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     

Support for the minimal essential MHC hypothesis: a parrot with a single,highly polymorphic MHC class II <Emphasis Type="Italic">B</Emphasis> gene

We characterized the MHC class II B gene in the green-rumped parrotlet, Forpus passerinus. Three approaches were used: polymerase chain reaction amplification using primers complementary to conserved regions of exon 2, sequencing clones from a genomic library, and amplification of exon 2 using species-specific primers. All three methods indicate that there is only a single class II B locus in this species and no pseudogenes. We suggest that this is the ancestral state for birds. The gene is highly polymorphic; 33 alleles were found in a sample of 25 individuals. Variation in exon 2 is concentrated in the peptide binding residues which show a significant excess of non-synonymous substitutions consistent with the operation of selection in maintaining this extraordinary polymorphism. Genomic clones show that major histocompatibility complex (MHC) gene organization is different from that of chickens; the class II A locus is close to II B. These data provide support for the hypothesis that the bird MHC constitutes a “minimal essential MHC” for responding to infectious disease.     

The MHC haplotypes of the chicken

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as standard B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The standard haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.     

Allelic differences in hemolytic activity and protein concentration of BF molecules are found in association with particular HLA haplotypes

After separating the *F and *S alleles by electrophoresis the allele-specific hemolytic activity was detected by agarose overlay method using the programmable densitometer for scanning. The hemolytic activity of BF allotypes was analyzed from 81 individuals. In thirteen FS heterozygous serum samples BF F had lower hemolysis than BF S. Four FF homozygous samples also exhibited lower hemolysis than a homozygous control sample. The low hemolytic activity of F in FS heterozygotes was not due to decreased protein concentrations relative to S. On the contrary, BF F was associated with higher protein concentration than BF S. The relative quantitation of the allele specific BF protein was done by crossed immunoelectrophoresis. BF F with low hemolytic activity but with high protein concentration associated strongly with HLA B35 phenotype and the family material confirmed the association with the haplotypes A3, Cw4, B35, DR1, BFFB, C4A3BQO (or A2BQO, A3,2BQO). The results suggest that particular MHC haplotypes contain a factor B allele with encoding for poor hemolytic activity or that MHC haplotype specific regulatory elements affect pre- or post-translational activity levels.     

Subdivision of the S region of the mouse major histocompatibility complex by identification of genomic polymorphisms of the class III genes

The S region of the mouse major histocompatibility complex (MHC) encodes the class III proteins, the second (C2) and fourth (C4) components of complement, and factor B. Previously, the assignment of S-region haplotypes was based on analysis of protein polymorphisms. The recent availability of C2, C4, and factor B cDNA probes prompted a search for restriction fragment length polymorphisms which would serve as additional genetic markers for these loci. DNA was isolated from livers of mice of all standard inbred H-2 haplotypes and of haplotypes pz and bs. These DNA samples were digested with restriction endonucleases and analyzed by Southern blot. By the pattern of restriction fragment length polymorphism observed, specific markers have been identified in factor B of haplotypes f, u, z, bs, r, and v, and in C4 of haplotypes b, q,f,j,p,s, pz, r, and v. These genetic markers were used in the analysis of S-region composition in strains B10.TFR5 (H-2 ^ap5) and C3H.LG (H-2 ^dx), and a possible intra-S-region recombinant was revealed in the H-2 ^dxhaplotype. The genetic markers identified here subdivide the S region and will be of value in defining further the composition of the complement gene complex of the mouse MHC.     

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4^F, C4^S, and C4^S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.Abbreviations used in this paper are as follows Bf locus for properdin factor B - MHC major histocompatibility complex - GPLA major histocompatibility complex of the guinea pig     

DNA polymorphism of the C2 and factor B genes

Factor B and the second component of complement (C2) in man are encoded within the major histocompatibility complex by single loci that are less than 1 kb apart. A 2.3 kb factor B-specific cDNA probe has been used to examine, by Southern blot analysis, the genomic DNA of individuals typed for C2 and factor B by protein electrophoresis. We have identified a restriction fragment length polymorphism using the endonuclease Taq I, which subdivides haplotypes carrying both the common variant of C2 (C2C) and the fast (F) variant of factor B. This DNA polymorphism has been mapped to lie in the C2 gene and represents a new genetic marker not defined by protein electrophoresis. This polymorphism may serve as a useful marker in the genetic analysis of diseases that are related to the major histocompatibility complex.     

