Novel polymorphism of the canine dopamine receptor D4 gene intron II region

Various dog breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of these characteristics are under genetic control. However, little is known about genes related to behavioral traits in canine species. In humans, it has been reported that the dopamine receptor D4 gene (DRD4) includes polymorphism at several regions that relate to personality or psychiatric disorders. In an earlier study by the authors of the present study, the polymorphisms in canine DRD4 exon III and exon I regions were reported. In the present study, a novel polymorphism in canine DRD4 intron II was found based on a 17 base pair insertion/deletion, and the two alleles detected were named P (shorter allele) and Q (longer allele). The allelic distribution in 28 breeds of dog, including a total of 1114 unrelated individuals, were then investigated. Both P and Q alleles were detected in most of the breeds investigated; however, the frequencies of P and Q differed greatly between breeds. With respect to classification based on breed origin, P and Q alleles were frequent in Occidental and Oriental breeds, respectively. Furthermore, two subspecies of wolves, the ancestors of dogs, were analyzed for the comparison of allele frequencies with dogs, and the P allele was predominant in both European and Chinese wolves.     

Breed Differences in Dopamine Receptor D4 Gene (DRD4) in Horses

Genetic polymorphisms in genes related to neurotransmitters or hormones affect personality or behavioral traits in many animal species including humans. In domestic animals, the allele frequency of such genes has been reported to be different among breeds and it may account for breed differences in behavior. In this study, we investigated breed differences in horses in the dopamine receptor D4 gene (DRD4), which has been reported to affect horse personality. We collected samples from seven horse breeds including those native to Japan and Korea, and compared the sequence of the DRD4 exon3 region among these breeds. We found that there were two types of polymorphisms (VNTR and SNPs) in the exon3 region, and some of them seemed to be breed-specific. In addition, we found that the allele frequency of G292A, reported to be associated with horse personality, differed greatly between native Japanese horses and Thoroughbred horses. The frequency of the A allele which is associated with low curiosity and high vigilance, was much lower in native Japanese horses (Hokkaido, 0.03; Taishu, 0.08) than in Thoroughbreds (0.62). This difference may account for breed differences in personality or behavioral traits. Further studies of the function of these polymorphisms and their effect on behavior are indicated.     

Allelic variation of the D4 dopamine receptor polymorphic region in two dog breeds, Golden retriever and Shiba

The D4 dopamine receptor (D4DR) polymorphic region, which is possibly related to the personality trait known as novelty seeking in humans, was examined in 34 dogs from two breeds (Golden retriever and the Japanese indigenous breed, Shiba) by the polymerase chain reaction (PCR), and the DNA sequences of each allele were determined. The polymorphic region of the dog D4DR gene was composed of 39- and 12- base pair (bp) units, and four alleles (A-D) were identified based on the number and/or order of these units. Intra- and inter-breed allele variations were observed. The frequency of the short A allele was dominant (78.9%) in the Golden retriever, while the long D allele was most common (46.7%) in the Shiba. These findings suggested that the allele frequency varied significantly between different breeds, and that analysis of the polymorphism in D4DR might be of use for understanding the behavioral traits of dogs.     

Identification of an cysteine-to-arginine substitution caused by a single nucleotide polymorphism in the canine monoamine oxidase B gene

Monoamine oxidase B catalytically oxidizes biogenic amines such as phenylethylamine and dopamine, and its activity is presumed to be related to particular behavioral traits. In this study, we first identified a single nucleotide polymorphism (T199C) located on the putative third exon of the canine monoamine oxidase B gene, which causes an amino acid substitution from cysteine to arginine. We then examined the allelic frequencies in five dog breeds (Golden Retriever, Labrador Retriever, Maltese, Miniature Schnauzer, and Shiba) and found significant variation among them. The present results suggest that analysis of the monoamine oxidase B polymorphism could be a useful means of elucidating the genetic background of breed-specific behavioral characteristics in dogs.     

Polymorphisms in the canine glutamate transporter-1 gene: identification and variation among five dog breeds

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.     

Breed differences in genotype and allele frequency of catechol O-methyltransferase gene polymorphic regions in dogs

Catechol O-methyltransferase (COMT) inactivates catecholamines and catechol-containing drugs such as L-DOPA. The common genetic polymorphism Val158Met in the human COMT gene is suspected to be associated with "persistence" or risk for schizophrenia. In this study, we attempted to identify the canine COMT gene fragment and to find a similar polymorphism and to reveal its genetic distribution among five representative canine breeds. We found that the amplified gene consisted of 663 bp nucleotides and was 84% homologous with the human COMT gene. The single nucleotide polymorphisms, guanine adenine substitution, were observed at the 39th, 216th and 482nd nucleotides. From the genotyping of the 216th polymorphism among 266 dogs by the polymerase chain reaction-restriction fragment length polymorphism method with restriction enzyme EagI, and that of the 482nd polymorphism with restriction enzyme SfcI, we found inter-breed variations of genotypes as well as of allelic frequencies for both of these polymorphic regions. These results suggest that the identified polymorphisms will be useful tools in elucidating the genetic background of canine behavioral traits.     

