Characterization of the canine desmin (DES) gene and evaluation as a candidate gene for dilated cardiomyopathy in the Dobermann

Canine-dilated cardiomyopathy (DCM) in dogs is a disease of the myocardium associated with dilatation and impaired contraction of the ventricles and is suspected to have a genetic cause. A missense mutation in the desmin gene (DES) causes DCM in a human family. Human DCM closely resembles the canine disease. In the present study, we evaluated whether DES gene mutations are responsible for DCM in Dobermann dogs. We have isolated bacterial artificial chromosome clones (BACs) containing the canine DES gene and determined the chromosomal location by fluorescence in situ hybrizidation (FISH). Using data deposited in the NCBI trace archive and GenBank, the canine DES gene DNA sequence was assembled and seven single nucleotide polymorphisms (SNPs) were identified. From the canine DES gene BAC clones, a polymorphic microsatellite marker was isolated. The microsatellite marker and four informative desmin SNPs were typed in a Dobermann family with frequent DCM occurrence, but the disease phenotype did not associate with a desmin haplotype.

We concluded that mutations in the DES gene do not play a role in Dobermann DCM. Availability of the microsatellite marker, SNPs and DNA sequence reported in this study enable fast evaluation of the DES gene as a DCM candidate gene in other dog breeds with DCM occurrence.     

Evaluation of the CTSL2 Gene as a Candidate Gene For Alopecia X in Pomeranians and Keeshonden

Alopecia X is a noninflammatory, progressive, bilateral symmetric alopecia in dogs. The disease is mainly found in Nordic breeds. The breed predisposition and a strong familial accumulation suggest a hereditary background. We analyzed the cathepsin L2 gene (CTSL2) as a candidate for alopecia X. The comparative sequencing of 14 affected and 18 control animals revealed ten polymorphisms; however, none of these polymorphisms affected the coding sequence. Haplotype analysis did not reveal an association of one particular CTSL2 haplotype with the disease phenotype; therefore, we conclude that the CTSL2 gene is probably not the causative gene for alopecia X.     

Evaluation of the CTSL2 gene as a candidate gene for alopecia X in Pomeranians and Keeshonden

Alopecia X is a noninflammatory, progressive, bilateral symmetric alopecia in dogs. The disease is mainly found in Nordic breeds. The breed predisposition and a strong familial accumulation suggest a hereditary background. We analyzed the cathepsin L2 gene (CTSL2) as a candidate for alopecia X. The comparative sequencing of 14 affected and 18 control animals revealed ten polymorphisms; however, none of these polymorphisms affected the coding sequence. Haplotype analysis did not reveal an association of one particular CTSL2 haplotype with the disease phenotype; therefore, we conclude that the CTSL2 gene is probably not the causative gene for alopecia X.     

Evaluation of 15 candidate genes for dilated cardiomyopathy in the Newfoundland dog

Dilated cardiomyopathy (DCM) is a disease of the myocardium, which causes heart failure and premature death. It has been described in humans and several domestic animals. In the Newfoundland dog, DCM is an autosomal dominant disease with late onset and reduced penetrance. We analyzed 15 candidate genes for their involvement in DCM in the Newfoundland dog. Polymorphic microsatellite markers and single Nucleotide Polymorphisms were genotyped in 4 families of Newfoundland dogs segregating dilated cardiomyopathy for the genes encoding alpha-cardiac actin (ACTC), caveolin (CAVI), cysteine-rich protein 3 (CSRP3), LIM-domain binding factor 3 (LDB3), desmin (DES), lamin A/C (LMNA), myosin heavy polypeptide 7 (MYH7), delta-sarcoglycan (SGCD), troponin I (TNNTI3), troponin T (TNNT2), alpha-tropomyosin (TPMI), titin (TTN) and vinculin (VCL). A Logarithm of the odds (LOD) score of less than -2.0 in 2-point linkage analysis indicated exclusion of all but 2 genes, encoding CSRP3 and DES. A (LOD) score between -1.5 and -2.0 for CSRP3 and DES makes these genes unlikely causes of DCM in this dog breed. For the phospholamban (PLN) and titin cap (TTN) genes, a direct mutation screening approach was used. DNA sequence analysis of all exons showed no evidence that these genes are involved in DCM in the Newfoundland dog.     