Structure,diversity and evolutionary patterns of expressed MHC class II<Emphasis Type="Italic">B</Emphasis> genes in chub (<Emphasis Type="Italic">Squalius cephalus</Emphasis>), a cyprinid fish species from Europe

The polymorphism of exon 2 of the DAB genes (major histocompatibility complex [MHC] class IIB) was investigated for the first time in the freshwater cyprinid fish species, Squalius cephalus, in the wide range of its distribution in Europe. We identified 111 different MHC class IIB variants in 15 chub populations distributed from Finland to Spain. The sequence analysis showed that many structurally important amino acid sites that were conserved among tetrapods were also conserved in chub. The analysis of recombination indicated that it does not play an important role in producing and maintaining the variation of DAB genes analyzed in the present study. The exon 2 was shown to be subjected to intense positive selection. Phylogenetic analysis and sequence identities suggest the presence of two class IIB loci (DAB1-like and DAB3-like) in chub. Nevertheless, the presence of three DAB3-like sequence variants in several individuals indicates the duplication of the DAB3 gene. A contrasting selection pattern was found in DAB1-like and DAB3-like genes, which suggests the potential functional differences between these genes. Some DAB sequence variants were shared among the populations of different mtDNA lineages. The phylogenetic analyses did not confirm any biogeographical pattern of the genetic structure of MHC IIB in chub, which is in line with balancing selection and trans-species polymorphism in MHC genes. Nevertheless, cluster analysis based on the presence/absence of DAB sequence variants in the populations showed the phylogeophraphical pattern corresponding to the mtDNA lineages, which indicates that neutral selection can partially explain the MHC IIB evolution in chub.     

Genetic resistance to fowl cholera is linked to the major histocompatibility complex

Chickens of the Iowa State S1 line have been selected for ability to regress Rous sarcoma virus-induced (RSV) tumors, humoral immune response to GAT (Ir-GAT), and erythrocyte antigen B. Sublines homozygous at the major histocompatibility complex (MHC), as well as F₁ heterozygotes and F₂ segregants, were tested for resistance to fowl cholera by challenge with Pasteurella multocida strain X73. Control of the response at high doses was associated in a preliminary study with Ir-GAT and response to RSV tumors. Genetic control of resistance to low doses of P. multocida was demonstrated via sublines and F₂ segregants to be linked with genes of the B-G region. Thus, genetic control of resistance to fowl cholera in chickens after exposure to Pasteurella multocida was shown to be linked to the major histocompatibility B complex, in this first demonstration of MHC-linked resistance to bacterial disease challenge.     

Influence of different B-complex recombinants on the outcome of Rous sarcomas in chickens

Seven major histocompatibility (B) complex recombinants were evaluated for anti-Rous sarcoma response. In experiment 1, the B^R5(F21-G19) recombinant haplotype both homozygous and in heterozygous combinations with B¹⁹ and B²¹ haplotypes were compared to B¹⁹/B¹⁹ and B²¹/B²¹ chickens to determine the relative influence of the B^F versus B^G chromosomal segments on regression of Rous sarcoma virus-induced tumours. In experiment 2, six recombinant haplotypes B^R1(F24-G23), B^R2(F2-G23), B^R3(F2-G23), B^R4(F2-G23), B^R6(F21-G23) and B^R8(F2-G2a,23) present in chickens heterozygous for normal haplotypes B¹⁹, B²³ or B²⁶ were compared for anti-sarcoma response. A total of 1328 chickens were blood typed for B alloanti-gens at 17 days of age, inoculated in the wingweb with Rous sarcoma virus at 6 weeks and monitored for anti-tumour immune response over a 10-week period. Genotypes which shared the same B^F haplotype, but differed in their B^G regions, had similar anti-tumour responses, implicating the B^F but not the B^G region in tumour regression. Chickens carrying B^F2 or B^F21 had a strong anti-tumour response, while B^F24 conferred a weaker response, regardless of the accompanying normal haplotype.     

Linkage disequilibrium between two genetic loci of the major histocompatibility complex in rats

The major histocompatibility complex (MHC) in natural populations of rats is composed of genetic phenotypes that are similar, if not identical, to those seen in inbred laboratory strains. Examination of individual wild rats from a single location in the city of Pittsburgh, Pennsylvania, resulted in the identification of seven different RT1.A histocompatibility serotypes and three RT1.B mixed lymphocytes responses. In this population of animals there is a significant association (p < 0.005) between four RT1.A and RT1.B phenotypic pairs: RT1.A⁸B¹, RTl.A^kBⁿ, RTl.A^dB^a and RT1.A¹B^a. The observed values for linkage disequilibrium (0.211,0.076,0.070 and 0.085, respectively) are very high and are close to the maximum expected, given the individual allelic frequencies. Although the animals included in this study were obtained from one location, agreement with Hardy-Weinberg equilibrium is demonstrated for other loci in the same population. The demonstration of equilibrium suggests that significant inbreeding is not affecting this population of rats. Not enough is known about the allelic frequencies in surrounding rat populations to determine how important the effect of migration is on these disequilibrium values. The large linkage disequilibria may indicate that, in the rat, environmental selective forces are operating to ensure the nonrandom association of separate components of the MHC.     

