Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing
Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing
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DOI：
10.1073/pnas.1007983107
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发表时间：
2010-07-13
影响因子：
11.1
通讯作者：
King, Mary-Claire
中科院分区：
文献类型：
--
作者：
Walsh, Tom;Lee, Ming K.;King, Mary-Claire
肿瘤抑制基因BRCA 1、BRCA 2和多种其他基因中的遗传性功能丧失突变使乳腺癌和/或卵巢癌的风险增高。这些基因中与癌症相关的遗传性突变总体上相当常见，但个体罕见，甚至是私人的。BRCA 1和BRCA 2突变的基因检测已成为临床实践的一个组成部分，但检测通常仅限于这两个基因和有严重乳腺癌或卵巢癌家族史的女性。为了确定大规模平行的“下一代”测序是否能够准确，彻底和具有成本效益地鉴定乳腺癌和卵巢癌的遗传突变，我们开发了一种基因组测定法，用于捕获，测序和检测21个基因中的所有突变，包括BRCA 1和BRCA 2，具有易患乳腺癌或卵巢癌的遗传突变。将来自具有已知遗传突变的受试者的组成基因组DNA（大小范围为1至> 100，000 bp）与定制寡核苷酸杂交，然后使用基因组分析仪测序。对每个样品中的突变进行盲分析。平均覆盖度为每个碱基对> 1200个读数。在对序列的质量和读数数量进行过滤后，检测到所有单核苷酸取代、小插入和缺失突变以及大基因组重复和缺失。在任何测试样品中，对于任何基因，无义突变、移码突变或基因组重排的假阳性检出为零。这种方法使广泛的基因检测和个性化的乳腺癌和卵巢癌风险评估成为可能。
Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, "next-generation" sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was > 1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer.