CTLA4 promoter polymorphisms are associated with canine diabetes mellitus.

Canine diabetes mellitus (DM) shares many similarities with human type 1 diabetes (T1D). It is a complex genetic disorder, which shows marked differences in breed susceptibility, with Samoyed dogs being highly susceptible, whereas the Boxer breed is relatively resistant. A number of immune response genes, which have been associated with human T1D, have also been implicated in determining susceptibility to canine DM, suggesting an immune-mediated component to the disease pathogenesis. Single nucleotide polymorphisms (SNPs) in the CTLA4 gene have consistently and reproducibly been associated with human T1D and other autoimmune diseases but the canine CTLA4 gene has not previously been investigated for involvement in canine DM. SNPs of particular interest in the human association studies are those in the promoter region which affect CTLA4 expression levels, and that of exon 1 which results in a non-synonymous amino acid change. We performed a canine SNP discovery investigation of CTLA4 on a region of DNA containing exon 1 and 1.5 kb upstream sequence in order to identify promoter region SNPs. Confirmed SNPs were used in a genetic association study of a canine diabetic cohort showing that CTLA4 promoter polymorphisms were associated with diabetes in crossbreed dogs and in five Pedigree breeds-Samoyed, Miniature Schnauzer, West Highland White Terrier, Border Terrier and Labrador. Meta-analysis of these breeds showed 9 out of 15 SNPs were associated with DM and genotype and haplotype analyses also confirmed the allelic associations in these breeds.

Association of canine anal furunculosis with TNFA is secondary to linkage disequilibrium with DLA-DRB1*.

Anal furunculosis (AF) is a chronic inflammatory disease of perianal tissues that particularly affects German Shepherd dogs (GSD). An immune-mediated aetiopathogenesis is suggested by T-cell infiltration, upregulated cytokine gene expression, clinical response to ciclosporin therapy and a strong genetic association with the DLA-DRB1*00101 allele. Given the close proximity of TNFA and DLA-DRB1 in the canine major histocompatibility complex (MHC), together with the strong linkage disequilibrium (LD) observed across this region, the primary disease association could be with either locus. We have investigated whether there may be an association of AF with TNFA gene polymorphism in GSDs. Cohorts of AF-affected and AF-unaffected GSDs of known dog leucocyte antigen (DLA) class II profile were genotyped for 10 single nucleotide polymorphisms (SNPs) in the canine TNFA locus using Sequenom iPLEX technology. Seven discrete TNFA haplotypes were identified in GSDs for combinations of these SNPs. TNFA haplotype frequencies were compared in cases and controls. The TNFA haplotype 3 (ATCGTTACGG), was at significantly increased frequency in cases (29% vs 15%, OR 2.5, 95% CI 1.4-4.8; P = 0.003). All seven discrete TNFA SNP haplotypes were examined for their association with DLA-DRB1/DQA1/DQB1 established haplotypes. TNFA haplotype 3 was preferentially associated with both DLA-DRB1*00101(3A)- and DLA-DRB1*00102(3B)-positive haplotypes. The DLA-DRB1* 00101/TNFA-3A haplotype was significantly associated with AF (19.3% vs 5.8%; OR 3.7, 95% CI: 1.5-8.9; P = 0.003), whereas the DLA-DRB1*00102/TNFA-3B haplotype was not (P = NS). These findings suggest that susceptibility to AF in GSDs is primarily associated with DLA-DRB1*00101 and any association with the TNFA locus is secondary and is likely to be because of LD.

Risk of anal furunculosis in German shepherd dogs is associated with the major histocompatibility complex.

Anal furunculosis (AF) is a chronic, progressive inflammatory disease of the perianal tissues most frequently affecting middle-aged or older German Shepherd dogs (GSD). Because this breed accounts for over 80% of all reported cases, there is likely to be a genetic association with disease susceptibility. Although there are some similarities with perianal fistulation that occurs in human Crohn's disease, the aetiology and pathogenesis of AF are still poorly understood. Recent research has suggested an immune-mediated aetiology, and evidence for this has been further provided by clinical responses to the immunosuppressive drug cyclosporin. The aim of the current study was to investigate canine major histocompatibility complex immune response genes. Dog leucocyte antigen class II alleles and haplotypes were characterised by sequence-based typing of 107 GSD affected with AF and 196 breed-matched controls collected in the UK. A highly significant association of DLA-DRB1*00101 with the presence of AF was observed (OR = 5.01, CI = 2.7-9.3, P < 0.00000001). This association was confirmed in a second cohort of GSD collected in Finland. Homozygosity for this allele is associated with an earlier disease onset.

Identification of susceptibility and protective major histocompatibility complex haplotypes in canine diabetes mellitus.

