Donald F. Patterson

Canine genetics comes of age

The dog, as humans favored companion, is unique among animal species in providing new insights into human genetic disease. In this review, we will discuss both the breed and the population structure of dogs and why that makes canines amenable to genetic studies. We will review the current state of the map and discuss the particular disease states in which canines stand to make the greatest contribution to medical genetics.

An integrated linkage-radiation hybrid map of the canine genome

Abstract. Purebred dogs are a unique resource for dissecting the molecular basis of simple and complex genetic diseases and traits. As a result of strong selection for physical and behavioral characteristics among the 300 established breeds, modern dogs are characterized by high levels of interbreed variation, complemented by significant intrabreed homogeneity. A high-resolution map of the canine genome is necessary to exploit the mapping power of this unusual resource. We describe here the integration of an expanded canine radiation hybrid map, comprised of 600 markers, with the latest linkage map of 341 markers, to generate a map of 724 markers—the densest map of the canine genome described to date. Through the inclusion of 217 markers on both the linkage and RH maps, the 77 RH groups are reduced to 44 syntenic groups, thus providing comprehensive coverage of most of the canine genome.

β-Glucuronidase Deficiency in a Dog: a Model of Human Mucopolysaccharidosis VII

Summary: This report describes a third mucopolysaccharidosis in animals: canine mucopolysaccharidosis VII. The affected dog was the offspring of a father-daughter mating. Weakness in the rear legs was evident at 8 weeks of age and became progressively worse. He had a large head, a shortened maxilla, and corneal granularities. Most joints were extremely lax, easily subluxated, with joint capsules that were swollen and fluctuant.The dog was alert and had apparently normal pain perception. At 13 months of age, there was radiographic evidence of extensive skeletal disease including bilateral femoral head luxation, abnormalities in the shape and density of the carpal and tarsal bones, radiolucent lesions of the epiphyseal regions of most long bones, and cervical vertebral dysplasia and platyspondylia. The electrophoretic pattern of precipitated glycosaminoglycans indicated a predominance of chondroitin sulfate.The animal died suddenly from gastric dilatation. There was generalized hepatomegaly, thickening of the atrioventricular heart valves, and generalized polyarthropathy. Vacuolated cytoplasm was observed in hepatocytes, keratocytes, fibroblasts, chondrocytes and cells of the synovial membrane, retinal pigment epithelium, and cardiac valves. Neurons had cytoplasmic vacuoles. Electron microscopy demonstrated membrane-bound cytoplasmic inclusions in polymorphonuclear leukocytes, hepatocytes, synovium, heart valves and spleen.The activities of 12 lysosomal hydrolases were determined in liver from the affected and control dogs: β-glucuronidase (EC 3.2.1.31), β-hexosaminidases A and B (EC 3.2.1.30), α-hexosaminidase (EC 3.2.1.-), α-L-iduronidase (EC 3.2.1.76), α-galactosidase A (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), arylsulfatases A and B (EC 3.1.6.1), acid α-mannosidase (EC 3.2.1.24), acid β-mannosidase (EC 3.2.1.25), and N-acetyl-D-galactosamine-6-sulfate sulfatase (EC 3.1.6.-). The activity of β-glucuronidase was reduced to less than 2% of the normal mean value of normal controls.

Inherited Selective Intestinal Cobalamin Malabsorption and Cobalamin Deficiency in Dogs

