Identification of radiation sensitivity alleles by whole exome sequencing

说明(申请人提供)：电离辐射(IR)对细胞有很大的毒性，是一种强大的诱变剂和致癌物。人类和其他哺乳动物对电离辐射的致死效应的反应表现出相当大的种群差异。从历史上看，人们对阐明这种变异的遗传基础很感兴趣，这既是因为对控制DNA损伤反应的细胞通路有深入的了解，也是因为利用这些信息有可能使放射治疗对个别患者的特定敏感度“个性化”。20多年来，我们一直在研究人类辐射敏感性的遗传学，重点研究导致最极端的IR超敏表型的隐性遗传疾病。这些研究包括ATM的定位和精细定位，A-T突变的基因定位和精细定位，NBS突变的NBN基因的定位和克隆，以及Ligase IV综合征的描述，即患者表现出与NBS相似的临床特征，但LIG4基因存在双等位基因失活突变。在这些研究过程中，我们积累了来自我们转介进行A-T或NBS诊断测试的患者的细胞系，这些患者经过详尽的筛查后，没有发现ATM或NBN基因突变。在与A-T细胞系相当的标准集落存活试验中，这些细胞系表现出显著的放射敏感性。这些对辐射敏感的淋巴母细胞系(RS-LCL)构成了鉴定和理解人类细胞辐射超敏机制的新资源。我们假设，单个RS-LCL在不同基因中包含单基因突变，这些突变对细胞DNA损伤反应的贡献是已知的，或者可能是未知的。我们进一步假设，通过与A-T或NBS等已知疾病的类比，RS-LCL来自于由单基因有害的双等位基因突变引起的尚未描述的隐性遗传病的个体。我们之前试图通过候选基因方法鉴定这些细胞系中的突变，结果与这一模型一致；我们鉴定出两个兄弟姐妹具有双等位LIG4突变，第二个尚未报道的患者具有RNF168的双等位突变。这些结果表明，对于DNA损伤反应中存在遗传缺陷的患者，RS面板是丰富的，但该面板中细胞系之间辐射敏感性的原因是多样的；候选基因研究将不是一种有效的方法来识别大多数这些细胞系中辐射敏感性的遗传损伤。幸运的是，现在可以通过新开发的外显子组测序方法识别由一个或几个受影响的个体所代表的单基因疾病的突变。在这里，我们建议应用外显子组测序来分阶段确定我们的RS-LCL小组中的致病突变；对具有代表性的RS-LCL样本进行测序，通过在小组的其余部分筛选已识别的基因来寻求确认，最后，通过转染突变基因的野生型副本来补充单个细胞系的辐射敏感性。

DESCRIPTION (provided by applicant): Ionizing radiation (IR) is significantly toxic to cells as well as being a potent mutagen and carcinogen. Humans and other mammals display considerable population variation in their response to the lethal effects of ionizing radiation. Historically, there has been much interest in elucidating the genetic bases for this variation both because of the insights to be gained into the cellular pathways controlling DNA damage responses and because of the potential to utilize this information to "personalize" radiation therapy to the specific sensitivities of individual patients. For more than 20 years, we have studied the genetics of radiosensitivity in humans, focusing on recessive genetic disorders that result in the most extreme IR hypersensitivity phenotypes. These studies have included the localization and fine mapping of ATM, the gene mutated in A-T, the mapping and cloning of the NBN gene mutated in NBS, and the description of Ligase IV Syndrome, in which patients display clinical features similar to NBS but have biallelic inactivating mutations in the LIG4 gene. During the course of these studies, we have accumulated cell lines from patients referred to us for diagnostic testing for A-T or NBS in whom no mutations in either the ATM or NBN genes could be found after exhaustive screening. These cell lines display significant radiosensitivity in a standard colony survival assay comparable to that of A-T cell lines. These radiosensitive lymphoblastoid cell lines (RS-LCL) constitute a novel resource for identifying and understanding the mechanisms of human cellular radiation hypersensitivity. We hypothesize that individual RS-LCLs contain monogenic mutations in different genes whose contributions to cellular DNA damage responses are known, or may as yet be unrecognized. We further postulate that, by analogy with known disorders such as A-T or NBS, the RS-LCLs derive from individuals with as yet undescribed recessive genetic disorders caused by deleterious biallelic mutations in single genes. Our previous attempts to identify mutations among these cell lines by candidate gene approaches have yielded results that are consistent with this model; we identified two siblings with biallelic LIG4 mutations and a second, as yet unreported, patient with biallelic mutations in RNF168. These results suggest that the RS panel is enriched for patients with inherited defects in DNA damage responses but that the causes of radiosensitivity among cell lines in the panel are diverse; candidate gene studies would be an inefficient approach to identify the genetic lesions underlying the radiosensitivity in the majority of these cell lines. Fortunately, it is now possible to identify mutations in monogenic disorders represented by one or a few affected individuals through newly developed exome sequencing approaches. We propose here to apply exome sequencing to identify the causative mutations in our panel of RS-LCLs in a staged approach; sequencing a representative sampling of the RS-LCLs, seeking confirmation by screening the identified genes in the remainder of the panel and finally, complementing the radiosensitivity of individual cell lines by transfecting wild type copies of the mutated genes.