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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.

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.

Some combinations of HLA-A, -B and -DR antigens occur more frequently than would be expected from their gene frequencies in the population. This phenomenon, referred to as Linkage Disequilibrium (LD) has been the origin of many speculations. One hypothesis to explain LD is that some haplotypes are protected from recombination. A second hypothesis is that these HLA antigens preferentially recombine after cross-over to create an LD haplotype. We tested these 2 hypotheses: from a pool of over 10,000 families typed in our department, we analyzed 126 families in which HLA-A:B or B:DR recombinant offspring was documented. To overcome a possible bias in our material, we used the non-recombined haplotypes from the same 126 families as a control group. Our results show that the number of cross-overs through LD haplotypes is not significantly lower then would be expected if recombination occurred randomly. Also the number of LD haplotypes created upon recombination was not significantly increased.

Analysis of the reaction patterns of 57 putative class II MHC alloantisera revealed 18 B-cell serum clusters, named Ds01-Ds18. These clusters were grouped together in such a manner that 17 provisional serological class II haplotypes were defined. Segregation in half-sib families of these class II haplotypes with serologically defined class I and 1D-IEF defined DRB3 types indicated that the 18 clusters identify class II polymorphisms in cattle and can be considered as local specificities.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

Eleven Ia or B-cell specificities of rhesus monkeys have been serologically identified. They are probably all controlled by the RhLa complex, but, according to results of a large population and family study, only five or six can be placed in a segregant series, designated as Ia1. Data from recombinant offspring in two rhesus families place the Ia1 locus in the close vicinity of the D or major MLC locus within RhLA. The genetic control of the Ia antigens not defined by the Ia1 locus has not yet been clarified.

A reassessment of HLA-A and B antigen associations in patients with severe aplastic anaemia confirmed the significantly elevated frequency of HLA-A2 in Caucasoid patients reported by Albert et al. (1976). In addition, a highly significant increase in the frequency of HLA-B14 in Italian patients was observed in an initial sample of 25 patients and confirmed in a second sample of 16 patients. This latter finding was cancelled out when the Italian Caucasoid patients were pooled with the non-Italian Caucasoid patients. These results suggest that the classification of populations as "Caucasoids", without examining their ethnic origins, may introduce a serious confounding error into HLA and disease studies.

The serology and genetics of 17 serologically defined tissue antigens of chimpanzees is described. Analysis of their distribution in 200 unrelated chimpanzees and their segregation in a large number of chimp families suggests that 14 of the antigens are controlled by two closely linked loci: seven by the A and seven by the B locus of ChLA. Typing of chimpanzees with human alloantisera revealed that several of those A and B locus antigens of ChLA are the chimp's serological counterparts of A and B locus antigens of the human HLA system, respectively. One of the newly discovered chimp antigens may be defined by a "third series" of ChLA, most likely the chimp's analogue of the human HLA-C series. The probable existence of a ChLA-linked D locus of chimpanzees and similarities between D-locus and Ia-locus (DRw) antigens of chimpanzee and man, are also briefly discussed.

Histocompatibility antigen testing has been carried out in 20 unrelated normotensive English Caucasoids with hypertrophic cardiomyopathy and in 17 relatives, six of whom also had the disease. A strong association with the HLA-DRw6 antigen complex was found (P = 0.0036) but it was no longer significant after correction for the 52 antigens tested (P = 0.1701).

Chimpanzees were allo-immunized with blood from donors, ChLA-A and -B identical to the recipient. The sera of some of these animals contained antibodies that reacted only with the platelets or granulocytes of the immunizing donor and of some related and unrelated chimpanzees. Both for the platelets and the granulocytes a di-allelic system of allo-antigens is described, provisionally called ChPL-1 and ChGR-1.

The use of specific cDNA probes and selected combinations of restriction endonucleases 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.

The status of the W4, W6 system as related to the HL-A system was re-evaluated by means of segregation and mating studies in 200 Dutch families with 755 children. Inclusion analyses were performed on the Fifth Workshop human populations and related to a reference analysis on the main file in Leiden. The results of the mating and segregation analyses showed a good fit for W4 and W6 as a genetic system. The inclusion analyses showed a high degree of concordance. Based on those results, and other cited evidence, it was suggested that W4 and W6 may represent the basic substance of the HL-A antigens in the FOUR series.

The combination of the Propidium Iodide method and automatic reading and scoring with a microscope fluorimeter is an accurate, reproducible and stable procedure for HLA-DR, MB and MT typing.