Identification of single nucleotide polymorphisms within exon 1 of the canine mu-opioid receptor gene

Objective  To determine the presence and frequency of single nucleotide polymorphisms (SNPs) within exon 1 of the canine mu-opioid receptor (MOR) gene.
Study design  Prospective genetic analysis.
Animals  Seventy-five dogs of various breeds.
Methods  DNA was isolated from dog blood. Polymerase chain reaction (PCR) was performed to amplify exon 1 of the canine MOR gene using primers derived from a published sequence. PCR products of anticipated size were identified by gel electrophoresis, isolated and sequenced.
Results  Two SNPs were found within the examined region. One is 15 base pairs (bp) upstream (C-15A) of the protein-coding portion of the gene. The second is at position 207 (C207T); a synonymous mutation predicting unaltered protein sequence. The overall prevalence of the C-15A SNP was 43% (64/150 alleles). The overall prevalence of the C207T SNP was 26% (39/150 alleles).
Conclusions and clinical relevance  Absence of haplotypes containing both an adenosine at position −15 and a thymine at position 207 suggests that these polymorphisms occurred independently from each other. How these SNPs influence variations in responses seen after opioid administration to dogs remain to be determined, however, our data indicates the C-15A SNP may play a role in opioid dysphoria.     

Lack of GNB3 exon 9 polymorphism in primary hypertensive and normotensive dogs

The 5500T allele variant of the C5500T single nucleotide polymorphism in the human G protein beta3 subunit (GNB3) has been reported to be associated with primary hypertension. In this study, the GNB3 gene of primary hypertensive and normotensive dogs was examined for an analogous nucleotide polymorphism associated with hypertension. The genomic GNB3 dna, with 10 exons and nine introns coding for 340 amino acids, is described. PCR product sequencing of the GNB3 exon 9 from 25 dogs (including five hypertensive animals) failed to detect any nucleotide polymorphism. In contrast to human beings, there was no polymorphism at either the analogous nucleotide or in the respective exon. Only the human hypertension-associated thymine was detected, regardless of whether the dogs were hypertensive or normotensive. Furthermore, examinations of 565 dogs of 85 distinct breeds for the presence of the human 5500C nucleotide at the analogous nucleotide side failed to detect a cytosine that is present with high allele frequency in normotensive man. Owing to the lack of allele variance, it is concluded that canine primary hypertension is not associated with a polymorphism at either the respective human hypertension-associated nucleotide site or in the entire exon.     

A truncated retrotransposon disrupts the GRM1 coding sequence in Coton de Tulear dogs with Bandera's neonatal ataxia

Background: Bandera's neonatal ataxia (BNAt) is an autosomal recessive cerebellar ataxia that affects members of the Coton de Tulear dog breed. Objective: To identify the mutation that causes BNAt. Animals: The study involved DNA from 112 Cotons de Tulear (including 15 puppies with signs of BNAt) and 87 DNA samples from dogs of 12 other breeds. Methods: The BNAt locus was mapped with a genome‐wide association study (GWAS). The coding exons of positional candidate gene GRM1, which encodes metabotropic glutamate receptor 1, were polymerase chain reaction (PCR)‐amplified and resequenced. A 3‐primer PCR assay was used to genotype individual dogs for a truncated retrotransposon inserted into exon 8 of GRM1. Results: The GWAS indicated that the BNAt locus was in a canine chromosome 1 region that contained candidate gene GRM1. Resequencing this gene from BNAt‐affected puppies indicated that exon 8 was interrupted by the insertion of a 5′‐truncated retrotransposon. All 15 BNAt‐affected puppies were homozygous for the insert, whereas all other Cotons de Tulear were heterozygotes (n = 43) or homozygous (n = 54) for the ancestral allele. None of the 87 dogs from 12 other breeds had the insertion allele. Conclusions and Clinical Importance: BNAt is caused by a retrotransposon inserted into exon 8 of GRM1. A DNA test for the GRM1 retrotransposon insert can be used for genetic counseling and to confirm the diagnosis of BNAt.     

