DNA sequence analysis of serologically detected ELA class II haplotypes at the equine DQP locus

The genetic diversity at the ELA DQβ locus was investigated using polymerase chain reaction and DNA sequencing. Based upon serological methods 16 class II homozygous animals were selected and their genomic DNA was used. A DQβ gene from an equine cDNA library was also sequenced. Our methology and the similarity between the genomic and the cDNA sequences suggest that the studied locus is expressed on equine lymphocytes. In the predicted amino acid sequence the most extensive variation is located at residues 56–60. The pattern of these five amino acids is strongly correlated to the serological ELA class II specificities (W13, W22, W23, Be200). The alleles corresponding to the W23 specificity are the most divergent among the equine DQβ alleles and also from other mammalian DQβ sequences.     

Joint Report of the Second International Workshop on Lymphocyte Alloantigens of the Horse, held 3–8 October 1982

The Second International Workshop on Lymphocyte Alloantigens of the Horse was held 3–8 October 1982. At this workshop, the 6 specificities identified at the first workshop were confirmed and an additional 5 new specificities were identified and given workshop nomenclature. Four of the new specificities, products of the ELA locus, were named ELA-W7, W8, W9, and W10. An additional specificity, designated ELY-2.1, is the product of a locus independent of the ELA locus.
Cell isolation methods were compared at this workshop, Technical variation in methods clearly affected reactivity of many reagents. However, when highly selected reagents were used, antigen assignment did not differ regardless of the cell isolation method. Based on the comparison of methods, isolation procedures in which thrombin was used were more effective than those relying on carbonyl iron or slow centrifugation.     

Joint Report of the Fourth International Workshop on Lymphocyte Alloantigens of the Horse, Lexington, Kentucky, 12–22 October 1985

Summary. The workshop consisted of 12 monthly cell exchanges of full-sibling families among the 10 participating laboratories. A total of 33 parents, 52 offspring and five unrelated horses were typed by each laboratory using local antisera. The raw data were submitted for central analysis before any identification of the animals was revealed.
Confidence derived from the consistent agreement between the laboratories on the assignment and segregation of the first 10 ELA-W specificities led to the removal of the W (workshop) notation and acceptance of full status as locus A antigens. The seemingly supertypic W11 specificity, however, remained unchanged.
Ten additional specificities were seen to segregate with the ELA system, suggesting either splits of previously described specificities or products of linked loci. The workshop (W) notation was given to the 10 specificities W12-W21, befitting their status as specificities under study.
The previously described ELY-1.1 specificity, characterized by segregation independent from the ELA system, was confirmed along with a new specificity, ELY-1.2, which behaves as an allele of ELY-1.1. For informative families, the two specificities showed codominant expression and appeared to constitute a closed, autosomal system.
The ELY-2.1 specificity was confirmed to segregate independently from the ELA-A and ELY-1 loci.     

Joint Report of the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, Baton Rouge, Louisiana, 31 October-1 November 1987

Six laboratories participated in the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, testing 132 alloantisera against lymphocytes of 880 horses chosen to represent different families and breeds. Most of the alloantisera were produced by lymphocyte immunization between horses matched at the ELA-A locus. All horses were also tested with antisera contributed to the workshop by participating laboratories which identified ELA specificities A1-A10 and W12-W21. Previously identified workshop specificities ELA-W14, W15 and W19 were accepted as products of the ELA-A locus based on family and population studies by the workshop. Their designations were changed to ELA-A14, ELA-A15 and ELA-A19, respectively. Two new specificities were identified, namely ELA-W22 (W22) and ELA-W23 (W23). Population and family studies indicated that W22 and W23 as well as W13 are products of an ELA locus other than ELA-A. The presence of these specificities was correlated with the presence of certain ELA-A locus specificities, e.g. W13 with A3, W22 with A2 and W23 with A5. However, the association was not complete and W13, W22 and W23 also segregated with other ELA-A specificities in some families. Evidence for recombination was found between the ELA-A locus and the locus or loci encoding these specificities resulting in seven recombinant haplotypes found among the data presented in this workshop. Further studies are required for definitive assignment of the specificities to a class I or class II locus.     

Lymphocyte alloantigens of the horse. III. ELY-2.1: a lymphocyte alloantigen not coded for by the MHC

A new polymorphic locus of the horse which has several unusual properties is described. The suggested name for the locus is ELY-2 . The gene product of one allele at this locus, designated ELY-2.1 , has been identified with antisera raised as a result of pregnancy. Antibody to ELY-2.1 was first detected on day 55 after conception in the serum of a mare in first pregnancy. This early onset of antibody is similar to that seen for antibody to ELA antigens, and suggests that the source of the antigenic stimulus may be the tissue of the equine endometrial cups.
The antisera identifying ELY-2.1 are cytotoxic and kill all peripheral blood lymphocytes from horses carrying the antigen. ELY-2.1 is a cell surface molecule expressed on lymphocytes, erythrocytes, and platelets. Other cell types have not been investigated. The overall phenotypic frequency of ELY-2.1 in several horse breeds was 16 %. The ELY-2.1 antigen is controlled by an autosomal, dominant gene which is not coded by the ELA region (the major histocompatibility complex of the horse), nor is it identical to the ELY-1 locus, which codes for another cell surface alloantigen of equine lymphocytes. Stimulator cells carrying ELY-2.1 did not induce proliferation of ELY-2.1 negative responder cells in mixed cultures of horse lymphocytes. Attempts to raise alloantisera to other alleles of the ELY-2 locus through immunization with lymphocytes were unsuccessful. It is possible that the alternate allele(s) does not code for a gene product which is expressed. The function and biochemical nature of the ELY-2.1 molecule are unknown.     

Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984

The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Joint Report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25–27 April 1984

Summary. The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25–27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Comparison of ELY-2.1 with blood group and ELY-1 markers in the horse*

The distribution of ELY-2 was compared to the distribution of blood group factors Aa, Ab, Ac, Ae, Ca, Da, Db, Dc, Dd, De, Df, Dh, Dk, Ka, Pa, Pb, X, Qa, Qc, Ua, and W in 2465 American Standardbred horses and to ELY-1 in 193 American Standardbred horses. The distribution patterns were different in each case. The segregation of ELY-2.1 and factors at the A, C, D, K, P, Q, U and T (W ) blood group loci and at the ELA locus indicated that ELY-2.1 is not a product of any of those loci. No segregation data were available for the ELY-I locus. Family studies indicated that the gene for ELY-2.1 is not sex-linked.     

Comparison of ELY-2.1 with blood group and ELY-1 markers in the horse

The distribution of ELY-2 was compared to the distribution of blood group factors Aa, Ab, Ac, Ae, Ca, Da, Db, Dc, Dd, De, Df, Dh, Dk, Ka, Pa, Pb, X, Qa, Qc, Ua, and W in 2465 American Standardbred horses and to ELY-1 in 193 American Standardbred horses. The distribution patterns were different in each case. The segregation of ELY-2.1 and factors at the A, C, D, K, P, Q, U and T (W) blood group loci and at the ELA locus indicated that ELY-2.1 is not a product of any of those loci. No segregation data were available for the ELY-1 locus. Family studies indicated that the gene for ELY-2.1 is not sex-linked.     

Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis

The equine leucocyte antigen (ELA) types and the clinical diagnosis for equine sarcoid and summer dermatitis were evaluated in 2026 horses representing five breeds. Data were analysed in unrelated animals and in family material. In the case of equine sarcoid, a strong association was observed between the ELA class II DW13 antigen and its effect on Swiss (cP < 0·001), French (cP < 0·0001) and Irish (cP < 0·01) Warmblood horses. The class I antigen A3 occurred more frequently in sarcoid-affected French horses (cP < 0·001). These results confirm our earlier findings (Gerber et al. 1988). Among Freiberger horses, which lack the ELA DW13 and A3 specificities, a breed-specific class I antigen, ABe108, displayed an increased frequency (cP < 0·05) in the affected group. Among Arabian horses, a tendency for increased frequency of the A1 antigen was observed in the affected animals, but the number of affected horses is too small for statistical significance. The Mendelian segregation in diseased half-siblings by ELA DW13 heterozygous stallions showed a strong association (P < 0·0001) between the inherited DW13 antigen and susceptibility to the sarcoid effect. In the case of summer dermatitis, previously published data (Marti et al. 1992) have been extended. The ELA types in four multiple-case families, founded by the same stallion, were analysed for an association with the effect of sarcoid. Eight out of nine ELA-typed affected offspring inherited the paternal haplotype A15, DW23 in contrast to nonaffected offspring where three out of 12 displayed these antigens (P < 0·005). Moreover, the ELA haplotypes of 11 out of 12 informative affected half-siblings sired by another stallion inherited the paternal haplotype A3, W12, DW23 (P < 0·05). Our findings demonstrate statistically significant associations between certain ELA antigens and two equine diseases. It is still unknown if the major histocompatibility complex (MHC) molecules themselves or another linked gene(s) play a role in the pathogenesis of these conditions.     

