The molecular basis for an animal model of inherited hyperuricosuria

遗传性高尿酸尿动物模型的分子基础

• 批准号: 7084992
• 负责人: DANIKA L BANNASCH
• 金额: $ 18.18万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084992
• 关键词: animal genetic material tag; autosomal recessive trait; complementary DNA; dogs; functional /structural genomics; gene expression; gene mutation; genetic models; hyperuricemia; linkage disequilibriums; liver metabolism; northern blottings; nucleic acid sequence; polymerase chain reaction; purine /pyrimidine metabolism; purine /pyrimidine metabolism disorder; single nucleotide polymorphism; southern blotting; urate

DESCRIPTION (provided by applicant): The Dalmatian is unique among dogs in that it excretes uric acid rather than allantoin, the normal byproduct of purine metabolism. Thus, the Dalmatian is a natural model for the human conditions hyperuricosuria and hyperuricemia (i.e., elevated levels of uric acid in the urine and blood, respectively). Tissue transplantation experiments have demonstrated that the defect is intrinsic to the liver, consistent with an inborn error in purine metabolism. Although the defect is fixed within the Dalmatian population, a multigenerational Pointer x Dalmatian backcross family segregates a single locus responsible for the autosomal recessive disorder. The proposed research will identify the gene responsible for hyperuricosuria/hyperuricemia (huu) in the Dalmatian breed. A genome scan has been performed which demonstrated linkage of huu to CFA 3 with a LOD score of 6.5. Further fine structure mapping using microsatellites derived from the canine genome sequence localized huu to a 3.3 Mb interval flanked by recombination breakpoints. Based on the recent availability of the canine genome sequence, there are twenty four candidate genes in this interval. Interestingly, none of these genes have previously identified functions in purine metabolism or urate transportation, confirming that this work will yield novel insights into hyperuricosuria. A three tiered approach will be taken to identify the mutation responsible for hyperuricosuria in the Dalmatian. First, SNPs obtained from the canine genome sequence will be evaluated for linkage disequilibrium in the Dalmatian breed. The area of LD may exclude additional candidate genes as well as establish our expectations for potential disease-causing alleles. Second, candidate genes from the critical interval that are expressed in the canine liver will be determined using RT-PCR. At the same time, expression levels will be compared between Dalmatians and non-Dalmatians using Northern Blots to identify any qualitative or quantitative differences in expression. Based on the results of the three approaches, we will begin sequencing cDNA from the remaining candidates as well as performing Southern blot analysis to identify any large rearrangements. Putative mutation-causing alleles will be evaluated by verification of homozygosity in the Dalmatian breed, lack of homozygosity in other dog breeds, as well as appropriate functional studies of the mutation. Identification of the gene and mutation that cause high levels of uric acid in the Dalmatian dog will further establish it as a model system for the human condition. Uric acid levels are a contributing factor for many human diseases including gout, kidney stones, cardiovascular and renal disease. The elucidation of a novel gene function should provide new avenues of investigation for the treatment of the human condition.

描述（由申请人提供）：达尔马提亚犬在狗中是独一无二的，因为它排泄尿酸而不是尿囊素，尿囊素是嘌呤代谢的正常副产物。因此，达尔马提亚犬是人类高尿酸血症和高尿酸血症（即尿液和血液中尿酸水平分别升高）的自然模型。组织移植实验表明，这种缺陷是肝脏固有的，与先天的嘌呤代谢错误相一致。虽然这种缺陷在达尔马提亚种群中是固定的，但一个多代的指针x达尔马提亚回交家族分离出一个负责常染色体隐性遗传病的单一位点。提出的研究将确定基因负责高尿酸尿/高尿酸血症（huu）在达尔马提亚品种。基因组扫描显示huu与CFA 3的关联，LOD评分为6.5。利用微卫星对犬基因组序列进行精细结构定位，将huu定位在3.3 Mb的区间内，两侧是重组断点。根据最近可获得的犬基因组序列，在这个区间有24个候选基因。有趣的是，这些基因之前都没有发现嘌呤代谢或尿酸转运的功能，这证实了这项工作将为高尿酸症提供新的见解。一个三层的方法将采取确定突变负责高尿酸尿在达尔马提亚。首先，从犬基因组序列中获得的snp将被评估为达尔马提亚品种的连锁不平衡。LD区域可以排除其他候选基因，并建立我们对潜在致病等位基因的期望。其次，将使用RT-PCR确定在犬肝脏中表达的临界区间的候选基因。同时，使用Northern Blots比较斑点狗和非斑点狗之间的表达水平，以确定表达的任何定性或定量差异。基于这三种方法的结果，我们将开始对剩余候选基因进行cDNA测序，并进行Southern blot分析以确定任何大的重排。将通过验证达尔马提亚犬种的纯合性、其他犬种的缺乏纯合性以及对突变进行适当的功能研究来评估可能引起突变的等位基因。确定导致达尔马提亚犬尿酸水平高的基因和突变将进一步建立它作为人类状况的模型系统。尿酸水平是许多人类疾病的一个促成因素，包括痛风、肾结石、心血管和肾脏疾病。新的基因功能的阐明将为人类疾病的治疗提供新的研究途径。