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. Within the animal colony, we have four breeding I-cell carrier females and two breeding I-cell carrier males. In addition, two young carrier females are being kept until maturity for breeding. In the past year, two cats affected with I-cell disease have been sacrificed: one at 8 months and another at 1 week. Also, we have archived formalin-fixed and frozen tissues in variable condition from affected and carrier as well as normal cats. Presently, we have two one-month old affected I-cell kittens, as well as a normal littermate which will serve as a control. Post-mortem analysis Affected kittens that die often have only mild gross lesions at necropsy, including dilated hearts, esophageal dilation, brittle bones, and severely thickened skin, with the age at death reflecting the severity of lesions due to the progressive nature of this disease. In I-cell affected cats that died at eight months, histological lesions were seen in a many tissues, including, but not limited to, the tongue, epiglottis, liver, skin, heart, aorta and other large arteries, smooth muscle in general, kidney, and bones. These lesions ranged from large clear vacuoles within cells that distorted the cellular and organ architecture (as variably seen in the epiglottis, tongue, aorta and other large arteries and cardiac valves), to small vacuoles that appear to have little effect on cellular morphology (liver, kidney), to abnormalities in the normal organization of tissue components (bone, skin). We have examined special stains that have been performed on tissue from human patients in the past. Biochemical identification of lysosomal contents within affected cells. Based on direct measurement, histological staining qualities, and ultrastructural appearance, oligosaccharides, lipids, and glycosaminoglycans (GAGs) are believed to accumulate within various cells in human and feline I-cell patients. However, specific substrates and their relative amounts in various tissues in affected, normal, and carriers are not known. Using a commercial kit (Blyscan), we have attempted to quantitate levels of GAGs within cultured fibroblasts from I-cell affected cats. Presently, we have grown, collected, and frozen cultured fibroblasts from mucopolysaccharidosis (MPS) I, III, and VI affected cats (as positive controls) as well as I-cell cultured cells. We have established an international collaboration with Dr. Marie Vanier, Toulouse University for specification and quantification of tissue gangliosides. Identification of the feline N-acetylglucosamine-1-phosphotransferase gene. The genetic sequence of N-acetylglucosamine-1-phosphotransferase (GNPTA) GNPTA has not yet been published in any species. Based on the unpublished human sequence, provided by our collaborator, William Canfield, Genzyme Corp., we were able to search the NCBI Trace Archives for recently available feline sequence from the Feline Genome Project (FGP) and found genetic sequences homologous to the human GNPTA gene. To this point, for all but three (exons 4, 5, and 6) of the 21 exons we have some whole genome shotgun sequences. Using the normal FGP sequence as a guide, we have designed primers to sequence gDNA and cDNA from an I-cell affected cat. Four exons have been sequenced so far from the gDNA (exons 2, 8, 12, and 13). Exon 2 sequencing is not complete, but appears to be homologous to the normal FGP sequence at this point. Also, exons 8 and 12 are identical to the FGP sequence, so do not appear to carry the mutation in this affected cat. Approximately half of exon 13 (the longest exon, at approximately 1300 bp) has been sequenced at this time. A single bp difference has been found in the 5 portion of this exon. however, its significance is not yet known. In addition, most of the GNPTA cDNA from a the normal and affected cat have been sequenced including the missing exons in the FGS and a few differences have been determined, which may relate to the disease-causing mutation.
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