A novel VWF variant associated with type 2 von Willebrand disease in German Wirehaired Pointers and German Shorthaired Pointers.

Von Willebrand disease (VWD), caused by deficiency of the von Willebrand factor (VWF), is the most common bleeding disorder in humans and dogs. The complete cDNA encoding VWF of a German Wirehaired Pointer with type 2 VWD was sequenced, and we found four variants that alter the amino acid sequence. These variants were: c.1657T>G corresponding to p.Trp553Gly; c.1777G>A (p.Glu593Lys); c.4937A>G (p.Asn1646Ser) and c.5544G>A (p.Met1848Ile). A haplotype of the c.1657G, c.1777A and c.4937G alleles co-segregated with the VWF antigen level in a four-generation pedigree with the disease. Healthy dogs of the breed were found that were homozygous for the c.1777A or the c.5544A allele, indicating that these variants do not cause VWD. Dogs that were homozygous for the c.4937G allele and had no signs of a bleeding disorder were observed in the Chinese Crested dog breed. Thus, only the c.1657G variant was found in the homozygous state exclusively in VWD affecteds, and this variant is the strongest candidate to be the cause of VWD type 2 in the German Wirehaired Pointer breed. A screen of German Shorthaired Pointers indicated that the variant also segregates with VWD in this breed.

High-resolution genetic mapping of mammalian motor activity levels in mice.

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

Evaluation of the serotonergic genes htr1A, htr1B, htr2A, and slc6A4 in aggressive behavior of golden retriever dogs.

Aggressive behavior displays a high heritability in our study group of Golden Retriever dogs. Alterations in brain serotonin metabolism have been described in aggressive dogs before. Here, we evaluate whether four genes of the canine serotonergic system, coding for the serotonin receptors 1A, 1B, and 2A, and the serotonin transporter, could play a major role in aggression in Golden Retrievers. We performed mutation screens, linkage analysis, an association study, and a quantitative genetic analysis. There was no systematic difference between the coding DNA sequence of the candidate genes in aggressive and non-aggressive Golden Retrievers. An affecteds-only parametric linkage analysis revealed no strong major locus effect on human-directed aggression related to the candidate genes. An analysis of 41 single nucleotide polymorphisms (SNPs) in the 1 Mb regions flanking the genes in 49 unrelated human-directed aggressive and 49 unrelated non-aggressive dogs did not show association of SNP alleles, genotypes, or haplotypes with aggression at the candidate loci. We completed our analyses with a study of the effect of variation in the candidate genes on a collection of aggression-related phenotypic measures. The effects of the candidate gene haplotypes were estimated using the Restricted Maximum Likelihood method, with the haplotypes included as fixed effects in a linear animal model. We observed no effect of the candidate gene haplotypes on a range of aggression-related phenotypes, thus extending our conclusions to several types of aggressive behavior. We conclude that it is unlikely that these genes play a major role in the variation in aggression in the Golden Retrievers that we studied. Smaller phenotypic effects of these loci could not be ruled out with our sample size.

Phenotyping of aggressive behavior in golden retriever dogs with a questionnaire.

Reliable and valid phenotyping is crucial for our study of genetic factors underlying aggression in Golden Retriever dogs. A mail questionnaire based on the Canine Behavioral Assessment and Research Questionnaire (CBARQ; Hsu and Serpell, 2003, JAVMA 223(9):1293-1300) was used to assess behavioral phenotypes. Owners of 228 Golden Retrievers completed the questionnaire. These dogs had been referred to our clinic for aggression problems several years earlier or they were related to aggressive dogs. In this paper, three sets of results are presented, which indicate that behavior scores from the CBARQ can be applied to genetic studies. First, factor analysis demonstrated that CBARQ items can be grouped into 10 behavioral traits, including three types of aggression: stranger-directed aggression, owner-directed aggression, and dog-directed aggression. The results were remarkably similar to those reported by Hsu and Serpell. The aggression scores showed considerable variation in our dog families, which is a prerequisite for genetic studies. Second, retrospective questions enabled us to study changes in the aggressive behavior of the dogs in the course of time. After an average time interval of 4.3 years, over 50% of the dogs had become less aggressive. Third, we analyzed data obtained with an aggression test of 83 dogs. Two out of the three CBARQ aggression factors were also found in the aggression test data.

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.

Structure and variation of three canine genes involved in serotonin binding and transport: the serotonin receptor 1A gene (htr1A), serotonin receptor 2A gene (htr2A), and serotonin transporter gene (slc6A4).

