ANTIBODY BASED ASSAY TO DEFECT BRCA1 PROTEIN TRUNCATIONS

描述（申请人描述）乳腺癌和卵巢癌排名第二 在美国，死亡率分别为第四和第四， 每年报告20万例新病例。 大约5%到10% 这些病例中的50%是由于生殖细胞的遗传易感性造成的， 常染色体显性遗传易感性的突变。 改建和 随后一个基因BRCA1的失活被认为存在于50 %和90%的癌症家庭， 早发性乳腺癌和卵巢癌。 女性携带者 这些家庭估计有85%的终生患病风险， 与BRCA1基因相关的癌症。 已经有超过100种突变 包括错义、移码和剪接位点改变，85 其中10%导致蛋白质形成的过早终止 导致截断。 目前，鉴定基因突变花费了大量的时间和费用 通过DNA测序方法。 需要采用更具成本效益的方法， 对这些家庭的女性和男性成员进行遗传性BRCA1筛查 改变。 我们使用了对两个氨基酸末端都有特异性的抗体， BRCA1蛋白，以证明BRCA1蛋白截短， 卵巢肿瘤组织与正常卵巢组织免疫组化分析 在 在正常组织中，BRCA1截短表明存在生殖系 突变 我们还提供了BRCA1蛋白表达的数据， 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遗传性BRCA1基因是否 可以通过使用定量的方法检测颊细胞中的变化 免疫组化分析，并将评估这一敏感性 在这项研究中，个体之间的分析。 突变将通过基因确认 匹配的血细胞DNA测序。 灵敏度大于75 %将为进一步开发该测定法奠定基础， 非侵入性，成本效益筛选测试的男性和女性携带者， 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.