MOLECULAR MAPPING AND GENE IDENTIFICATION OF THE VCFS CRITICAL REGION

大多数的腭心面综合征（VCFS）患者， 显示具有22q11.2的微缺失。 删除的区域很大， 很可能编码几个相邻的基因。 开始理解 这一染色体片段的半合子性是如何产生多个 缺陷，包括腭裂和颅面特征 在VCFS中看到的畸形，必须仔细分析该区域， 基因识别 为了实现这一目标，我们建议使用我们的详细 分离重叠克隆基因组DNA的区域物理图谱 来自YAC和粘粒文库的片段。 DNA样本从一个选定的 将使用VCFS患者的子集来缩小临界区域。 在 特别是，存在孤立性的缺失患者的断点 VCFS的特征，如腭裂、VPI或颅面畸形， 将发现。 多种方法将用于识别基因， 地区 这些方法将包括绘制新分离的染色体 22-特异性基因，直接选择定位于VCFS的cDNA 关键区域使用YACs和cosmetics和计算机分析的DNA 从VCFS关键的大规模测序中获得的序列 地区 以这种方式识别的编码区域将得到确认， 通过杂交或基于PCR的策略分离的全长cDNA。 的 这些基因的表达模式将使用RT-PCR确定， 从各种人体组织中分离的cDNA。 老鼠的基因 在适当的时间和地点发展表达将是 在人类中进行映射，以确定是否有任何与VCFS中的基因同源， 关键区域。 基因的鉴定和表征 负责VCFS的人应该大大增加我们对 这种发展障碍的病因，也将有助于我们 了解正常颅面发育

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.