Novel Muscle-Specific Genes TCAP,TNNI1, and FHL1 in Cattle: SNVs,Linkage Disequilibrium,Combined Genotypes,Association Analysis of Growth Performance,and Carcass Quality Traits and Expression Studies

TCAP, TNNI1, and FHL1 regulate muscle growth and development. In this study, four single nucleotide variants (SNVs) were discovered in almost all of the exon and intron regions of the TCAP, TNNI1, and FHL1 genes using DNA pooled sequencing, polymerase chain reaction (PCR)-RFLP, and forced-PCR-RFLP methods in 576 cattle. Four SNVs were significantly associated with the growth performance and carcass quality traits of the cattle. In addition, the haplotype, haplotype frequency, and linkage disequilibrium coefficient of three sequence variants were also evaluated in the cattle population. Haplotype analysis demonstrated that eight haplotypes were present in the Qinchuan cattle population and no haplotypes were present in the Chinese Holstein population; haplotype 1 had the highest frequency in the Qinchuan (42.7%) population. Statistical analyses of 12 combined genotypes indicated that some were significantly associated with the growth performance and carcass quality traits of the Qinchuan cattle population. Moreover, the quantitative real-time polymerase chain reaction results demonstrated that the bovine TCAP, TNNI1, and FHL1 genes were exclusively expressed in muscle tissue. These data support the high potentials of the TCAP, TNNI1, and FHL1 as marker genes to improve the growth performance and carcass quality traits of Qinchuan cattle or other animals selection programs.     

Ancestral matrilineages and mitochondrial DNA diversity of the Lidia cattle breed

To clarify the genetic ancestry and the mitochondrial DNA (mtDNA) diversity of the Lidia cattle breed, a 521-bp D-loop fragment was sequenced in 527 animals belonging to 70 herds distributed across 29 lineages. The mtDNA diversity recorded was similar to that seen for Middle Eastern breeds and greater than that recorded for the majority of European breeds. Haplotype T3 was the most common (81%), followed by the African T1 haplotype (17%); very low frequencies were recorded for haplotypes T and T2. The results agree with there being two major ancestral lines for the Lidia breed, European and African, similar to that seen for other Mediterranean breeds. A wide range of variation in haplotype frequencies was seen between the examined lineages. Haplotype T3 was present in all those analysed; in five it was the only one present, and in only one lineage (Miura) was its frequency lower than that of T1. T1*, a haplotype reported in Criollo breeds and to date in only a single European breed (the Retinta breed from Spain), was found in a single animal belonging to the Concha y Sierra lineage. Network analysis of the Lidia breed revealed the presence of two major haplotypes: T3 and T1. The Lidia breed appears to be more closely related to prehistoric Iberian and Italian than to British aurochs.     

Origin and phylogenetic analysis of Tibetan Mastiff based on the mitochondrial DNA sequence

At present, the Tibetan Mastiff is the oldest and most ferocious dog in the world. However, the origin of the Tibetan Mastiff and its Phylogenetic relationship with other large breed dogs such as Saint Bernard are unclear. In this study, the primers were designed according to the mitochondrial genome sequence of the domestic dog, and the 2,525 bp mitochondrial sequence, containing the whole sequence of Cytochrome b, tRNA-Thr, tRNA-Pro, and control region of the Tibetan Mastiff, was obtained. Using grey wolves and coyotes as outgroups, the Tibetan Mastiff and 12 breeds of domestic dogs were analyzed in phylogenesis. Tibetan Mastiff, domestic dog breeds, and grey wolves were clustered into a group and coyotes were clustered in a group separately. This indicated that the Tibetan Mastiff and the other domestic dogs originated from the grey wolf, and the Tibetan Mastiff belonged to Carnivora, Canidae, Canis, Canis lupus, Canis lupus familiaris on the animal taxonomy. In domestic dogs, the middle and small breed dogs were clustered at first; German Sheepdog, Swedish Elkhound, and Black Russian Terrier were clustered into one group, and the Tibetan Mastiff, Old English Sheepdog, Leonberger, and Saint Bernard were clustered in another group. This confirmed the viewpoint that many of the famous large breed dogs worldwide Such as Saint Bernard possibly had the blood lineage of the Tibetan Mastiff, based on the molecular data. According to the substitution rate, we concluded that the approximate divergence time between Tibetan Mastiff and grey wolf was 58,000 years before the present (YBP), and the approximate divergence time between other domestic dogs and grey wolf was 42,000 YBP, demonstrating that the time of origin of the Tibetan Mastiff was earlier than that of the other domestic dogs.     

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.     

Complex segregation analysis of dilated cardiomyopathy (DCM) in Irish wolfhounds

The objective of the present study was to analyse the mode of inheritance for dilated cardiomyopathy (DCM) in Irish wolfhounds using regressive logistic models by testing for mechanisms of genetic transmission. Insights from this spontaneous animal model should aid importantly in understanding basic pathogenic mechanisms with regard to genetics and molecular biology of DCM in humans. Moreover, a procedure for the simultaneous prediction of breeding values and the estimation of genotype probabilities for DCM is expected to markedly improve breeding programmes. Results of cardiovascular examinations of 1018 dogs carried out between 1987 and 2003 by one veterinarian were analysed. Data of 878 dogs from 531 litters in 147 different kennels were used for complex segregation analyses. Pedigree information was available for more than 15 generations. Male dogs were affected significantly more often by DCM than female dogs. The segregation analysis showed that among all other tested models a mixed monogenic-polygenic model including a sex-dependent allele effect best explained the segregation of affected animals in the pedigrees. A pure monogenic inheritance of DCM could be significantly rejected in favour of the major gene and most general model. The gene action of the major gene was significantly different between female and male dogs.     

