Background: Autosomal recessive hereditary nephropathy (ARHN) was diagnosed in 2 English Springer Spaniels (ESS), a breed not previously reported to be affected by hereditary nephropathy (HN).Objective: To identify and characterize the genetic cause of ARHN in ESS.Animals: Sixty-three ESS (2 with ARHN, 2 obligate carriers, and 59 others), 2 mixed-breed dogs with X-linked HN, and 2 English Cocker Spaniels (ECS) with ARHN were included.Methods: ARHN was diagnosed based on transmission electron microscopy and immunostaining of kidney. DNA from affected dogs was screened for the mutation known to cause ARHN in ECS. Quantities of COL4A3, COL4A4, and COL4A5 mRNA transcripts in renal cortex were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for ARHN-affected dogs and 7 other dogs. The coding regions of COL4A3 and COL4A4 were sequenced for the 2 ARHN-affected ESS and an unaffected dog. Exon 30 of COL4A4 was sequenced for all 63 ESS.Results: qRT-PCR indicated a significant reduction in transcript levels of both COL4A3 and COL4A4 mRNA in the kidney of ARHN-affected ESS. Sequencing identified a single nucleotide substitution in COL4A4 at base 2806 resulting in a premature stop codon. Thirteen of 25 related dogs were identified as carriers.Conclusions and Clinical Importance: A mutation highly likely to cause ARHN in ESS has been identified.

Table S1 Primers and melting temperatures used for polymerase chain reaction amplification of canine COL2A1. Table S2 Allele frequencies, chi-square statistics, p-values, and odds ratios for SNPs surrounding the COL2A1 locus. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Deafness in dogs is frequently associated with the pigment genes piebald and merle. Little is known about the prevalence of deafness in dogs carrying the merle allele.OBJECTIVE:To determine the prevalence of deafness in dogs heterozygous and homozygous for the merle allele of the mouse Silver pigment locus homolog (SILV) gene.ANIMALS:One hundred and fifty-three privately owned merle dogs of different breeds and both sexes.METHODS:Hearing was tested by brainstem auditory-evoked response and classified as bilaterally hearing, unilaterally deaf, or bilaterally deaf. DNA from buccal cells was genotyped as either heterozygous or homozygous for the merle allele. Deafness association tests among merle genotype, eye color, and sex were performed by the chi(2) test.RESULTS:Deafness prevalence in merles overall was 4.6% unilaterally deaf and 4.6% bilaterally deaf. There was a significant association between hearing status and heterozygous versus homozygous merle genotype. For single merles (Mm), 2.7% were unilaterally deaf and 0.9% were bilaterally deaf. For double merles (MM), 10% were unilaterally deaf and 15% were bilaterally deaf. There was no significant association with eye color or sex.CONCLUSIONS:Deafness prevalence in merle dogs was greater than that in some dog breeds homozygous for the piebald gene, such as the English Cocker Spaniel, but comparable to, or lower than, that in the Dalmatian and white Bull Terrier. Dogs homozygous for the merle allele were significantly more likely to be deaf than heterozygotes.

Alport syndrome (AS) and hereditary nephropathy (HN) are glomerular nephropathies caused by mutations in the genes encoding the type IV collagens. In a mixed breed of dog, termed Navasota (NAV) dogs, X-linked hereditary nephropathy (XLHN) is caused by a 10-bp deletion in exon 9 of COL4A5. Males harboring this mutation succumb to end-stage renal disease before 18 months of age. In contrast, female carriers of this disease survive much longer, most have a normal life-span, and vary in disease progression as compared with XLHN-affected males. X chromosome inactivation (XCI) patterns have been studied in human X-linked AS carriers and some have been shown to have a high degree of skewed XCI. However, similar studies have never been reported in an animal model of this disease. Therefore, patterns of XCI were examined in XLHN-carrier NAV dogs. The variation in XCI among the 26 XLHN-carrier and seven normal female NAV dogs studied was low and only three were found to preferentially inactivate one X chromosome, all of which were XLHN-carriers. The average skewedness among all dogs was 59% and 57% among the XLHN-carriers. No significant difference in XCI was found between the two groups (P = 0.477). It is clear from these data that genotype does not seem to have an effect on inactivation; the majority of these dogs have random patterns of XCI. Highly skewed X chromosome inactivation also appears to be random, given that no difference was observed between the XLHN-carriers and normal females. Because of the apparent rarity of skewed XCI, these dogs may not be a suitable model for studying a potential correlation between this phenomenon and disease progression.

