Functional Analysis of Breast Cancer Susceptibility Genes in Mice

小鼠乳腺癌易感基因的功能分析

• 批准号: 9153561
• 负责人: SHYAM SHARAN
• 金额: $ 90.66万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153561
• 关键词: Adult; Age; Amino Acid Sequence; Arginine; BRCA1 Mutation; BRCA1 gene; BRCA2 Mutation; BRCA2 gene; BRCT Domain; Base Sequence; Binding; Biochemical; Biological Models; Biological Process; Cancer-Predisposing Gene; Case Study; Cell Survival; Cells; Chemoprevention; Child; Chromosome abnormality; DNA Damage; DNA Repair; Databases; Defect; Development; Diagnosis; Disease; Early Diagnosis; Embryo; Engineering; Exhibits; Family; Family history of; Genes; Genetic screening method; Glutamine; Hereditary Breast Carcinoma; Human; Individual; Inherited; Knock-in Mouse; Laboratories; Lead; Length; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Modeling; Mus; Mutant Strains Mice; Mutation; Nephroblastoma; Nucleotides; Oligonucleotides; Oligosaccharides; Operative Surgical Procedures; Organ; Pancytopenia; Penetrance; Play; Positioning Attribute; Predisposition; Prevention; Radial; Reporting; Risk; Role; Siblings; Structure; T-Lymphocyte; Testing; Transgenic Mice; Tryptophan; Tumor Suppressor Proteins; United States; Variant; Woman; base; cancer diagnosis; carcinogenesis; design; early onset; embryonic stem cell; improved; in vivo Model; inhibitor/antagonist; malignant breast neoplasm; mortality; mouse model; mutant; mutation carrier; prophylactic; screening; tumor

My laboratory focuses on the functional analysis of the human breast cancer susceptibility genes, BRCA1 and BRCA2. Breast cancer is the most frequently diagnosed cancer in women in the United States. It has been estimated that about 178,480 new cases of invasive breast cancer were diagnosed and more than 40,400 individuals died from this disease in 2007. Among the various factors responsible for the development of this cancer, a family history of the disease seems to play a major role. Mutations in BRCA1 and BRCA2 are linked to increased risk of early onset familial breast and ovarian cancers. Individuals with mutations in either of these genes are also at risk for developing cancer in other organs as well. The penetrance of the disease in BRCA1 and BRCA2 mutation carriers has been estimated to be 35-80%. In an effort to reduce the mortality from breast cancer through prevention and early diagnosis, BRCA1 and BRCA2mutation carriers are encouraged to undergo intensive screening and, in some cases, prophylactic surgery or chemoprevention. Sequencing based genetic tests are available to identify BRCA1 and BRCA2 mutation carriers. Currently, association analyses in families are used to determine whether a mutation poses a risk. One of the main aims of this project is to understand how deleterious mutations in BRCA1 and BRCA2 result in tumor development. By generating such mutations in mice, we hope to improve our understanding of the role of BRCA1 and BRCA2 as tumor suppressor. To understand the effect of human BRCA1/2 variants, we have generated humanized mouse models where desired mutations are engineered in the BRCA1 or BRCA2 gene in BAC and the phenotypic effect of the mutation is analyzed in transgenic mice that lack both copies of the endogenous gene. We have previously shown that wild type human BRCA1 and the BRCA2 present in BAC clones are able to rescue the lethality of Brca1ko/ko and Brca2ko/ko mice, respectively. Due to the relatively poor nucleotide as well as amino acid sequence conservation between mouse and human BRCA1 and BRCA2, these mice provide an ideal in vivo model system to examine the effect of any mutation identified in the human gene. Over the years we have generated a number of transgenic mice harboring mutations in human BRCA1 (C64G, C61G, A1708E, I26A, R1699Q, M1652I). With the exception of M1652I, which is a neutral variant of BRCA1 in the BRCT domain, all others have resulted in embryonic lethality, consistent with our observation that they are essential for ES cell viability. We examining the effect of these variants on tumor susceptibility in adults. For BRCA1, we have focused on I26A variant, and used M1652I as a control line as described below. We have also generated three knock-in mouse models expressing BRCA2 variants that can rescue the lethality of Brca2ko/ko ES cells but show a defect in DNA repair function and therefore, likely to be hypomorphic. BRCA2 G25R Biochemical studies have shown that G25R BRCA2 has a moderate effect on the binding of BRCA2 with PALB2, which is essential for DNA repair function of BRCA2. Survival of Brca2ko/ko ES cells expressing G25R is comparable to WT BRCA2 expressing cells. However, the G25R expressing cells exhibited mild sensitivity to DNA damaging agents, 50% reduction in HR and accumulation of chromosomal aberrations. There is one reported case of G25R in the BIC database. BRCA2 R3052Q The crystal structure of the C-terminus of BRCA2 depicts arginine at position 3052 in an apparently important position at the interface between two oligonucleotide/ oligosaccharide-binding folds. The BIC database contains two different variants involving this residue; arginine is changed to glutamine (R3052Q) in one variant and to tryptophan (R3052W) in the other. Homology-based modeling revealed R3052W to severely disrupt the structure due to the loss of interaction with surrounding residues while R3052Q had a moderate effect on the structure as this change retainedsome of the interactions. We tested these variants using ES cells and found that R3052W failed to rescue the lethality of Brca2ko/ko ES cells whereas R3052Q expressing cells are viable but exhibit mild to moderate sensitivity to DNA damaging agents. We are generating a knock-in mouse model expressing this variant to determine whether the moderate defect in BRCA2 function associated with the R3052Q variant contributes to cancer development. BRCA2 L2510P In contrast to G25R and R3052Q variants, L2510P results in reduced viability of Brca2ko/ko ES cells and the cells were hypersensitive to DNA-damaging agents, and deficient in IR-induced RAD51 foci formation and HR efficiency. We also observed an increase in chromosomal aberrations, such as breaks, gaps, or radial structures in mutant cells. While we predict homozygous mutant mice expressing this variant may not be viable, one family with two siblings inheriting this variant along with a truncating mutation c.4876GT p.E1550X have been reported to be born alive. These children did not exhibit bone marrow failure but one child developed a Wilms tumor at the age of six months and a year later developed AML. The other child developed T-cell ALL at the age of about 5 years.

