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The alkaline phosphatases comprise a multigene enzyme family that hydolyze phosphate esters and are widely distributed in nature. Three main classes have been isolated from humans, the placental, intestinal, and liver/bone/kidney forms. We have mapped the placental and intestinal alkaline phosphatase genes to 2q34-q37 by using chromosomal in situ hybridization and a somatic-cell hybrid panel.

We have used three different methods to map the human liver/bone/kidney alkaline phosphatase (ALPL) locus: (1) Southern blot analysis of DNA derived from a panel of human-rodent somatic cell hybrids; (2) in situ hybridization to human chromosomes; and (3) genetic linkage analysis. Our results indicate that the ALPL locus maps to human chromosome bands 1p36.1-p34 and is genetically linked to the Rh (maximum lod score of 15.66 at a recombination value of 0.10) and fucosidase A (maximum lod score of 8.24 at a recombination value of 0.02) loci. These results, combined with restriction fragment length polymorphisms identified by ALPL DNA probes, provide a useful marker for gene mapping studies involving the short arm of chromosome 1. In addition, our results help to elucidate further the structure and evolution of the human alkaline phosphatase multigene enzyme family.

The Australian Terrier breed is the breed at highest risk for naturally-occurring diabetes mellitus in the United States, where it is 32 times more likely to develop diabetes compared to mixed breed dogs. However, the heritability and mode of inheritance of spontaneous diabetes in Australian Terriers has not been reported. The aim of this study was therefore to investigate the heritability and mode of inheritance of diabetes in Australian Terriers. A cohort of related Australian Terriers including 383 Australian Terriers without diabetes, 86 Australian Terriers with spontaneous diabetes, and 14 Australian Terriers with an unknown phenotype, was analyzed. A logistic regression model including the effects of sex was formulated to evaluate the heritability of diabetes. The inheritance pattern of spontaneous diabetes in Australian Terriers was investigated by use of complex segregation analysis. Six possible inheritance models were studied, and the Akaike Information Criterion was used to determine the best model for diabetes inheritance in Australian Terriers, among the models deemed biologically feasible. Heritability of diabetes in Australian Terriers was estimated at 0.18 (95% confidence interval 0.0-0.67). There was no significant difference in the effect of males and females on disease outcome. Complex segregation analysis suggested that the mode of diabetes inheritance in Australian Terriers is polygenic, with no evidence for a large effect single gene influencing diabetes. It is concluded that in the population of Australian Terriers bred in the United States, a relatively small degree of genetic variation contributes to spontaneous diabetes. A genetic uniformity for diabetes-susceptible genes within the population of Australian Terriers bred in the Unites States could increase the risk of diabetes in this cohort. These findings hold promise for future genetic studies of canine diabetes focused on this particular breed., Competing Interests: The authors have declared that no competing interests exist.

Background: Evidence for an autoimmune etiology in canine diabetes is inconsistent and could vary based on breed. Previous studies demonstrated that small percentages of diabetic dogs possess autoantibodies to antigens known to be important in human type 1 diabetes, but most efforts involved analysis of a wide variety of breeds. The objective of this study was to evaluate the presence of glutamic acid decarboxylase 65 (GAD65), insulinoma-associated protein 2 (IA-2), and zinc transporter 8 (ZnT8) autoantibodies in diabetic and non-diabetic Australian Terriers and Samoyeds, two breeds with comparatively high prevalence of diabetes, in the United States., Results: There was no significant difference in the proportion of samples considered positive for GAD65 or ZnT8 autoantibodies in either breed evaluated, or for IA-2 autoantibodies in Australian Terriers (p > 0.05). The proportion of IA-2 autoantibody positive samples was significantly higher in diabetic versus non-diabetic Samoyeds (p = 0.003), but substantial overlap was present between diabetic and non-diabetic groups., Conclusions: The present study does not support GAD65, IA-2, or ZnT8 autoantibodies as markers of autoimmunity in canine diabetes in Samoyeds or Australian Terriers as measured using human antigen sandwich enzyme-linked immunosorbent (ELISA) assays. Future studies using canine specific assays as well as investigation for alternative markers of autoimmunity in these and other canine breeds are warranted.