Diabetes mellitus occurs spontaneously in dogs, which is believed to have an autoimmune component and to be a model of human latent autoimmune diabetes of adults (LADA). Some dog breeds (e.g. Samoyed) are particularly predisposed, whereas others (e.g. Boxer) are highly resistant. With the completion of the Dog Genome Assembly, comparative genomic studies of complex diseases in dogs, including diabetes, could provide an important investigative approach into such disorders. Type 1 diabetes in humans is strongly associated with major histocompatibility complex (MHC) class II polymorphisms. We have investigated whether canine dog leucocyte antigen (DLA) class II haplotypes are associated with diabetes. DNA from 460 cases and 1047 controls were genotyped for DLA-DRB1, DLA-DQA1 and DLA-DQB1 using sequence-based typing. Three DLA haplotypes, DRB1*009/DQA1*001/DQB1*008, DRB1*015/DQA1*0061/DQB1*023 and DRB1*002/DQA1*009/DQB1*001, were found at significantly increased frequency in cases with diabetes compared with controls. One DLA-DQ haplotype, DQA1*004/DQB1*013, was significantly reduced in cases with diabetes. Further analysis showed that DQA1 alleles carrying arginine at codon 55 of DQA1 were increased in dogs with diabetes. To our knowledge, this is the first report of a comparative study of MHC and diabetes in a non-rodent species. Since no laboratory model of LADA exists and dogs and humans share similar environments, further research into canine diabetes is warranted.

The Tabby cat locus maps to feline chromosome B1.

The Tabby markings of the domestic cat are unique coat patterns for which no causative candidate gene has been inferred from other mammals. In this study, a genome scan was performed on a large pedigree of cats that segregated for Tabby coat markings, specifically for the Abyssinian (Ta-) and blotched (tbtb) phenotypes. There was linkage between the Tabby locus and eight markers on cat chromosome B1. The most significant linkage was between marker FCA700 and Tabby (Z = 7.56, theta = 0.03). Two additional markers in the region supported linkage, although not with significant LOD scores. Pairwise analysis of the markers supported the published genetic map of the cat, although additional meioses are required to refine the region. The linked markers cover a 17-cM region and flank an evolutionary breakpoint, suggesting that the Tabby gene has a homologue on either human chromosome 4 or 8. Alternatively, Tabby could be a unique locus in cats.

White spotting in the domestic cat (Felis catus) maps near KIT on feline chromosome B1.

Five feline-derived microsatellite markers were genotyped in a large pedigree of cats that segregates for ventral white spotting. Both KIT and EDNRB cause similar white spotting phenotypes in other species. Thus, three of the five microsatellite markers chosen were on feline chromosome B1 in close proximity to KIT; the other two markers were on feline chromosome A1 near EDNRB. Pairwise linkage analysis supported linkage of the white spotting with the three chromosome B1 markers but not with the two chromosome A1 markers. This study indicates that KIT, or another gene within the linked region, is a candidate for white spotting in cats. Platelet-derived growth factor alpha (PDGFRA) is also a strong candidate, assuming that the KIT-PDGFRA linkage group, which is conserved in many mammalian species, is also conserved in the cat.

Characterization of the COMMD1 (MURR1) mutation causing copper toxicosis in Bedlington terriers.

Copper toxicosis is an autosomal recessive disorder affecting Bedlington terriers, characterized by elevated liver copper levels and early death of affected dogs. Genetic linkage mapping studies initially identified linkage between the disease and the microsatellite marker C04107. Subsequently, the deletion of exon 2 of the copper metabolism domain containing 1 (COMMD1) gene (formerly MURR1) was shown to be the major cause of copper toxicosis, although the deletion breakpoints were not defined. In this investigation, polymerase chain reaction (PCR)-based techniques and sequencing were used to isolate the deletion breakpoints, utilizing the newly available dog genome sequence. The breakpoints were positioned at 65.3091 and 65.3489 Mb of dog chromosome 10, in intron 1 and intron 2 of COMMD1 respectively, a deletion of 39.7 kb. The two breakpoints share sequence homology suggesting that homologous recombination may have been responsible for the deletion. Using this information, a genomic diagnostic test for the COMMD1 deletion was developed and compared with microsatellite C04107 genotypes of 40 Bedlington terriers. Results from the 40 samples showed allele 2 of C04107 to be in linkage disequilibrium with the COMMD1 deletion.

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications.

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.

High-resolution characterization of the canine DLA-DRB1 locus using reference strand-mediated conformational analysis.

