Discovery and Characterization of Mutations in Breast and Ovarian Cancers

癌症是一种生殖细胞和体细胞水平的遗传疾病。随着科技的进步， 测序在对抗癌症的斗争中变得越来越重要，无论是在确定发展风险方面， 疾病和治疗决定。在过去的几年里，我的主要职责是苏珊博士 诺伊豪森的单位一直是高通量测序卵巢癌和乳腺癌病例和健康 使用Illumina的下一代测序（NGS）技术进行控制。诺伊豪森博士一直在研究 自1992年以来，她一直在研究乳腺癌和卵巢癌的遗传性致病突变，当时她是 定位并鉴定BRCA 1和BRCA 2。从那时起，其他基因也被鉴定出来，但这些基因的组合 所有基因的作用解释了不到一半的乳腺癌遗传风险。目前，人们对 其他基因在癌症进展中的作用，甚至更少的是关于变异的影响 在这些基因中。这种知识的缺乏限制了我们识别那些高风险个体的能力， 针对性预防癌症。我们希望能够确定一部分缺失的遗传风险。到目前为止， 对一千多个乳腺癌和卵巢癌样本进行了靶向和全外显子组测序。这 这个过程需要为Illumina的测序平台创建文库。我做了一些修改， 显著降低成本--整合基因组学核心（IGC）已在 希望之城我目前正在研究诺伊豪森博士的R 01 CA 184585。这是第一次大规模的 测序研究，以确定基因，易患乳腺癌的西班牙裔，并使用联合 生殖系和体细胞方法来鉴定致病突变。根据我们飞行员提供的信息 测序研究，全基因组关联研究（GWAS），和BRCA突变筛查在西班牙裔，我们 将可能发现新的基因和新的复发突变。我们已经确定了PALB 2的创始人 突变我负责整个外显子组测序，以及后期的靶向测序。我在工作 我将与IGC进行合作，以更好地识别大规模的重排，然后我将测试结果是否正确。除了 鉴定已知的致病突变，我进行分子生物学测定，包括：a）酵母双杂交 分析以检查错义突变对蛋白质-蛋白质相互作用的影响; B）荧光素酶测定， 研究可能影响microRNA结合和转录的3'和5' UTR突变的影响 c）PCR和亚克隆以评估剪接变体的影响。我也工作 与合作者一起鉴定生殖细胞和体细胞突变。这个研究专家奖将使我能够 我将继续为当前和未来的NCI资助的基因组项目提供我在分子生物学方面的专业知识。我会 不断开发和测试新方法，以更好地实现我们的目标。通过发现导致 癌症和了解突变的影响，我们可以更好地识别高风险的个体， 最终可以在诊断时提供预防选择和更有效的治疗。

Cancer is a genetic disease on both the germline and somatic levels. With advances in technology, sequencing is becoming increasingly important in the fight against cancer, both for determining risk to develop disease and for treatment decisions. For the past several years, my primary responsibility in Dr. Susan Neuhausen’s unit has been the high-throughput sequencing of ovarian and breast cancer cases and healthy controls using Illumina’s next generation sequencing (NGS) technology. Dr. Neuhausen has been studying inherited pathogenic mutations in breast and ovarian cancers since 1992 when she was part of the team who localized and identified BRCA1 and BRCA2. Since then, other genes have been identified but the combined effect of all genes explains less than half of the genetic risk for breast cancer. Currently, little is known about the role of additional genes in the progression of cancer, and even less is known about the effects of variants within these genes. This lack of knowledge limits our ability to identify those individuals at high risk to develop cancer for targeted prevention. We hope to identify a proportion of the missing genetic risk. To date, I have performed targeted and whole exome sequencing of over a thousand breast and ovarian cancer samples. This process requires creating libraries for Illumina’s sequencing platform. I have made several modifications to significantly reduce costs—modifications that have been adapted by the Integrative Genomics Core (IGC) at City of Hope. I am currently devoting my time to Dr. Neuhausen’s R01 CA184585. This grant is the first large sequencing study to identify genes that predispose to breast cancer in Hispanics, and to use a combined germline and somatic approach to identify pathogenic mutations. Based on what is known from our pilot sequencing study, genome-wide association studies (GWAS), and BRCA mutation screening in Hispanics, we will likely identify both novel genes and novel recurrent mutations. We have already identified a founder PALB2 mutation. I am responsible for the whole exome sequencing, and the later targeted sequencing. I am working with the IGC to better identify large rearrangements and then I will test if the findings are correct. In addition to identifying known pathogenic mutations, I perform molecular biology assays including: a) yeast-two-hybrid analysis to examine the effects of missense mutations on protein-protein interactions; b) luciferase assays to investigate the effects of 3’ and 5’ UTR mutations that may affect binding of microRNAs and transcription factors to regulatory elements; and c) PCR and subcloning to assess effects of splicing variants. I also work with collaborators to identify germline and somatic mutations. This Research Specialist award will allow me to continue to provide my expertise in molecular biology for current and future NCI-funded genomic projects. I will keep on developing and testing new methods to better achieve our goals. By discovering genes that cause cancers and understanding the effects of the mutations, we can better identify individuals who are at high risk, and ultimately can offer preventive options and more effective treatments at diagnosis.