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（由申请方提供）：达尔马提亚犬在犬中是独特的，因为它分泌尿酸而不是尿囊素，后者是嘌呤代谢的正常副产物。因此，达尔马提亚是人类高尿酸尿和高尿酸血症（即，分别在尿液和血液中尿酸水平升高）。组织移植实验表明，缺陷是内在的肝脏，符合先天性错误嘌呤代谢。虽然缺陷是固定在达尔马提亚人口，一个多代指针x达尔马提亚回交家庭分离一个单一的基因座负责常染色体隐性遗传病。这项拟议中的研究将确定达尔马提亚品种中导致高尿酸尿/高尿酸血症（huu）的基因。已经进行了基因组扫描，其证明huu与CFA 3的连锁，LOD评分为6.5。进一步的精细结构映射使用来自犬基因组序列的微卫星定位huu到两侧重组断点的3.3 Mb间隔。根据最近获得的犬基因组序列，在这个区间内有24个候选基因。有趣的是，这些基因中没有一个先前在嘌呤代谢或尿酸盐转运中鉴定出功能，证实了这项工作将产生对高尿酸尿的新见解。一个三层的方法将被用来确定突变负责高尿酸血症的达尔马提亚。首先，从犬基因组序列中获得的SNP将在达尔马提亚品种的连锁不平衡进行评估。LD区域可能排除了其他候选基因，并建立了我们对潜在致病等位基因的预期。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，使用北方印迹比较斑点狗和非斑点狗之间的表达水平，以鉴定表达的任何定性或定量差异。基于三种方法的结果，我们将开始对剩余候选者的cDNA进行测序，并进行Southern印迹分析以鉴定任何大的重排。将通过验证斑点狗品种的纯合性、其他犬品种的纯合性缺乏以及突变的适当功能研究来评价推定的致突变等位基因。在达尔马提亚狗中鉴定导致高水平尿酸的基因和突变将进一步将其建立为人类状况的模型系统。尿酸水平是许多人类疾病的促成因素，包括痛风、肾结石、心血管和肾脏疾病。新基因功能的阐明将为人类疾病的治疗提供新的研究途径。