Several methods exist for genotyping class II DLA gene polymorphisms in the dog. The most accurate method is sequence-based typing, which involves direct sequencing of polymerase chain reaction products. However, this method is expensive and unsuitable for large-scale studies. Recently, reference strand-mediated conformation analysis (RSCA) has been shown to be effective for characterizing major histocompatibility complex genes in humans, sheep, horse, and cats. RSCA is a cheap and rapid method, ideal for large epidemiological studies. We have developed RSCA for typing DLA-DRB1 in the dog. Control panels including dogs typed by sequence-based typing and cloned major histocompatibility complex class II alleles in plasmids were used to establish migration patterns for each allele using 20 different fluorescent labeled references, of which 5 were selected to allow for clear identification and discrimination of all known DLA-DRB1 alleles. We have compared 168 dogs typed by RSCA for DLA-DRB1 and characterized by sequence-based typing, with less than 1% discrepancy. These differences were due to missing alleles because of a weak polymerase chain reaction. To date, we have RSCA-typed 1,394 dogs. RSCA is likely to become the method of choice for characterizing DLA genes in the dog and will prove a useful tool for dissecting the immune response of dogs in clinical studies.

Localizing the X-linked orange colour phenotype using feline resource families.

Many genes influencing mammalian coat colours are well conserved. While genes responsible for pelage phenotypes in one species provide strong evidence for a candidate gene in a different species, the X-linked orange phenotype of the domestic cat is unique within mammals. The orange locus (O) undergoes X-inactivation, producing females that express both wildtype black (wt) and orange (variant) phenotypes when heterozygous (tortoiseshell). The orange locus has not yet been localized on the X chromosome. Tortoiseshell male cats have been identified but have been shown to be sex chromosome trisomies (XXY). To localize the cat orange locus, 10 feline-derived X-linked microsatellites were analysed in two extended cat pedigrees consisting of 79 and 55 individuals, respectively, segregating for the orange phenotype. Linkage analyses excluded close association of orange in the vicinity of the nine informative X-linked microsatellites. One marker was not polymorphic within either family. Several markers suggested exclusion (Z < -2.0) at distances of 7.5-33 cM. Exclusion analyses suggested a possible location for orange a 14 cM region near Xcen. Recombination distances of markers in the segregating feline pedigrees were reduced as compared with the feline interspecies backcross family. Thus, the presented pedigrees may be useful as reference families for the domestic cat because more accurate recombination rates for domestic cats can be determined.

A physical and transcript map based upon refinement of the critical interval for PPH1, a gene for familial primary pulmonary hypertension. The International PPH Consortium.

Primary pulmonary hypertension (PPH), an often fatal disorder, is characterized by sustained elevation of pulmonary artery pressure of unknown cause. In its familial form (FPPH), the disorder segregates as an autosomal dominant and displays markedly reduced penetrance. A gene for FPPH was previously localized to a 25-cM interval on the long arm of chromosome 2 (2q31-q33). We now report a complete yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC)/P1 artificial chromosome contig (PAC), assembled by STS content mapping, across a newly identified minimum nonrecombinant interval containing the gene designated PPH1. The physical map has served to establish polymorphic marker order unequivocally, enabling the establishment of detailed haplotypes for the region. Together with the identification of novel recombination events in affected individuals from six newly ascertained kindreds, these data have allowed the significant reduction of the minimum PPH1 critical interval to a 4.8-cM region. The region, flanked by the polymorphic markers D2S115 (centromeric) and D2S1384 (telomeric), corresponds to a minimum physical distance of 5.8 Mb at 2q33. Numerous expressed sequence tags and known genes were placed on the YAC/BAC contig spanning the PPH1 gene critical region.

Genetic relationships of Asians and Northern Europeans, revealed by Y-chromosomal DNA analysis.

We have identified a new T-->C transition on the human Y chromosome. C-allele chromosomes have been found only in a subset of the populations from Asia and northern Europe and reach their highest frequencies in Yakut, Buryats, and Finns. Examination of the microsatellite haplotypes of the C-allele chromosomes suggests that the mutation occurred recently in Asia. The Y chromosome thus provides both information about population relationships in Asia and evidence for a substantial paternal genetic contribution of Asians to northern European populations such as the Finns.

Evaluation of Y-chromosomal STRs: a multicenter study.

