Discovery and Characterization of Mutations in Breast and Ovarian Cancers

癌症是一种在生殖系和躯体水平上的遗传病。随着技术的进步， 测序在抗击癌症的斗争中变得越来越重要，这既是为了确定发生的风险 用于疾病和治疗决策。在过去的几年里，我在苏珊博士身上的主要责任 纽豪森的单位已经对卵巢癌和乳腺癌病例进行了高通量测序，并 控制使用Illumina的下一代测序(NGS)技术。纽豪森博士一直在研究 自1992年以来乳腺癌和卵巢癌的遗传性致病突变，当时她是 已本地化并识别BRCA1和BRCA2。从那时起，除了组合在一起的基因外，还发现了其他基因 所有基因的作用解释了乳腺癌遗传风险的不到一半。目前，人们对此知之甚少。 其他基因在癌症进展中的作用，对变异的影响更是知之甚少 在这些基因中。这种知识的缺乏限制了我们识别高危人群的能力。 有针对性地预防癌症。我们希望找出一部分缺失的基因风险。到目前为止，我有 对1000多个乳腺癌和卵巢癌样本进行了定向和完整的外显子组测序。这 这一过程需要为Illumina的测序平台创建文库。我对以下内容做了几处修改 显著降低成本-集成基因组学核心(IGC)已在 希望之城。我目前正致力于Neuhausen博士的R01 CA184585。这笔赠款是第一笔 对西班牙裔美国人乳腺癌易感基因的测序研究，并使用联合 用种系和体细胞方法鉴定致病突变。根据我们从飞行员那里得知的情况 拉美裔美国人的测序研究、全基因组关联研究和BRCA突变筛查 可能会发现新基因和新的反复出现的突变。我们已经确定了创始人PALB2 突变。我负责整个外显子组测序，以及后来的靶向测序。我在工作 与IGC一起更好地确定大的重新安排，然后我将测试结果是否正确。除了……之外 鉴定已知的致病突变，我进行分子生物学分析，包括：a)酵母双杂交 分析错义突变对蛋白质相互作用的影响；b)荧光素酶分析 研究3‘和5’非编码区突变对microRNAs结合和转录的影响 影响调控元件的因素；以及c)聚合酶链式反应和亚克隆以评估剪接变异体的效果。我也在工作 与合作者一起鉴定生殖系和体细胞突变。这个研究专家奖将使我能够 继续为NCI目前和未来资助的基因组项目提供我在分子生物学方面的专业知识。这就做 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危个体， 最终可以在诊断时提供预防选择和更有效的治疗方法。

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.