Linkage-disequilibrium mapping without genotyping.

Genomic mismatch scanning (GMS) is a technique that enriches for regions of identity by descent (IBD) between two individuals without the need for genotyping or sequencing. Regions of IBD selected by GMS are mapped by hybridization to a microarray containing ordered clones of genomic DNA from chromosomes of interest. Here we demonstrate the feasibility and efficacy of this form of linkage-mapping, using congenital hyperinsulinism (HI), an autosomal recessive disease, whose relatively high frequency in Ashkenazi Jews suggests a founder effect. The gene responsible (SUR1) encodes the sulfonylurea receptor, which maps to chromosome 11p15.1. We show that the combination of GMS and hybridization of IBD products to a chromosome-11 microarray correctly maps the HI gene to a 2-Mb region, thereby demonstrating linkage-disequilibrium mapping without genotyping.

A second-generation screen of the human genome for susceptibility to insulin-dependent diabetes mellitus.

During the past decade, the genetics of type 1 (insulin-dependent) diabetes mellitus (IDDM) has been studied extensively and the disorder has become a paradigm for genetically complex diseases. Previous genome screens and studies focused on candidate genes have provided evidence for genetic linkage between polymorphic DNA markers and 15 putative IDDM susceptibility loci, designated IDDM1-IDDM15. We have carried out a second-generation screen of the genome for linkage and analysed the data by multipoint linkage methods. An initial panel of 212 affected sibpairs (ASPs) was genotyped for 438 markers spanning all autosomes, and an additional 467 ASPs were used for follow-up genotyping. Other than the well-established linkage with the HLA region at chromosome 6p21.3, there was only one region, located on chromosome 1q and not previously reported, where the log likelihood ratio (lod) was greater than 3. Lods between 1.0 and 1.8 were found in six other regions, three of which have been reported in other studies. Another reported region, on chromosome 6q and loosely linked to HLA, also had an elevated lod. Little or no support was found for most reported IDDM loci (lods were less than 1), despite larger sample sizes in the present study.

A genome-wide search for human non-insulin-dependent (type 2) diabetes genes reveals a major susceptibility locus on chromosome 2.

Non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is a common disorder of middle-aged individuals characterized by high blood glucose levels which, if untreated, can cause serious medical complications and lead to early death. Genetic factors play an important role in determining susceptibility to this disorder. However, the number of genes involved, their chromosomal location and the magnitude of their effect on NIDDM susceptibility are unknown. We have screened the human genome for susceptibility genes for NIDDM using non-and quasi-parametric linkage analysis methods in a group of Mexican American affected sib pairs. One marker, D2S125, showed significant evidence of linkage to NIDDM and appears to be a major factor affecting the development of diabetes mellitus in Mexican Americans. We propose that this locus be designated NIDDM1.

Monoclonal antibody (SBU-1 and SBU-3) identification of cells dissociated from the sheep placentomal trophoblast.

We studied the localization of alpha-keratin in the sheep placenta using an alpha-keratin-specific monoclonal antibody (MAb) SBU-1, and examined the feasibility of using this MAb as a marker for determining the purity of isolated uninucleate cells from the placentomal trophoblast. At about 30-50 days of gestation the placentomal and interplacentomal uninucleate cells and some binucleate cells were stained by SBU-1, whereas only the apical region of the syncytial cytoplasm was stained with this MAb. Other cells stained included the uterine and endometrial glandular epithelial cells and fibroblast-like cells in the endometrium and chorionic villi. At about 100-130 days of gestation only the trophoblast uninucleate cells were stained by SBU-1. Approximately 60% of cells isolated from placentomes at 100-130 days of gestation were stained by SBU-1, and they had similar morphological features to the trophoblast uninucleate cells. The number of binucleate cells present was confirmed by their affinity for MAb SBU-3. These results show that MAb SBU-1 is an excellent marker for trophoblast uninucleate cells from placenta of sheep at the later stages of pregnancy.

Characterization of a sheep trophoblast-derived antigen first appearing at implantation.

A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. Lee et al (1985) have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the maternal syncytial layer. Immunoprecipitation of 125I-labelled microvilli by SBU-3, characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.

Localization and purification of SBU-4--a pregnancy specific protein of the ovine uterus.

In order to elucidate the function of molecules of the ovine maternal-fetal interface a monoclonal antibody was produced to intact interplacentomal trophoblast membranes. Extensive immunohistological studies revealed that the monoclonal antibody recognizes a protein designated SBU-4 which originates in the intercaruncular regions of the gravid sheep uterus at about the time of implantation and increases in concentration throughout gestation. The data suggest that SBU-4 is produced by endometrial epithelial cells and that adjacent uninucleate cells of the trophoblast acquire the antigen by endocytosis. Initial biochemical analysis of the purified SBU-4 molecule prepared by monoclonal antibody immunoaffinity chromatography indicates that SBU-4 is high molecular weight glycoprotein complex comprising several sub-units.

Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct subpopulations of class II molecules.

The availability of a panel of monoclonal antibodies to sheep MHC class I and class II gene products has allowed for the first time an assessment of the relative complexity of the sheep MHC. By using four monoclonal antibodies to MHC class I, and seven monoclonal antibodies to MHC class II molecules together with one-dimensional SDS-PAGE, sequential immunoprecipitation and 2-dimensional gel analysis, three class I gene products and four distinct subsets of class II molecules have been identified. Sheep class I molecules showed heterogeneity on 2-dimensional gels and as in mouse and man, represented the products of at least three different non-allelic class I genes. Interestingly, the sheep beta 2 microglobulin molecule also displayed heterogeneity, consistent with either two primary gene products or allelic variation. Four sheep class II monoclonal antibodies identified distinct, non-overlapping subsets of sheep class II molecules of Mr 32-36 K (alpha chain) and 25-28 K (beta chain). These class II molecules were co-expressed on sheep B lymphocytes and represented the primary products of different sheep MHC class II genes. The class II molecules within three of these subsets displayed allelic polymorphism essentially restricted to their beta polypeptides, while the fourth subset of class II molecules showed allelic variation in both their alpha and beta polypeptides. The results of this study represent the first evidence for gene duplication and heterogeneity within the sheep MHC. The identification of three primary class I gene products and four distinct subsets of class II molecules suggests three class I loci and up to four distinct class II subregions within the sheep MHC. Potentially large numbers of allelic variants of these different gene products may be expressed in normal sheep.

Genetic selection for disease resistance and traits of economic importance in animal production.

Significant advances have been made in recent years in improving animal stocks by selective breeding. However, existing selection techniques still rely on laborious and time-consuming progeny-testing programs and often depend on subjective assessment of the phenotype. New techniques in molecular genetics are being developed, aimed at the isolation and identification of DNA markers linked to genes for economically important production traits and disease resistance. When available, these markers will provide animal breeders with an objective test system to identify, at birth or even earlier, animals carrying desirable genes. This review outlines some of these new techniques and how they may be applied to the animal industries. Consideration is also given to some of the recent advances in our understanding of the immune system and of possible mechanisms of genetic control of animal disease resistance or susceptibility. The current knowledge of major histocompatibility complex (MHC) and non-MHC associated disease resistance/susceptibility in domestic animals is summarised and mechanisms which may be responsible for these associations are presented. Genes that control such factors as macrophage activation, cytokines, cytokine receptors and gamma delta-T cell receptors are also presented as potential candidates for analysis in genetic disease association studies. Ultimately, the goal will be to identify genes or DNA markers which can be used to select for or to genetically engineer disease resistance and enhanced production traits.

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB.

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrates considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dC.dA)15, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.

Sheep lymphocyte antigens (OLA). I. Major histocompatibility complex class I molecules.

Three monoclonal antibodies, SBU.I 41-17, 41-19 and 41-28, have been produced which recognize sheep Class I major histocompatibility complex antigens (OLA). All three antibodies are able to precipitate a heavy chain of 44,000 MW and a smaller beta 2-microglobulin of 12,000 MW from 125I-surface labelled lymphocytes. The antibodies have been used to localize OLA Class I antigens in lymphoid and non-lymphoid tissues using indirect immunoperoxidase histological staining and cytofluorograph analyses. Evidence suggests that the three antibodies are directed against monomorphic determinants but that they recognize different epitopes.

Expression of major histocompatibility complex antigens on the bovine placenta.

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.

Nucleotide sequence, polymorphism, and evolution of ovine MHC class II DQA genes.

The nucleotide sequence of all exons and introns, excluding exon 1, of the ovine major histocompatibility complex (MhcOvar) genes analogous to the HLA-DQA1 and -DQA2 genes has been determined and the gene structure found to be similar to that reported for other species. The predicted amino acid sequences of the Ovar-DQA genes have been compared with the equivalent DQA genes in man, mouse, rat, rabbit, and cattle and used to determine the evolutionary relationships of the sheep class II genes to these other species. Northern blot analysis of sheep mRNA using exon specific probes for each of the two Ovar-DQA genes show that both genes are transcribed, whereas in humans there is no evidence that HLA-DQA2 is transcriptionally active. Restriction fragment length polymorphisms (RFLPs) have been used to define a polymorphic series of alleles in both Ovar-DQA genes and have indicated that the number of DQA genes is not constant in sheep as it is in humans, but varies with the haplotype.