Table S1 Primer sequences for the six exons of DLA-DRB1. Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Merle is a pattern of coloring observed in the coat of the domestic dog and is characterized by patches of diluted pigment. This trait is inherited in an autosomal, incompletely dominant fashion. Dogs heterozygous or homozygous for the merle locus exhibit a wide range of auditory and ophthalmologic abnormalities, which are similar to those observed for the human auditory-pigmentation disorder Waardenburg syndrome. Mutations in at least five genes have been identified as causative for Waardenburg syndrome; however, the genetic bases for all cases have not been determined. Linkage disequilibrium was identified for a microsatellite marker with the merle phenotype in the Shetland Sheepdog. The marker is located in a region of CFA10 that exhibits conservation of synteny with HSA12q13. This region of the human genome contains SILV, a gene important in mammalian pigmentation. Therefore, this gene was evaluated as a candidate for merle patterning. A short interspersed element insertion at the boundary of intron 10/exon 11 was found, and this insertion segregates with the merle phenotype in multiple breeds. Another finding was deletions within the oligo(dA)-rich tail of the short interspersed element. Such deletions permit normal pigmentation. These data show that SILV is responsible for merle patterning and is associated with impaired function of the auditory and ophthalmologic systems. Although the mutant phenotype of SILV in the human is unknown, these results make it an intriguing candidate gene for human auditory-pigmentation disorders.

Although recent endeavors to discover the mechanisms of the aging process have been numerous and successful, there is still much to be learned. Genes implicated in the aging process were mapped to the canine genome and will serve as additional framework markers for the assignment of contiguous segments from the canine genome sequence to chromosomes. The 54 genes were selected because of their demonstrated contribution to longevity in other organisms or based upon their proximity to a marker, D4S1564, on human chromosome 4 (Puca et al., 2001). This effort lays the necessary groundwork for our utilization of the domestic dog as a model organism to define the genes that govern aging and longevity. Within the species, naturally diverse life expectancies and highly homogeneous populations create an ideal population structure for studying the genetic components of aging (Patronek et al., 1997).

OBJECTIVE:To identify microsatellite markers linked to progressive retinal atrophy (PRA) in American Eskimo Dogs.SAMPLE POPULATION:Blood samples or buccal epithelial cells from 66 American Eskimo Dogs, including 53 PRA-unaffected and 13 PRA-affected dogs.PROCEDURE:The genotypes of unaffected and affected dogs were determined by use of microsatellite markers spanning canine chromosome 9 (CFA09). Homozygosity mapping was used to detect linkage between markers and the gene locus for PRA.RESULTS:Significant allelic association between marker alleles and the gene locus for PRA was detected for GALK1 and TK1, indicating linkage between these markers and the causative gene locus for PRA.CONCLUSIONS AND CLINICAL RELEVANCE:These data indicate that PRA in American Eskimo Dogs is located on CFA09 and allow for the development of a microsatellite-based test to identify carrier (unaffected) and affected dogs before clinical signs appear.

Hereditary loss of hearing affects many breeds of the domestic dog, but the Dalmatian has the highest prevalence. Approximately 30% are affected in the United States (U.S.) population. It is widely accepted that a relationship exists between deafness and pigmentation in the dog and also in other animals. While the Dalmatian exemplifies this relationship, the genetic origin and mode of inheritance of deafness in this breed are unknown. The goals of this study were to: (1) estimate the heritability of deafness in an extended kindred of U.S. Dalmatians and (2) determine, through complex segregation analysis, whether there is a major segregating locus that has a large effect on the expression of deafness. A kindred of 266 Dalmatians was assembled, of which 199 had been diagnosed using the brainstem auditory evoked response to determine auditory status. Of these, 74.4% (N = 148) had normal hearing, 18.1% (N = 36) were unilaterally deaf, and 7.5% (N = 15) were bilaterally deaf. A heritability of 0.73 was estimated considering deafness a dichotomous trait and 0.75 considering it as a trichotomous trait. Although deafness in the Dalmatian is clearly heritable, the evidence for the presence of a single major gene affecting the disorder is not persuasive.