ABSTRACT: Inherited selective intestinal malabsorption of cobalamin (Cbl) was observed in a family of giant schnauzer dogs. Family studies and breeding experiments demonstrated simple autosomal recessive inheritance of this disease. Affected puppies exhibited chronic inappetence and failure to thrive beginning between 6 and 12 wk of age. Neutropenia with hypersegmentation, anemia with anisocytosis and poikilocytosis, and megaloblastic changes of the bone marrow were present. Serum Cbl concentrations were low, and methylmalonic atiduria and homocysteinemia were present. Parenteral, but not oral, cyanocobalamin administration rapidly eliminated all signs of Cbl deficiency except for low serum Cbl concentrations. Cbl malabsorption in affected dogs was documented by oral administration of [57Co]cyanocobalamin with or without simultaneous oral administration of intrinsic factor or normal dog gastric juice. Quantitation and function studies of intrinsic factor and transcobalamin-II from affected dogs revealed no abnormality. Other gastrointestinal functions and ileal morphology were normal, indicating a selective defect of Cbl absorption at the level of the ileal enterocyte. Immunoelectron microscopy of ileal biopsies showed that the receptor for intrinsic factor-Cbl complex was absent from the apical brush border microvillus pits of affected dogs. This canine disorder resembles inherited selective intestinal Cbl malabsorption (Imerslund-Grasbeck syndrome) in humans, and is a spontaneously occurring animal model of early onset Cbl deficiency.

Histologic studies on normal and persistent ductus arteriosus in the dog

The process of anatomic closure of the ductus arteriosus was studied at the ultrastructural level in 15 normal beagles (age 0 hour to 13 days) and in 18 specimens from a strain of dogs with hereditary persistent ductus arteriosus (age 4 hours to 27 days). Normal ductal closure takes place from the pulmonary artery to the aortic end. It is accompanied by a series of histologic changes: 1) separation of the endothelial cells from the internal elastic lamina resulting in a wide region of subendothelial edema; 2) ingrowth and infolding of endothelial cells and migration of undifferentiated smooth muscle cells from the inner media into the subendothelial region; 3) apposition of endothelial cells bordering the lumen; and 4) degenerative changes. In persistent ductus arteriosus, these changes do not occur. The endothelial cells remain closely adhered to the internal elastic lamina and the underlying media is abnormal in structure. In the case of partial persistent ductus arteriosus (ductus diverticulum), both the normal and the abnormal type of wall are found in a single ductus arteriosus. The histologic features of the normal and the persistent ductus arteriosus in the dog resemble those of the normal and the persistent ductus arteriosus in humans, suggesting a similar pathogenesis.

Alpha-L-iduronidase deficiency in a cat: a model of mucopolysaccharidosis I.

Summary: A 1-yr-old male domestic shorthair cat was referred to the University of Pennsylvania Veterinary Hospital with a history of progressive lameness. The cat had a short, broad face, frontal bossing, a depressed nasal bridge, small ears, bilateral corneal clouding, and thickened skin over the dorsal aspect of his neck. Radiographically, the cervical vertebrae were wide, assymetrical, and appeared nearly fused; there was bilateral coxofemoral sub-luxation and pectus excavatum. Electrophoresis of glycosamino-glycans (GAG) from the urine revealed an excess of both dermatan sulfate and heparan sulfate. The incorporation of 35SO4 into the GAG of fibroblasts revealed an exaggerated accumulation of [35S] -glycosaminoglycans. By light microscopy, neurons swollen with vacuolated cytoplasm were observed. By electron microscopy, the spinal cord neurons contained membrane-bound “zebra bodies”. Membrane-bound inclusions containing granular material or an occasional myelin-like figure were present in hepatocytes. The activities of seven lysosomal hydrolases (α-L-iduronidase EC. 3.2.1.76, β-D-glucuronidase EC. 3.2.1.31, arylsulfatase A EC. 3.1.6.1, arylsulfatase B EC. 3.1.6.1, α-D-glucosaminidase EC. 3.2.1.50, β-D-glucosaminidase EC. 3.2.1.30, and β-D-galactosidase EC. 3.2.1.33) were investigated in cells from the affected cat. The activity of α-L-iduronidase was deficient in both cultured fibroblasts and peripheral leucocytes, while the activity of the other enzymes was similar to that in normal cats. It is apparent that the pattern of GAG excretion, evidence of lysosomal storage in various tissues, evidence of defective GAG degradation in cultured fibroblasts, and the specific deficiency in activity of α-L-iduronidase in the affected cat parallel closely the findings in mucopolysaccharidosis (MPS) I of man.Speculation: This feline model of MPS I (Hurler, Scheie, or Hurler/Scheie Syndrome) should allow advances in the understanding of the pathogenesis and approaches to therapy for this and related genetic storage diseases.

