MOLECULAR MAPPING AND GENE IDENTIFICATION OF THE VCFS CRITICAL REGION

VCFS关键区域的分子图谱和基因鉴定

• 批准号: 3732358
• 负责人: MARCIA BUDARF
• 金额: --
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3732358
• 关键词: DNA; animal genetic material tag; artificial chromosomes; autosomal dominant trait; chromosome deletion; chromosome disorders; chromosomes; cleft palate; complementary DNA; computer assisted sequence analysis; congenital oral /facial /cranial defect; developmental genetics; gene expression; genetic library; genetic mapping; human genetic material tag; human subject; hybrid cells; molecular genetics; plasmids; polymerase chain reaction; syndrome

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.

大多数 velo-cardio-facial 综合征 (VCFS) 患者已接受过 显示有 22q11.2 微缺失。 删除的区域很大， 可能编码几个连续的基因。 开始了解 该染色体片段的半合性如何产生多个 缺陷，包括腭裂和特征性颅面 VCFS 中出现的畸形，必须仔细分析该区域， 确定的基因。 为了实现这一目标，我们建议使用我们详细的 用于分离重叠克隆基因组 DNA 的区域物理图 来自 YAC 和粘粒文库的片段。 DNA 样本来自选定的 VCFS 患者的子集将用于缩小关键区域。 在 特别是，被删除的患者的断点呈现出孤立的状态 VCFS 的特征，如腭裂、VPI 或颅面畸形， 将会发现。 将使用多种方法来鉴定基因 地区。 这些方法将包括新分离的染色体的图谱 22个特定基因，直接选择映射到VCFS的cDNA 使用 YAC 和粘粒以及 DNA 计算机分析的关键区域 从 VCFS 关键的大规模测序获得的序列 地区。 以这种方式识别的编码区域将被确认并 通过杂交或基于 PCR 的策略分离的全长 cDNA。 这 这些基因的表达模式将通过 RT-PCR 确定 从各种人体组织中分离出 cDNA。 小鼠基因是 在适当的时间和地点，会得到发展性的表达 在人类中绘制图谱以确定是否有与 VCFS 中的基因同源的 临界区。 基因的鉴定和表征 负责VCFS的应该大大增加我们对 这种发育障碍的病因学，也将有助于我们 了解正常颅面发育。