Discovery of New Genes for Inherited Predisposition to Breast Cancer by Exome Seq

描述(由申请者提供)：该项目的目标是确定乳腺癌遗传易感性的新基因。DNA捕获和大规模并行测序技术将被用来在1822个家庭中识别这些基因，每个家庭至少有四个患有乳腺癌的亲属。在这些家庭中，有1500多个乳腺癌的遗传原因仍未解决。在这些家族中发现新的乳腺癌基因涉及三个目标。在AIM 1中，所有已知的乳腺癌遗传易感性基因将在150个家系的所有受影响亲属中进行完全测序。所有未分辨家系的先证者都有BRCA1、BRCA2、CHEK2、PALB2、CHEK2、BRIP1、P53和PTEN的野生型序列。然而，其他受影响的家庭成员可能携带这些基因的突变，先证者可能是零星病例。受影响亲属的构成DNA将被用来准备成对的末端文库，这些文库将与定制的寡核苷酸池杂交，以捕获所有已知的乳腺癌易感基因，然后对丰富的文库进行条形码编码，并在Illumina GAIIx基因组分析仪上以多重设计进行测序。突变将通过桑格测序进行验证。在AIM 2中，Illumina平台将再次被使用，以完整地对屏幕上所有女性的整个外显子进行测序，其中包括所有已知乳腺癌基因的野生型序列。将对变异进行筛选，以识别罕见的无义突变、移码、剪接位点突变以及扰乱基因的基因组缺失和复制。筛选出的突变将被验证和测试与乳腺癌的共同遗传。多重有害突变是所有已知的遗传性乳腺癌基因的标志，也是新乳腺癌基因功能后果的最有力证据。在AIM 3A中，最有希望的候选基因将在一系列独立的家族性乳腺癌患者的DNA样本中完全测序，以揭示更多的突变。在AIM 3B中，这些基因的潜在功能突变将在大型后续系列中通过TaqMan分析进行基因分型，以估计个人和基因范围的突变频率和相对风险。新的乳腺癌易感基因的发现将使预防策略扩展到目前原因基因尚不清楚的家庭，更好地识别处于危险中的妇女，允许对这些妇女进行更密切的医学监测，促进基于遗传证据的新预防策略的设计，并更好地了解乳腺癌发生的生物途径。 公共卫生相关性：该项目的目标是利用DNA捕获和下一代测序技术确定乳腺癌遗传易感性的新基因。新的乳腺癌易感基因的发现将使预防策略扩展到目前因果基因尚不清楚的家庭。

DESCRIPTION (provided by applicant): The goal of this project is to identify new genes for inherited susceptibility to breast cancer. DNA capture and massively parallel sequencing technologies will be exploited to identify these genes in a cohort of 1822 families, each with at least four relatives with breast cancer. The genetic causes of breast cancer in more than 1500 of these families remain unresolved. Discovery of new breast cancer genes in these families involves three aims. In AIM 1, all known genes for inherited predisposition to breast cancer will be fully sequenced in all affected relatives from 150 of the families. The probands of all unresolved families have wildtype sequences at BRCA1, BRCA2, CHEK2, PALB2, CHEK2, BRIP1, p53, and PTEN. However, other affected family members may carry mutations in these genes, and the proband may be a sporadic case. Constitutional DNA of affected relatives will be used to prepare paired-end libraries, which will be hybridized to custom oligonucleotide pools to capture all known breast cancer susceptibility genes, then the enriched libraries will be bar-coded and sequenced in a multiplex design on an Illumina GAIIx genome analyzer. Mutations will be validated by Sanger sequencing. In AIM 2, the Illumina platform will be used again, to fully sequence the entire exomes of all women in the screen with wildtype sequences at all known breast cancer genes. Variants will be filtered to identify rare nonsense mutations, frameshifts, splice site mutations, and genomic deletions and duplications that disrupt genes. Filtered mutations will be validated and tested for co-inheritance with breast cancer. Multiple deleterious mutations are a hallmark of all known genes for inherited breast cancer and are the strongest proof of functional consequence of new breast cancer genes. In Aim 3A, the most promising candidate genes will be fully sequenced in DNA samples from an independent series of familial breast cancer patients to reveal additional mutations. In Aim 3B, potentially functional mutations in these genes will be genotyped by TaqMan assays in large follow-up series to estimate individual and gene-wide mutation frequencies and relative risks. Discovery of new breast cancer susceptibility genes will allow prevention strategies to be extended to families for which causal genes are currently unknown, to better identify women at risk, to allow closer medical surveillance of these women, to stimulate design of new prevention strategies based on genetic evidence, and to offer a better understanding of the biological pathways involved in breast cancer development. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify new genes for inherited susceptibility to breast cancer using DNA capture and next generation sequencing technologies. Discovery of new breast cancer susceptibility genes will allow prevention strategies to be extended to families for which causal genes are currently unknown.