我的实验室专注于人类乳腺癌易感基因 BRCA1 和 BRCA2 的功能分析。乳腺癌是美国女性最常诊断出的癌症。据估计，2007 年大约有 178,480 例新诊断的浸润性乳腺癌病例，超过 40,400 人死于这种疾病。在导致这种癌症发生的各种因素中，这种疾病的家族史似乎起着重要作用。角色。 BRCA1 和 BRCA2 突变与早发家族性乳腺癌和卵巢癌的风险增加有关。这些基因中任何一个发生突变的个体也有其他器官患癌症的风险。 BRCA1 和 BRCA2 突变携带者的疾病外显率估计为 35-80%。为了通过预防和早期诊断降低乳腺癌死亡率，鼓励 BRCA1 和 BRCA2 突变携带者进行强化筛查，在某些情况下进行预防性手术或化学预防。基于测序的基因测试可用于识别 BRCA1 和 BRCA2 突变携带者。目前，家族中的关联分析用于确定突变是否构成风险。该项目的主要目标之一是了解 BRCA1 和 BRCA2 的有害突变如何导致肿瘤发展。通过在小鼠中产生此类突变，我们希望加深对 BRCA1 和 BRCA2 作为肿瘤抑制因子的作用的理解。为了了解人类 BRCA1/2 变异的影响，我们构建了人源化小鼠模型，其中在 BAC 中的 BRCA1 或 BRCA2 基因中设计了所需的突变，并在缺乏内源基因两个拷贝的转基因小鼠中分析突变的表型效应。我们之前已经证明，BAC 克隆中存在的野生型人类 BRCA1 和 BRCA2 能够分别挽救 Brca1ko/ko 和 Brca2ko/ko 小鼠的致死性。由于小鼠和人类 BRCA1 和 BRCA2 之间的核苷酸和氨基酸序列保守性相对较差，这些小鼠提供了理想的体内模型系统来检查人类基因中发现的任何突变的影响。多年来，我们培育了许多携带人类 BRCA1 突变（C64G、C61G、A1708E、I26A、R1699Q、M1652I）的转基因小鼠。除了M1652I（它是BRCT结构域中BRCA1的中性变体）之外，所有其他都导致胚胎致死，这与我们的观察结果一致，即它们对于ES细胞的活力至关重要。我们研究了这些变异对成人肿瘤易感性的影响。对于 BRCA1，我们重点关注 I26A 变体，并使用 M1652I 作为对照线，如下所述。我们还生成了三种表达 BRCA2 变体的敲入小鼠模型，这些变体可以挽救 Brca2ko/ko ES 细胞的杀伤力，但显示出 DNA 修复功能缺陷，因此可能是低效型的。 BRCA2 G25R 生化研究表明，G25R BRCA2 对 BRCA2 与 PALB2 的结合具有中等影响，这对于 BRCA2 的 DNA 修复功能至关重要。表达 G25R 的 Brca2ko/ko ES 细胞的存活率与表达 WT BRCA2 的细胞相当。然而，表达 G25R 的细胞对 DNA 损伤剂表现出轻度敏感性，HR 降低 50%，并且染色体畸变积累。 BIC 数据库中报告了 1 例 G25R 病例。 BRCA2 R3052Q BRCA2 C 末端的晶体结构描绘了位于两个寡核苷酸/寡糖结合折叠之间的界面处的明显重要位置的位置3052处的精氨酸。 BIC 数据库包含涉及该残基的两种不同变体；精氨酸在一种变体中变为谷氨酰胺 (R3052Q)，在另一种变体中变为色氨酸 (R3052W)。基于同源性的建模显示，由于与周围残基失去相互作用，R3052W 严重破坏了结构，而 R3052Q 对结构具有中等影响，因为这种变化保留了一些相互作用。我们使用 ES 细胞测试了这些变体，发现 R3052W 未能挽救 Brca2ko/ko ES 细胞的致死性，而表达 R3052Q 的细胞是可行的，但对 DNA 损伤剂表现出轻度至中度敏感性。我们正在构建表达该变体的敲入小鼠模型，以确定与 R3052Q 变体相关的 BRCA2 功能中度缺陷是否会导致癌症的发展。 BRCA2 L2510P 与 G25R 和 R3052Q 变体相反，L2510P 导致 Brca2ko/ko ES 细胞活力降低，并且细胞对 DNA 损伤剂高度敏感，并且缺乏 IR 诱导的 RAD51 病灶形成和 HR 效率。我们还观察到染色体畸变的增加，例如突变细胞中的断裂、间隙或放射状结构。虽然我们预测表达该变体的纯合突变小鼠可能无法生存，但据报道，一个有两个兄弟姐妹继承该变体以及截短突变 c.4876GT p.E1550X 的家庭可以存活。这些孩子没有表现出骨髓衰竭，但其中一个孩子在六个月大时患上肾母细胞瘤，一年后患上急性髓系白血病。另一个孩子在大约 5 岁时患上 T 细胞 ALL。