Hereditary β-mannosidosis causing progressive lysosomal neuropathy and other clinical signs, has been previously described in humans, Nubian goats, and Salers cattle. Here we report the clinicopathological, metabolic, and molecular genetic features of canine beta-mannosidase (MANBA, EC 3.2.1.25) deficiency. A 1-year-old male mix-breed dog from St. Kitts was presented with progressive stumbling, weakness, and regurgitation. Vacuolated lymphocytes were observed on the blood film. Postmortem findings included marked enlargement of nerves, megaesophagus, and internal hydrocephalus. Vacuolated macrophages, neurons, and secretory epithelial cells suggested an oligosaccharide storage disease. Plasma concentration of the β-mannosidosis specific oligosaccharide was approximately 75 fold that of controls. The plasma beta-mannosidase activity was severely reduced to ~5% of controls; five other lysosomal acid hydrolase activities were increased or within their normal reference interval. Genomic sequencing of this dog's MANBA gene identified a homozygous exonic five bp tandem duplication in the penultimate exon of the MANBA gene (c.2377_2381dupTATCA) which results in a reading frame shift, altering the subsequent amino acid sequence and creating a premature stop codon. The truncated beta-mannosidase enzyme is expected to be dysfunctional. This enzyme deficiency causes the accumulation of un-degraded oligosaccharides in cells, which affect the myelination of the peripheral and central nervous systems. This insertion was not encountered in 121 and 80-screened samples from dogs on St. Kitts (all were homozygous for wild-type) and Philadelphia region (wild-type), respectively. In conclusion, canine β-mannosidosis has similar clinicopathological features with some human patients, but milder signs than in ruminants and more severe than in knockout mice. Hence, dogs with β-mannosidosis could become a valuable disease model for the human disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two domestic shorthair cats, 1 intact female and 1 intact male, presented with progressive limb lameness and digital deformities at 4 and 6 months of age. Stiffness and swelling of the distal thoracic and pelvic limb joints progressed to involve hip and shoulder joints, resulting in reduced mobility. Radiographs in both cats and computed tomography of the male cat revealed ankylosing, polyarticular deposits of extracortical heterotopic bone spanning multiple axial and appendicular joints, extending into adjacent musculotendinous tissues. All findings supported fibrodysplasia ossificans progressiva (FOP), a disorder characterized by toe malformations and progressive heterotopic ossification in humans. In both cats, molecular analyses revealed the same heterozygous mutation in the activin A receptor type I ( ACVR1 ) gene that occurs in humans with FOP. Several reports of heterotopic ossification in cats exist, but this is the first one to identify clinical FOP in 2 cats with the identical mutation that occurs in >95% of humans with FOP.

Background: GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β-hexosaminidase A (Hex-A) and β-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency., Objectives: To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog., Animals: One affected Shiba Inu and a clinically healthy dog., Methods: Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes., Results: A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT)., Conclusions and Clinical Importance: Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Plectin, a large linker protein found in many tissues, acts to connect components of the cytoskeleton to each other. In the epidermis, plectin binds keratin intermediate filaments to hemidesmosomes. A deficiency of plectin in the skin leads to blister formation in the basal layer and the disease epidermolysis bullosa simplex (EBS)., Hypothesis/objectives: To describe a novel blistering disease that arose spontaneously in a litter of puppies., Animals: Two female and one male 20-day-old Eurasier puppies, from a litter of six, were presented for evaluation of failure to thrive and then euthanized due to poor prognosis. The puppies had ulcers on the lips, tongue, nasal planum, paw pads and abdomen., Results: Immunolabelling on frozen skin for basement membrane proteins revealed patchy and weak to absent staining for plectin as compared with strong linear staining in normal dogs. Ultrastructurally, hemidesmosomes were irregularly shaped and had loss of distinction between inner and outer plaques. Pedigree analysis supported an autosomal recessive mode of inheritance. A premature stop codon was discovered in exon 27 of PLEC that resulted in the production of a severely truncated protein., Conclusion: The study describes the first documented spontaneous EBS associated with a PLEC variant in domestic animals., (© 2016 ESVD and ACVD.)