β-Glucuronidase mediated pathway essential for retinal pigment epithelial degradation of glycosaminoglycans. Disease expression and in vitro disease correction using retroviral mediated cDNA transfer

A beta-glucuronidase mediated pathway for the degradation of glycosaminoglycans is present in the retinal pigment epithelium. The pathway has been defined using ocular tissues and cultured cells from mutant animals having a recessively inherited deficiency of the lysosomal enzyme. In situ, storage products accumulate in secondary lysosomes of the retinal pigment epithelium, the cytoplasm fills with inclusions and the cells hypertrophy; severity of the disease increases with aging. Deficient activity of beta-glucuronidase is present in primary and second passage cultures. Radiolabel studies with 35SO4 show a significant retention of cell layer label by mutant retinal pigment epithelial cells during a 72-hr pulse or 24-hr chase period. The labels is in newly synthesized chondroitin sulfate and heparan sulfate, which are natural substrates for the deficient enzyme. There is no difference from normal in the total radioactivity and electrophoretic profile of the glycosaminoglycans that are synthesized and released into the media. A retroviral vector was used to transfer normal rat beta-glucuronidase cDNA into the mutant cells. The vector treatment results in restoration of enzyme activity and correction of the degradative defect; 35SO4 labeling shows that chondroitin sulfate and heparan sulfate levels return to normal. The vector treatment studies indicate that a single gene defect determines the abnormal beta-glucuronidase mediated pathway in the mutant retinal pigment epithelium.

Hereditary defects of the conotruncal septum in keeshond dogs: Pathologic and genetic studies☆

Abstract Pathologic studies of a hereditary cardiovascular defect in Keeshond dogs demonstrated a spectrum of malformations, primarily involving the ventricular outflow tracts. Lesions ranged from subclinicai defects of the crista supraventricularis to end-stage tetralogy of Fallot and included isolated ventricular septal defects and pulmonic stenosis. The spectrum can be explained by assuming that the anatomic variations represent different thresholds along a continuum of maldevelopment producing hypoplasia and malpositioning of the conotruncal septum. The results of breeding experiments conducted in a colony of Keeshond dogs with conotruncal septal defects confirmed the hereditary nature of the abnormality but were not consistent with any simple genetic hypothesis. Both the incidence and the severity of the conotruncal lesions increased with the severity of the parental defect. The results are shown to fit a polygenic model with three developmental thresholds, in which multiple genes act additively to produce a continuous distribution of maldevelopment involving the conotruncal septum. Lesions of increasing severity occur with an increasing “dose” of genes predisposing to conotruncal septal defects.

ANCHORING OF CANINE LINKAGE GROUPS WITH CHROMOSOME-SPECIFIC MARKERS

Abstract. A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest.

Glycogen Storage Disease Type IV: Inherited Deficiency of Branching Enzyme Activity in Cats

ABSTRACT: Glycogen storage disease type IV due to branching enzyme deficiency was found in an inbred family of Norwegian forest cats, an uncommon breed of domestic cats. Skeletal muscle, heart, and CNS degeneration were clinically apparent and histologically evident in affected cats older than 5 mo of age, but cirrhosis and hepatic failure, hallmarks of the human disorder, were absent. Beginning at or before birth, affected cats accumulated an abnormal glycogen in many tissues that was determined by histochemical, enzymatic, and spectral analysis to be a poorly branched α-1,4-D-glucan. Branching enzyme activity was less than 0.1 of normal in liver and muscle of affected cats and partially deficient (0.17–0.75 of normal) in muscle and leukocytes of the parents of affected cats. These data and pedigree analysis indicate that branching enzyme deficiency is a simple autosomal recessive trait in this family. This is the first reported animal model of human glycogen storage disease type IV. A breeding colony derived from a relative of the affected cats has been established.