Development of Cell-based Functional Tests for Rare Germline ATM Gene Variants in Hereditary Ovarian Cancer Families

对癌症易感性高危人群的DNA全基因组测序导致了 大量未知意义的变种的鉴定，VUS。我们评估了48名高危女性 卵巢癌家系生殖系突变的全外显子组测序 生殖系DNA。这些受试者之前被发现对任何已知的致病突变都是阴性的。 BRCA1或BRCA2。在相关途径中有中到高影响的大量遗传变异 都被发现出现在这个队列中。特别是，我们确定了2种有害的和5种潜在的破坏性的 这些患有卵巢癌的高危女性的正常DNA中ATM基因的变异。的作用 ATM在卵巢癌中的作用一直被低估，因为缺乏对其影响的功能性分析 这些基因变化。在这个项目中，我们将试行开发一种通用方法来确定 使用基于细胞的功能分析研究DNA修复相关基因的私人和罕见突变的性质。 将对基因共济失调-毛细血管扩张突变(ATM)的新生殖系变异进行功能分析 在永生化的正常卵巢和乳腺上皮细胞中进行了研究。这种方法比使用 已经存在DNA修复缺陷的已建立的肿瘤细胞。PI的实验室在研究 癌症遗传学及其突变型遗传癌基因对正常细胞生物学的影响 基因组不稳定性以前主要集中在Li-Fraumeni综合征。自动柜员机编码一种蛋白激酶， 调节DNA损伤反应，启动激活细胞周期检查点的信号级联反应 以及DNA双链断裂的修复。我们将开发基于细胞的分析方法，可用于 ATM基因中未知意义遗传变异(VUS)的功能分析。ATM突变 与乳腺癌的风险有关；然而，关于卵巢癌的信息很少。 因此，我们将比较永生化对正常乳腺和永生化的影响。 卵巢上皮细胞的ATM基因经过编辑后，含有我们在 病人的DNA。我们将确定这些明显有害和具有潜在破坏性的变种是否 以特定细胞类型对细胞周期或DNA修复机制产生重大影响。有害的和 ATM中潜在的破坏性VUS在我们的队列中被统计得过高(表1)，而不是 1000个基因组数据库中的MAF。然而，每个VU都需要一些功能分析，以 向患者提供临床上有用的信息。该项目将以两个目标进行： 目的1：我们将在hTERT永生化正常人中对内源性ATM基因进行基因编辑 乳腺和卵巢上皮细胞使用CRISPR/Cas9产生潜在的有害ATM变体。 目标2：在已知与卵巢癌遗传风险相关的途径中使用功能分析 我们将确定这些VUS对细胞周期控制和DNA修复机制的影响。

Whole genome sequencing of DNA from subjects at high risk for cancer predisposition has led to the identification of numerous variants of unknown significance, VUSs. We evaluated 48 women from high-risk families diagnosed with ovarian cancer for germline mutations by whole exome sequencing of their germline DNA. These subjects were previously found to be negative for any known pathogenic mutations in either BRCA1 or BRCA2. Numerous genetic variants with a moderate to high effect in relevant pathways were found to be present in this cohort. In particular, we identified 2 deleterious and 5 potentially damaging variants in the ATM gene in the normal DNA from these high-risk women with ovarian cancer. The role of ATM in ovarian cancer has been underestimated because of a lack of functional assays for the effects of these genetic changes. In this project we will pilot the development of a general approach to determine the nature of private and rare mutations in DNA repair associated genes using cell-based functional assays. The functional analysis of novel germline variants in the gene ataxia-telangiectasia mutated (ATM) will be studied in immortalized normal ovarian and mammary epithelial cells. This approach is superior to using established tumor cells that already have DNA repair defects. The PI's lab is experienced in studying the genetics of cancer and the ramifications of mutant hereditary cancer genes on normal cell biology and genomic instability focusing previously on Li-Fraumeni Syndrome. ATM codes for a protein kinase that regulates the DNA damage response initiating the signaling cascades that activate cell cycle checkpoints and the repair of DNA double-strand breaks. We will develop cell-based assays that can be used for the functional analysis of genetic variants of unknown significance (VUSs) in the ATM gene. ATM mutations are associated with the risk of breast cancer; however little information is available on ovarian cancer. Therefore, we will compare the biological and biochemical impact on immortalized normal mammary and ovarian epithelial cells that have had their ATM genes edited to contain the SNPs that we have identified in patients' DNA. We will determine whether these clearly deleterious and potentially damaging variants have significant impact on cell cycle or DNA repair mechanisms in a cell type specific fashion. Deleterious and potentially damaging VUSs in ATM were statistically overrepresented in our cohort (Table 1) compared to the MAF in the 1000 genomes database. However each VUS will require some functional analysis to provide clinically useful information to patients. This project will be pursued in two aims: AIM 1: We will employ gene editing of the endogenous ATM gene in hTERT immortalized normal mammary and ovarian epithelial cells using CRISPR/Cas9 to produce potentially deleterious ATM variants. AIM 2: Using functional assays in pathways known to be associated with inherited risk of ovarian cancer we will determine the impact of these VUSs on cell cycle control and DNA repair mechanisms.