GENETIC DEFECT OF COBALAMIN ABSORPTION IN DOGS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。自从我们对巨型雪纳瑞的钴胺素吸收不良引起的甲基丙二酸尿症进行了初步研究以来，我们已经在边境牧羊犬、小猎犬、金毛猎犬、微型雪纳瑞以及最近的一只猫和一个澳大利亚牧羊犬家庭中发现了类似的缺陷。这些动物表现出不同的临床体征，从进行性发育不良到间歇性酮症酸中毒和高氨血症性腹泻样发作。除蛋白尿外，父母给予钴胺素完全逆转了临床和代谢变化。研究正在进行中，以比较巨型雪纳瑞与其他品种的狗和猫的缺陷。这些动物是Imerslund-Gr <$sbeck综合征（I-GS）的模型，正在与密歇根州立大学副教授John Fyfe DVM博士合作进行进一步研究。 Cubilin是肠道内内源性因子钴胺素的受体，研究表明Cubilin蛋白在患病犬的肠道和肾脏中异常表达，因此Cubilin是犬选择性钴胺素吸收不良的主要生物学候选基因。两年前，我们曾报道过cubilin已被连锁分析排除在进一步考虑之外。我们继续通过合作努力来检查这种疾病背后的缺陷。与John Fyfe博士一起，我们启动了一项基因组扫描，以确定该疾病与DNA标记的联系，这些标记提供了比较位置的候选基因。转介中心还提供了用于异交交配的动物，其将更大的标记异质性插入I-GS连锁家族。基因组扫描在该过程的早期结出果实，与狗8号染色体（CFA 8）上的标记高度显著连锁。该区域与人类染色体14 q的一个区域高度同源。为了补充该区域犬基因组的可用标记，对该区域的犬基因版本进行扩增和测序，以鉴定I-GS家族中提供信息的多态性。使用这些新的标记，犬I-GS基因座被确定为在CFA 8的端粒附近的5 Mb区域中，并且与KNS 2基因无重组地连锁。这种高分辨率的连锁分析是可能的，因为来自巨型雪纳瑞的I-GS家族现在相当大。虽然数量少得多，但我们已经从一个表现出I-GS的澳大利亚牧羊犬家庭中收集了DNA样本。他们的疾病也与cubilin基因座无关，并且在可能的解决方案范围内，考虑到家庭规模与巨型雪纳瑞I-GS的CFA 8相同区域有关。这些研究的结果已提交出版。 在人类14 q的这一区域中寻找到了一个无标记基因（AMN）。AMN是一种功能未知的基因，其在肾脏和肠中高度且几乎完全表达，这两种组织中表达cubilin并且在犬I-GS中发生功能障碍。我们克隆了犬AMN cDNA，并在巨型雪纳瑞犬和澳大利亚牧羊犬家族中搜索受影响的犬AMN cDNA突变。在巨型雪纳瑞家系中发现AMN基因第10外显子有33 bp的缺失，但在澳大利亚牧羊犬中尚未发现突变。为了进一步研究澳大利亚牧羊犬疾病，进行了额外的交配以扩大连锁家族并提供受影响的犬组织用于分析。就在最近，AMN突变导致I-GS在一些人类激酶已被证明。进一步的研究，只可能在犬I-GS模型将追求，以确定AMN的功能和AMN缺陷如何废除cubilin的表达。这些研究将在授予Fyfe博士的NIH资助下进行（#DK 064161，极化Cubilin表达的分子机制，2003年4月1日-2005年3月31日）。迄今为止的结果表明，cubilin和AMN作为异源二聚体多配体受体复合物的强结合亚基协作，该复合物在许多组织中具有重要功能

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