GENETIC DEFECT OF COBALAMIN ABSORPTION IN DOGS

狗体内钴胺素吸收的遗传缺陷

• 批准号: 7391953
• 负责人: JOHN C FYFE
• 金额: $ 0.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391953
• 关键词: GENETIC; DEFECT; COBALAMIN; ABSORPTION; DOGS

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

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。自从我们对巨型雪纳瑞因钴胺素吸收不良引起的甲基丙二酸尿症进行初步研究以来，我们在边境牧羊犬、比格犬、金毛猎犬、小型雪纳瑞以及最近的一只猫和一个澳大利亚牧羊犬家族中发现了类似的缺陷。这些动物表现出不同的临床症状，从进行性生长障碍到间歇性酮症酸中毒和高氨血症癫痫样发作。肠外给予钴胺素可完全逆转除蛋白尿之外的临床和代谢变化。目前正在进行研究，将巨型雪纳瑞的缺陷与其他品种的狗和猫进行比较。这些动物是伊默斯伦德-格贝克综合征 (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 是肠道中内因子钴胺素的受体，是犬选择性钴胺素吸收不良的主要生物学候选基因，因为研究表明，cubilin 蛋白在受影响的狗的肠道和肾脏中异常表达。两年前，我们报告说，cubilin 已通过连锁分析从进一步考虑中排除。我们继续通过合作努力检查这种疾病背后的缺陷。我们与 John Fyfe 博士一起启动了基因组扫描，以寻找该疾病与 DNA 标记的联系，这些标记提供了相对位置候选基因。转介中心还提供动物进行异型交配，将更大的标记异质性插入到 I-GS 连锁家族中。基因组扫描在此过程的早期就取得了成果，与狗 8 号染色体 (CFA 8) 上的标记具有高度显着的连锁性。该区域与人类染色体 14q 的一个区域高度同源。为了补充该区域的犬基因组的可用标记，对该区域的犬基因进行了扩增和测序，以鉴定在 I-GS 家族中提供信息的多态性。使用这些新标记，确定犬 I-GS 基因座位于 CFA 8 端粒附近的 5 Mb 区域，并且与 KNS2 基因无重组连接。这种高分辨率的连锁分析是可能的，因为源自巨型雪纳瑞的 I-GS 家族现在相当大。尽管数量较少，但我们还是从一个表现出 I-GS 的澳大利亚牧羊人家庭中收集了 DNA 样本。它们的疾病也与 cubilin 基因座无关，并且考虑到家族规模与巨型雪纳瑞 I-GS 与 CFA 8 的同一区域有关，因此在可能的分辨率范围内。这些研究的结果已提交发表。 对人类 14q 这个区域的注释基因的搜索显示无羊膜 (AMN)。 AMN 是一种功能未知的基因，在肾和肠中高度且几乎完全表达，这两个组织表达 cubilin，但在犬 I-GS 中出现功能障碍。我们克隆了犬 AMN cDNA，并在巨型雪纳瑞和澳大利亚牧羊犬家族中搜索了受影响的狗 AMN cDNA 中的突变。在巨型雪纳瑞家族中发现了 AMN 外显子 10 的 33 bp 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狗组织进行分析。就在最近，在一些人类家族中引起 I-GS 的 AMN 突变已被证实。为了确定 AMN 的功能以及 AMN 缺陷如何消除 cubilin 表达，将进行只能在犬 I-GS 模型中进行的进一步研究。这些研究将在 NIH 授予 Fyfe 博士的资助下进行（#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）。迄今为止的结果表明，cubilin 和 AMN 作为异二聚体、多配体受体复合物的强结合亚基进行协作，该复合物在许多组织中具有重要功能