanism may be that presentation of bcr-abl breakpoint peptides in these HLA molecules can induce a protective immune response.

Selective co-evolution of the D6STNFa microsatellite region with HLA class I and II loci.

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.

Validation of haplotype frequency estimation methods.

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.

Bone Marrow Donors Worldwide and cord blood stem cell transplantation.

Stem cell transplantation using cord blood donors mismatched for one or two HLA antigens can be successful. A simulation study was performed to assess the percentage of patients for whom a matched and/or a one or two antigen mismatched donor is available. Using either bone marrow donors worldwide (BMDW) (n = 925109) or the cord blood donor file in BMDW (n = 11049). As a donor source, for 97% of the Dutch patients a 0 or 1 antigen mismatched cord blood donor was available, for patients from the minority groups this percentage was 55%. These percentages compare favourably with the fraction of 0 mismatched donors found in BMDW.

HLA-DRB1, DQA1, and DQB1 genotypes and risk of vasculitis in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the association of rheumatoid vasculitis (RV) with the expression of the shared epitope, HLA-DRB1*04, DQA1, and DQB1 alleles. METHODS: A case-control study was designed to compare the frequency of the shared epitope, DRB1*04 alleles, DQA1, and DQB1 alleles of 31 patients with RV with that of 76 patients with rheumatoid arthritis (RA) without vasculitis. RV cases were discerned in subgroups according to presence or absence of minor skin vasculitis and major organ lesions. HLA-DRB1, DQA1, and DQB1 typing was performed by a polymerase chain reaction amplification and oligonucleotide hybridization. RESULTS: Overall, no associations were found between RV and the shared epitope, the DRB1*04 alleles, DQA1, or DQB1 alleles. The risk of developing RV with minor skin vasculitis (i.e., purpura or petechiae) was 10-20-fold increased in patients with DRB1*04, in particular in those homozygous for DRB1*04, and also in those with DRB1*0401. No such association was found for patients with major organ lesions. CONCLUSION: The occurrence of vasculitis in RA is not associated with the shared epitope. DQA1, or DQB1 alleles. However, the risk of minor skin vasculitis is higher in patients carrying DRB1*04.

HLA gene haplotype frequencies in bone marrow donors worldwide registries.

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.

Minimal phenotype panels. A method for achieving maximum population coverage with a minimum of HLA antigens.

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.

HLA gene and haplotype frequencies in Dutch blood donors.

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.

Validation of large data sets, an essential prerequisite for data analysis: an analytical survey of the Bone Marrow Donors Worldwide.

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.

The probability of finding a suitable related donor for bone marrow transplantation in extended families.

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).