Autosomal recessive congenital ichthyosis in the American bulldog is characterized by generalized scaling and erythema with adherent scale on the glabrous skin. We had previously linked this disorder to NIPAL4, which encodes the protein ichthyin. Sequencing of NIPAL4 revealed a homozygous single base deletion (CanFam3.1 canine reference genome sequence NC_06586.3 g.52737379del), the 157th base (cytosine) in exon 6 of NIPAL4 as the most likely causative variant in affected dogs. This frameshift deletion results in a premature stop codon producing a truncated and defective NIPAL4 (ichthyin) protein of 248 amino acids instead of the wild-type length of 404. Obligate carriers were confirmed to be heterozygous for this variant, and 150 clinically non-affected dogs of other breeds were homozygous for the wild-type gene. Among 800 American bulldogs tested, 34% of clinically healthy dogs were discovered to be heterozygous for the defective allele. More importantly, the development of this canine model of autosomal recessive congenital ichthyosis will provide insight into the development of new treatments across species., Competing Interests: The authors have declared that no competing interests exist.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, muscle-wasting disorder caused by mutations in the large, 2.4-Mb dystrophin gene. The majority of DMD-causing mutations are sporadic, multi-exon, frameshifting deletions, with the potential for variable immunological tolerance to the dystrophin protein from patient to patient. While systemic gene therapy holds promise in the treatment of DMD, immune responses to vectors and transgenes must first be rigorously evaluated in informative preclinical models to ensure patient safety. A widely used canine model for DMD, golden retriever muscular dystrophy, expresses detectable amounts of near full-length dystrophin due to alternative splicing around an intronic point mutation, thereby confounding the interpretation of immune responses to dystrophin-derived gene therapies. Here we characterize a naturally occurring deletion in a dystrophin-null canine, the German shorthaired pointer. The deletion spans 5.6 Mb of the X chromosome and encompasses all coding exons of the DMD and TMEM47 genes. The sequences surrounding the deletion breakpoints are virtually identical, suggesting that the deletion occurred through a homologous recombination event. Interestingly, the deletion breakpoints are within loci that are syntenically conserved among mammals, yet the high homology among this subset of ferritin-like loci is unique to the canine genome, suggesting lineage-specific concerted evolution of these atypical sequence elements.

OBJECTIVE To sequence exons and splice consensus sites of the dynactin subunit 1 (DCTN1) gene in Leonbergers and Labrador Retrievers with clinical laryngeal paralysis. ANIMALS 5 unrelated Leonbergers with laryngeal paralysis, 2 clinically normal Leonbergers, 7 unrelated Labrador Retrievers with laryngeal paralysis, and 2 clinically normal Labrador Retrievers. PROCEDURES Primers were designed for the entire coding regions of the DCTN1 gene, a noncoding exon at the 5´ end of the gene, and a 900-bp single-nucleotide polymorphism (SNP)-rich region located 17 kb upstream of the DCTN1 gene by use of the CanFam3 assembly of the canine genome sequence. Sequences were generated and compared between clinically normal and affected dogs. The SNPs flanking the DCTN1 gene as well as a previously identified nonsynonymous SNP in exon 32 were genotyped in affected and clinically normal Leonbergers and Labrador Retrievers. RESULTS None of the affected dogs were homozygous for any mutation affecting coding regions or splicing consensus sequences. Of the 16 dogs tested for the missense SNP in exon 32, all were homozygous for the reference allele, except for 2 affected and 1 clinically normal Labrador Retriever and 1 clinically normal Leonberger. The DCTN1 gene sequences (5 dogs) and haplotypes of polymorphic markers surrounding the DCTN1 gene (all dogs) were not consistent with the hypothesis that laryngeal paralysis was associated with inheritance of the same DCTN1 disease-causing allele within all Labrador Retrievers or Leonbergers evaluated. CONCLUSIONS AND CLINICAL RELEVANCE Mutations in the DCTN1 gene did not appear to cause laryngeal paralysis in Leonbergers or Labrador Retrievers.

Background: Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds., Hypothesis/objectives: To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds., Animals: Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs., Methods: Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA., Results: In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown., Conclusions and Clinical Importance: These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds., (Copyright © 2013 by the American College of Veterinary Internal Medicine.)

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β3), GPIIb (αIIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon-intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.

Dogs of the soft-coated wheaten terrier breed (SCWT) are predisposed to adult-onset, genetically complex, protein-losing nephropathy (average onset age = 6.3 ± 2.0 years). A genome-wide association study using 62 dogs revealed a chromosomal region containing three statistically significant SNPs (p(raw) ≤ 4.13 × 10(-8); p(genome) ≤ 0.005) when comparing DNA samples from affected and geriatric (≥14 years) unaffected SCWTs. Sequencing of candidate genes in the region revealed single nucleotide changes in each of two closely linked genes, NPHS1 and KIRREL2, which encode the slit diaphragm proteins nephrin and Neph3/filtrin, respectively. In humans, mutations in nephrin and decreased expression of Neph3 are associated with podocytopathy and protein-losing nephropathy. The base substitutions change a glycine to arginine in the fibronectin type 3 domain of nephrin and a proline to arginine in a conserved proline-rich region in Neph3. These novel mutations are not described in other species, nor were they found in 550 dogs of 105 other breeds, except in 3 dogs, including an affected Airedale terrier, homozygous for both substitutions. Risk for nephropathy is highest in dogs homozygous for the mutations (OR = 9.06; 95 % CI = 4.24-19.35). This is the first molecular characterization of an inherited podocytopathy in dogs and may serve as a model for continued studies of complex genetic and environmental interactions in glomerular disease.