The frequencies of the HLA-A, B, C, D, DR and MB antigens have been determined in a homogeneous Shanghai Chinese population. Comparisons with the HLA-A and -B frequencies in other subsets of the Chinese population revealed some marked differences. No comparisons were possible for the D, DR and MB antigens since there were no previous studies of the antigens in those loci. We suggest that studies of the Chinese population should be confined to clearly defined homogeneous subsets. In this manner, the confounding effect of population heterogeneity may be avoided, and it is this heterogeneity which calls for extensive surveys of the huge Chinese population.

HLA-DQ alpha DNA polymorphism was studied by Southern blot analysis in a panel of 117 individuals, consisting of 56 randomly selected and 61 HLA-DR homozygous individuals. Hybridizing fragments representing DQ alpha genes correlate with DR specificities owing to linkage disequilibrium between DQ and DR. Two fragments representing DX alpha genes were identified with the restriction enzymes PvuII and TaqI and a DQ alpha cDNA probe. The two fragments of PvuII-DQ alpha hybridization (a 7200 and a 7000 basepair fragment) and the two of TaqI-DQ alpha (2200 and 1900 basepairs) have an identical distribution in the panel, and reveal gene frequencies of 49.1% and 50.9%; they behave as alleles of the putative DX alpha locus. The panel study shows that the DX alpha fragments are not linked with the DR or DQ specificities but segregate along with HLA as shown in family studies.

A group of 43 Australian Aborigines was typed for HLA--DR determinants and other antigens on B cells, using pregnancy sera from Caucasoid women. The group could not be considered as random, since a selection was made for an equal number of Au antigen positive and Au antigen negative individuals The typing results showed that the specificities DRw2, 4 and 8 were as well defined in this population as in Caucasoids, whereas the specificities DRw1, 3, 5 and 7 could not be defined. Even more interesting was the finding that a cluster of six sera, defining the local B cell specificity LB12, gave positive reactions in 41 out of 43 Aborigines. Although LB12 is found to be highly associated with DRw1, 2 and 6 in the Caucasoid population, in 36 cases the LB12 positive Aborigines could not be demonstrated to carry the specificities DRw1 or 2. The recognition of DRw6 is difficult at the moment and for the reason its presence or absence in this material is uncertain. However, three individuals typed positive for DRw4 and 8 and for LB12 as well. These findings strongly support the hypothesis that the genes coding for LB12 and its postulated allele LB13 are separate from the genes coding for the known DR determinants. LB12 is very likely identical to the recently described determinants DC1 (Tosi & Tanigaki 1979) and MB1 (Duquesnoy et al. 1979a).

We have shown that the application of Piazza's formula for the estimation of the "three-locus interaction delta" should not be used for that purpose. In addition, the method of Porta & McHugh, for the demonstration of haplotype interaction in associations between HLA and disease, leads to completely erroneous conclusions. Other methods of calculation that have been used to analyse haplotype interactions also appear to be based on erroneous concepts. We suggest that nothing more than the estimation of heterogeneity in simple 2 X 2 contingency tables should be used for the analysis of linkage disequilibria. This principle is applied to the HLA-A, -B and -C haplotype frequency tables of Baur & Danilovs. For the greater part of the HLA-B alleles, three-locus haplotype frequencies can be explained from the A, B and the B, C disequilibria, without any further haplotype interactions. The predominant exceptions in that respect are haplotypes containing the B44 allele, which has been shown to contain two subgroups and therefore, we are not justified to conclude that this exceptional behaviour of haplotypes with B44 should be attributed to three-locus haplotype interactions.

Seventy-seven patients with rheumatoid arthritis were investigated to examine the frequency of HLA antigens and their relationship to clinical and serological manifestations of extra-articular disease. The phenotype frequencies of DR4, DRw53, Bw62 and Cw3 were significantly increased, compared to normal controls, and there were negative associations with DR2 and DR7. The HLA antigen in strongest association with rheumatoid arthritis was DR4 (73.6%) and the relationship with DRw53 appeared to be secondary. The frequency of DR4 rose to 92% in seropositive patients with extra-articular disease manifestations whose serum contained immune complexes. A high frequency of DR4 was also seen in male patients (86%), reaching 100% in the small group of seropositive male patients with immune complexes. It is suggested that extra-articular disease represents a manifestation of severe classical rheumatoid arthritis and is not an 'overlap' syndrome. We propose that the HLA haplotype Cw3-Bw62-Dw4-DR4-DRw53 makes a greater genetic contribution to disease susceptibility in both extra-articular and male rheumatoid arthritis patients than in other subsets of RA.

