A unique long-read genome sequencing approach to identify structural variants predisposing to hereditary breast cancer

DNA修复基因BRCA1和BRCA2以及一些相互作用伙伴和下游靶点的突变易导致遗传性乳腺癌和卵巢癌（HBOC）。然而，只有大约35%符合分子检测临床标准的HBOC病例与所描述的HBOC基因的致病突变有关。令人惊讶的是，在包括许多受影响个人在内的大量大家庭中，以及在发病非常早的病例中，检测结果也为阴性。此外，最近的分子研究表明存在基因组结构突变，例如在已知和未知的hboc基因的调控区域，这些突变无法通过短读测序方法检测到。基于以下观察：(i)已确定的HBOC相关基因仅在约35%的患者中解释家族性风险；（ii）目前用于鉴定新型HBOC易感基因（即大型患者队列中的WES）的致病突变的策略还不是很成功，我们新成立的下萨克森州联盟开发了一种创新的新策略，用于鉴定HBOC中的新基因和新分子机制。假设新生或隐性突变在极早发性HBOC的分子发病机制中起重要的致病作用，我们将选择4个早发性乳腺癌（<28岁）且家族史阴性的三人组（母亲、父亲、HBOC指数[所有已知HBOC基因均阴性]）进行低覆盖率长读（Pacific Biosciences）和高覆盖率短读（Illumina） WGS。长读WGS旨在检测结构变异，短读WGS将检测致病的单核苷酸变异。除了这三种方法外，我们还选择了四个大的hboc基因阴性家族，其中不同代中有几个受影响的个体，我们的目标是通过PacBio-和Illumina-WGS的组合来识别新的致病突变和基因。这两件事都将由四个家族中不同世代的两个人来完成。生物信息学分析将由位于杜塞尔多夫和科隆的团队提供，其中还包括在科学合作的基础上使用SMRT Link管道进行二次分析。为了确保WGS数据集的成功变异解释，我们在Göttingen人类遗传学研究所的MutationMining （MM）团队将参与该项目。MM团队是一个独特的团队，由21名临床医生和科学家组成，他们在临床遗传学、分子遗传学、分子生物学、生物化学和生物信息学等领域拥有不同的专业知识，并在基于ngs的基因鉴定研究方面拥有长期的经验。新的HBOC相关基因和突变谱随后将在更大的HBOC队列中进行测试，以确定突变的频率和相对风险。

Mutations in the DNA repair genes BRCA1 and BRCA2 as well as several interaction partners and downstream targets predispose to hereditary breast and ovarian cancer (HBOC). However, only approximately 35% of cases with HBOC fulfilling the clinical criteria for molecular testing can be associated with causative mutations in described HBOC-genes. Surprisingly, negative testing results are also obtained in a substantial number of large families including numerous affected individuals, and also in cases presenting with a very early onset of the disease. In addition, recent molecular studies suggest the presence of genomic structural mutations, e.g. in regulatory regions of known and unknown HBOC-genes, which cannot be detected by short-read sequencing approaches. Based on the observation that (i) identified HBOC-associated genes explain the familial risk in only ~35% of patients and (ii) current strategies for the identification of causative mutations in novel HBOC-predisposing genes, namely WES in large cohorts of patients, have not been very successful, our newly established Lower Saxony consortium developed an innovative, new strategy for the identification of novel genes and novel molecular mechanisms in HBOC. Under the hypothesis that de novo or recessive mutations play an important causative role in the molecular pathogenesis of very early onset HBOC, we will perform both, low-coverage long-read (Pacific Biosciences) WGS and high-coverage short-read (Illumina) WGS in four selected trios (mother, father, HBOC index [negative for all known HBOC-genes]) with an early onset of breast cancer (<28 years of age) and negative family history. Long-read WGS aims to detect structural variants, and short-read WGS will detect causative single nucleotide variants. In addition to this trio approach, we have selected four large, HBOC-gene negative families with several affected individuals in different generations, and we aim to identify novel causative mutations and genes by the combination of PacBio- and Illumina-WGS. Both will be done in two individuals from different generations in each of the four families. The bioinformatics analysis will be provided by the teams in Düsseldorf and Cologne, which also includes the secondary analysis using the SMRT Link pipeline on the basis of a scientific cooperation. To ensure successful variant interpretation of WGS data sets, our MutationMining (MM) team at the Institute of Human Genetics in Göttingen will be involved in the project. The MM team is a unique team consisting of 21 clinicians and scientists with different expertise in e.g. clinical genetics, molecular genetics, molecular biology, biochemistry and bioinformatics, and it has a long-standing experience in NGS-based gene identification studies. Novel HBOC-associated genes and mutations profiles will be subsequently tested in larger HBOC cohorts in order to determine the frequency and relative risk of mutations.