Hereditary C2 deficiency: diagnosis and HLA gene complex associations.

Nine families with genetically controlled C2 deficiency have been described where the propositii and family members are heterozygous C2 deficient. The diagnosis of hereditary C2 heterozygous deficiency was suspected on the basis of analysis for CH50, C2 protein, and C2 function, and then confirmed by family studies. Analysis of HLA antigens in these families supported the close association of C2 defiency and HLA-A10 and/or B18, particularly the latter. Analysis by MLC studies revealed recombination in one family between the HLA and B and D loci and in another family probable recombination between the D and C2 complement loci. Therefore, the order of the loci on the sixth chromosome is likely to be C2 complement, HLA-D, HLA-B, HLA-A.     

Genetics of cell-mediated lympholysis in man.

Cell-mediated lympholysis (CML) was studied in a family containing two siblings in who genetic recombinaiton had occurred in the HLA comples. In one sibling, recombination occurred between the HLA-A locus and the HLA-B locus. In the second sibling recombination occurred between the HLA-B locus and the HLA-D locus. Strong CML activity was generated in mixed lymphocyte cultures (MLC) when stimulator and responder cells differed in HLA-A, B, and D antigens. MLC involving HLA-D differences alone did not generate CML. Weak, but definite CML activity was generated during MLC with cells differing at HLA-A and HLA-B but sharing HLA-D. HLA-B antigens were good targets for lysis in all combinations studied. HLA-A antigens were poor targets in some but not in all combinations. However, combinations where HLA-A antigens seemed to be good targets could have involved HLA-B differences due to polymorphism of HLA-B7 antigens each inherited from a different parent. HLA-D antigens did not serve as targets for lysis. In three cell experiments, excellent CML activity was generated when responder cells were stimulated by HLA-D antigens and by HLA-A and B antigens present on separate stimulator cells.     

The genetics of cell-mediated lympholysis.

The role of HLA antigens in the generation of cytotoxic cells in CML has been investigated. Cytotoxic effector cells were generated in MLC among HLA-A or HLA-A and HLA-B disparate, HLA-D identical siblings, and among HLA-A and HLA-B disparate, MLC identical (%RR less than or equal to 2 3.6) unrelated individual. The data indicate that HLA-D differences and poliferative MLC responses as measured by 3H-thymidine incorporation are not requisite for the in vitro generation of cytotoxic cells and suggest the existence of a CML-S locus (loci) distinct from HLA-A, HLA-B and HLA-D. The degree of cytotoxicity generated in a proliferative versus a "nonproliferative" MLC was comparable. In addition, these studies demonstrate that antigens other than the currently definable HLA-A, HLA-B, HLA-C, and HLA-D can serve as target determinants in cell-mediated lympholysis.     

Lack of linkage between gene(s) controlling the synthesis of the seventh component of complement and the HLA region on chromosome No. 6 in man

Summary The family of an individual was studied who lacks the seventh component of complement in his serum (C7 homozygous deficiency). Both parents are C7 heterozygousdeficient. In this investigation, the following parameters were determined: complement components in functional and immunochemical tests; HLA-A,B antigens, HLA-D (MLC) determinants; the Bf system; glyoxalase I and B cell antigens. No evidence for linkage between the immunogenetic linkage group on chromosome 6 and gene(s) controlling the synthesis of the seventh component of complement was obtained. This is in accordance with the assumption that only genes controlling components of the initiating rather than the membrane attack unit of complement are linked to the HLA region.     

