Modulation of NOD Strain Diabetes by ENU-Induced Mutations

ENU 诱导突变对 NOD 菌株糖尿病的调节

• 批准号: 10642549
• 负责人: BRUCE A BEUTLER
• 金额: $ 221.49万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-13 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642549
• 关键词: 18 year old; Acceleration; Affect; Alleles; Animals; Apoptotic; Archives; Autoantigens; Autoimmune Diabetes; Autoimmune Diseases; B-Lymphocytes; Beta Cell; Biological Assay; Blood; Breeding; CRISPR/Cas technology; CTLA4 gene; Cell Count; Cells; Child; Childhood; Code; Collaborations; Complex; Cryopreservation; DNA; DNase-I Footprinting; Data; Databases; Date of birth; Development; Diabetes Mellitus; Disease; Disease remission; Documentation; Epitopes; Ethylnitrosourea; Etiology; Exhibits; Expenditure; Female; Frequencies; Funding; Genes; Genetic; Genetic Diseases; Genome; Genomic DNA; Genotype; Guidelines; Hematopoietic stem cells; Human; Immune; Immune system; Immunologics; Inbred NOD Mice; Inbreeding; Individual; Induced Mutation; Influentials; Insulin-Dependent Diabetes Mellitus; Intellectual Property; Ions; Islet Cell; Islets of Langerhans; Knock-out; Laboratories; Light; Maps; Measures; Medical; Meiosis; Metabolic; Methodology; Methods; Missense Mutation; Modification; Molecular; Monitor; Mus; Mutagenesis; Mutagens; Mutant Strains Mice; Mutation; N-terminal; Non obese; North America; Nucleotides; Orthologous Gene; Paris, France; Pathogenesis; Pathogenicity; Patients; Penetrance; Peptides; Persons; Pharmaceutical Preparations; Phenotype; Pilot Projects; Point Mutation; Predisposition; Progress Reports; Protein Overexpression; Proteins; Proteomics; Publications; RNA Splicing; Recovery; Regulatory T-Lymphocyte; Research Design; Resolution; Resource Sharing; Sampling; Signal Transduction; Site; Splice-Site Mutation; Stress; Sum; T cell receptor repertoire sequencing; T cell response; T-Lymphocyte; T-cell receptor repertoire; Technology; Testing; Therapeutic; Thymus Gland; Tissues; Tumor Necrosis Factor Receptor; United States National Institutes of Health; Untranslated RNA; Work; Writing; animation; autoreactivity; cell type; conditional knockout; data dissemination; diabetes pathogenesis; diabetic; epigenetic regulation; exome sequencing; forward genetics; genetic pedigree; insight; insulin sensitivity; insulitis; male; meetings; member; molecular sequence database; mouse genome; mutant; novel; phosphoproteomics; prevent; protein protein interaction; response; screening; sperm cryopreservation; success; technology platform; transcriptome; transcriptomics

