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. Since our initial studies of methylmalonic aciduria due to cobalamin malabsorption in giant schnauzers, we have identified similar defects in border collies, beagles, golden retrievers, miniature schnauzers, and most recently, in a cat and a family of Australian shepherds. These animals present with variable clinical signs from progressive failure to thrive, to intermittent ketoto-acidosis and hyperammonemic seizure-like episodes. Parental administration of cobalamin completely reverses the clinical and metabolic changes except for proteinuria. Studies are in progress to compare the defect in giant schnauzers to other breeds of dogs and cats. These animals are models for the Imerslund-Gr sbeck syndrome (I-GS) and are being further studied in collaboration with John Fyfe DVM, PhD, Associate Professor at Michigan State University. Cubilin is the receptor for intrinsic factor-cobalamin in the intestine and was the primary biological candidate gene for canine selective cobalamin malabsorption because studies demonstrated that cubilin protein is abnormally expressed in intestine and kidney in affected dogs. Two years ago we reported that cubilin, had been eliminated by linkage analysis from further consideration. We have continued through collaborative efforts to examine the defect underlying this disorder. Together with Dr. John Fyfe, we initiated a genome scan for linkage of the disorder to DNA markers that has provided comparative positional candidate genes. The Referral Center also provided animals for outcross matings that interjected greater marker heterogeneity into the I-GS linkage family The genome scan bore fruit early in the process with highly significant linkage to a marker on dog chromosome 8 (CFA 8). The region was highly homologous to a region of human chromosome 14q. In order to supplement the available markers of the canine genome in this region, the canine version of genes in the region were amplified and sequenced to identify polymorphisms that were informative in the I-GS family. Using these new markers, the canine I-GS locus was determined to be in a 5 Mb region near the telomere of CFA 8 and was linked with no recombination to a the KNS2 gene. This high resolution linkage analysis was possible because the I-GS family derived from giant schnauzers is now quite large. Though many fewer, we have collected DNA samples from a family of Australian shepherds exhibiting I-GS. Their disorder is also not linked to the cubilin locus and within the resolution possible given the family size is linked to the same region of CFA 8 as the giant schnauzer I-GS. Results of these studies have been submitted for publication. A search of annoted genes in this region of human 14q revealed amnionless (AMN). AMN is a gene of unknown function that is highly and almost exclusively expressed in kidney and intestine, the two tissues in which cubilin is expressed and which malfunction in canine I-GS. We cloned the canine AMN cDNA and searched affected dog AMN cDNA for mutations in both the giant schnauzer and Australian shepherd families. A 33 bp deletion in exon 10 of AMN was found in the giant schnauzer family, but no mutation has yet been found in Austalian shepherds. In order to further investigate the Australian shepherd disorder, additional matings have been perform to enlarge the linkage family and to provide affected dog tissues for analysis. Just recently, AMN mutations causing I-GS in some human kindreds have been demonstrated. Further studies that are only possible in the canine I-GS models will be pursued in order to determine the function of AMN and how AMN defects abrogate cubilin expression. These studies will be conducted under an NIH grant awarded to Dr. Fyfe, (#DK 064161, Molecular Mechanism of Polarized Cubilin Expression, April 1, 2003- March 31, 2005). Result to date indicate that cubilin and AMN collaborate as strongly bound subunits of a heterodimeric, multiligand receptor complex that has essential function in a number of tissues
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