Canine hypoadrenocorticism is an endocrine disorder characterised by inadequate secretion of steroid hormones from the adrenal glands. Pathology results from immune-mediated destruction of the adrenal cortex, which is similar to that seen in the human Addison's disease. Both the canine and human diseases have similar clinical presentation, with the diagnosis based on performing a dynamic adrenocorticotropic hormone stimulation test. MHC class II has previously been associated with the human and canine diseases. In the current study, we conducted an MHC class II association study in eight breeds of dog with diagnoses of hypoadrenocorticism. We demonstrated significant differences in dog leukocyte antigen (DLA) haplotype frequencies in six of these breeds: Cocker spaniel, Springer spaniel, Labrador, West Highland white terrier (WHWT), Bearded collie, and Standard poodle. In the Springer spaniel, the DLA-DRB1*015:01--DQA1*006:01--DQB1*023:01 haplotype was significantly associated with disease risk (p = 0.014, odds ratio (OR) = 5.14) and showed a similar trend in the Cocker spaniel. This haplotype is related to one associated with hypoadrenocorticism in the Nova Scotia duck tolling retriever. Similar haplotypes shared between breeds were demonstrated, with DLA-DRB1*001:01--DQA1*001:01--DQB1*002:01 more prevalent in both affected Labrador (p = 0.0002, OR = 3.06) and WHWT (p = 0.01, OR = 2.11). Other haplotypes that have not previously been associated with the disease were identified. The inter-breed differences in DLA haplotypes associated with susceptibility to canine hypoadrenocorticism could represent divergent aetiologies. This could have implications for clinical diagnosis and future comparative studies. Alternatively, it may suggest that the gene of interest is closely linked to the MHC.

We recently reported autosomal recessive fetal-onset neuroaxonal dystrophy (FNAD) in a large family of dogs that is not caused by mutation in the PLA2G6 locus (Fyfe et al., J Comp Neurol 518:3771-3784, 2010). Here, we report a genome-wide linkage analysis using 333 microsatellite markers to map canine FNAD to the telomeric end of chromosome 2. The interval of zero recombination was refined by single-nucleotide polymorphism (SNP) haplotype analysis to ~200 kb, and the included genes were sequenced. We found a homozygous 3-nucleotide deletion in exon 14 of mitofusin 2 (MFN2), predicting loss of a glutamate residue at position 539 in the protein of affected dogs. RT-PCR demonstrated near normal expression of the mutant mRNA, but MFN2 expression was undetectable to very low on western blots of affected dog brainstem, cerebrum, kidney, and cultured fibroblasts and by immunohistochemistry on brainstem sections. MFN2 is a multifunctional, membrane-bound GTPase of mitochondria and endoplasmic reticulum most commonly associated with human Charcot-Marie-Tooth disease type 2A2. The canine disorder extends the range of MFN2-associated phenotypes and suggests MFN2 as a candidate gene for rare cases of human FNAD.

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (naïve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Neuroaxonal dystrophy in brainstem, spinal cord tracts, and spinal nerves accompanied by cerebellar hypoplasia was observed in a colony of laboratory dogs. Fetal akinesia was documented by ultrasonographic examination. At birth, affected puppies exhibited stereotypical positioning of limbs, scoliosis, arthrogryposis, pulmonary hypoplasia, and respiratory failure. Regional hypoplasia in the central nervous system was apparent grossly, most strikingly as underdeveloped cerebellum and spinal cord. Histopathologic abnormalities included swollen axons and spheroids in brainstem and spinal cord tracts; reduced cerebellar foliation, patchy loss of Purkinje cells, multifocal thinning of the external granular cell layer, and loss of neurons in the deep cerebellar nuclei; spheroids and loss of myelinated axons in spinal roots and peripheral nerves; increased myocyte apoptosis in skeletal muscle; and fibrofatty connective tissue proliferation around joints. Breeding studies demonstrated that the canine disorder is a fully penetrant, simple autosomal recessive trait. The disorder demonstrated a type and distribution of lesions homologous to that of human infantile neuroaxonal dystrophy (INAD), most commonly caused by mutations of phospholipase A2 group VI gene (PLA2G6), but alleles of informative markers flanking the canine PLA2G6 locus did not associate with the canine disorder. Thus, fetal-onset neuroaxonal dystrophy in dogs, a species with well-developed genome mapping resources, provides a unique opportunity for additional disease gene discovery and understanding of this pathology.

