HLA antigen and gene frequencies in Eskimos of East Greenland.

A population of 78 Ammassallik Eskimos was tested for HLA-A, B, Cw, DR and DQ specificities using serological techniques and HLA-Cw, DRB, DQB1 and DPB1 using three different genomic techniques. The application of two new genomic techniques for HLA-Cw (PCR-SSP phototyping) and HLA-DPB1 (PCR-RLFP) typing confirmed serological results and gave a higher resolution, with more heterozygotes than previously found. High gene frequencies of HLA-A24 (0.80), B51 (0. 21), B61 (0.30), B62 (0.21), Cw*0303 (0.27), Cw*0304 (0.51), DRB1*0401 (0.45), DRB1*1402 (0.24), DQ7 (0.88), DPB1*0201 (0.18), DPB1*0401 (0.40) and DPB1*0402 (0.30) were found. A limited number of alleles were found at all HLA loci. The Ammassallik Eskimos of East Greenland are genetically homogenous with little, if any, admixture with European populations. This contrasts with other Greenlandic populations formerly tested. The pattern of alleles indicates a close genetic relationship with other Eskimo populations.

Population studies of the human V kappa A18 gene polymorphism in Caucasians, blacks and Eskimos. New functional alleles and evidence for evolutionary selection of a more restricted antibody repertoire.

Immunoglobulin gene polymorphisms are interesting because they reflect differences in the available antibody repertoire which may affect the susceptibility to specific infections. Until recently, the human V kappa gene, A18, was known as a nonfunctional gene only. In this study, we cloned and sequenced four apparently functional alleles and determined the gene frequencies in three well-defined populations: Danish Caucasians, eastern Greenland Eskimos and Mozambican blacks. The A18b allele that was recently described in Native American Navajos by Atkinson et al. was found in all three populations with gene frequencies of 8%, 45% and 23% in Caucasians, Eskimos and blacks, respectively. Conversely, the frequencies of the nonfunctional A18a allele were 92%, 55% and 57%. Further, three new A18 alleles, c, d, and e were found exclusively in blacks, among whom they had an total frequency of 19%. These data indicate that both the A18a and A18b alleles originated before the diversification of Africans and non-Africans 90,000 years ago, whereas the A18c, A18d and A18e alleles may have a more recent origin. The functionality of the A18b allele was documented by the demonstration of properly rearranged and somatically hypermutated A18b messenger RNA present in the blood lymphocytes of individuals carrying this allele. The expression clearly exceeded that of a known functional V gene, A2, indicating that functional A18 alleles contribute significantly to the available antibody repertoire. In this context, it is surprising that the functional A18b allele apparently has been negatively selected in the Caucasian population, among whom 85% completely lack a functional gene.

Antigenic characteristics and cDNA sequences of HLA-B73.

The cDNA sequence and serological data for HLA-B73 are reported. Anti-B73 sera are found relatively frequently, considering the rarity of the antigen. It was noted early that in some cases the antibodies in sera of multiparous women did not react with the eliciting cells (fathers) and thus all behaved as a naturally occurring antibody. We report on 18 B73 antisera found during the screening of 55,000 Danish sera. Only one of the 17 stimulators typed also had the B73 tissue type. Ten of the stimulators had antigens from the B7 CREG (B7, B22, B27, B42, B67, B73), whereas none of the responders had such tissue types. In seven cases the serum was not able to react with the stimulator's lymphocytes in a cytotoxicity assay and in four cases the stimulator lymphocytes could not deplete the anti-B73 activity from the serum in absorption experiments. The cDNA of B73 was expressed correctly in COS cells and was recognized on the cell surface by a monospecific serum. The alpha 1 alpha 2 domains of B73 are most similar to those of the HLA-B22 family. Interestingly, the alpha 3 and transmembrane domains of HLA-B73 are not standard human domains, but are most similar to the corresponding domains of some gorilla and chimpanzee HLA-B genes.

Clinical applicability of the immunomagnetic beads technique for serological crossmatching in renal transplantation.

The aim of the present prospective study was to investigate the clinical applicability of the immunomagnetic (IM) beads technique for serological crossmatching (XM) in renal transplantation. The IM XM were read after various periods of incubation, and the results were compared with those obtained by the conventional Kissmeyer-Nielsen (KN) technique. A total of 132 sera from 96 potential recipients were tested against cells from 62 donors. Eight-nine KN T-cell XM-negative renal allograft transplantations were performed, and the IM XM results were related to clinical 3-month follow-up data (incidence of primary non-function, never functioning grafts, graft losses and rejection episodes). The IM technique was clearly more sensitive than KN, and sensitivity increased markedly with increasing duration of incubation. KN-, IM+ reactions were predominantly found among sera from patients with panel-reactive antibodies (PRA, 2p less than 0.01), and thus probably caused by HLA antibodies. However, positive IM XM, appearing after more than 35 min of incubation, did not influence the overall clinical outcome in the observation period. With reading after exactly 35 min of incubation, XM results obtained by IM and KN techniques correlated well. Thus, we believe, that the IM XM technique will be as safe and effective in avoidance of hyperacute rejections as the conventional assay. In the present material, the incidence of primary nonfunction was significantly (2p = 0.0023) higher among PRA+ recipients compared to PRA- patients. To conclude, we recommended the IM technique with reading after exactly 35 min of incubation for easy, fast (70 min) and reliable XM, that is always possible to perform using peripheral blood.