PROJECT SUMMARY/ABSTRACT This P01 proposal offers a fresh approach to understanding the genetics of an extremely important polygenic autoimmune disease: type 1 diabetes (T1D), which affects nearly three children out of every thousand in North America, and many more around the world. T1D occurs with variable penetrance in Non-Obese Diabetic (NOD) mice, which exhibit a phenotype remarkably similar to that of human patients. Both environmental and genetic factors determine penetrance, but most of the influential mutations and the genes they affect remain unknown. We have developed a powerful technology platform that permits instantaneous identification of point mutations that cause phenotype. Using this platform, we have already identified two spontaneous mutations that cause high and low frequency of disease development in the NOD/NckH and NOD/NckL sublines, respectively. Noting that these sublines, isolated by selective breeding over a period of only seven years, had approximately the same mutational distance from one another as one finds in a pedigree of ENU mutagenized mice as compared to the parental reference strain, we performed a pilot study in which mice were mutagenized on the NOD/NckH background. In a sample of 14 pedigrees, we unambiguously identified twelve ENU-induced mutations with modifying effects on T1D: some accelerating the disease and others suppressing it. Stressing the precision of these studies, which do not merely identify intervals or candidates, but resolve the exact nucleotide change responsible for T1D modification, we propose to expand our efforts, analyzing 21,000 coding/splicing mutations for modifier effects over a period of five years. Our preliminary work suggests that T1D is “balanced on a knife’s edge” from a genetic point of view. Mutations in many genes are clearly capable of influencing T1D development, since randomly induced coding/splicing mutations affecting approximately 1% of the mouse genome caused unambiguous modifier phenotypes. We expect to identify scores if not hundreds of individual modifier mutations during the period of funding. Some of these will have important new facts to tell us about what it takes to develop T1D. Concentrating on those modifier mutations that show large effect sizes, may be amenable to targeting with therapeutic drugs, and/or are particularly surprising in light of what we presently know about T1D pathogenesis, we will rigorously verify causation by re-creating the mutations and/or deleting the causative genes on clean backgrounds (NOD/NckH or NOD/NckL devoid of ENU-induced mutations). We will then systematically examine the mechanism of phenotype modification, both at the level of cellular immuno-pathogenesis, and at the level of molecular pathogenesis. Ultimately, we hope to understand how T1D can be prevented or driven into remission, and we expect many new insights to emerge from the studies planned. A close collaboration between the Bach/Chatenoud group, with its great expertise in the study of T1D pathogenesis in NOD mice, and the Beutler group, with its strength in forward genetics, will contribute to the success of this P01. The Core laboratories and Projects are exceptionally synergistic, assuring that this P01 will dramatically exceed the sum of its parts.

项目摘要/摘要 这一P01建议为理解一种极其重要的多基因的遗传学提供了一种新的途径 自身免疫性疾病：1型糖尿病(T1D)，在北方每1000名儿童中就有近3名儿童受到影响 美国，以及世界上更多的国家。非肥胖型糖尿病(NOD)患者发生T1D外显率不同 小鼠，表现出与人类患者非常相似的表型。无论是环境还是遗传 外显性决定因素，但大多数有影响的突变及其影响的基因仍不清楚。 我们已经开发了一个强大的技术平台，可以即时识别点突变 这会导致表型。利用这一平台，我们已经确定了两种自发突变，它们会导致 NOD/Nck H和NOD/Nck L亚系发生疾病的频率分别较高和较低。备注 这些亚系，通过仅七年的选择性育种而分离出来，大约有 与在ENU致突变小鼠家系中发现的相同的突变距离比较 对于亲本参考菌株，我们进行了一项初步研究，在NOD/Nck H上对小鼠进行了诱变 背景资料。在一个14个家系的样本中，我们明确地识别了12个ENU诱导的突变 对T1D的调节作用：有的加速疾病，有的抑制疾病。强调产品的精确性 这些研究不仅确定了间隔或候选，而且解决了准确的核苷酸变化 负责T1D修饰的我们建议进一步努力，分析21,000个编码/剪接突变 用于五年内的修改器效果。我们的初步工作表明，T1D在刀子上是平衡的 从遗传学的角度来看，“边缘”。许多基因的突变显然能够影响T1D的发育， 由于随机诱导的影响大约1%的小鼠基因组的编码/剪接突变导致 明确的修饰语表型。我们希望确定分数，如果不是数百个单独的修饰突变 在资助期间。其中一些将有重要的新事实告诉我们需要什么才能发展 T1D。集中在那些显示出大效应大小的修饰物突变上，可能会服从于 治疗药物，和/或特别令人惊讶的是，鉴于我们目前对T1D发病机制的了解， 我们将通过在CLEAN上重新创建突变和/或删除致病基因来严格验证因果关系 背景(无ENU诱导突变的NOD/Nck H或NOD/Nck L)。然后我们将系统地研究 表型修饰的机制，既在细胞免疫发病水平上，也在细胞免疫水平上 分子发病机制。最终，我们希望了解如何预防或缓解T1D， 我们预计，计划中的研究将产生许多新的见解。两国之间的密切合作 Bach/Chatenoud小组，其在NOD小鼠T1D发病机制研究方面的专业知识，以及Beutler 集团凭借其在正向遗传学方面的实力，将为本次P01的成功做出贡献。核心实验室和 项目具有特别的协同性，确保P01将大大超过其各部分的总和。