Successful genetic treatment of most primary immunodeficiencies or hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSC) rather than their progeny to achieve enduring production of genetically corrected cells and durable immune reconstitution. Current ex vivo transduction protocols require manipulation of HSC by culture in cytokines for various lengths of time depending upon the retroviral vector that may force HSC to enter pathways of proliferation, and possibly differentiation, which could limit their engraftment potential, pluripotentiality and long-term repopulating capacity. We have compared the ability of normal CD34(+) cells cultured in a standard cytokine cocktail for 18hours or 4.5 days to reconstitute XSCID dogs following bone marrow transplantation in the absence of any pretransplant conditioning with that of freshly isolated CD34(+) cells. CD34(+) cells cultured under standard gamma-retroviral transduction conditions (4.5 days) showed decreased engraftment potential and ability to sustain long-term thymopoiesis. In contrast, XSCID dogs transplanted with CD34(+) cells cultured for 18hours showed a robust T cell immune reconstitution similar to dogs transplanted with freshly isolated CD34(+) cells, however, the ability to sustain long-term thymopoiesis was impaired. These results emphasize the need to determine ex vivo culture conditions that maintain both the engraftment potential and "stem cell" potential of the cultured cells.

The way in which veterinary scientists think about and approach the study of genetic disease has not changed, but the tools available to veterinary scientists have and will continue to change, allowing us to study increasingly complex problems and to make more rapid advances in the context of simple problems. To put these advances in perspective, this article first gives a historical perspective on the approaches to studying genetic diseases, particularly in human beings, and then outlines the advances that have become possible with the availability of the dog genome sequence. The article then discusses two inherited defects that are associated with urolithiasis, in particular, those responsible for cystine and purine (uric acid and its salts) stone formation. Together, these two conditions illustrate the contemporary use of a broad range of genetic approaches.

Dilated cardiomyopathy (DCM), the most common form of cardiomyopathy, often leads to heart failure and sudden death. While a substantial proportion of DCMs are inherited, mutations responsible for the majority of DCMs remain unidentified. A genome-wide linkage study was performed to identify the locus responsible for an autosomal recessive inherited form of juvenile DCM (JDCM) in Portuguese water dogs using 16 families segregating the disease. Results link the JDCM locus to canine chromosome 8 with two-point and multipoint lod scores of 10.8 and 14, respectively. The locus maps to a 3.9-Mb region, with complete syntenic homology to human chromosome 14, that contains no genes or loci known to be involved in the development of any type of cardiomyopathy. This discovery of a DCM locus with a previously unknown etiology will provide a new gene to examine in human DCM patients and a model for testing therapeutic approaches for heart failure.

Dogs with X-linked severe combined immunodeficiency (XSCID) can be successfully treated by bone marrow transplants (BMT) resulting in full immunologic reconstitution and engraftment of both donor B and T cells without the need for pretransplant conditioning. In this study, we evaluated the T cell diversity in XSCID dogs 4 months to 10.5 years following BMT. At 4 months posttransplantation, when the number of CD45RA+ (naïve) T cells had peaked and plateaued, the T cells in the transplanted dogs showed the same complex, diverse repertoire as those of normal young adult dogs. A decline in T cell diversity became evident approximately 3.5 years posttransplant, but the proportion of Vbeta families showing a polyclonal Gaussian spectratype still predominated up to 7.5 years posttransplant. In 2 dogs evaluated at 7.5 and 10.5 years posttransplant, >75% of the Vbeta families consisted of a skewed or oligoclonal spectratype that was associated with a CD4/CD8 ratio of <0.5. The decline in the complexity of T cell diversity in the transplanted XSCID dogs is similar to that reported for XSCID patients following BMT. However, in contrast to transplanted XSCID boys who show a significant decline in their T cell diversity by 10 to 12 years following BMT, transplanted XSCID dogs maintain a polyclonal, diverse T cell repertoire through midlife.

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).

Deficiency of glycogen branching enzyme (GBE) activity causes glycogen storage disease type IV (GSD IV), an autosomal recessive error of metabolism. Abnormal glycogen accumulates in myocytes, hepatocytes, and neurons, causing variably progressive, benign to lethal organ dysfunctions. A naturally occurring orthologue of human GSD IV was described previously in Norwegian forest cats (NFC). Here, we report that while most affected kittens die at or soon after birth, presumably due to hypoglycemia, survivors of the perinatal period appear clinically normal until onset of progressive neuromuscular degeneration at 5 months of age. Molecular investigation of affected cats revealed abnormally spliced GBE1 mRNA products and lack of GBE cross-reactive material in liver and muscle. Affected cats are homozygous for a complex rearrangement of genomic DNA in GBE1, constituted by a 334 bp insertion at the site of a 6.2 kb deletion that extends from intron 11 to intron 12 (g. IVS11+1552&#95;IVS12-1339 del6.2kb ins334 bp), removing exon 12. An allele-specific, PCR-based test demonstrates that the rearrangement segregates with the disease in the GSD IV kindred and is not found in unrelated normal cats. Screening of 402 privately owned NFC revealed 58 carriers and 4 affected cats. The molecular characterization of feline GSD IV will enhance further studies of GSD IV pathophysiology and development of novel therapies in this unique animal model.