HLA and IgA deficiency

The distribution of 29 HLA-A and B antigens was compared in 50 Caucaso?ds with an IgA deficit and in 300 healthy controls. The patients were divided in 3 groups: 1) Partial selective IgA deficit (40); 2) Total selective Iga deficit (7); 3) IgA deficit associated with hypogammaglobulinemia (3). The patients viewed as a whole, we observed an increased frequency for the antigens HLA-Aw19, HLA-B5 and HLA-BW17. Yet, the modifications are not cleanly significant, with p less than 0.05, but p corrected not significant. We also considered the 3 groups separated and we did not remark any particular association with HLA. The data concerning HLA and congenital immune insufficiencies are reviewed. The most authors at once studied several immune defects. Only one Hungarian work was performed on IgA deficit. We do not confirm HLA-A1 and HLA-B8 increased frequencies, as it was reported, in Hungary, by Bajtai and al. There is no evident association between one HLA-A or B gene and the IgA deficit. The possible relation of IgA insufficiency with autoimmunity and allergy would justify complementary investigations, especially about HLA-D and Ia genes repartition in this disease.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Genetic control of immune response to the L-Glu, L-Lys, L-Phe terpolymer in man.

We have demonstrated that human lymphocytes can respond to the synthetic polypeptide GLPhe upon in vitro challenge by the antigen similar to that of (H,G)-A--L, (T,G)-A--L, (Phe,G)-A--L, and GAT. Family studies further support our postulation that responses to these synthetic polymers are under dual gene control. Three families with intra-HLA-A/B recombinants provided mapping information for Ir-GLPhe genes. The response phenotype of the recombinant of family 21 localized the Ir-GLPhe genes toward the HLA-B of D regions, whereas recombinants of family 24 and 27 placed the Ir-GLPhe genes distal to HLA-B, toward the A region. This discrepant gene assignment can be explained by assuming that recombination occurred at different positions between HLA-A and HLA-B. In family 21, crossover occurred distal to the Ir genes, while for the other two, proximal to them. A second possibility is that as in the mouse the two complementing genes are situated in different regions of the human major histocompatibility complex (MHC) and all three of the crossovers occurred between them with the putative Ir-GLPhe-1 located near the HLA-A region and Ir-GLPhe-2 on the HLA-D region or vice versa. A third possibility is that immune response required interaction between a complete HLA-D-like molecule encoded in the A region and another encoded elsewhere, perhaps in HLA-D.     

HLA polymorphism in the Havasupai: evidence for balancing selection.

The characterization and analysis of genetic variation at the HLA loci provides important insight for population geneticists trying to understand the evolutionary forces that have shaped human populations. This study describes the HLA-A and HLA-B loci serotyping and statistical analysis on an isolated Native American population, the Havasupai of Arizona. Four alleles at the HLA-A locus were identified, while eight alleles were found at the HLA-B locus. These variants were present as 20 of 32 potential two-locus haplotypes, with five of the six most common haplotypes exhibiting high positive linkage disequilibrium. Significant homozygote deficiency (heterozygosity excess) was detected both at HLA-A and at HLA-B. This deviation from Hardy-Weinberg proportions was not attributable to nonselective causes such as different allele frequencies in males and females or avoidance of consanguineous matings. In addition, the distribution of alleles at both HLA-A and HLA-B was more even than expected from neutrality theory; that is, the observed Hardy-Weinberg homozygosity was only 62.4% of that expected under neutrality. These observations suggest that balancing selection is of major importance in maintaining genetic variation at HLA-A and HLA-B.     

Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3)

The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific.     

A public HLA antigen associated with HLA-A9, Aw32, and Bw4

The HLA complex codes for three distinct 44000 dalton molecules associated with beta2 microglobulin--HLA-A, B and C--each with its own multiallelic series of private antigens. The HLA-B molecule is exceptional in that it also carries a diallelic system, Bw4 and Bw6. One of these, Bw4, is often associated with the A-locus specificity A9. This finding has usually been ascribed to linkage disequilibrium between A- and B-locus antigens. We have shown, however, that an epitope called LHe is actually shared by A-locus and B-locus molecules. This epitope is found on all HLA-B molecules bearing the Bw4 determinant and is also found on all HLA-A molecules carrying the A9 (Aw23 and Aw24) or Aw32 specificities. We consider this a "public" HLA antigen; the possible molecular basis for both subtypic and public antigens on a single glycoprotein is discussed.     

HLA antigens in Japanese populations.