To expedite linkage studies and positional cloning efforts in the dog, Minimal Screening Set 2 (MSS-2) of 327 canine microsatellite markers has been multiplexed into chromosome-specific panels. MSS-2 provides 9 Mb coverage of the canine genome with no gaps larger than 17.1 Mb and is the most recent and comprehensive set of microsatellites available for whole-genome scans. Markers were labeled with fluorescent dyes based on locations and expected product sizes to facilitate the multiplexing of a maximum number of markers for each chromosome. All markers are amplified using a single thermal cycling program and PCR mix and are optimized for resolution on an ABI 3100 genetic analyzer. Sixty-nine chromosome-specific panels were created by coamplification of a maximum number of markers and subsequent coloading of the remaining markers.

Recent advances in mapping the canine genome have led to an increase in the number of linkage studies aimed at dissecting the genetic causes of many hereditary diseases that affect the domestic dog. The first step in developing molecular tools for a whole genome scan was the characterization of a set of microsatellite markers, termed minimal screening set 1 (MSS1), that provided an estimated coverage of 10 cM. A limiting factor in use of the MSS1 is not all of the 172 MSS1 markers have been localized to specific chromosomes. Seventy-five of the markers were positioned on a total of 15 chromosomes with the original publication of the MSS1. The localization based on linkage data of 14 additional MSS1 markers to chromosomes using CRIMAP v. 2.4 to build a linkage map of 113 MSS1 markers that were polymorphic in a kindred of Dalmatians is reported here.

Our interest is in understanding the genetic bases for hereditary renal diseases of the domestic dog (Canis familiaris) and in characterizing gene loci for placement on the map of the canine genome. We report here on the cloning, sequencing and radiation hybrid mapping of the canine cDNA encoding uromodulin, a renal-specific glycoprotein. The cDNA is 2.3 kb in length and, as expected, comparisons of nucleotide sequences reveal that canine umod is quite similar to umod of other mammals. The predicted amino acid sequence of canine uromodulin has at least 70% identity with other mammalian uromodulin proteins. Canine umod has been mapped on the RHDF5000 radiation hybrid panel and positioned on the most recent canine genome map. Data indicate that umod is linked to the marker CZP2 (canine zona pellucida gene) on an RH group not yet assigned to a canine chromosome. The human umod and CZP2 genes are located on chromosome 16p13.

The proto-oncogene, c-kit (KIT), encodes a tyrosine kinase receptor, and mutations in this gene are causative for several mammalian diseases, including cancer and a form of pigmentation-associated hereditary deafness. Our laboratories are interested in a form of hereditary deafness that is associated with abnormalities in pigmentation and is common in the Dalmatian. Thus, KIT is being analyzed as a candidate gene for deafness in this breed. In addition to our interest in deafness, we are involved in mapping gene loci in the canine genome. Reported here is the identification of two isoforms of canine C-kit and radiation hybrid mapping of KIT to CFA13.

There is incredible morphological and behavioral diversity among the hundreds of breeds of the domestic dog, CANIS FAMILIARIS. Many of these breeds have come into existence within the last few hundred years. While there are obvious phenotypic differences among breeds, there is marked interbreed genetic homogeneity. Thus, study of canine genetics and genomics is of importance to comparative genomics, evolutionary biology and study of human hereditary diseases. The most recent version of the map of the canine genome is comprised of 3,270 markers mapped to 3,021 unique positions with an average intermarker distance of approximately 1 Mb. The markers include approximately 1,600 microsatellite markers, about 1,000 gene-based markers, and almost 700 bacterial artificial chromosome-end markers. Importantly, integration of radiation hybrid and linkage maps has greatly enhanced the utility of the map. Additionally, mapping the genome has led directly to characterization of microsatellite markers ideal for whole genome linkage scans. Thus, workers are now able to exploit the canine genome for a wide variety of genetic studies. Finally, the decision to sequence the canine genome highlights the dog's evolutionary and physiologic position between the mouse and human and its importance as a model for study of mammalian genetics and human hereditary diseases.