Aggressive behavior is the most frequently encountered behavioral problem in dogs. Abnormalities in brain serotonin metabolism have been described in aggressive dogs. We studied canine serotonergic genes to investigate genetic factors underlying canine aggression. Here, we describe the characterization of three genes of the canine serotonergic system: the serotonin receptor 1A and 2A gene (htr1A and htr2A) and the serotonin transporter gene (slc6A4). We isolated canine bacterial artificial chromosome clones containing these genes and designed oligonucleotides for genomic sequencing of coding regions and intron-exon boundaries. Golden retrievers were analyzed for DNA sequence variations. We found two nonsynonymous single nucleotide polymorphisms (SNPs) in the coding sequence of htr1A; one SNP close to a splice site in htr2A; and two SNPs in slc6A4, one in the coding sequence and one close to a splice site. In addition, we identified a polymorphic microsatellite marker for each gene. Htr1A is a strong candidate for involvement in the domestication of the dog. We genotyped the htr1A SNPs in 41 dogs of seven breeds with diverse behavioral characteristics. At least three SNP haplotypes were found. Our results do not support involvement of the gene in domestication.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Evaluation of canine COL4A3 and COL4A4 as candidates for familial renal disease in the Norwegian elkhound.

The collagen type IV alpha3 and alpha4 chains (COL4A3 and COL4A4) are part of the specialized glomerular basement membrane in the kidney. In human these genes are responsible for Alport syndrome (a type of hereditary nephritis). Histopathological similarities between kidneys of Norwegian elkhound dogs affected with familial renal disease and human Alport syndrome were the basis for a candidate gene approach in Norwegian elkhounds. Three microsatellites-tightly linked to canine COL4A3 and COL4A4--were developed. The microsatellites were used to analyze linkage between COL4A3 and COL4A4 and familial renal disease in a Norwegian elkhound pedigree segregating this disease. Presence of one recombinant between familial renal disease and COL4A3/COL4A4 suggests that these genes are not likely candidates for familial renal disease in this breed.

Non-pruritic granuloma in Norwegian forest cats.

The eosinophilic granuloma complex is a group of skin disorders common in cats. This paper describes the clinical, haematological and histopathological features of 17 related Norwegian forest cats, six of which had a linear granuloma on the caudal thigh, three of which also had a granuloma on the lower lip, and one of which had a granuloma in combination with an indolent ulcer. The high prevalence of the disease in this population is suggestive of a genetic background.

Chicken single nucleotide polymorphism identification and selection for genetic mapping.

Single nucleotide polymorphisms (SNP) are the ideal markers for high-density genome wide mapping. A total of 327,000 expressed sequence tag (EST) sequences, obtained from the ChickEST project, were examined for the presence of SNP. A total of 32,268 potential chicken SNP were identified and stored in a customized Microsoft Access database and evaluated in silico for their usability for a high-density genetic map. Based on a minimum of 3 for the minor allele occurrence and a minimum of 30% for the minor allele frequency, 5,332 reliable SNP were selected, of which both SNP alleles were present in the database at a high frequency. To test the usefulness of the in silico SNP identification, 24 SNP affecting a BglII site were used for a genotyping study. A functional PCR assay could be designed for 21 of the 24 SNP. It was possible to validate 90% of this marker subset (21 SNP) by BglII restriction analysis. The high percentage of validated markers demonstrates the reliability of the 5,332 chicken SNP markers. Furthermore, the limited number of genomic DNA samples necessary to validate 90% of the SNP markers confirmed the prediction of the high frequency at which both alleles of the selected SNP were present in the tested chicken populations.

Analysis of the inheritance of white spotting and the evaluation of KIT and EDNRB as spotting loci in Dutch boxer dogs.

The genetic basis of the white spotting pattern in Dutch boxer dogs is not known. We studied whether the segregation of white spotting in boxers follows a Mendelian inheritance pattern. Blood samples were collected, along with digital photographs in standard directions of (grand)parents (n=16) and offspring (n=52) from eight litters of Dutch boxers. In order to select heterozygous parents, we selected nonuniform litters, in which at least one puppy was extreme white. On the basis of criteria for the location, the extent of white spotting, and the mean percentage of pigmented area of the foot soles, we classified 10 dogs as solid colored, 27 as flashy, and 15 as extreme white. This was not a significant deviation from the expected 1:2:1 ratio. Because the flashy phenotype seems to be an intermediate between the two homozygotes, white spotting in the Dutch boxer can be considered to be due to a single gene effect, with incomplete dominance. We have evaluated candidate genes c-KIT (KIT) and EDNRB for segregation with white spotting phenotype in these litters. Using polymorphic markers, very near the KIT and EDNRB genes, we found that segregation of the white spotting pattern did not coincide with segregation of these polymorphic markers. Thus neither KIT nor EDNRB are likely to be responsible for white spotting in the Dutch population of boxers.

Development of a single nucleotide polymorphism map of porcine chromosome 2.