HLA antigens in 841 healthy, unrelated Japanese from nine widely separated geographic localities were studied. The five most common antigens observed in order of decreasing frequency were for the HLA-A locus: HLA-A9, A2, A10, AW32 and A11; and for the HLA-B locus: HLA-'B5' (= HLA-B5+B17), BW40, B12, B14 and B8. The allelic frequency of undetected antigens of the HLA-A locus was .14-.37, and that of the HLA-B locus, .32-.67, indicating that there were serological difficulties in typing for Japanese antigens using antisera from Caucasians. Marked gene frequency clines were observed for HLA-A9 and HLA-A2 from south (Okinawa) to north (Nagoya). Two haplotypes, HLA-A9, B5 and HLA-A10, BW40 were shown to be in linkage disequilibrium in four of the nine subpopulations.     

Linkage between C2 deficiency and theHLA-A10,B18,Dw2/BfS haplotype in a French family

A linkage between C2 deficiency and the HLA-A10,B18/BfS antigens has been found in a French family from the Strasbourg area. The propositus, suffering from a chronic glomerulonephritis, is homozygous forHLA-A10,B18/BfS and totally C2-deficient. The parents and the brother are heterozygous for C2 deficiency and share theHLA-A10,B18/BfS haplotype. MLC tests and HLA-D typing revealed that the homozygous C2-deficient patient is also homozygous at theHLA-D locus for the w2 specificity. Evidence was obtained for a heterogeneity of the HLA-Dw2 specificity. This observation confirms the remarkable association between C2 deficiency and theHLA-A10,B18,Dw2 haplotype.     

Evidence for linkage between HLA antigens and receptors for complement components C3b and C3d in human-mouse hybrids

A linkage between HLA-A and HLA-B antigens and receptors for complement components C3b and C3d has been demonstrated by means of human-mouse somatic cell hybridization. In 26 hybrid clones formed between the human lymphoblastoid cell lines RPMI 6410 and Raji and the murine fibroblast line 1T22, HLA-A and HLA-B antigens and receptors for C3b and C3d segregated conjointly. No segregation of the C3b and C3d receptors from each other or from HLA antigens was observed. The results suggest that the genes for the C3b and C3d receptors are located on the same chromosome that encodes the major histocompatibility complex of the human.     

Human MHC class III genes, Bf and C4. Polymorphism, complotypes and association with MHC class I genes in the Finnish population

Electrophoretically detected genetic polymorphism of human MHC class III genes, factor B (Bf) and complement C4A and C4B, was studied in the Finnish population. Bf alleles were determined in a panel of sera from 70 unrelated individuals. The common Bf alleles, Bf*S and Bf*F, had frequencies of 73% and 26%, respectively. Only in 1 individual was another allele, Bf*F1, detected. The frequencies of the C4A and C4B alleles were based on studies of 254 unrelated individuals. In this panel, five different alleles were detected at the C4A locus and four at the C4B locus. At both loci an allele without a gene product, i.e. a 'null' allele, was observed with high frequency, 11% for C4A 'null' and 17% for C4B 'null'. The association of complotypes to HLA haplotypes was analyzed in 70 chromosomes. The most common combination, defined by class I and class III alleles, was HLA-B7-S31 (13%), followed by HLA-B35-F20 (8.4%) and HLA-B8-S03 (7.1%). Some HLA-B specificities, for example B15, B27 and B40, were associated with a variety of complotypes. The importance of complotyping in HLA genetics is discussed.     