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain (gammac) gene and is identical clinically and immunologically to human XSCID, making it a true homologue of the human disease. Bone marrow-transplanted (BMT) XSCID dogs not only engraft donor T cells and reconstitute normal T-cell function but, in contrast to the majority of transplanted human XSCID patients, also engraft donor B cells and reconstitute normal humoral immune function. Shortly after our initial report of successful BMT of XSCID dogs, it soon became evident that transplanted XSCID dogs developed late-onset severe chronic cutaneous infections containing a newly described canine papillomavirus. This is analogous to the late-onset cutaneous papillomavirus infection recently described for human XSCID patients following BMT. Of 24 transplanted XSCID dogs followed for at least 1 year post-BMT, 71% developed chronic canine papillomavirus infection. Six of the transplanted dogs that developed cutaneous papillomas were maintained for >3 1/2 years post-BMT for use as breeders. Four of these six dogs (67%) developed invasive squamous cell carcinoma (SCC), with three of the dogs (75%) eventually developing metastatic SCC, an extremely rare consequence of SCC in the dog. This finding raises the question of whether SCC will develop in transplanted human XSCID patients later in life. Canine XSCID therefore provides an ideal animal model with which to study the role of the gammac-dependent signaling pathway in the response to papillomavirus infections and the progression of these viral infections to metastatic SCC.

During the last 15 years, breeders have reported an increase in the proportion of Irish Wolfhounds with seizure disorders. Clinical data and pedigrees from closely related Irish Wolfhounds were collected retrospectively and analyzed. Idiopathic epilepsy was diagnosed, by exclusion of other causes for seizures, in 146 (18.3%) of 796 Irish Wolfhounds from 115 litters. The first seizure occurred by the age of 3 years in 73% of all dogs. Males were more commonly affected than females (61.6% versus 38.4%), with males having a later average age of seizure onset. The life expectancy of affected dogs was decreased by 2 years when compared with the average Irish Wolfhound population. The heritability index for the affected dogs, their littermates, and unaffected parents was 0.87. No simple mode of inheritance explains the pattern of affected dogs in pedigrees. Hallmarks of dominant and sex-linked inheritance were notably absent, and the segregation ratio was less than would be expected for simple autosomal recessive inheritance. Assuming all affected dogs have the same form of epilepsy, the simplest description of the complex pattern of inheritance observed is autosomal recessive, with incomplete penetrance and male dogs at increased risk.

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-kappaB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.

Earlier studies in the keeshond breed of dogs established that isolated conotruncal defects (CTDs) are a group of genetically and embryologically related cardiac malformations, including sub-clinical defects of the conal septum, conal ventricular septal defects, tetralogy of Fallot, and persistent truncus arteriosus. The same spectrum occurs in some human families. In both species, inheritance of non-syndromic CTDs is usually complex and multifactorial inheritance has been assumed. Previous studies in the keeshond suggested that susceptibility to CTD is an autosomal recessive trait, with alleles at modifying loci affecting severity. Here we report results of a genome-wide scan for CTD linked loci in a keeshond x beagle F1 backcross pedigree in which 46 of 101 offspring had CTDs. Two-point linkage analysis identified regions of suggestive linkage on each of three chromosomes CFA2, CFA9, and CFA15. No single locus accounted for segregation of CTDs in the pedigree, ruling out a single autosomal susceptibility locus. Multipoint analysis with Genehunter resulted in a corrected LOD score of 3.7 at the locus on CFA9 and supported linkage to the loci on CFA2 and CFA15 (LOD scores of 2.71 and 3.03). Genehunter Twolocus analysis suggested that CTD-predisposing alleles of these three loci are necessary, at least in pairs, to produce CTD. The canine CTD-linked chromosome regions are orthologous to human regions HSA5q11-13, HSA5q31, HSA17q11-24, and HSA4q31. We excluded from the linked regions in the dog, a number of genes known to have a role in the etiology of CTDs and predict that continuing studies will identify CTD-predisposing genes not previously recognized.

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.

