CLONING OF THE Hol MUTATION

Hol 突变的克隆

• 批准号: 7932518
• 负责人: Licia Selleri
• 金额: $ 11.08万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932518
• 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; Development; Developmental Process; DiGeorge Syndrome; Ear; Ear ossicles; Embryo; Embryonic Development; Ethylnitrosourea; Exhibits; 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; Palate; Pathogenesis; Pathway interactions; Pattern; Phenocopy; Phenotype; Planet Mars; Polymorphic Microsatellite Marker; Primordium; Proteins; Recombinants; Regulation; Research Personnel; Resolution; Single Nucleotide Polymorphism; 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; public health relevance; skeletal

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（由申请人提供）：我们的实验室使用遗传和发育方法，利用小鼠作为模型系统，研究发育过程中的骨骼模式和形态发生。为此，我们正在小鼠中通过乙基亚硝基脲（ENU）诱变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，筛选产生了四种具有骨骼缺陷的突变小鼠品系。其中两条线表现出明显的颅面畸形。具体来说，来自04/014系的突变体发育出短下颌骨、发育不全或缺失的舌骨以及未发育成熟的颈部软骨。此外，他们的上颚裂开，基蝶骨与基枕骨融合。此外，04/014突变体表现出低位和发育不全的耳廓，并且中耳小骨受到严重影响。最后，颞骨岩部的软骨原基缺失，留下一个空腔（因此这条线在下文中将被定义为“空心耳”，或 Hol），并且耳囊（侧软骨颅骨的一部分，包含正在发育的内耳器官）发育不良且畸形。总体而言，Hol 颅面突变表型复制了小鼠中的 Tbx1 纯合突变，该突变模拟了迪乔治综合症 (DGS)。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，通过利用高分辨率遗传图谱，我们忽略了 Hol 突变映射到 16 号染色体上的 Tbx1 或 Crkl 位点。此外，我们通过利用基于使用单核苷酸多态性 (SNP) 面板的全基因组扫描的新型作图技术，将 Hol 突变基因映射到小鼠 11 号染色体上大约 8 Mb 的间隔。最后，自该提案上次修改提交以来，我们通过高分辨率作图和多态性标记，进一步将 Hol 轴承区间缩小到约 3.9 Mb。我们假设 Hol 基因在颅面发育中的 Tbx1 途径中发挥作用，但 Hol 不是 Tbx1 或 Crkl。我们计划通过以下具体目标来揭示这种突变表型的分子基础：1）通过进一步进行高分辨率作图和微阵列分析来“寻找基因”，识别Hol候选基因； 2)通过对候选基因进行分析来鉴定Hol基因，并通过对候选基因进行测序来鉴定Hol分子病变。这些研究的完成将增进我们对颅面模式和形态发生的遗传调控的理解，并导致发现可能在 Tbx1 通路中起作用的新基因。从更广泛的角度来看，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，例如迪乔治综合症（DGS）。公共卫生相关性：对颅面和耳朵结构的模式和形态发生以及与其发育过程有关的基因的了解仍然很基础。这里提出的研究的完成将代表我们对脊椎动物颅骨模式和形态发生的遗传调控的理解向前迈进了一步。此外，这项工作将揭示一种新基因，该基因可能与 Tbx1 和 Crkl 一起在遗传途径中发挥作用，并且是关键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发病机制的认识，特别是关于迪乔治综合症的畸形。