Diversity and diversification of HLA-A,B,C alleles

The nucleotide sequences encoding 14 HLA-A,B,C and 5 ChLA-A,B,C molecules have been determined. Combining these sequences with published data has enabled the polymorphism in 40 HLA-A,B,C and 9 ChLA-A,B,C alleles to be analyzed. Diversity is generated through assortment of point mutations by recombinational mechanisms including gene and allelic conversions. The distribution and frequency of silent and replacement substitutions indicate that there has been positive selection for allelic diversity in the 5' part of the gene (exons 1 to 3) and for allelic homogenization and locus specificity in the 3' part of the gene (exons 4 to 8). These differences may correlate with the lengths of converted sequences in the two parts of the gene and frequency of the CpG dinucleotide. Locus-specific divergence of HLA-A,B, and C demonstrates that recombinational events involving alleles of a locus have been more important than conversion between loci. This contrasts with the predominance of gene conversion events in the evolution of mutants of the H-2Kb gene. However, a striking example of gene conversion involving HLA-B and C alleles of an oriental haplotype has been found. Comparison of human and chimpanzee alleles reveals extensive sharing of polymorphisms, confirming that diversification is a slow process, and that much of contemporary polymorphism originated in ancestral primate species before the emergence of Homo sapiens. There is less polymorphism at the HLA-A locus compared to HLA-B, with greater similarity also being seen between HLA-A and ChLA-A alleles than between HLA-B and ChLA-B alleles. Although greater diversity is seen in the 5' "variable" exons of HLA-B compared to HLA-A, there is increased heterogeneity in the 3' "conserved" exons of HLA-A compared to HLA-B.     

Regulation of HLA class I expression by non-coding gene variations

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC’s suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNβ, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.     

MHC-linked class III genes. Analysis of C4 gene frequencies,complotypes and associations with distinct HLA haplotypes in German Caucasians

The class III complement components, C4, C2 and factor B (BF), are encoded in the human major histocompatibility complex (MHC). The two genes determining C4 (C4A and C4B) display considerable polymorphism and, thus, are important markers for HLA. In combination with alleles of C2 and BF they can be grouped into unique complotypes. We have analyzed the C4 alleles in a panel of 204 unrelated German Caucasians and studied their segregation with HLA haplotypes in 24 normal families. Inclusion of the class III markers with the class I and 11 alleles provides a more refined picture of the genetic structure of the MHC in these families. When charted according to the HLA-B locus specificities the MHCs can be clustered into groups showing distinctly homogenous or heterogenous complotypes. The identification of such groups is valuable for the selection of genetic material to analyze the molecular genetics of the human MHC.Abbreviations BF factor B - C2 second component of complement - C4 fourth component of complement - EDTA ethylenediamine tetraacetate - GLO glyoxalase-I - MHC major histocompatibility complex     

"Silent" alleles at the HLA-C locus.

Defined specificities of the HLA-C locus possess a significantly lower immunogenicity as compared with the HLA-A and HLA-B specificities. HLA-C "blanks" are not immunogenic at all. Among the possible explanations, the most likely one appears to be a low phenotypic expression of these alleles. C locus antigens as a whole are thus likely to be of minor relevance in donor/recipient matching for transplantation.     

HLA-J, a second inactivated class I HLA gene related to HLA-G and HLA-A. Implications for the evolution of the HLA-A-related genes.

Ragoussis and co-workers (Genomics 4:301) previously described a class I HLA gene (now designated HLA-J) that maps to within 50 kb of HLA-A. The nucleotide sequences of three HLA-J alleles are reported here. Comparison of the nucleotide sequences of HLA-J alleles shows this gene is more related to HLA-G, A, and H than to HLA-B, C, E, and F. All four alleles of HLA-J are pseudogenes because of deleterious mutations that produce translation termination either in exon 2 or exon 4. Apart from these mutations, the predicted proteins have structures similar to those of HLA-A, B, and C molecules. There is, however, little polymorphism at HLA-J and none at functional positions of the Ag-recognition site. The polymorphism is less than found for HLA-H another HLA-A-related pseudogene. HLA-J appears, like HLA-H, to be an inactivated gene that result from duplication of an Ag-presenting locus related to HLA-A. Nucleotide sequence comparisons show that the HLA-A, H, J, and G genes form a well defined group of "HLA-A-related" loci. Evolutionary relationships as assessed by construction of trees suggest the four modern loci: HLA-A, G, H, and J were formed by successive duplications from a common ancestral gene. In this scheme one intermediate locus gave rise to HLA-A and H, the other to HLA-G and J.