Evaluation for type 1 diabetes associated autoantibodies in diabetic and non-diabetic Australian terriers and Samoyeds.

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.

Heritability and complex segregation analysis of naturally-occurring diabetes in Australian Terrier Dogs.

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.

Hereditary ß-mannosidosis in a dog: Clinicopathological and molecular genetic characterization.

Hereditary ß-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 ß-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 ß-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 ß-mannosidosis could become a valuable disease model for the human disease.

Identification of the Identical Human Mutation in ACVR1 in 2 Cats With Fibrodysplasia Ossificans Progressiva.

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.

Mechanism of Deletion Removing All Dystrophin Exons in a Canine Model for DMD Implicates Concerted Evolution of X Chromosome Pseudogenes.

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.

A Defect in NIPAL4 Is Associated with Autosomal Recessive Congenital Ichthyosis in American Bulldogs.

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.

Epidermolysis bullosa simplex in sibling Eurasier dogs is caused by a PLEC non-sense variant.

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.

Evaluation of the dynactin 1 gene in Leonbergers and Labrador Retrievers with laryngeal paralysis.

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.

Polymorphisms in canine platelet glycoproteins identify potential platelet antigens.

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 (ß3), GPIIb (αIIb), and GPIbß, 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.

MHC class II association study in eight breeds of dog with hypoadrenocorticism.

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.

Glomerulopathy and mutations in NPHS1 and KIRREL2 in soft-coated Wheaten Terrier dogs.

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.

A novel mitofusin 2 mutation causes canine fetal-onset neuroaxonal dystrophy.

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.

Ex vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma.

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.

Inherited neuroaxonal dystrophy in dogs causing lethal, fetal-onset motor system dysfunction and cerebellar hypoplasia.

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.

Effect of ex vivo culture of CD34+ bone marrow cells on immune reconstitution of XSCID dogs following allogeneic bone marrow transplantation.

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.

Changing paradigms in diagnosis of inherited defects associated with urolithiasis.

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.

A novel locus for dilated cardiomyopathy maps to canine chromosome 8.

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.

T cell repertoire development in XSCID dogs following nonconditioned allogeneic bone marrow transplantation.

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.

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model.

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).

A complex rearrangement in GBE1 causes both perinatal hypoglycemic collapse and late-juvenile-onset neuromuscular degeneration in glycogen storage disease type IV of Norwegian forest cats.

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_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.