鸡IGFBP-2基因的遗传多态性及其遗传效应分析

采用PCR-SSCP方法检测京海黄鸡、AA鸡、尤溪麻鸡、边鸡4个鸡品种胰岛素样生长因子结合蛋白2(IGFBP-2)第2内含子部分序列和第3外显子的多态性,并分析其对京海黄鸡生长和繁殖性能的遗传效应。结果表明:在该区域中,共检测到4个等位基因,10种基因型。χ2检验结果表明,除边鸡外其余3个品种群体在该座位均达到Hardy-Weinberg平衡(P>0.05)。关联分析结果显示,除开产蛋重和12周龄体重外,其他生长和繁殖性状在不同基因型间均存在显著差异(P<0.05)。因此,推测IGFBP-2基因对个体的生长和繁殖性能有一定影响,将IGFBP-2基因应用于鸡育种过程中的标记辅助选择可以加快鸡的育种进程。     

Microsatellite polymorphism in intron 14 of the canine BRCA1 gene

Microsatellite polymorphism due to differences in CT dinucleotide repeats was demonstrated in intron 14 of the canine BRCA1 gene. Genotype analysis of 103 unrelated dogs from 30 different breeds detected the presence of five alleles, including 10 of the expected 15 genotypes. Gene frequencies were biased and all alleles with the exception of one were below 0.1. This polymorphism, which occurs at the intron of canine BRCA1 should prove to be a useful marker for detecting the loss of heterozygosity (LOH). One of the more notable findings of the present study was the detection of homozygotes of rare alleles. This finding identified an accumulation of rare alleles in specific canine breeds and demonstrated the usefulness of this characteristic for the biological study of dog evolution.     

Evaluation of canine heat shock transcription factor 4 (HSF4) as a candidate gene for primary cataracts in the Dachshund and the Entlebucher Mountain dog

OBJECTIVE: Testing of the cataract-causing insertion/deletion mutation in the canine HSF4 gene for its linkage and association with primary cataracts (CAT) in Dachshunds and Entlebucher Mountain dogs. MATERIALS: Exon 9 with flanking intronic regions of the canine HSF4 gene was sequenced in 24 Dachshunds and 20 Entlebucher Mountain dogs. The HSF4 cDNA sequence of lens tissue was analyzed in a CAT-unaffected mixed-breed dog and in three CAT-affected dogs of different breeds, including a Wire-haired Dachshund, a Dachshund-mix and a German Shepherd dog. RESULTS: In all dogs investigated here, the previously reported CAT-causing mutation did not exist. We found a single nucleotide polymorphism (SNP) in intron 9, which was neither associated nor linked with the CAT phenotype in the two dog breeds. CONCLUSION: The CAT phenotype in the two dog breeds investigated here was not caused by the same mutation found to be associated with early-onset CAT in the Staffordshire Bull Terrier and Boston Terrier. The intronic SNP may be useful to test HSF4 for linkage with CAT in further dog breeds.     

马生长激素基因多态性与体尺指标之间的关联性分析

为研究广西德保矮马、百色马和新疆伊犁马的生长激素(GH)基因多态性与体尺指标之间的相关性,本实验利用PCR-SSCP技术分析3个品种277匹马GH基因5′侧翼区、第3外显子、第4外显子、第5外显子的遗传多态性。结果表明:只有GH基因第5外显子出现多态性,表现为AA、BB和AB 3种基因型,其他片段未发现多态性。群体遗传学分析表明,等位基因A和B的基因频率相等;Hardy-Weinberg平衡检验显示,3个品种马均处于非平衡状态;它们的多态信息含量均为中度多态。统计分析表明,GH基因第5外显子的基因型与3个品种马体尺指标之间无显著相关性。     

Haplotype analysis of the MDR1 flanking region in the dog breed Elo

A deletion mutation in the canine multidrug resistance (MDR1) gene provokes drug sensitivity in several dog breeds from the Collie lineage. A haplotype of four microsatellites containing this mdr1-1Delta mutation was conserved among affected breeds. In this study, we analysed the haplotypes of the MDR1 flanking region of 177 dogs of the breed Elo which is composed of several dog breeds including the Old English sheepdog from the Collie lineage. We detected a haplotype in the Elo breed which had previously been associated with the mutant mdr1-1Delta allele in Old English sheepdogs. Using a regression analysis for the probability of the haplotype on the proportion of genes of the founder breeds, we could exclude the Old English sheepdog as origin of this haplotype for the Elo breed. The MDR1 flanking region could be traced back to the Japanese Spitz as one of the founder dog breeds of the Elo and thus, the introgression of the mdr1-1Delta mutation into the dog breed Elo through the Collie lineage is very unlikely.     