Evidence of a second polymorphic ELA class I (ELA-B) locus and gene order for three loci of the equine major histocompatibility complex

Two antisera, B-442 and R-2046, were produced by immunizing offspring with purified peripheral blood lymphocytes from a parent matched for the ELA-A specificity carried on the unshared haplotype. Absorption analysis demonstrated that these antisera contained at least two families of cytotoxic antibodies, one directed against antigens present on T and B cells, and a second directed preferentially against antigens present on surface Ig positive cells. Immunoprecipitation studies using these antisera demonstrated that both antisera contain antibodies specific for glycoproteins with molecular weights characteristic of class I and class II MHC antigens. In lymphocyte typing tests of unfractionated lymphocytes, only the class I activity was readily detectable since the class II activity killed less than 25% of the cells. Family studies demonstrated that these antisera recognize products of genes linked to the ELA system. Based on two recombinants in an extended family it became apparent that the specificities detected by B-442 and R-2046 are not products of the ELA-A locus, but rather they are products of at least one other locus, defined in this paper as ELA-B. In this family a third recombinant was found between the A blood group system and the ELA-A locus. Based on these three recombinants, the most probable linear relationship of the following genes is: A blood group system/ELA-A/ELA-B.     

Equine lymphocyte antigens and reproduction in the Standardbred mare

Summary. Equine lymphocyte antigen (ELA) gene frequencies were estimated for pacing and trotting Standardbred mares residing on a breeding farm in central Ohio. The ELA gene frequencies for Ohio Standardbreds did not differ significantly from the ELA gene frequencies of Kentucky Standardbreds, determined by Bailey (1983). No significant differences were found in the distribution of ELA class I antigens in horses with lower overall fertility or a history of abortion on the investigated breeding farm. Likewise, no significant association was observed when the ELA types of both the mare and the stallion to which she was mated were compared with the reproductive efficiency of the mare.     

Detection of a novel HLA-DQ specificity: serological and immunochemical analyses by a monoclonal antibody

A monoclonal antibody (mAb) with a novel human B-cell allospecificity was produced by immunizing a C3H/He mouse with the human B lymphoblastoid cell line EBV-Wa (HLA-DR4/Dw15/DQblank homozygous). The mAb, termed HU-46, reacted with B cells from not only DR4/Dw15-positive individuals but also certain DRw8/Dw8-positive ones whose DQ phenotypes had not yet been defined. Two-dimensional gel analyses indicated that the mAb recognized class II antigens which were encoded by the HLA-DQ locus. Furthermore, in genetic analysis, the gene encoding the class II antigen detected by HU-46 met the Hardy-Weinberg condition as a fourth allele of the DQ locus. We provisionally labeled this novel DQ specificity DQWa.     

Gene organization of haplotypes expressing two different C4A allotypes

Summary The gene organization of C4 haplotypes expressing two different C4A allotypes with a C4B null allele (C4A3A2-BQ0 and C4A3A6BQO) was studied using Southern blot analysis with cDNA probes and restriction enzymes which give C4A and C4B locus-specific restriction fragments. These haplotypes were shown to have both a C4A and a C4B locus present, suggesting that the C4B locus expresses a C4A protein. The finding of a 21-OH A and a 21-OH B gene on the C4A3A6BQO haplotype further suggests that this haplotype has the common gene organization C4A, 21-OH A, C4B, 21-OH B. A model explaining C4 null alleles on haplotypes found to have two C4 loci is presented.     

Equine lymphocyte antigens and reproduction in the Standardbred mare

Equine lymphocyte antigen (ELA) gene frequencies were estimated for pacing and trotting Standardbred mares residing on a breeding farm in central Ohio. The ELA gene frequencies for Ohio Standardbreds did not differ significantly from the ELA gene frequencies of Kentucky Standardbreds, determined by Bailey (1983). No significant differences were found in the distribution of ELA class I antigens in horses with lower overall fertility or a history of abortion on the investigated breeding farm. Likewise, no significant association was observed when the ELA types of both the mare and the stallion to which she was mated were compared with the reproductive efficiency of the mare.     

