Cloning of a type 2 diabetes modifier in obese mice

描述（由申请人提供）：2型糖尿病（T2DM）影响了超过5%的美国人口，造成了巨大的痛苦，每年直接医疗费用超过1000亿美元。在人类和啮齿动物中，肥胖背景下对2型糖尿病的易感性受到尚未确定的基因的强烈影响。在人类中识别这些易感基因的固有复杂性促使我们在遗传肥胖小鼠中使用双等位基因系统来识别候选基因和2型糖尿病的途径，然后在人类中进行测试。在B6/DBA小鼠Lep-ob杂交分离的F2后代中，我们将T2DM位点定位到小鼠Chr1的一个区域（p<10[-8]），与人类lq23一致。该小鼠区间位于人类lq23区间的中间，约为人类lq23区间的1/10，在人类连锁研究中多次被确定为含有糖尿病易感基因。该杂交的肥胖F2后代的糖尿病内表型在遗传上肥胖的B6成员中维持。DBA N12基因系在86年从小鼠区分离出约5mb的DBA DNA。基因动物的相关表型是：低胰岛素血症高血糖症、HbA1c升高和发育不良/营养不良的β细胞。我们建议使用我们开发的信息学和分子生物学技术，系统地识别和分析DBA基因区间中的所有基因。步骤包括：1.)B6所有DNA序列的加入和分析。通过精细映射将进一步减小DBA的遗传间隔。2)通过计算技术鉴定所有转录本，并通过混合器官rna的表达分析证实。3) B6和DBA之间的序列比较，以确定这些转录本中所有非同义编码变体。1500 bp 5′编码序列典型启动子元件的鉴定与序列比较。4)编码和非编码变异对二级蛋白结构/功能和转录影响的计算评估。5.)间隔DBA剂量对基因动物体内/体外β细胞功能和骨骼肌胰岛素反应的影响。6.)在体外体内研究的基础上，利用定量PCR对选定器官中选定转录本进行定量基因表达分析（B6 v. DBA）。7)候选编码和调控序列变异的体外功能分析。8.)对于最引人注目的序列变异，制备DBA等位基因的转基因B6敲入蛋白，并评估后代的相关糖尿病表型。9.)在5000多名参与与1q23相关的T2DM研究的个体中，检查同源候选基因。如果成功，该项目将确定肥胖背景下糖尿病易感性的主要基因（可能是一种新的途径）。该基因可用于预测诊断和预防，以及治疗药物的设计。

DESCRIPTION (provided by applicant): Type 2 diabetes mellitus (T2DM) affects over 5% of the US population, causing tremendous suffering with annual direct medical costs of over $100 billion. In both humans and rodents, susceptibility to T2DM in the context of obesity is powerfully influenced by genes that have not been identified. The inherent complexity of identifying such susceptibility genes in humans led us to use a biallelic system in enetically obese mice to identify candidate genes and pathways for T2DM that can then be tested in humans. In F2 progeny of a B6/DBA murine cross segregating for Lep-ob, we mapped a T2DM locus to a region of murine Chr1 (p<10[-8]), syntenic to human lq23. This mouse interval lies in the middle of, and is about 1/10th the physical size of, the human interval at lq23 repeatedly identified as containing diabetes susceptibility genes in human linkage studies. The diabetic endophenotypes of the obese F2 progeny of this cross are maintained in the genetically obese members of a B6.DBA N12 congenic line segregating about 5 Mb of DBA DNA from the mouse region 1at 86. The associated phenotypes in the congenic animals are: hypoinsulinemic hyperglycemia, elevated HbA1c, and hypoplastic/hypotrophic beta cells. We propose to systematically identify and analyze all genes in the DBA congenic interval, using informatics and molecular biological techniques that we have developed. The steps will include: 1.) accession and analysis of all DNA sequence for the B6.DBA congenic interval that will be further reduced by fine mapping. 2.) identification of all transcripts by computational techniques, confirmed by expression analysis in pooled organ RNAs. 3.) sequence comparisons between B6 and DBA to identify all non-synonymous coding variants in these transcripts. Identification and sequence comparison of canonical promoter elements in 1500 bp 5' of coding sequences. 4.) computational assessment of effects of coding and non-coding variants on secondary protein structure/function and transcription. 5.) analysis of in vivo/in vitro beta cell function and skeletal muscle insulin response at 30 and 60 days in congenic animals by interval DBA dose. 6.) based upon in vitro in vivo studies, quantitative gene expression analysis (B6 v. DBA) of selected transcripts in selected organs using quantitative PCR. 7.) in vitro analysis of the functional consequences of candidate coding and regulatory sequence variants. 8.) for the most compelling sequence variants, preparation of transgenic B6 knock-ins of the DBA alleles and assessment of the relevant diabetes phenotypes in the progeny. 9.) in over 5000 individuals who have participated in studies of T2DM linked to 1q23, examine orthologous candidate genes. If successful, this project could identify a major gene (and possibly a novel pathway) for diabetes susceptibility in the context of obesity. Such a gene could be used for anticipatory diagnosis and prevention, and the design of therapeutic agents.