CORE--MUTATION DETECTION & CHARACTERIZATION OF TUMOR SUPPRESSOR GENES

这个核心的主要功能是检测肉瘤家族中的p53突变并对其进行功能鉴定。这些分析对于项目1、4和5的目标是必不可少的。斯特朗博士(项目1)使用从这个核心获得的数据来表征儿童肉瘤患者家族中额外的癌症风险，评估P53胚系突变对儿童肉瘤的相对贡献，并确定从头突变的父亲来源。此外，由于我们发现了非P53癌易感家族，她也将表征这些家族的癌症风险。她进行这些分析的能力取决于确定哪些人携带p53胚系突变。这个核心获得的数据对于Lozano博士的目标(项目4)绘制非p53癌症易感性的一个或多个基因座也是至关重要的，因为识别更多的家庭和个人加强了连锁分析所需的力量。为了确定与P53突变相关的明显的定量微卫星不稳定表型(项目5，西西里亚诺博士)，显然有必要确定具有胚系P53突变的患者。这个核心的具体目标是：1)对软组织肉瘤和骨肉瘤队列先证者的P53编码外显子和侧翼剪接连接进行测序，以确定是否存在P53胚系突变2)检查(1)以上未发现P53突变的样本，以排除其中一种P53等位基因的可能大体缺失3)检测LFS患者肿瘤和永生化成纤维细胞中的杂合性缺失4)建立SSCP和ASO方法，在鉴定出特定的P53突变后筛查家族成员，并筛选其他已知的抑癌基因5)使用酵母菌功能试验和组织培养细胞的瞬时转基因实验对p53突变进行功能分析，以证明突变之间的功能差异。这一核心的功能最终将演变为包括对其他一个或多个易患癌症的基因的突变分析(见项目4和核心E)。

The main functions of this core are the detection and functional characterization of p53 mutation in sarcoma kindreds. These analyses are essential for the aims of projects 1, 4, and 5. Dr. Strong (project 1) uses the data obtained from this core to characterize excess cancer risk in kindreds of childhood sarcoma patients, to assess the relative contribution of p53 germline mutations to childhood sarcoma, and to identify paternal origin of de novo mutations. Additionally, since our discovery of non-p53 cancer prone kindreds, she will characterize cancer risk in these kindreds as well. Her ability to do these analyses is dependents of establishing which individuals carry p53 germline mutations. The data obtained by this core are also essential for Dr. Lozano's aim (project 4) to map the locus or loci responsible for cancer predisposition in non p53 cancer prone kindreds since the identification of additional families and individuals strengthens the power needed for linkage analysis. To determiner where there is a distinct quantitative microsatellite instability phenotype associated with p53 mutations (project 5, Dr. Siciliano), it is clearly essential to identify the patients with germline p53 mutations. The specific aims of this core are: 1) to sequence the p53 coding exons and flanking splice junctions from the probands of the soft tissue sarcoma and osteosarcoma cohorts to identify the presence or absence of germline p53 mutations 2) to examine those samples in which a p53 mutation is not identified by (1) above by southern analysis to exclude possible gross deletions of one of the p53 alleles 3) to examine LOH in tumors and immortalized fibroblasts from LFS patients with and without p53 mutations 4) to develop SSCP and ASO methods to screen family members upon identification of the specific p53 mutation and to screen o6ther known tumor suppressor genes in non p53 kindreds 5) to perform functional analyses of p53 mutations using the yeast functional assay and transient transfection experiments in tissue culture cells to document functional differences between mutations. The function of this core will eventually evolve to include mutational analyses of the other gene or genes that predispose to cancer (see project 4 and core E).