This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MPS IIIB in Schipperke Dogs A naturally occurring canine form of MPS IIIB has been identified through the metabolic screening laboratory. The initial description of the dogs identified with this condition has been published. We have isolated and sequenced the entire protein-coding region of the canine NAGLU gene which has been presented in abstract form. Additionally the mutation has been identified and was found to be an unusual L1 retrotransposon-mediated insertion of a polyadenylate sequence of approximately 45 A s. The insertion occurs in the sixth exon and is predicted to lead to the insertion of 15 lysine residues after amino acid 704 of the normal protein sequence. The insertion of so large a series of lysines would be predicted to result in the elimination of normal enzyme activity, whether or not the lysines were followed by in frame or frame shifted translations. A DNA diagnostic for this mutation has been developed and is used to diagnose animals produced in the colony as well as in the Skipperke dog population. While the DNA test was still in development, four new affetced animals were diagnosed through the metabolic screening laboratory. Urine, serum and blood samples were requested from relatives of these animals to determine carrier status and identify other affected animals. A total of 96 samples were analyzed from 28 animals. Four additional affected dogs were diagnosed and 11 carriers were detected. This study allowed preliminary accuracy testing of the newly developed DNA test in the breeding community. Preliminary evaluations of the brains of the two original affected dogs indicated striking accumulation of GM2 and GM3 gangliosides. Evalutions underway include neurologic testing, histopathologic evaluation of the CNS and biochemical analysis of the CNS, with a specific focus on ganglioside elevations. Part of the canine colony for this lysosomal storage disease in now present at Iowa State University, where it will be the focus of study by Dr. N.M. Ellinwood. A further effort associated with this model is the assessment of gene therapy for MPS IIIB. Specifically, recombinant adeno-associated vectors, of various serotypes, are being constructed, and will be evaluated in the brains of MPS IIIB affected dogs following intracerebral injection Canine MPS VI After the original discovery of mucopolysaccharidosis (MPS) VI or arylsulfatase deficiency in Siamese cats and their clinicopathologic and molecular characterization and use in gene transfer studies, we have recently identified several dogs with MPS VI. The first affected breed was the Miniature Pinscher, followed by the Welsh Corgi, Chesapeake Bay retriever, and Miniature Schnauzer. MPS VI is caused by a deficiency of the enzyme N-acetylgalactosamine-4-sulfatase, also known as 4S (EC # 3.1.6.12). 4S deficiency results in the lysosomal accumulation of glycosaminoglycans (GAG), specifically dermatan sulfate, that is found in urine. MPS VI is inherited as an autosomal recessive trait, and is clinically characterized by short stature, facial dysmorphism, corneal dystrophy, secondary neurological disturbances, and various orthopedic abnormalities. The normal canine ARSB cDNA sequence was determined using primers developed based on conserved sequences of the human and feline ARSB, and by screening clones from a canine cDNA library. When the DNA sequence from Miniature Pinschers affected with MPS VI was compared to the normal canine sequence, a single missense mutation (G to A) was identified. This mutation, occurring in exon V, replaces the tiny hydrophobic amino acid glycine with a bulky positively charged basic amino acid arginine in a highly conserved region of the protein. The same mutation (G302R) has been described in humans to cause a severe form of the disease. A DNA test based on restriction enzyme analysis of PCR products was developed. Samples were solicited for testing from AKC registered Miniature Pinschers. DNA obtained from cheek swab or EDTA blood samples from 130 Miniature Pinschers has been analyzed. One affected and 20 carrier dogs were identified. The affected dog and all identified carrier dogs were closely related. In this biased population, the frequency of the mutant allele was approximately 0.0846. To identify the mutation that is responsible for MPS VI in Miniature Schnauzers, primers were developed from intronic sequence provided by The Insititute for Genome Research (TIGR) and the Whitehead Genomic Institute as well as 5' utr sequence and 3' utr sequence generated from the normal canine cDNA. The mutation responsible for MPS VI in the Miniature Schnauzer was identified as a 56 bp deletion that eliminates the last 26 bp of the 5' utr and the first 10 codons of exon I including the initiator ATG. Since the next ATG is located 426 bp downstream in the cDNA, this truncated protein is predicted to be non-functional and unstable. Canine MPS VII A German shepherd puppy was presented to the University of Georgia because of difficulties ambulating. Laboratory diagnostic tests were pursued and a diagnosis of MPS VII was reached. The same mutation was found in this German shepherd dog as previously described in a mixed breed dog 20 years ago thus confirming that this disease originated in the German shepherd breed. No relatives have become available for further study. A juvenile female Rat terrier was presented to because of severe deformities and gait abnormalities. The puppy exhibited a dome shaped head, severe underbite and multiple limb deformities. Skeletal radiographs revealed the classical bone changes observed with many MPS disorders. White blood cells contained granular material which stained with toluidine blue. Urine contained large amounts of chondroitin and dermatin sulfate. The serum beta-glucuronidase activity was completely lacking documenting an MPS VII disorder. This dog does not have the same mutation seen in the original mixed breed and recent German Shepherd dog. Relatives of the affected dog have been examined and some were found to be carriers. At this time there are no plans to capture this model because of the close similarities of the disease in the established canine m
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