Y-chromosome microsatellite variation in Italian wolves: A contribution to the study of wolf-dog hybridization patterns

One major concern in wolf (Canis lupus) conservation is the risk of genetic contamination due to crossbreeding with domestic dogs. Although genetic monitoring of wolf populations has become widely used, the behavioural mechanisms involved in wolf-dog hybridization and the detrimental effects of genetic introgression are poorly known. In this study we analysed Y-chromosome microsatellite variation in the recovering Italian wolf population and detected strikingly different allele frequencies between wolves and dogs. Four Y haplotypes were found in 74 analysed male wolves, and all of them were present in a focus wolf population in the Apennines. On the other hand, only 1 haplotype was found in the recolonizing wolf population from the Western Alps. The most common haplotype in a sample of domestic dogs, was also found in 5 wolves, 2 of which revealing a signature of recent hybridization. Moreover, another suspect hybrid carried a private haplotype of possible canine origin. These results give support to the idea that female wolves can breed with male stray dogs in the wild. The Y-chromosome variation in Italian wolves contrasts with the previously observed lack of mitochondrial variation. Further investigations are needed to clarify at what extent historical or recent wolf-dog hybridization events may have contributed to the observed haplotype diversity. In conclusion, the two molecular markers employed in this study represent effective means to trace directional genetic introgression into the wolves male lineage and have the noteworthy advantage of being suitable for analyses on low-quality DNA samples.     

Mitochondrial DNA Cleavage Patterns Distinguish Independent Origin of Chinese Domestic Geese and Western Domestic Geese

It has generally been assumed, based on morphology, that Chinese domestic goose breeds were derived from the swan goose (Anser cygnoides) and that European and American breeds were derived from the graylag goose (Anser anser). To test the validity of this assumption, we investigated the mtDNA cleavage patterns of 16 Chinese breeds and 2 European breeds as well as hybrids produced between a Chinese breed and a European breed. After 224 mtDNAs, isolated from the Chinese and European breeds, were digested by 19 restriction endonucleases, variations of the cleavage patterns were observed for four enzymes (EcoRV, HaeII, HincII, and KpnI). All Chinese breeds and their maternal hybrids except the Yili breed showed an identical haplotype, named haplotype I or the Chinese haplotype; the European breeds and the Yili breed showed another haplotype, named haplotype II or the western haplotype. None of the haplotype found in the Chinese type was detectable in the western type and vice versa. The two haplotypes were found to differ from each other at 8.0% of the sites surveyed and with a 0.72% sequence divergence. Using 2% substitution per million years calibrated from the genera Anser and Branta, the two domestic geese haplotypes were estimated to have diverged approximately 360,000 years ago, well outside the 3000-6000 years in domestic history. Our findings provide the first molecular genetic evidence to support the dual origin assumption of domestic geese in the world. Meanwhile, the four mtDNA restriction fragment length polymorphisms can be used as maternal genetic markers to distinguish the two types of domestic geese.     

Evaluation of tafazzin as candidate for dilated cardiomyopathy in Irish wolfhounds

Dilated cardiomyopathy (DCM) is a common disease in humans and dogs. Large-breed dogs and especially Irish wolfhounds belong to the frequently affected breeds. Male Irish wolfhounds show a significantly higher prevalence of DCM than females. Therefore, we evaluated X chromosome markers for linkage with DCM as well as a human candidate gene on the X chromosome. A set of X chromosomal microsatellites was genotyped in Irish wolfhound families segregating for DCM. In addition, exon and intron sequences of the tafazzin (TAZ) gene were assayed for polymorphisms segregating in these families. Statistical analysis of the microsatellite markers did not reveal linkage to DCM. Furthermore, all Irish wolfhounds included in this study were monomorphic for TAZ, and only 8 sequence differences to the Dog Genome Assembly 2.1 could be found. The results indicate that due to the lack of mutations, TAZ is unlikely to cause DCM in Irish wolfhounds.     