taurus (cattle) 10, 16, 25, 32, 39, 42, 49, 53

OBJECTIVETo assess the heritability of pancreatic acinar atrophy (PAA) in German Shepherd Dogs (GSDs) in the United States.ANIMALS135 GSDs belonging to 2 multigenerational pedigrees.PROCEDURETwo multigenerational pedigrees of GSDs with family members with PAA were identified. The clinical history of each GSD enrolled in the study was recorded, and serum samples for canine trypsin-like immunoreactivity (cTLI) analysis were collected from 102 dogs. Dogs with a serum cTLI concentration < or = 2.0 microg/L were considered to have exocrine pancreatic insufficiency (EPI) and were assumed to have PAA.RESULTSPedigree I consisted of 59 dogs and pedigree II of 76 dogs. Serum cTLI concentrations were measured in 48 dogs from pedigree I and 54 dogs from pedigree II. A total of 19 dogs (14.1%) were determined to have EPI, 9 in pedigree I (15.3%) and 10 in pedigree II (13.6%). Of the 19 dogs with EPI, 8 were male and 11 were female.CONCLUSIONS AND CLINICAL RELEVANCEEvaluation of data by complex segregation analysis is strongly suggestive of an autosomal recessive mode of inheritance for EPI in GSDs in the United States.

In order to better define the cornification process in dogs and to understand how it is similar or different from cornification in other mammals, we are sequencing the major molecules of cornification in the dog. In this report, we describe the complete genomic sequence of three superficial type 2 canine keratins: K1, K2e and K2p. Oligonucleotide primers for PCR were designed from homologous regions of previously reported human or murine cDNA sequences of the genes of interest. DNA sequencing and evaluation with blast confirmed that the primers amplified the respective canine genes. These primers were then used to screen a subset of a canine genomic library known to contain basic keratin genes. Selected clones were sequenced to obtain the complete DNA sequence. K1, K2e and K2p each had nine exons and eight introns characteristic of the type 2 keratins that code for proteins with variable glycine‐rich head and tail regions and a central α‐helical rod domain. K2p encoded a protein of 659 amino acids, larger than both K1 and K2e (620 and 634 amino acids, respectively). Amino acid sequence homology with humans was 81, 74 and 81% for K1, K2e and K2p, respectively, with homology greatest at the central rod region and decreasing dramatically at the head and tail. Comparisons of the amino acid sequences of these keratins within the canine species defined K2e and K2p as having a slightly higher degree of similarity. In contrast, in humans K2e has greater sequence homology with K1. This study was funded in part by the Morris Animal Foundation.

The transglutaminase 1 gene (TGM1) encodes an enzyme necessary for cross-linking the structural proteins that form the cornified envelope, an essential component of the outermost layer of the skin, the stratum corneum. Reported here is the complete coding region of canine TGM1, its chromosome localization, and its map position in the integrated canine linkage-radiation hybrid map. Canine TGM1 consists of 2,448 nucleotides distributed over 15 exons. The nucleotide sequence has 90% identity to human TGM1. The deduced canine TGM1 protein is 816 amino acids long and is 92% identical to human TGM1. Using fluorescence in situ hybridization, we localized canine TGM1 to dog (Canis familiaris) chromosome 8 (CFA 8q). Canine TGM1 localized to CFA 8 on the integrated linkage-radiation hybrid map in the interval FH2149–MYH7. Characterizing the coding region of canine TGM1 is a first step in examining the role of this enzyme in normal and defective cornification in the dog.

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67–78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.

The integrin family of receptors serves as major receptors for extracellular matrix-mediated cell adhesion and migration, cytoskeletal organisation, cell proliferation, survival, and differentiation. The alpha-V integrins consist of a subset which share a common alpha-V subunit combined with one of five beta subunits (beta-1, 3, 5, 6, or 8). The alpha-V integrins have been implicated in a number of developmental processes, including vasculogenesis and angiogenesis, and are therapeutic targets for inhibition of angiogenesis and osteoporosis. The human cDNA for alpha-V integrin (ITGAV) consists of a 5,717-bp transcript with a coding sequence (CDS) of 3,146 bp encoding a 150-kDa mature peptide. Here we describe the gene structure of ITGAV.