Glomerular disease was diagnosed by histopathologic examination in 11 related Bullmastiff dogs, and clinical and laboratory data were collected retrospectively. Four female and seven male dogs between the ages of 2.5 and 11 years were affected. Clinical signs, including lethargy and anorexia, were nonspecific and occurred shortly before death or euthanasia. In five affected dogs serial blood samples were obtained, and dramatically elevated blood urea nitrogen and creatinine levels were demonstrated up to 2.75 years before death. Protein-creatinine ratios were elevated in six of six dogs and were above normal 3.5 years before death in one dog. The kidneys appeared grossly normal to slightly smaller than normal at necropsy. Histologic abnormalities of the kidneys were consistent with chronic glomerulonephropathy with sclerosis. Examination of the pedigrees of related affected dogs yielded evidence supporting an autosomal recessive mode of inheritance.

Selective malabsorption of cobalamin (vitamin B(12)) accompanied by proteinuria, known as Imerslund-Gräsbeck syndrome or megaloblastic anemia 1 (I-GS, MGA1; OMIM 261100), is a rare autosomal recessive disorder. In Finnish kindreds, I-GS is caused by mutations in the cubilin gene ( CUBN), located on human Chromosome (Chr) 10. However, not all patients have CUBN mutations, and three distinct mutations in the amnionless gene, AMN, were very recently identified in patients from Norwegian and Israeli families. The present study demonstrates that in a large canine I-GS pedigree, the disease is genetically linked (peak multipoint LOD score 11.74) to a region on dog Chr 8 that exhibits conserved synteny with human Chr 14q. Multipoint analysis indicates that the canine disease gene lies in an interval between the echinoderm microtubule-associated, protein-like 1 ( EML1) gene and the telomere. A single critical recombinant further suggests that the disease gene is between markers in EML1 and the G protein-coupled receptor ( G2A) gene, defining an I-GS interval in the human genome that contains the AMN gene. Thus, these comparative-mapping data provide evidence that canine I-GS is a homologue of one form of the human disease and will provide a useful system for understanding the molecular mechanisms underlying the disease in humans.

We have initially sequenced approximately 8,000 canine expressed sequence tags (ESTs) from several complementary DNA (cDNA) libraries: testes, whole brain, and Madin-Darby canine kidney (MDCK) cells. Analysis of these sequences shows that they provide partial sequence information for about 5%-10% of the canine genes. An analysis pipeline has been created to cluster the ESTs and to map individual ESTs as well as clustered ESTs to both the human genome and the human proteome. Gene ontology (GO) terms have been assigned to the ESTs and clusters based on their top matches to the International Protein Index (IPI) set of human proteins. The data generated is stored in a MySQL relational database for analysis and display. A Web-based Perl script has been written to display the analyzed data to the scientific community.

Our laboratory has identified an X-linked severe combined immunodeficiency (XSCID) in dogs that is the result of mutations in the common gamma chain (gammac) subunit of the interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. Canine XSCID, unlike genetically engineered gammac-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID, suggesting species-specific differences exist in the role of the gammac and its associated cytokines in mice in comparison to their role in humans and dogs. This review compares and contrasts thymopoiesis and postnatal T cell development in gammac-deficient (XSCID) dogs raised in a conventional environment, with gammac-deficient dogs raised in a gnotobiotic environment. Therapy to accelerate T cell regeneration following hematopoietic stem cell transplantation or gene therapy is also discussed.

Five adult Samoyed dogs from two unrelated litters were diagnosed with diabetes mellitus. Two full-sibling male dogs (Family A) were raised in the same household. The other three dogs, two female and one male, were also full siblings (Family B) raised in different households. All five dogs developed polyuria and polydipsia and demonstrated fasting hyperglycemia and glucosuria. Diabetes mellitus was diagnosed in all five dogs and responded to appropriate therapy with insulin. The occurrence of insulin-dependent diabetes mellitus in multiple, closely related Samoyed dogs suggests a familial predisposition in this breed.

Hypophosphatasia, a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum, reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP. High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but, as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser, Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability of hypophosphatasia.