Site-directed differences in the immune response to the fetus.

Major differences in the maternal immune response to the fetus were observed in the placentomes and in the interplacentomal regions of the pregnant sheep uterus. Firstly, fewer lymphocytes were detected in the placentomes compared to the interplacentomal regions and to non-pregnant uterine tissue (Lee, Gogolin-Ewens & Brandon, 1988). Secondly, a large population of CD45R+ granulated lymphocytes was uniformly distributed in the interplacentomal uterine epithelium throughout pregnancy but never in the syncytial layer of the placentomes. Thirdly, monoclonal antibodies specific for the CD5 antigen consistently stained the endothelium of blood vessels within the placentomes but never blood vessels in the interplacentomal areas. Finally, OLA class I antigens were present on the interplacentomal uterine epithelial cells and on the maternal stromal cells, but no staining of the trophoblast or syncytium was observed. These observations suggest that different mechanisms to prevent immune rejection of the fetus may operate in the placentomes where trophoblast invasion of the maternal tissue occurs compared to the interplacentomal regions.

Characterization of two sheep lymphocyte differentiation antigens, SBU-T1 and SBU-T6.

The monoclonal antibodies 25.91 and 20.27 define two lymphocyte cell surface antigens of sheep. 25.91 is reactive with 60-80% of lymphocytes and 98% of thymocytes, and only stains surface immunoglobulin-negative peripheral lymphocytes. 25.91 immuno-precipitates a 67,000 MW protein from lymphocyte lysates under both reducing and non-reducing conditions, whereas immunoprecipitation of thymocyte lysates reveals a 67,000, 62,000 MW complex. The tissue distribution and molecular weight analysis reported here for the antigen recognized by 25.91 indicate that this antigen is the sheep homologue of the human T1 and mouse Ly 1 antigens. The monoclonal antibody 20.27 is reactive with 80% of thymocytes and the majority of cell surface immunoglobulin-positive peripheral blood lymphocytes (B cells), but is unreactive with peripheral blood T cells. 20-27 also stains Langerhans cells in skin tissue sections and large dendritic-like cells in the paracortex sections and large dendritic-like cells in the paracortex of lymph node tissue sections. Immunoperoxidase staining of thymus tissue sections with 20.27 shows intense staining of cortical thymocytes and an absence of staining within the medulla. Molecular weight analysis of the 20.27 antigen reveals two major bands of 46,000 and 12,000 MW under both reducing and non-reducing conditions. The 20.27 antigen has properties resembling MHC class I-like antigens such as T6 in the human and TL in the mouse.

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes.

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine lambda gt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class II A sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.

Characterization of placentation-specific binucleate cell glycoproteins possessing a novel carbohydrate. Evidence for a new family of pregnancy-associated molecules.

The ovine binucleate cell-specific glycoproteins recognized by the monoclonal antibody SBU-3 first appear at the initiation of placentation, and their expression continues throughout gestation. These placenta-specific proteins have not been detected in any other adult or fetal sheep tissues and are specific to the materno-fetal interface. The SBU-3 monoclonal antibody recognizes the carbohydrate epitope common to a group of proteins ranging in molecular mass from 30 to 200 kDa whose function during pregnancy remains undefined. The biochemical properties of these uniquely expressed glycoproteins were investigated by analyzing both the carbohydrate and protein portion of the molecules. Analysis of phytohemagglutinin and concanavalin A binding to electrophoretically separated SBU-3 proteins revealed that the major proteins between 40 and 70 kDa bind phytohemagglutinin. In contrast, concanavalin A bound only to minor proteins in the SBU-3 glycoprotein preparation. Analysis of the carbohydrate conjugated to the SBU-3 glycoproteins revealed that the major chains are sialylated O-linked and complex partially sialylated multiple antennary N-linked chains. The presence of N-glycolylneuraminic acid in an N-linked structure indicates the unique nature of this carbohydrate epitope. The differential binding to phytohemagglutinin and concanavalin A provided a method for further purification and characterization of the major protein components with monoclonal antibody immunoaffinity-purified SBU-3 proteins being further separated by concanavalin A-Sepharose chromatography. Microsequence analysis of the major non-concanavalin A-binding proteins (69, 62, and 57 kDa) revealed partial homology to ovine and bovine pregnancy-associated glycoprotein and rabbit pepsinogen F. Immunoblot analysis of the SBU-3 proteins showed cross-reactivity with polyclonal antisera directed against ovine placental-associated glycoprotein and pregnancy-specific glycoprotein B. These results suggest that together these glycoproteins represent members of a binucleate cell-derived family of pregnancy-associated molecules in the ruminant placenta.