5个黄牛群体垂体转录因子基因变异研究

 为了阐明云南黄牛垂体转录因子(POU1F1)基因的群体变异特征,采用DNA序列分析和PCR-RFLP技术对云南地区5个黄牛群体（3个本地群体和2个引进品种）的垂体转录因子(POU1F1)基因的第5内含子和第6外显子进行了基因克隆测序和群体变异检测分析。在5个群体中均发现存在A和B两个等位基因,在国外引进的短角牛和安格斯牛中,A等位基因为优势等位基因,其基因频率分别为0.944和0.700;而在云南的昭通黄牛、迪庆黄牛和荷斯坦奶牛中B等位基因占优势,其基因频率分别为0.645,0.727和0.917;在短角牛和安格斯牛中,BB基因型频率为0;而在云南荷斯坦奶牛中,AA基因型频率为0。在所检测的座位中,昭通黄牛、迪庆黄牛和安格斯牛具有较高的杂合度,而其他群体该座位杂合度较低。     

Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy

OBJECTIVE: To evaluate the role of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy (DCM). ANIMALS: 6 dogs with DCM, including 2 Doberman Pinschers, 2 Newfoundlands, and 2 Great Danes. PROCEDURE: All dogs had clinical signs of congestive heart failure, and a diagnosis of DCM was made on the basis of echocardiographic findings. Blood samples were collected from each dog, and genomic DNA was isolated by a salt extraction method. Specific oligonucleotides were designed to amplify the promoter, exon 1, the 5'-part of exon 2 including the complete coding region, and part of intron 1 of the canine phospholamban gene via polymerase chain reaction procedures. These regions were screened for mutations in DNA obtained from the 6 dogs with DCM. RESULTS: No mutations were identified in the promoter, 5' untranslated region, part of intron 1, part of the 3' untranslated region, and the complete coding region of the phospholamban gene in dogs with DCM. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that mutations in the phospholamban gene are not a frequent cause of DCM in Doberman Pinschers, Newfoundlands, and Great Danes.     

Genetic polymorphism as a background of animal behavior

Various studies have shown the associations between differences in human behavioral traits and genetic polymorphism of neurotransmitter-related proteins such as receptors, transporters and monoamine oxidase. To clarify the genetic background of animal behavior, corresponding regions in animals have been analyzed. The study has been especially focused on primates, as the evolutionally closest animal to humans, and on dogs, as the socially closest animal to humans. In primates, polymorphisms were discovered between or within species, and the functional effects on neural transmission were found to be different by alleles. Even in apes, the closest species to humans, function was different from that in humans. In dogs, allele distributions of several genes were different among breeds showing different behavioral traits, and genes associated with individual differences in aggressiveness and aptitude of working dogs were surveyed. The survey of behavior-related genes has also been carried out in other mammals such as horses and cetaceans. Genes controlling various behaviors in birds have also been reported. The marker genes for behavior will provide useful information for human evolution, welfare of zoo animals and effective selection of working dogs and industry animals.     

Canine tyrosine hydroxylase (TH) gene and dopamine beta -hydroxylase (DBH) gene: their sequences, genetic polymorphisms, and diversities among five different dog breeds

Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.     

PrP genotype frequencies of Quebec sheep breeds determined by real-time PCR and molecular beacons

The allele and genotype frequencies of the prion protein gene (PrP), known to have an impact on scrapie susceptibility, were determined by real-time PCR for 500 Quebec purebred rams. Molecular beacons were very efficient in discriminating the 5 alleles investigated. Polymorphisms at coding positions 136, 154, and 171 of the PrP gene were analyzed using 3 separate real-time PCR reactions and a total of 7 molecular beacons. A total of 4 different alleles (ARQ, ARR, AHR, and VRQ) were observed at different frequencies among the 7 breeds of sheep investigated. Results show that more than 50% of the rams in every breed carried at least one ARR allele, which is considered the most resistant to scrapie. The susceptibility ARQ allele was also present in every breed and together with the ARR allele, they were the most frequent alleles found in Quebec rams. The VRQ allele associated with the highest susceptibility to scrapie occurred in 5 of the 7 breeds, although at low frequencies. Overall, the results indicate that the frequencies of PrP alleles and genotypes in common breeds of sheep in Quebec make it feasible to reduce scrapie risk by selective breeding.     

猪PLSCR4基因外显子区PCR-RFLP遗传多态性分析

本研究利用PCR-RFLP技术对我国6个地方猪种、中国野猪以及杜洛克、约克夏和长白3个国外猪品种,共307头猪的磷脂爬行酶基因(PLSCRs)外显子区的遗传变异进行了研究。结果表明:PLSCR4第7外显子处存在T68C的同义突变,第8外显子处存在G4A的错义突变;在T68C突变位点处,杜洛克、约克夏、长白猪、二花脸、五指山和民猪6个猪群优势等位基因为T,荣昌和野猪群体中优势等位基因为C;在G4A突变位点处,杜洛克、约克夏、长白猪、二花脸、五指山和民猪群体中优势等位基因为G,藏猪、荣昌、金华及野猪群体中优势等位基因A。