The main purpose of Eurotransplant, an international organ exchange organisation, is to maximize the survival time of transplanted organs through tissue typing and matching. Regular proficiency testings are necessary to assess the reproducibility and the reliability of the HLA typings performed by the individual typing centres. Two different protocols have been used: 1) since 1978, 35 samples of blood and donor spleen were used for cell exchanges (CE) and typed by all participating laboratories; 2) since 1977 samples of donor spleen have been shipped to the reference laboratory in Leiden for retyping (RD) resulting in over 2600 comparable HLA typings. The discrepancy rates of the HLA-A, B, C and DR specificities were analysed over the years. A large number of discrepancies was due to false negative assignments which led to assumed homozygosity at one or more HLA-loci. The recognition of HLA-DR has improved dramatically but with much variation per DR specificity. The concordancy rates for HLA-AB + DR have improved from 40% in 1981 to 91% for CE and 80% for RD typings in 1984. The reproducibility of DR typings in individual laboratories also has improved greatly but there is still considerable variation between the different laboratories.

A study of the distribution of histocompatibility (HLA) antigens in patients with Raynaud's phenomenon (RP) is presented, based upon the analysis of RP patients with and without connective tissue disease (CTD). In all patients, the phenomenon preceded the other symptoms of CTD by several years. The RP patients with CTD showed a significantly increased frequency (59%) of HLA-B8 compared with the frequency (23%) of that same antigen in the Dutch population. The RP patients without CTD showed no significant difference in frequency of HLA-B8. At least 50% of the patients with RP and HLA-B8 develop a CTD versus 18% of the patients without HLA-B8. It is suggested that HLA testing in patients with isolated RP may help to distinguish the patient in whom RP is the first symptom of CTD from the patient with truly primary RP.

Thirty Ethiopian malnutrition-related diabetes mellitus (MRDM) patients were HLA typed and their HLA antigen frequencies were compared to those of 31 previously typed insulin-dependent diabetes mellitus (IDDM) patients and to 84 controls from the same ethnic background. In comparison to controls, a striking association between MRDM and HLA-DR3 (X2 = 15.15, p = 0.0001) was observed, whereas the frequency of HLA-DR4 was non-significantly increased (RR = 1.72). The frequency of DR2, DQw1, and DQw6 was decreased among MRDM. In comparison to IDDM that is associated with both DR3 and DR4 in this population, MRDM showed no significant differences in HLA class II antigens frequencies. Therefore, the genetic basis of susceptibility to MRDM and IDMM in Ethiopia is at least partially identical.

A population study to test whether associations between HLA and sporadic--i.e. non-familial--tuberculoid leprosy exist was undertaken in a hyperendemic area in India. Since previous family studies in the same area had shown both non-random haplotype segregation in the family members affected with tuberculoid leprosy and the preferential segregation of HLA-DR2 into tuberculoid leprosy patients, an increased frequency of DR2 among the "sporadic" patients was expected. However, no heterogeneity for HLA was detected between patients and controls. These findings could indicate that tuberculoid leprosy is a heterogeneous disease with regard to genetic background.

Five new serologically defined (SD) tissue antigens of rhesus monkeys are described. Results of a population study and a segregation analysis in families were consistent with their control by the major histocompatibility complex (MHC), as alleles of the two previously established SD loci of RhL--A. The number of identifiable SD specificities of the rhesus monkeys' MHC is now twenty-five, thirteen controlled by the SD1 locus and twelve by SD2. The recombination frequency between SD1 and SD2 is estimated to be 0.3%. No evidence of a third SD series, as analogue of the human HLA--C locus, has yet been found.

Sixty-two Dutch patients with the diagnosis paranoid schizophrenia (SCH) were HLA-A-, -B- and -C-typed. An increase in the frequencies of A9 (P = 0.02, relative risk 1.8) and B5 (P = 0.04, relative risk 1.9) was found. Although these correlations do not remain significant after correction for the number of antigens tested, both findings confirm other data from the literature, including the first published report from a population in Sweden. From all hitherto published literature data, the combined relative risks for A9 and B5 is significantly increased. These data strongly indicate that the distribution of HLA antigens among SCH patients is different from the control population.