Single nucleotide polymorphism markers are developed on SSC2, predominantly on the p-arm. Several studies reported a quantitative trait loci (QTL) for backfat thickness in this region. Single nucleotide polymorphisms were identified by comparative re-sequencing of polymerase chain reaction (PCR) products from a panel of eight individuals. The panel consisted of five Large Whites (each from a different Dutch breeding company), a Meishan, a Pietrain and a Wild Boar. In total, 67 different PCR products were sequenced and 301 SNPs were identified in 32,429 bp of consensus sequence, an average of one SNP in every 108 bp. After correction for sample size, this polymorphism rate corresponds to a heterozygosity value of one SNP in every 357 bp. For 63% of the SNPs, there was variation among the five Large Whites, and these SNPs are relevant for linkage and association studies in commercial populations. Comparing the Whites with other breeds revealed higher variation rates with: (i) Meishan, 89%; (ii) Pietrain, 69%; (iii) Wild Boar, 70%. Because many of the experimental populations to identify QTL are based on crosses between these breeds, these SNPs are relevant for the fine mapping of the QTL identified within these crosses.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.

Isolation and characterization of the canine serotonin receptor 1A gene (htr1A).

Although the serotonergic system and htr1A have been studied extensively, little is known about the canine serotonin receptor 1A. We are interested in this receptor in the dog because it is likely to be involved in behavioral disorders such as anxiety. Therefore, we isolated a canine bacterial artificial chromosome (BAC) clone containing htr1A, and, with the help of this clone, the complete canine coding sequence of this gene was determined. Radiation hybrid (RH) mapping showed that htr1A is part of a conserved linkage group also including the survival of motor neuron 1 (smn1) gene. Htr1A is estimated to be located about 7.3 Mb from smn1 on cfa02. In addition, we report a possible breed-specific variant of the gene in four golden retrievers.

Familial non-rcd1 generalised retinal degeneration in Irish setters.

Four Irish setters were diagnosed with bilateral retinal degeneration and cataracts at an age ranging from six to 11 years. In three of these dogs, progressive night blindness was reported from an age of eight to 11 years. In the fourth dog, aged six, no signs of visual impairment had been noticed. In all four dogs, the rod-cone dysplasia type 1 (rcd1) mutation was excluded as a cause, using an allele-specific PCR. From their three-generation pedigrees, a familial relationship was detected in three out of four dogs, which were also related to four additional Irish setter dogs with a history and clinical signs suggestive of late-onset progressive retinal degeneration. These results suggest the existence of a possibly hereditary, late-onset, progressive retinal atrophy in the Irish setter breed, that is distinct from rcd1.

Exclusion of the lim homeodomain gene LHX4 as a candidate gene for pituitary dwarfism in German shepherd dogs.

Pituitary dwarfism in the German shepherd dog is an autosomal recessive inherited abnormality. We tested the hypothesis that a variant of the LIM homeodomain gene LHX4 is responsible for the dwarfism phenotype. To this end, we isolated Bacterial Artificial Chromosome clones for the canine LHX4 gene. Southern blotting experiments showed that the LHX4 gene is a single copy gene in the canine genome. A complex CA-repeat was isolated from the BAC clones and was found to be polymorphic in German shepherd dogs. Genotyping 5 litters in which the dwarfism was segregating showed disconcordance between the inheritance of the dwarfism phenotype and the DNA marker. It is concluded that the LHX4 gene does not play a primary role in the pituitary dwarfism in the German shepherd dogs.

Quantitative trait loci influencing hepatic copper in rats.

Significant differences in liver copper content have been observed between rat inbred strains. To define loci controlling this trait, the offspring (n = 190) from an (LEW/OlaHsd x BC/CpbU) F(2)-intercross was genetically analyzed. From each F(2) animal, liver copper content was determined and genomic DNA was screened with polymorphic DNA markers. We found a major quantitative trait locus (QTL) for liver copper content in females on chromosome 2 and in males on chromosome 10. Both QTLs accounted for approximately 20% of the genetic variance. In addition, suggestive linkage for liver copper content was found on rat chromosomes 1, 8, 10, 12, 14, and 19. The regions on these chromosomes contain genes that are responsible for 9.0-15.5% of the genetic variance of liver copper content.

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris).

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR(3000) and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1-ALAS2-AR-IL2RG-XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.

Genetic and correlation analysis of hepatic copper content in the rat.

Thirty recombinant inbred (RI) strains derived from the spontaneous hypertensive rat (SHR/OlaIpcv) and the Brown Norway (BN-Lx/Cub) progenitors were used to search for quantitative trait loci (QTLs) that are responsible for differences in liver copper between these two strains. The heritability of liver copper concentration (expressed as microg/g liver wet wt and microg/g liver dry wt) and liver copper store (microg/whole liver) was estimated to be 57, 57, and 46%, respectively. In a total genome scan of the RI strains, involving over 600 genetic markers, suggestive association was found between liver copper store (microg/whole liver) and the D16Wox9 marker on chromosome 16 (lod score = 2.8), and between liver copper concentration (microg/g dry wt) and the D10Cebrp1016s2 marker on chromosome 10 (lod score = 3.0). These putative QTLs are responsible for nearly 34 and 40% of the additive genetic variability for liver copper store and concentration, respectively.

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs.