CLONING OF THE Hol MUTATION

Hol 突变的克隆

• 批准号: 7470947
• 负责人: Licia Selleri
• 金额: $ 25.2万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470947
• 关键词: Affect; Alleles; Animals; Applications Grants; Biological Models; Breeding; Candidate Disease Gene; Cardiac; Cartilage; Chondrocranium; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 16; Cleaved cell; Cleft Palate; Cloning; Congenital Disorders; Defect; Depth; Development; Developmental Process; DiGeorge Syndrome; Ear; Ear ossicles; Embryo; Embryonic Development; Essential Genes; Ethylnitrosourea; Exhibits; External Ear; Future; Gene Expression; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic Screening; Genomics; Goals; Head; Human; Institutes; Knowledge; Laboratories; Labyrinth; Lateral; Lead; Left; Lesion; Mandible; Maps; Microarray Analysis; Modeling; Molecular; Molecular Genetics; Morphogenesis; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Nature; Neck; Numbers; Palate; Pathogenesis; Pathway interactions; Pattern; Phenocopy; Phenotype; Planet Mars; Polymorphic Microsatellite Marker; Primordium; Proteins; Public Health; Recombinants; Regulation; Research Personnel; Resolution; Single Nucleotide Polymorphism; Skeletal system; Structure; Technology; Temporal bone structure; Work; base; body system; bone; capsule; craniofacial; cranium; developmental genetics; discount; external ear auricle; gene complementation; genetic linkage analysis; genome wide association study; insight; interest; loss of function; malformation; mandible/maxilla; middle ear; mutant; novel

DESCRIPTION (provided by applicant): Our laboratory uses genetic and developmental approaches, exploiting the mouse as a model system, to study skeletal patterning and morphogenesis during development. To this end, we are performing a phenotype- based forward genetic screen by ethylnitrosourea (ENU) mutagenesis in the mouse to uncover novel genes important in mammalian craniofacial development. Within the past two years, the screen produced four mutant mouse lines with skeletal defects. Two of these lines exhibit remarkable craniofacial malformations. Specifically, mutants from line 04/014 develop a short mandible, a hypoplastic or absent hyoid, and rudimentary neck cartilages. Also, their palate is cleft and the basisphenoid is fused to the basioccipital bone. Additionally, 04/014 mutants exhibit low-set and hypoplastic ear pinnae and their middle ear ossicles are severely affected. Finally, the cartilage primordium of the petrous part of the temporal bone is absent, leaving a hollow space (therefore this line will be defined hereafter as "Hollow ear", or Hol), and the Otic Capsule (part of the lateral chondrocranium, containing the developing inner ear apparatus) is hypoplastic and dysmorphic. Overall, the Hol craniofacial mutation phenocopies the Tbx1 homozygous mutation in the mouse, which models DiGeorge Syndrome (DGS). We propose that the Hol mutation disrupts a gene essential for vertebrate craniofacial and ear development. Indeed, by exploiting high-resolution genetic mapping, we discounted that the Hol mutation maps to either the Tbx1 or Crkl loci on chromosome 16. Furthermore, we mapped the Hol mutant gene to an interval of approximately 8 Mb on mouse chromosome 11, by exploiting novel mapping technology based on whole genome scanning using single nucleotide polymorphisms (SNP) panels. Finally, since the last amended submission of this proposal, we have further narrowed the Hol-bearing interval to approximately 3.9 Mb, by high-resolution mapping and polymorphic markers. We hypothesize that the Hol gene acts in the Tbx1 path- way in craniofacial development, but Hol is not Tbx1 or Crkl. We plan to uncover the molecular basis of this mutant phenotype through the following specific aims: 1) Identify Hol candidate genes, by performing further high-resolution mapping and microarray analysis for "gene finding"; and 2) Identify the Hol gene, by conducting analysis of candidate genes and identification of the Hol molecular lesion by sequencing of candidate genes. Completion of these studies will advance our understanding of the genetic regulation of craniofacial patterning and morphogenesis, as well as lead to the discovery of a new gene that likely acts in the Tbx1 pathway. Under a broader perspective, this work will have an impact on the pathogenesis of human congenital disorders that affect craniofacial and ear development and function, such as DiGeorge Syndrome (DGS). PUBLIC HEALTH RELEVANCE: Knowledge of the patterning and morphogenesis of craniofacial and ear structures and of the genes implicated in their developmental processes is still elementary. Completion of the studies proposed here will represent a step forward in our understanding of the genetic regulation of patterning and morphogenesis of the vertebrate cranium. Furthermore, this work will uncover a novel gene that likely acts in a genetic pathway together with Tbx1 and Crkl and is required for critical craniofacial developmental processes. Under a broader perspective, these studies will have an impact on our knowledge of the pathogenesis of human congenital disorders that affect normal craniofacial and ear development and function, in particular with regard to the malformations of DiGeorge Syndrome.