Marek's disease and major histocompatibility complex haplotypes in chickens selected for high or low antibody response

Sublines of chickens differing in genotypes at the major histocompatibility complex (MHC) were developed from lines selected for high (HA) and low (LA) antibody response to sheep erythrocytes. To evaluate the influence of MHC genotypes in diverse background genomes on resistance to Marek's disease, chicks with MHC genotypes B13B13, B13B21 and B21B21 from both background genomes were exposed naturally commencing at 1 day of age. Individuals which died up to 120 days of age were autopsied to determine cause of death. Mortality due to Marek's disease was greater for HA than LA chickens and greater for males than females. Interactions of MHC genotypes with background genome and with sex suggest a complex picture of the influence of MHC genotypes. A heterozygous advantage for resistance to Marek's disease was noted, as would be predicted by genetic theory concerning maintenance of polymorphism at the MHC.     

BF types and the mode of inheritance of insulin-dependent diabetes mellitus (IDDM)

Insulin-dependent diabetes mellitus (IDDM) has been found to be highly associated with a rare allele of the complement protein, properdin factor B (BF). Assuming that there is a susceptibility gene for IDDM tightly linked to the genetic locus forBF and the major histocompatibility complex (MHC), the distribution of BF types in more than 1100 North American IDDM patients strongly argues for the rejection of dominant, epistatic, and overdominant modes of inheritance. Other evidence suggesting complex modes of inheritance for IDDM is reviewed and it is concluded that our observations and published data are consistent with the idea of susceptibility to IDDM being inherited as a simple autosomal recessive trait. — C4 and C2 types, also linked toBF and theMHC, were investigated too. C4 Fs⁰ was found to be increased in association with BF F1, while C4 f⁰S and C2 B were each found to occur twice as frequently as in a control population and will be of value in defining haplotypes associated with susceptibility to IDDM.     

Cross-reactivity between RSV-induced tumor antigen and B5 MHC alloantigen in the chicken

Lymphocytes from chickens homozygous (B ² B ²) at the major histocompatibility complex (MHC) were tested for cytotoxic activity against five types of target chicken embryo fibroblasts (CEF). Lymphocytes from B²B² chickens bearing RSV-induced tumors lysed in vitro targets of B ² B ² and B ⁵ B ⁵ RSV-infected CEF and B ⁵ B ⁵ normal CEF, but did not lyse B ² B ² and B ²⁴ B ²⁴ normal CEF. Lymphocytes from normal B ² B ² chickens did not lyse any of the five types of CEF targets. Alloantisera absorption studies showed that both RSV-infected and uninfected CEF shared alloantigens, in particular B-F alloantigens, with syngeneic erythrocytes. Absorption with B ² B ² RSV-infected CEF significantly lowered the titer of B ² B ² anti-B ⁵ B ⁵ alloantisera. Cross-reactivity between B ₅ antigen(s) and tumor-associated antigen was suggested and the nature of the cross-reactivity was discussed. It is hypothesized that this cross-reactivity prevents B ⁵ B ⁵ chickens from recognizing RSV-induced tumors as foreign, enhances tumor growth and leads to death of the host.     

Recombination between genes coding for immune response and the serologically determined antigens in the chickenB system

Evidence is presented for a crossover between the genes coding for the serologically determined (SD) antigens on erythrocytes and an immune response gene (Ir-GAT) controlling immune response to the synthetic polypeptide GAT within theB complex, the MHC of chickens. TheIr-GAT ¹ andIr-GAT ¹⁹ alleles control low and high immune response to GAT, respectively. Both low and high responders were recovered as recombinants fromB ¹ B ¹ andB ¹⁹ B ¹⁹ birds. The low-responder haplotypes are homozygous for theIr-GAT ¹ allele and the high-responder haplotypes carry theIr-GAT ¹⁹ allele. Mortality forB ¹ B ¹ nonresponder birds was 39%, compared with 19% for theB ¹ B ¹ high responders; this suggests the possibility that genes located within the immune response region of theB complex exert some genetic control over viability and survival.The following abbreviations are used in this paper MHC major histocompatibility complex - Ir immune response - SD serologically determined - GA (L-glutamic acid⁶⁰, L-alanine⁴⁰) n - DNP-GL dinitrophenyl-(L-glutamic acid⁶⁰, L-lysine⁴⁰) - PLL poly-L-lysine - (T,G)-A--L poly-(L-tyrosine-L-glutamic acid)-poly-D, L-alanine-poly-L-lysine - GAT, GAT¹⁰ (L-glutamic acid⁶⁰ L-alanine³⁰ L-tyrosine¹⁰) n - CFA complete Freund's adjuvant - PBS phosphate-buffered saline     

