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; 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.

我的实验室重点是对人乳腺癌易感基因BRCA1和BRCA2的功能分析。乳腺癌是美国女性最常见的癌症。据估计，诊断出约178,480例新的侵入性乳腺癌病例，并于2007年因这种疾病而死于该疾病。 BRCA1和BRCA2中的突变与早期发作家族性乳腺癌和卵巢癌的风险增加有关。这两个基因中的突变的个体也有其他器官患癌症的风险。该疾病在BRCA1和BRCA2突变载体中的渗透率估计为35-80％。为了通过预防和早期诊断来降低乳腺癌的死亡率，鼓励BRCA1和BRCA2Mot携带者进行密集筛查，在某些情况下是预防性手术或化学预防。基于测序的基因检测可用于鉴定BRCA1和BRCA2突变载体。当前，家庭中的关联分析用于确定突变是否构成风险。该项目的主要目的之一是了解BRCA1和BRCA2中有害突变如何导致肿瘤发育。通过在小鼠中产生这种突变，我们希望能够提高对BRCA1和BRCA2作为肿瘤抑制剂的作用的理解。为了了解人BRCA1/2变体的效果，我们已经生成了人性化的小鼠模型，其中在BRCA1或BRCA2基因中设计了所需的突变，并且在缺乏内源基因的两个副本的转基因小鼠中分析了突变的表型效应。我们先前已经表明，BAC克隆中存在的野生型人BRCA1和BRCA2能够分别挽救BRCA1KO/KO和BRCA2KO/KO小鼠的致死性。由于小鼠与人BRCA1和BRCA2之间相对较差的核苷酸以及氨基酸序列保守，因此这些小鼠为体内模型系统提供了理想的体内模型系统，可检验人基因中鉴定出的任何突变的效果。多年来，我们产生了许多具有人类BRCA1突变的转基因小鼠（C64G，C61G，A1708E，I26A，R1699Q，M1652i）。除M1652i（BRCT域中BRCA1的中性变体）外，所有其他人都导致了胚胎致死性，这与我们的观察到它们对于ES细胞生存能力至关重要。我们检查了这些变体对成人肿瘤易感性的影响。对于BRCA1，我们专注于I26A变体，并将M1652i用作控制线，如下所述。我们还产生了三种表达BRCA2变体的敲门小鼠模型，这些模型可以挽救BRCA2KO/KO ES细胞的杀伤力，但显示出DNA修复功能的缺陷，因此可能是型型。 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细胞的存活与WT BRCA2表达细胞相当。然而，G25R表达细胞对DNA损伤剂表现出轻度的敏感性，HR降低50％，染色体畸变的积累。 BIC数据库中有一个G25R的案例。 BRCA2 R3052Q BRCA2的C末端的晶体结构在3052的位置描绘了精氨酸在两个寡核苷酸/寡核苷酸 - 寡糖结合褶皱之间的界面上显然重要的位置。 BIC数据库包含涉及该残基的两个不同变体。精氨酸在一种变体中更改为谷氨酰胺（R3052Q），另一个变体（R3052W）更改为另一个变体。基于同源的建模揭示了R3052W，由于与周围残基的相互作用的丧失，而R3052Q对结构产生了中等影响，因为这种变化保留了相互作用的相互作用，因此严重破坏了结构。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R3052Q表达细胞的可行性可行，但对DNA损害剂的敏感性轻度至中度敏感性。我们正在生成一种表达这种变体的敲门小鼠模型，以确定BRCA2功能中的中度缺陷是否与R3052Q变体相关联是否有助于癌症的发展。与G25R和R3052Q变体相反，L2510p的BRCA2 L2510p导致BRCA2KO/KO ES细胞的生存能力降低，并且细胞对DNA损伤剂过敏，并且缺乏IR诱导的RAD51 FOCI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，间隙或径向结构。虽然我们预测表达这种变体的纯合突变小鼠可能不可行，但据报道，一个具有两个兄弟姐妹的兄弟姐妹的兄弟姐妹以及截断的突变c.4876gt p.e1550x均活着。这些孩子没有表现出骨髓衰竭，但一个孩子在六个月零一年后出现了Wilms肿瘤。另一个孩子在大约5岁的时候就出现了T细胞。