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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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而导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、比格猎犬、金毛猎犬、微型雪纳瑞中发现了类似的缺陷，最近在一只猫和一个澳大利亚牧羊犬家庭中也发现了类似的缺陷。这些动物表现出不同的临床症状，从进行性生长失败到间歇性酮症酸中毒和高氨血症发作样发作。父母给予钴胺素完全逆转临床和代谢变化，除了蛋白尿。目前正在进行研究，将巨型雪纳瑞犬的缺陷与其他品种的狗和猫进行比较。这些动物是Imerslund-Gr¿sbeck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蛋白在患病犬的肠道和肾脏中异常表达。两年前，我们报道了cubilin，在进一步的考虑中被连锁分析淘汰了。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，我们发起了一项基因组扫描，以寻找该疾病与DNA标记的联系，这些标记提供了比较位置候选基因。转诊中心还为异交交配提供了动物，这些异交交配在I-GS连锁家族中插入了更大的标记异质性。基因组扫描在这一过程的早期就发现了与狗8号染色体上的一个标记高度显著的连锁（CFA 8）。该区域与人类14q染色体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，我们对该区域的犬基因进行了扩增和测序，以确定在I-GS家族中具有信息意义的多态性。利用这些新的标记，确定犬I-GS位点位于CFA 8端粒附近的5mb区域，与KNS2基因无重组关联。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，但我们从一个澳大利亚牧羊犬家庭中收集了DNA样本，显示出I-GS。他们的疾病也与立方体位点无关，并且在可能的分辨率内，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发表。通过对人类14q区域的注释基因的搜索，发现了羊膜缺失（AMN）。AMN是一种功能未知的基因，在肾脏和肠道中高度表达，几乎完全表达，这两个组织表达cubilin，在犬I-GS中功能失调。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寻找受影响的犬AMN cDNA突变。在巨型雪纳瑞家族的AMN外显子10上发现了一个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，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进行分析。就在最近，AMN突变在一些人类中引起了I-GS。为了确定AMN的功能以及AMN缺陷是如何抑制cubilin表达的，我们将继续进行只可能在犬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- 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在许多组织中具有重要功能