CLONING OF THE Hol MUTATION

描述（由申请人提供）：我们的实验室采用遗传和发育方法，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。在过去的两年中，筛选产生了四个具有骨骼缺陷的突变小鼠系。其中两条线表现出明显的颅面畸形。具体来说，来自04/014系的突变体发育为短下颌骨，舌骨发育不全或缺失，颈部软骨发育不全。此外，他们的上颚是裂的，基底突骨与基底枕骨融合。此外，04/014突变体耳廓低、发育不全，中耳小骨受到严重影响。最后，颞骨岩部的软骨原基缺失，留下一个中空的空间（因此这条线将被定义为“空心耳”或Hol），耳囊（外侧软骨颅骨的一部分，包含发育中的内耳器官）发育不良和畸形。总的来说，Hol颅面突变表型与小鼠的Tbx1纯合突变相似，后者模拟了迪乔治综合征（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，通过利用高分辨率的遗传图谱，我们认为Hol突变映射到16号染色体上的Tbx1或Crkl位点。此外，我们利用基于单核苷酸多态性（SNP）面板的全基因组扫描的新定位技术，在小鼠11号染色体上定位了大约8 Mb的Hol突变基因。最后，自上次修改提交该提案以来，我们通过高分辨率作图和多态标记进一步将含孔层段缩小到约3.9 Mb。我们假设Hol基因在颅面发育中以Tbx1途径起作用，但Hol不是Tbx1或Crkl。我们计划通过以下具体目标揭示这种突变表型的分子基础：1)通过进一步的高分辨率定位和微阵列分析来确定Hol候选基因，以进行“基因发现”；2)通过候选基因分析鉴定Hol基因，通过候选基因测序鉴定Hol分子病变。这些研究的完成将促进我们对颅面模式和形态发生的遗传调控的理解，并导致发现一个可能在Tbx1途径中起作用的新基因。从更广泛的角度来看，这项工作将对影响颅面和耳部发育和功能的人类先天性疾病，如迪乔治综合征（DGS）的发病机制产生影响。公共卫生相关性：颅面和耳部结构的模式和形态发生以及与它们的发育过程有关的基因的知识仍然是初级的。完成这里提出的研究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，这项工作将发现一个可能与Tbx1和Crkl一起在遗传途径中起作用的新基因，该基因在颅面发育的关键过程中是必需的。从更广泛的角度来看，这些研究将对我们对影响正常颅面和耳部发育和功能的人类先天性疾病发病机制的认识产生影响，特别是对diggeorge综合征畸形的认识。

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.