Keith E. Murphy

A cat cloned by nuclear transplantation.

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Cell biology: A cat cloned by nuclear transplantation

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog

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.

Understanding hereditary diseases using the dog and human as companion model systems

Animal models are requisite for genetic dissection of, and improved treatment regimens for, human hereditary diseases. While several animals have been used in academic and industrial research, the primary model for dissection of hereditary diseases has been the many strains of the laboratory mouse. However, given its greater (than the mouse) genetic similarity to the human, high number of naturally occurring hereditary diseases, unique population structure, and the availability of the complete genome sequence, the purebred dog has emerged as a powerful model for study of diseases. The major advantage the dog provides is that it is afflicted with approximately 450 hereditary diseases, about half of which have remarkable clinical similarities to corresponding diseases of the human. In addition, humankind has a strong desire to cure diseases of the dog so these two facts make the dog an ideal clinical and genetic model. This review highlights several of these shared hereditary diseases. Specifically, the canine models discussed herein have played important roles in identification of causative genes and/or have been utilized in novel therapeutic approaches of interest to the dog and human.

Linkage and Segregation Analysis of Black and Brindle Coat Color in Domestic Dogs

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red–yellow pheomelanin vs. black–brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador × greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (KB) > brindle (kbr) > yellow (ky), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.

Genetic cause of X-linked Alport syndrome in a family of domestic dogs.

Alport syndrome is a hereditary disease of type IV (basement membrane) collagens that occurs spontaneously in humans and dogs. In the human, X-linked Alport syndrome (XLAS) is caused by mutations in COL4A5, resulting in absence of type IV collagen α5 chains from the glomerular basement membrane (GBM) of affected individuals. The consequence of this defect is progressive renal failure, for which the only available treatments are dialysis and transplantation. Recent studies support the prospect of gene transfer therapy for Alport syndrome, but further development of required technologies and demonstration of safety and efficacy must be accomplished in a suitable animal model. We previously identified and have propagated a family of mixed-breed dogs with an inherited nephropathy that exhibits the clinical, immunohistochemical, pathological, and ultrastructural features of human XLAS. To identify the causative mutation, COL4A5 cDNAs from normal and affected dogs were sequenced in their entirety. Sequence analyses revealed a 10-bp deletion in exon 9 of affected dogs. This deletion causes a frame-shift that results in a premature stop codon in exon 10. Characterization of the causative mutation was followed by development of an allele-specific test for identification of dogs in this kindred that are destined to develop XLAS.

Linkage analysis and gene expression profile of pancreatic acinar atrophy in the German Shepherd Dog

Pancreatic acinar atrophy (PAA) is a degenerative disease of the exocrine pancreas and is the most common cause of exocrine pancreatic insufficiency in the German Shepherd Dog. Analyses of inheritance have shown that a single gene segregating in an autosomal recessive fashion is causative for PAA. To date the gene and causative mutation have not been determined. To identify a region of interest and/or candidate genes, we conducted linkage and gene expression studies. Analysis of 384 microsatellite markers resulted in a maximum two-point LOD score of 2.5 for FH2107 on CFA03. We used an oligonucleotide array to generate gene expression profiles for normal and affected pancreata. It revealed 244 genes with greater than two-fold difference in expression levels. Five genes of interest were further assessed by TaqMan quantitative real-time RT-PCR that confirmed trends observed using the microarray. One gene, gp25L, located on CFA03, was found to be downregulated by more than 500-fold in affected pancreata and was further investigated as a candidate gene. Sequence data did not reveal a mutation in the coding sequence that segregates with PAA.

Heritability and Segregation Analysis of Deafness in U.S. Dalmatians

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.

Heritability and transmission analysis of necrotizing meningoencephalitis in the Pug

Necrotizing meningoencephalitis (NME) in the Pug is an invariably fatal disease with an early age of onset whose cause remains unknown. Breed predilection strongly suggests genetic component(s), and viral etiology proves negative in studied cases. The current study was undertaken as the first analysis of the heritable component(s) involved in NME in the Pug. Complete medical records, individual characteristics, and pedigree information were collected for 58 affected dogs with data pertaining to 4698 dogs analyzed. A high inbreeding coefficient with differences across gender and significant differences across coat color classes and variable expression was evident. Median onset age was 19months and median survival time 23days. Screening for herpes-, adeno-, and parvoviruses was negative. The data demonstrate a strong familial inheritance of NME in the Pug. This investigation provides parameters of disease from the largest Pug NME cohort analyzed to date and offers evidence of previously unrecognized familial inheritance.

Genetic cause of autosomal recessive hereditary nephropathy in the english cocker spaniel

BACKGROUND Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown. ANIMALS One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included. METHODS Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels. RESULTS Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status. CONCLUSIONS AND CLINICAL IMPORTANCE The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.