Increased growth of RSV-induced tumors in chickens partially tolerant to MHC alloantigens

Chickens withB ² B ² MHC genotypes were made partially tolerant to B₅ MHC cell-surface antigens and the fate of their Rous-sarcoma-virus (RSV)-induced tumors was determined.B ² B ² chickens partially tolerant to viable or lysed white blood cells (WBC) or viable red blood cells (RBC) fromB ⁵ B ⁵ chickens had a significantly higher incidence of tumor progression than untreated, PBS-treated, orB ² B ² chickens inoculated with WBC from otherB ² B ² chickens. The criteria for tolerance were absence of antibody titer to the cell type inoculated and acceptance of allografts fromB ⁵ B ⁵ donors byB ² B ² chickens. Graft-vs-host reactions occurred only inB ² B ² chickens inoculated with viable WBC fromB ⁵ B ⁵ chickens. It appears thatB ² B ² chickens partially tolerant to B₅ antigens failed to mount a successful immune response to RSV-induced tumors partly because a B₅ MHC antigen(s) cross-reacted with a tumor associated antigen(s) thereby severely limitingB ² B ² host recognition of the tumor as foreign. Since WBC and RBC cell-surface antigens appear to contribute similarly to the effect, theB-F- region of the MHC may be involved.     

Studies on the genetic polymorphism of lactate dehydrogenase B (phenotype B−) in rodent erythrocytes

While the common red cell phenotype of lactate dehydrogenase in mammals includes A and B subunits, in some species, especially of the rodent suborder Myomorpha, LDH B subunits are absent in erythrocytes (phenotype B^–). A polymorphism involving LDH B⁺ and LDH B^– phenotypes was observed in Mus musculus, and it was found that LDH B^– is recessive in relation to LDH B⁺ (Shows and Ruddle, 1968). A similar polymorphism is described here in the field mouse, Apodemus sylvaticus. In family studies, the recessive mode of inheritance is confirmed. Induction of reticulocytosis reveals that the LDH B^– phenotype is not the consequence of degradation of the B subunits in aging red cells. The nature of the mutation giving rise to this polymorphism is discussed.Supported by the Deutsche Forschungsgemeinschaft.     

Limited polymorphism at major histocompatibility complex (MHC) loci in the Swedish moose A. alces

The Swedish moose was analysed for genetic variability at major histocompatibility complex (MHC) class I and class II DQA, DQB and DRB loci using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Both methods revealed limited amounts of polymorphism. Since the SSCP analysis concerned an expressed DRB gene it can be concluded that the level of functional MHC class II polymorphism, at least at the DRB locus, is low in Swedish moose. DNA fingerprinting was used to determine if the unusual pattern of low MHC variability could be explained by a low degree of genome-wide genetic diversity. Hybridizations with two minisatellite probes gave similarity indices somewhat higher than the average for other natural population, but the data suggest that the low MHC variability cannot be explained by a recent population bottleneck. However, since minisatellite sequences evolve more rapidly than MHC sequences, the low levels of MHC diversity may be attributed to a bottleneck of more ancient origin. The selection pressure for MHC variability in moose may also be reduced and we discuss the possibility that its solitary life style may reduce lateral transmission of pathogens in the population.     

Heterogeneity of human C4 gene size

In this article we present a study showing that the human C4 genes differ in length because of the presence or absence of a 6.5 kb intron near the 5 end of the gene. DNA from individuals of known HLA, factor B, and C4 haplotypes was analyzed for restriction fragment length polymorphism (RFLP) by Southern blot analysis with C4-specific cDNA probes. The RFLP patterns obtained showed that the C4 genes are either 22.5 kb or 16 kb in length. They are referred to as long and short C4 genes, respectively. A population study was carried out to examine the distribution of the gene size according to C4 allotypes and haplotypes. Long C4 genes included all C4A genes studied and also some C4B allotypes, e. g., B1 on most C4 A3B1 haplotypes. Similarly, C4B null genes were found to be of the long form. Other C4B allotypes tested were found to be coded for by short C4 genes, including B2, B1 in C4 A6B1 and C4 AQOB1 (with a single C4B gene haplotype).Abbreviations used in this paper C4 fourth component of complement - C2 second component of complement - BF factor B - MHC major histocompatibility complex - RFLP restriction fragment length polymorphism - EDTA ethylenediaminetetraacetic acid - SDS lauryl sulfate, sodium salt