描述(申请人提供)：我们的实验室使用遗传学和发育方法，以小鼠为模型系统，研究发育过程中的骨骼模式和形态发生。为此，我们正在通过乙基亚硝胺(ENU)突变在小鼠身上进行基于表型的正向遗传筛选，以发现在哺乳动物颅面发育中重要的新基因。在过去的两年里，该筛查培育出了四个骨骼缺陷的突变小鼠品系。其中两条线显示出明显的颅面畸形。具体地说，来自品系04/014的突变体发育出短小的下颌骨、发育不良或缺失的舌骨和发育不全的颈部软骨。此外，他们的腭裂，基蝶骨与基枕骨融合。此外，04/014突变体表现出耳廓低位和发育不良，其中耳听小骨受到严重影响。最后，颞骨岩部的软骨原基缺失，留下一个中空的空间(因此这条线在下文中将被定义为“中空耳”，或Hol)，Otic囊(外侧软骨颅骨的一部分，包含发育中的内耳器官)发育不全和畸形。总体而言，Hol颅面突变在小鼠中复制了TBX1纯合子突变，这是DiGeorge综合征(DGS)的模型。我们认为Hol突变破坏了脊椎动物颅面和耳朵发育所必需的基因。事实上，通过利用高分辨率的遗传作图，我们排除了Hol突变映射到16号染色体上的TBX1或Crk1基因座。此外，我们利用基于单核苷酸多态(SNP)全基因组扫描的新作图技术，将Hol突变基因定位到小鼠11号染色体上大约8Mb的间隔。最后，自上一次修改本提案以来，通过高分辨率作图和多态标记，我们进一步缩小了Hol的携带间隔，约为3.9Mb。我们推测Hol基因在头面部发育过程中起着Tbx1途径的作用，但Hol不是Tbx1或Crk1。我们计划通过以下具体目标来揭示该突变表型的分子基础：1)通过进行进一步的高分辨率定位和微阵列分析来寻找Hol候选基因；以及2)通过对候选基因的分析和通过对候选基因的测序来识别Hol分子损伤来识别Hol基因。这些研究的完成将促进我们对颅面纹饰和形态发生的遗传调控的理解，并导致一个可能作用于TBX1途径的新基因的发现。在更广泛的视角下，这项工作将对影响颅面和耳朵发育和功能的人类先天性疾病的发病机制产生影响，如DiGeorge综合征(DGS)。与公共卫生相关：关于颅面和耳朵结构的构型和形态发生以及与其发育过程有关的基因的知识仍然是基本的。这里提出的研究的完成将代表着我们在理解脊椎动物头骨的图案化和形态发生的遗传调节方面向前迈进了一步。此外，这项工作将发现一个新的基因，它可能与TBX1和Crk1一起在遗传途径中发挥作用，并且是关键的颅面发育过程所必需的。从更广泛的角度来看，这些研究将影响我们对影响正常颅面和耳朵发育和功能的人类先天性疾病的发病机制的认识，特别是关于DiGeorge综合征的畸形。