MOLECULAR MAPPING AND GENE IDENTIFICATION OF THE VCFS CRITICAL REGION

大多数速度-心脏-面部综合征(VCFS)患者 显示有22q11.2的微缺失。删除的区域较大且 很可能编码几个相邻的基因。开始理解 这个染色体片段的半合性是如何引起多个 缺陷，包括腭裂和特征性的颅面 在VCFS中看到的变形障碍，必须仔细分析这个区域，并 确定了基因。为了实现这一点，我们建议使用详细的 分离重叠克隆基因组DNA的区域物理图谱 来自YAC和COSMID文库的片段。选定的DNA样本 VCFS患者的亚群将被用来缩小临界区域。在……里面 具体地说，出现隔离的删除患者的断点 VCFS的特征，如腭裂、VPI或颅面畸形， 威尔的发现。将使用多种方法来识别 区域。这些方法将包括绘制新分离的染色体 22-特异基因，直接选择与VCFs相对应的基因 YAC和Cosmids的临界区与DNA的计算机分析 从VCFS关键序列的大规模测序中获得的序列 区域。以这种方式识别的编码区将被确认并 通过杂交或基于聚合酶链式反应的策略分离的全长cDNA。这个 这些基因的表达模式将使用RT-PCR来确定 从各种人体组织中分离出的cDNA。小鼠基因是 在适当的时间和地点表达发展性的将是 在人类中定位以确定是否有与VCFS中的基因同源的基因 临界区。该基因的鉴定与鉴定(S) 负责VCFS的人应该大大增加我们对 这种发育障碍的病因，也将有助于我们的 了解正常的头面部发育。

The majority of velo-cardio-facial syndrome (VCFS) patients have been shown to have microdeletions of 22q11.2. The region deleted is large and is likely to code for several contiguous genes. To begin to understand how hemizygosity of this chromosomal segment gives rise to multiple defects, including cleft palate and the characteristic craniofacial dysmorphia seen in VCFS, this region must be carefully analyzed and the genes identified. To accomplish this we propose to use our detailed physical map of the region to isolate overlapping cloned genomic DNA fragments from YAC and cosmid libraries. DNA samples from a selected subsets of VCFS patients will be used to narrow the critical region. In particular, the breakpoint of deleted patient who present with isolated features of VCFS, such as cleft palate, VPI or craniofacial dysmorphia, will findings. Multiple methods will be used to identify genes in region. These methods will include mapping of newly isolated chromosome 22-specific genes, directly selecting for cDNA which map to the VCFS critical region using YACs and cosmids and computer analysis of the DNA sequence obtained from large scale sequencing of the VCFS critical region. Coding regions identified in this manner will be confirmed and full-length cDNAs isolated by hybridization or PCR-based strategies. The pattern of expression of these genes will be determined using RT-PCR of cDNA isolated from various human tissues. Murine genes which are developmentally expressed at the appropriate time and place will be mapped in humans to determine if any are homologous to genes in the VCFS critical region. The identification and characterization of the gene(s) responsible for VCFS should greatly increase our understanding of the etiology of this developmental disorder and will also contribute to our understanding of normal craniofacial development.