中国部分地方黄牛品种线粒体D-loop区的遗传变异与进化分析

测定了13个黄牛品种125个个体的线粒体D-loop区段的全序列,包括12个中国地方黄牛品种的123个个体和德国黄牛2个个体,并进行了分析。结果显示,共检测到93个变异位点,57个单倍型,平均核苷酸差异(average number ofnucleotide differences,k)为22.708,核苷酸多样度(nucleotide diversity,π)为0.0251±0.00479,单倍型多样度(haplotypediversity,Hd)为0.888±0.026,表明我国黄牛品种遗传多样性非常丰富。构建的Neighbor-Joining进化树显示这13个品种主要分成两大类型:普通牛和瘤牛;新发现的特殊类型Ⅲ只有一个西藏阿沛甲咂牛的个体,它与牦牛D-loop序列最相近,证明西藏地区的黄牛与牦牛之间存在基因渗入现象。普通牛和瘤牛在日喀则驼峰牛中占的比例分别是64.3%和35.7%,在阿沛甲咂牛中占的比例分别是50.0%和50.0%,证明了西藏的黄牛也有瘤牛类型。云南牛品种的单倍型非常丰富证明了云南在中国黄牛起源上的重要地位;在27个中国黄牛品种中(本研究11个品种以及GenBank上的16个品种)找到了中国瘤牛的核心单倍型i1,并且对它进行了讨论。同时证明了西藏瘤牛独立于中国瘤牛核心类群的特殊性。     

Canine population structure: assessment and impact of intra-breed stratification on SNP-based association studies

Background

In canine genetics, the impact of population structure on whole genome association studies is typically addressed by sampling approximately equal numbers of cases and controls from dogs of a single breed, usually from the same country or geographic area. However one way to increase the power of genetic studies is to sample individuals of the same breed but from different geographic areas, with the expectation that independent meiotic events will have shortened the presumed ancestral haplotype around the mutation differently. Little is known, however, about genetic variation among dogs of the same breed collected from different geographic regions.

Methodology/Principal Findings

In this report, we address the magnitude and impact of genetic diversity among common breeds sampled in the U.S. and Europe. The breeds selected, including the Rottweiler, Bernese mountain dog, flat-coated retriever, and golden retriever, share susceptibility to a class of soft tissue cancers typified by malignant histiocytosis in the Bernese mountain dog. We genotyped 722 SNPs at four unlinked loci (between 95 and 271 per locus) on canine chromosome 1 (CFA1). We showed that each population is characterized by distinct genetic diversity that can be correlated with breed history. When the breed studied has a reduced intra-breed diversity, the combination of dogs from international locations does not increase the rate of false positives and potentially increases the power of association studies. However, over-sampling cases from one geographic location is more likely to lead to false positive results in breeds with significant genetic diversity.

Conclusions

These data provide new guidelines for association studies using purebred dogs that take into account population structure.     

Canine RD3 mutation establishes rod-cone dysplasia type 2 (rcd2) as ortholog of human and murine rd3

Rod-cone dysplasia type 2 (rcd2) is an autosomal recessive disorder that segregates in collie dogs. Linkage disequilibrium and meiotic linkage mapping were combined to take advantage of population structure within this breed and to fine map rcd2 to a 230-kb candidate region that included the gene C1orf36 responsible for human and murine rd3, and within which all affected dogs were homozygous for one haplotype. In one of three identified canine retinal RD3 splice variants, an insertion was found that cosegregates with rcd2 and is predicted to alter the last 61 codons of the normal open reading frame and further extend the open reading frame. Thus, combined meiotic linkage and LD mapping within a single canine breed can yield critical reduction of the disease interval when appropriate advantage is taken of within-breed population structure. This should permit a similar approach to tackle other hereditary traits that segregate in single closed populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. V. Kukekova and O. Goldstein contributed equally to this work. Nucleotide sequence data reported here are available in the GenBank database under accession numbers EU687743, EU687744, and EU687745.     

Mutations of presenilin genes in dilated cardiomyopathy and heart failure

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.     

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.     

Mitochondrial DNA sequence variation in Portuguese native dog breeds: diversity and phylogenetic affinities

In an extensive survey of the genetic diversity in Portuguese dogs, we have examined an 887-bp fragment of the mitochondrial DNA (mtDNA) from 8 Portuguese, 1 Spanish, and 2 North African native dog breeds, including village dogs from Portugal and Tunisia. Forty-nine haplotypes were found in the 164 individuals analyzed, with private haplotypes being found in several breeds. For example, the Castro Laboreiro Watchdog, a rare breed from a small and isolated region in Portugal, was monomorphic for mtDNA and possessed a new haplotype, which may be provisionally considered a breed-specific marker. Phylogenetic analyses recapitulated 4 major clades identified in other studies, but new haplotypes, grouping within a clade that was previously thought as geographically restricted, were detected in Portugal and Morocco. Portuguese village dogs showed no genetic differentiation from nonnative dogs or from local breeds of the areas in which the village dogs were sampled. Although Iberian and North African dog breeds possessed breed-specific mtDNA haplotypes, no significant geographic structure could be detected among them. There is no evidence for introgression of North African haplotypes in Iberian dogs, contrary to previous results for other domestic animals.     

Pre-Columbian origins of Native American dog breeds,with only limited replacement by European dogs,confirmed by mtDNA analysis

Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.