Lymphocytotoxic cross-matching performed on spleen cells: immunomagnetic technique versus current KN (Kissmeyer-Nielsen) technique.

This paper compares the immunomagnetic (IM) and the KN lymphocytotoxic cross-matching techniques. Only sera from patients with panel reactive antibodies and spleen cells from cadaveric donors were used. A panel study involving 60 combinations revealed 11 (18%) discrepancies. Four T-cell cross-matches were KN negative while IM positive. Inversely, six T-cell cross-matches were IM negative while KN positive (two) or doubtfully positive (four). Regarding HLA class II antibodies, one combination was found IM positive but KN negative. Out of 106 cadaveric renal allograft transplantations performed during 1987 with assistance from our laboratory, IM cross-matching was retrospectively performed on 33 KN T-cell cross-match negative donor-recipient pairs: two combinations were found T-cell cross-match positive. The corresponding allografts were not lost owing to hyperacute rejection involving performed antibodies. We recommend the IM technique for HLA typing, but more experience needs to be gained before we can recommend the technique for routine cross-matching prior to transplantation.

HLA-DR typing using restriction fragment length polymorphism (RFLP) with one enzyme and two probes.

A panel of 43 homozygous and 36 heterozygous highly selected cells, representing the most common DR-specificities, were investigated with the DNA hybridization technique. By using a single restriction enzyme, TaqI, and two probes, DR beta and DQ alpha, it was possible to construct assignment criteria giving a reasonable definition of DR1, 3, 4, 5, 7 + w9, w8, w10 and w11. The criteria sometimes require that certain bands must not be present. Therefore, in certain genotypic combinations, the presence or absence of the particular specificity on one haplotype cannot be decided. This is a problem only for DR2 and DRw6, which for this reason cannot be assigned in about 1/3 to 1/4 of the cases. The association between RFLP assignment and serological assignment is not absolute, the correlation coefficients ranking from 0.62 to 1.0. In the case of false negative RFLP assignment, this may be due to genetic heterogeneity, as in the case of a DR2 individual who proved to be Dw12 and not Dw2 associated. It is often stated that interpretation of the RFLP pattern is particularly difficult in random or heterozygous individuals compared to proven homozygotes. This is not the case in the present study, where in fact correlation coefficients between RFLP and serologically determined DR specificities were higher in the heterozygotes (range 0.79-1.00).

The impact of HLA-DR antigen matching on the survival of cadaveric renal allografts. A prospective one-center analysis.

The impact of HLA-DR antigen matching on the survival of cadaveric renal allografts was assessed in 158 consecutive transplants performed in our unit since early 1978. In 41 donor-recipient pairs with two shared HLA-DR antigens, the actuarial graft survival rate at 6 months was 73% as compared with 51% in 76 transplants with one HLA-DR antigen shared and 32% in 41 transplants with zero shared HLA-DR antigens. This finding is highly significant (P for heterogeneity [PH] = 0.0005 and P for trend, [PT] = 0.0001). Our data clearly indicate that HLA-DR antigen sharing is more beneficial than merely avoiding HLA-DR incompatibility. But, the frequent antigen HLA-DRw6 was not taken into account in this study due to difficulties in its identification. We found no evidence that the observed beneficial effect of HLA-DR matching could be explained by interaction of other prognostic factors, such as sex, age, previous transplantation, diabetes mellitus or pretransplant blood transfusion. Patients who did not receive blood transfusion prior to transplantation had a significantly lower graft survival rate than those who did (13% vs. 56% at 6 months). HLA-DR matching was found to have a powerful effect on graft survival even among pretransplant blood transfused recipients (PH = 0.002, PT = 0.0006). We conclude that selection of recipients for transplantation should attempt to achieve HLA-DR identical combinations.

HLA-DR genes and antigens in the Danish population. A study of 500 unrelated Danes.

Five hundred unrelated Danes, including 202 healthy individuals, 35 cadaveric kidney donors, and 263 patients in terminal uraemia were typed for the HLA-DR antigens DR1-w8. The HLA-DR gene frequencies of healthy Danes are in good agreement with frequencies estimated during The Eighth International Histocompatibility Workshop of the Scandinavian population, but differ to some extent from other groups of European Caucasians. This indicates geographical variation in the distribution of DR genes within Europe. Very close associations were found between the HLA-DR antigens and their corresponding HLA-D specificities, except for Dw4 and Dw6, which were included in DR4 and DRw6, respectively. Linkage disequilibrium was calculated between alleles of two loci (HLA-A, -DR; -B, -DR and -C, -DR alleles was assessed for multiple loci by using haplotype data from 32 HLA-A, -B, -C, -D, -DR, and Bf typed Danish families. The haplotype data showed that HLA-DR4 is strongly associated with HLA-B15 only in haplotypes together with HLA-Dw4. This indicates genetic heterogeneity of HLA-DR4 which, however, does not appear serologically in this study.