Identification and functional analyses of concomitant monoallelic mutations in Fanconi anemia/BRCA pathway genes in the germline of children with cancer

生活方式因素在儿童癌症中基本上可以忽略不计，因此，癌症易感基因（CPGs）的遗传种系突变可能在一定程度上起到了被低估的作用。最近的研究表明，相当比例的儿童癌症（8.5%）显示CPGs突变。然而，儿童恶性肿瘤的确切比例可归因于潜在的CPS仍不清楚。此外，CPGs中遗传与新生种系突变的相对贡献，以及其他儿童的复发风险，仍不清楚。父母-孩子三人组的全外显子组测序（WES）被证明是一种有前途的最新策略，可以识别罕见疾病儿童蛋白质编码基因的致病遗传变异。为了找到相关的单核苷酸变异（snv），我们将WES应用于癌症儿童及其父母的种系。有趣的是，我们的WES分析发现了一种遗传现象，其特征是在25%的儿童癌症（包括白血病和实体瘤）中存在（至少）两个独立的单等位基因种系突变，这些突变涉及FA/BRCA途径。这些突变要么是由（迄今为止）临床健康的父母遗传的，要么是一个SNV是从母亲/父亲遗传的，而第二个SNV是从头发生的。这种基因单等位基因FA/BRCA通路突变的组合可能会严重损害DNA修复并导致基因组不稳定，这是癌症的一个标志。从临床角度来看，这种导致FA/BRCA通路失调的协同/协同效应与这些儿童和家长的临床表型完全吻合。因此，我们的初步发现可能对患者及其高危家庭成员具有重要的临床意义。在这项研究计划中，我们打算解决以下目标：(1)我们将系统地分析患有癌症的儿童和青少年，通过使用父母-孩子三人WES来识别FA/BRCA通路基因的畸变/突变；(2)我们将使用患者细胞和异位细胞模型对已识别的单等位基因FA/BRCA通路突变（可能在DNA损伤反应和DNA修复中发挥作用）进行功能分析；(3)我们将阐明两者的功能相互作用。利用分子细胞生物学和生物化学在细胞和分子水平上研究单等位基因FA/BRCA通路突变。我们的研究结果有望为遗传性儿童癌症的发展提供基本的新见解，并将有助于确定新的诊断和治疗策略。

Lifestyle factors are largely negligible in childhood cancer and therefore, inherited germline mutations in cancer predisposition genes (CPGs) presumably contribute to a yet underestimated extent. Recent studies indicate that a considerable proportion of childhood cancers (8.5%) show mutations in CPGs. However, the exact proportion of children with a malignancy that is attributable to an underlying CPS is still unclear. Additionally, the relative contribution of inherited versus de novo germline mutations in CPGs, and thus the risk of recurrence in other children, still remains unclear. Whole-exome sequencing (WES) of parents-child trios proofed as a promising state-of-the-art strategy to identify causative genetic variants in protein-coding genes in children with rare diseases. To find relevant single nucleotide variants (SNVs) we applied WES on the germline of children with cancer and their parents. Interestingly, our WES analyses identified a genetic phenomenon characterized by the presence of (at least) two independent, monoallelic germline mutations in genes involved in the FA/BRCA pathway in 25% of pediatric cancers including leukemias and solid tumors. The mutations were either inherited by the (so far) clinically healthy parents or one SNV was transmitted from the mother/or father, whereas the second SNV occurred de novo. Such combinations of digenic monoallelic FA/BRCA pathway mutations may substantially compromise DNA repair and lead to genomic instability, which is a hallmark of cancer. From the clinical point of view, such cooperative/synergistic effects leading to deregulation of the FA/BRCA pathway perfectly matches with the clinical phenotype of these children and parents. Thus, our preliminary findings could have important clinical implications for patients and their at-risk family members. In this research proposal we intend to address the following aims: (1) we will systematically analyze children and adolescents with cancer by using parents-child trio WES to identify aberrations/mutations in FA/BRCA pathway genes, (2) we will functionally analyze identified monoallelic FA/BRCA pathway mutations (which likely play a role in DNA damage response and DNA repair) using patient cells and ectopic cell models and (3) we will elucidate the functional interplay of the combined, monoallelic FA/BRCA pathway mutations on the cellular and molecular level using molecular cell biology and biochemistry. Our results are expected to provide fundamental novel insights into the development of inherited childhood cancers and will contribute to identify novel strategies for diagnosis and treatment.