GENETIC DEFECT OF COBALAMIN ABSORPTION IN DOGS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。自从我们对巨型雪纳瑞吸收钴胺不良所致的甲基丙二酸尿症进行初步研究以来，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在一只猫和一家澳大利亚牧羊犬中发现了类似的缺陷。这些动物表现出不同的临床症状，从进行性无法生长，到间歇性酮症酸中毒和高氨性癫痫样发作。父母给予钴胺可完全逆转除蛋白尿外的临床和代谢变化。将巨型雪纳瑞与其他品种的狗和猫的缺陷进行比较的研究正在进行中。这些动物是Imerslund-Grésbeck综合征(I-GS)的模型，目前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是肠道内固有因子钴胺的受体，是犬选择性钴胺吸收不良的主要生物候选基因，因为研究表明Cubilin蛋白在患病犬的肠道和肾脏中异常表达。两年前，我们报道了Cubilin，已被连锁分析从进一步考虑中剔除。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，以寻找这种疾病与DNA标记的联系，DNA标记提供了比较的位置候选基因。参考中心还提供了将更大的标记异质性注入I-GS连锁家族的异种杂交动物。基因组扫描在这一过程的早期就结出了果实，与狗8号染色体上的一个标记(CFA 8)有非常显着的连锁。该区域与人类染色体14Q的一个区域高度同源。为了补充该区域犬类基因组的现有标记，对该区域的犬类基因进行了扩增和测序，以确定在I-GS家族中具有信息的多态。利用这些新标记，犬的I-GS基因座被确定位于CFA8端粒附近的5Mb区域，并且与KNS2基因没有重组。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，在可能的分辨率内，考虑到家庭大小与巨型雪纳瑞I-GS的CFA 8区域有关。这些研究的结果已提交出版。对人类14Q这一区域的一个著名基因的搜索发现了羊膜无性系(AMN)。AMN是一个功能未知的基因，在犬I-GS中表达Cubilin的两个组织肾和肠中高度且几乎唯一表达。我们克隆了犬AMN基因，并搜索了巨型雪纳瑞和澳大利亚牧羊犬家族中受影响的犬AMN基因的突变。在巨型雪纳瑞家系中发现了AMN基因外显子10的33bp缺失，但在澳大利亚牧羊犬中尚未发现突变。为了进一步调查澳大利亚牧羊犬疾病，已经进行了额外的交配，以扩大连锁家族并提供受影响的狗组织用于分析。就在最近，在一些人类家族中发现了导致I-GS的AMN突变。只有在犬I-GS模型中才有可能的进一步研究将继续进行，以确定AMN的功能以及AMN缺陷如何取消cubilin的表达。这些研究将在NIH授予Fyfe博士的拨款下进行(#DK 064161，极化Cubilin表达的分子机制，2003年4月1日至2005年3月31日)。到目前为止的结果表明，Cubilin和AMN作为异二聚体、多配体受体复合体的强结合亚单位相互协作，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