X-linked severe combined immunodeficiency (X-SCID) is the most common form of human SCID and is caused by mutations in the common gamma chain (gammac), a shared component of the interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. BMT for human X-SCID results in engraftment of donor T-cells and reconstitution of normal T-cell function but engraftment of few, if any, donor B-cells and poor reconstitution of humoral immune function. Canine X-SCID is also caused by mutations in the yc and has an immunological phenotype identical to that of human X-SCID. We have previously reported that transplantation of nonconditioned X-SCID dogs with unfractionated histocompatible bone marrow results in engraftment of both donor B- and T-cells and reconstitution of normal T-cell and humoral immune function. In this study, we assessed the ability of purified canine CD34+ bone marrow cells to reconstitute lymphoid populations after histocompatible BMT in 6 nonablated X-SCID dogs. All dogs showed engraftment of donor T-cells, with T-cell regeneration occurring through a thymic-dependent pathway, and had reconstituted normal T-cell function. In contrast to our previous studies, only 3 dogs had engraftment of donor B-cells and reconstituted normal antigen-specific B-cell function post-BMT. The variable donor B-cell engraftment and reconstitution of normal humoral immune function observed in this study are similar to the outcomes observed in the majority of human X-SCID patients following BMT. This study demonstrates that canine CD34+ cells contain progenitors capable of immune reconstitution and is the first study to document the ability of CD34+ bone marrow cells to reconstitute normal B- and T-cell function in a nonablated large-animal model of BMT. This study also demonstrates that the quality of immune reconstitution following CD34+ BMT may be dosage dependent Thus canine X-SCID provides a large-animal preclinical model that can be used not only to determine the optimal conditions for both donor B- and T-cell engraftment following CD34 BMT, but also to develop and evaluate strategies for gene therapy protocols that target CD34 cells.

Dilated cardiomyopathy recently has been recognized in juvenile Portuguese Water Dogs. The purpose of this study was to evaluate unaffected and affected puppies by physical examination, electrocardiogram (ECG), echocardiogram, specific biochemical assays, and ultrastructure to document disease progression and to develop a method of early detection. Results of segregation analysis were consistent with autosomal recessive inheritance. Of 124 puppies evaluated clinically and echocardiographically, 10 were affected. No significant differences were found between unaffected and affected puppies for blood and myocardial carnitine or taurine concentrations, serum chemical variables, results of ophthalmological examinations, ECGs, or measurement of urine metabolites. Ultrastructural examination of myocardium from affected dogs revealed myofibrillar atrophy and small regions of myofibrillar degeneration, most prominently at the region of the intercalated discs. Only echocardiography allowed detection of affected puppies before clinical signs became evident. Echocardiography revealed a significant difference in the shortening fraction, E point to septal separation, and the end systolic and diastolic left ventricular internal diameters. Affected puppies were detected 1-4 weeks before the development of acute congestive heart failure.

Cystinuria is an inherited renal and intestinal disease characterized by defective amino acid reabsorption and cystine urolithiasis. Different forms of the disease, designated type I and non-type I in cystinuric humans, can be distinguished clinically and biochemically, and have been associated with mutations in the SLC3A1 (rBAT) and SLC7A9 genes, respectively. Type I cystinuria is the most common form and is inherited as an autosomal recessive trait in humans. Cystinuria has been recognized in more than 60 breeds of dogs and a severe form, resembling type I cystinuria, has been characterized in the Newfoundland breed. Here we report the cloning and sequencing of the canine SLC3A1 cDNA and gene, and the identification of a nonsense mutation in exon 2 of the gene in cystinuric Newfoundland dogs. A mutation-specific test was developed for the diagnosis and control of cystinuria in Newfoundland dogs. In cystinuric dogs of six other breeds, either heterozygosity at the SLC3A1 locus or lack of mutations in the coding region of the SLC3A1 gene were observed, indicating that cystinuria is genetically heterogeneous in dogs, as it is in humans. The canine homologue of human type I cystinuria provides the opportunity to use a large animal model to investigate molecular approaches for the treatment of cystinuria and other renal tubular diseases.

Two related female Norwegian Elkhounds were evaluated at 6 and 8 months of age for enlarged clitori. Both had a 78 XX karyotype. Histology of their internal reproductive tracts demonstrated 1 to be an XX true hermaphrodite with bilateral ovotestes and the other to be an XX male with bilateral aspermatogenic testes. Polymerase chain reaction-based tests of genomic DNA showed that both dogs lacked Sry, the testis-determining gene. Pedigree analysis was consistent with an autosomal recessive mode of inheritance, as has been reported in the American Cocker Spaniel and the German Shorthaired Pointer. This is the 1st reported case of familial Sry-negative XX sex reversal in the Norwegian Elkhound. A summary of 34 previously unreported cases of dogs with masculinized external genitalia and a normal 78 XX karyotype seen from 1980 to 1997 is given.

We describe four pregnancies in two families in which mild hypophosphatasia, apparently transmitted as an autosomal dominant trait, manifested in utero as severe long bone bowing. Postnatally, there was spontaneous improvement of the skeletal defects. Recognition of this presentation for hypophosphatasia by family investigation and assessment of the fetal skeleton for degree of ossification and chest size using ultrasonography is important. The prognosis for this condition is considerably better than for more severe forms of hypophosphatasia and for many other disorders that cause skeletal defects with long bone bowing in utero., (Copyright 1999 Wiley-Liss, Inc.)