ANTIBODY BASED ASSAY TO DEFECT BRCA1 PROTEIN TRUNCATIONS

描述(申请人的描述)乳腺癌和卵巢癌位居第二 在美国死亡率分别为第四位和第四位 每年报告的新增病例为20万例。大约5%到10% 这些病例中有一部分是由生殖系遗传倾向引起的。 常染色体显性遗传易感性突变。更改和修改 随后，一种名为BRCA1的基因被认为在50%出现失活 90%和90%的癌症家族 分别为早发性乳腺癌和卵巢癌。中国女性携带者 据估计，这些家庭有85%的终身感染风险。 与BRCA1基因相关的癌症。已经有100多个基因突变 已发现包括错义、移码和剪接位点改变，85 导致蛋白质形成提前终止的百分比 导致截断。 目前，鉴定基因突变需要花费大量的时间和费用。 通过DNA测序的方法。需要更具成本效益的方法来 筛查这些家庭的男性和女性成员是否可遗传BRCA1 改装。我们使用了针对两个氨基酸末端的抗体 BRCA1蛋白，通过以下方式证明BRCA1蛋白截断 卵巢肿瘤与正常组织配对的免疫组织化学分析。在……里面 在正常组织中，BRCA1被截断表明存在生殖系 突变。我们还提供了BRCA1蛋白在细胞中表达的数据 人颊上皮细胞使用相同的抗体和BRCA1 mRNA的存在 RT-PCR法。这项拟议的研究将确定可遗传的BRCA1基因 用定量的方法可以检测到口腔黏膜细胞的改变 免疫组织化学分析，并将评估这一敏感性 在这项研究中的个体之间的化验。突变将由基因证实 对匹配的血细胞DNA进行测序。敏感度大于75 百分比将为进一步发展这一检测方法奠定基础 对男性和女性携带者进行无创、成本效益高的筛查测试 BRCA1突变。

DESCRIPTION (Applicant's Description) Breast and ovarian cancer rank second and fourth respectively in mortality in the United States with greater than 200,000 new cases reported each year. Approximately 5 percent to 10 percent of these cases result from a hereditary predisposition with germline mutations conferring autosomal dominant susceptibility. The alteration and subsequent inactivation of one gene, BRCA1, is believed to be present in 50 percent and 90 percent of cancer families with increased incidence of early-onset breast and ovarian cancer respectively. Female carriers in these families have an estimated 85 percent life long risk of contracting cancers associated with the BRCA1 gene. Over 100 mutations have been identified including missense, frameshifts and splice-site alterations, 85 percent of which result in premature termination of protein formation resulting in truncation. Presently, much time and expense is incurred to identify gene mutations through DNA sequencing methods. More cost-effective methods are required to screen female and male members of these families for heritable BRCA1 alterations. We used antibodies specific for both amino acid terminals of the BRCA1 protein, to demonstrate BRCA1 protein truncations by immunohistochemical analysis of matched ovarian tumor and normal tissue. In normal tissue, BRCA1 truncation is indicative of the presence of a germline mutation. We also present data demonstrating expression of BRCA1 protein in human buccal cells using the same antibodies and presence of BRCA1 mRNA by RT-PCR. The proposed study will determine whether heritable BRCA1 gene alterations may be detected in buccal cells by using quantitative immunohistochemical analysis, and will evaluate the sensitivity of this assay among individuals in this study. Mutations will be confirmed by gene sequencing of matched blood cell DNA. A sensitivity of greater than 75 percent would establish a basis for further development of this assay as a noninvasive, cost effective screening test for male and female carriers of BRCA1 mutations.