A multicenter study has been carried out to characterize 13 polymorphic short tandem repeat (STR) systems located on the male specific part of the human Y chromosome (DYS19, DYS288, DYS385, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, YCAI, YCAII, YCAIII, DXYS156Y). Amplification parameters and electrophoresis protocols including multiplex approaches were compiled. The typing of non-recombining Y loci with uniparental inheritance requires special attention to population substructuring due to prevalent male lineages. To assess the extent of these subheterogeneities up to 3825 unrelated males were typed in up to 48 population samples for the respective loci. A consistent repeat based nomenclature for most of the loci has been introduced. Moreover we have estimated the average mutation rate for DYS19 in 626 confirmed fatherson pairs as 3.2 x 10(-3) (95% confidence interval limits of 0.00041-0.00677), a value which can also be expected for other Y-STR loci with similar repeat structure. Recommendations are given for the forensic application of a basic set of 7 STRs (DYS19, DYS3891, DYS389II, DYS390, DYS391, DYS392, DYS393) for standard Y-haplotyping in forensic and paternity casework. We recommend further the inclusion of the highly polymorphic bilocal Y-STRs DYS385, YCAII, YCAIII for a nearly complete individualisation of almost any given unrelated male individual. Together, these results suggest that Y-STR loci are useful markers to identify males and male lineages in forensic practice.

Chromosome Y microsatellites: population genetic and evolutionary aspects.

By means of a multicenter study, a large number of males have been characterized for Y-chromosome specific short tandem repeats (STRs) or microsatellites. A complete summary of the allele frequency distributions for these Y-STRs is presented in the Appendix. This manuscript describes in more detail some of the population genetic and evolutionary aspects for a restricted set of seven chromosome Y STRs in a selected number of population samples. For all the chromosome Y STRs markedly different region-specific allele frequency distributions were observed, also when closely related populations were compared. Haplotype analyses using AMOVA showed that when four different European male groups (Germans, Dutch, Swiss, Italians) were compared, less than 10% of the total genetic variability was due to differences between these populations. Nevertheless, these pairwise comparisons revealed significant differences between most population pairs. Assuming a step-wise mutation model and a mutation frequency of 0.21%, it was estimated that chromosome Y STR-based evolutionary lines of descent can be reliably inferred over a time-span of only 1950 generations (or about 49,000 years). This reduces the reliability of the inference of population affinities to a historical, rather than evolutionary time scale. This is best illustrated by the construction of a human evolutionary tree based on chromosome Y STRs in which most of the branches connect in a markedly different way compared with trees based on classical protein polymorphisms and/or mtDNA sequence variation. Thus, the chromosome Y STRs seem to be very useful in comparing closely related populations which cannot probably be separated by e.g. autosomal STRs. However, in order to be used in an evolutionary context they need to be combined with more stable Y-polymorphisms e.g. base-substitutions.

Recurrent duplication and deletion polymorphisms on the long arm of the Y chromosome in normal males.

Deletion of the 50f2/C (DYS7C) locus in interval 6 of Yq has previously been reported as a polymorphism in three males. We describe a survey of worldwide populations for further instances of this deletion. Of 859 males tested, 55 (approximately 6%) show absence of the 50f2/C locus; duplication of the locus was also detected in eight out of 595 males (approximately 1.4%). Populations having the deletion are confined to Asia, Australasia, and southern and northern Europe; of those of reasonable sample size, Finns had the highest deletion frequency (55%; n = 21). The deletions vary in size and the larger ones remove some of the RBM (RNA Binding Motif) genes, but none of the deletion males lack DAZ (Deleted in AZoospermia), a candidate gene for the azoospermia factor. On a tree of Y haplotypes, 28 deletion and eight duplication chromosomes fall into six and four haplotypic groups respectively, each of which is likely to represent an independent deletion or duplication event. Microsatellite and other haplotyping data suggest the existence of at least two further classes of deletion. Thus duplications and deletions in this region of Yq have occurred many times in human evolution, but remain useful markers for paternal lineages.

Mutation processes at human minisatellites.

Minisatellites provide one of the most experimentally tractable systems for studying tandem repeat instability in man. Analysis of mutation processes has been greatly aided by the development of single molecule methods for recovering de novo mutants, and of techniques for exploring allele structure in detail. Application of these approaches to man has shown that minisatellites do not primarily mutate by processes such as replication slippage and unequal crossover intrinsic to the tandem repeat array. Instead, germline repeat instability is largely regulated by cis-acting elements near the array and involves unexpectedly complex processes of gene conversion, of potential relevance to the biology of meiosis. These processes can be explored both in humans and, in principle, in transgenic mouse models of human repeat instability.

Minisatellite mutation rate variation associated with a flanking DNA sequence polymorphism.

Human minisatellite mutation in the male germline frequently involves complex interallelic gene conversion events restricted to one end of the tandem repeat array. Some alleles at minisatellite MS32 show reduced variability in human populations and are associated with a G to C transversion upstream of the array. Analysis of single sperm demonstrated a frequently profound reduction in mutation rate at alleles carrying the C variant. This mutation suppression acts in cis, but does not affect the ability of an allele to act as sequence donor during gene conversion. This mutation rate polymorphism provides strong evidence for elements near the minisatellite that regulate tandem repeat instability.