Equine lymphocyte antigens in four major Belgian horse populations. Contribution to serology and antigen distribution

158 Belgian Saddlebreds, 130 Belgian Trotters, 108 Belgian Draft horses and 92 Shetland ponies have been typed for serologically defined antigens at the ELA and ELY systems. Gene frequencies were estimated in each breed for the internationally established ELA, ELY-1 and ELY-2 alleles as well as for locally assigned additional ELA markers and for subtypes of ELA-W3, W9 and W11. The distribution of ELA alleles was in agreement with the expected Hardy-Weinberg equilibrium for the 4 horse breeds described here. Differences in gene frequencies between these main Belgian horse populations were observed.     

Equine lymphocyte antigens in four major Belgian horse populations. Contribution to serology and antigen distribution

158 Belgian Saddlebreds, 130 Belgian Trotters, 108 Belgian Draft horses and 92 Shetland ponies have been typed for serologically defined antigens at the ELA and ELY systems. Gene frequencies were estimated in each breed for the internationally established ELA, ELY-1 and ELY-2 alleles as well as for locally assigned additional ELA markers and for subtypes of ELA-W3, W9 and W11. The distribution of ELA alleles was in agreement with the expected Hardy-Weinberg equilibrium for the 4 horse breeds described here. Differences in gene frequencies between these main Belgian horse populations were observed.     

Diversity of the CYP21P-like gene in CYP21 deficiency

More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by mutations of the CYP21 gene. The occurrence of defective CYP21 genes, including 15 mutations, has been attributed to intergenic recombination of DNA sequences from CYP21P, and shows no influence on the RP1-C4A-CYP21P-XA-RP2-C4BCYP21- TNXB gene locus on chromosome 6p21.3. However, multiple gene deletions in this region produce at least three categories of gene arrangements: (a) C4A-CYP21P/CYP21-TNXB, in which there is a CYP21P/CYP21 fusion gene; (b) C4A-XCYP21-TNXB, where XCYP21 indicates that the CYP21 gene contains mutations of IVS2 (-12A/C>G and 707-714delGAGACTAC); and (c) C4A-CYP21P-TNXA/TNXB, in which the TNX A and B genes are fused. Among them, seven different structures of the CYP21 haplotype were found at these three loci. Formation of the C4A-CYP21P/CYP21-TNXB locus produced four distinct CYP21P/CYP21 chimeras. The C4A-XCYP21-TNXB locus contained the IVS2 mutation -12A/C>G and 707-714delGAGACTAC from the XCYP21 gene; and two kinds of TNXA/TNXB hybrids were found in the C4A-CYP21P-TNXA/TNXB locus. The seven different CYP21 alleles produced 3.2 kb Taq I fragments caused by deletion of the RP2-XA-C4B locus. Therefore, production of a 3.2-kb CYP21 allele shows diversity, but is not a unique feature of the CYP21P gene. Most of these gene arrangements probably exist in the C4A-XCYP21-TNXB and C4A-CYP21P/CYP21-TNXB gene loci. The existence of the C4A-CYP21P-TNXA/TNXB locus might not be common in CAH patients with 21-hydroxylase deficiency.     

Self-compatible B mutants in coprinus with altered pheromone-receptor specificities

A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.     

Analysis of the duplicated human C4/P450c21/X gene cluster

Gene duplications, deletions and rearrangements occur with an unusually high frequency in the region of the P450c21 genes encoding 21-hydroxylase. In the human genome, the locus contains at least 6 genes, oriented 5′ C4A, P450c21A, XA, C4B, P450c21B, XB 3′. Sequence analysis of the XA gene, of the 5′ flanking DNA of the C4A gene, and of part of the XB gene revealed that this gene cluster was duplicated by nonhomologous recombination at a CAAG tetranucleotide. The location of this duplication suggests that it may have occurred after mammalian speciation. The XA gene is abundantly expressed in the human adrenal as a stable 2.6 kb RNA, but it is not known if that RNA serves a biological function. Knowledge of the anatomy of the XA gene facilitates genetic analysis of disease-causing lesions in the P450c21B gene. Southern blotting data show that about 76% of disordered P450c21B alleles bear gene microconversions that resemble point mutations; the remaining alleles are equally distributed between gene deletions and large gene conversions.