Identification of radiation sensitivity alleles by whole exome sequencing

描述（由申请方提供）：电离辐射（IR）对细胞具有显著毒性，并且是一种强效诱变剂和致癌物。人类和其他哺乳动物在对电离辐射致死效应的反应方面表现出相当大的群体差异。从历史上看，人们对阐明这种变异的遗传基础很感兴趣，这既是因为要了解控制DNA损伤反应的细胞途径，也是因为有可能利用这些信息来“个性化”放射治疗，以适应个体患者的特定敏感性。20多年来，我们研究了人类放射敏感性的遗传学，重点是导致最极端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突变基因，NBS突变NBN基因的定位和克隆，以及连接酶IV综合征的描述，其中患者显示出与NBS相似的临床特征，但在LIG 4基因中具有双等位基因失活突变。在这些研究的过程中，我们已经积累了来自转介给我们进行A-T或NBS诊断测试的患者的细胞系，在这些患者中，在彻底筛选后，在ATM或NBN基因中均未发现突变。这些细胞系在标准集落存活测定中显示出与A-T细胞系相当的显著辐射敏感性。这些放射敏感性淋巴母细胞系（RS-LCL）构成了一个新的资源，用于识别和理解人类细胞辐射超敏反应的机制。我们假设单个RS-LCL在不同基因中含有单基因突变，其对细胞DNA损伤反应的贡献是已知的，或者可能尚未被识别。我们进一步假设，通过与已知的疾病如A-T或NBS类比，RS-LCL来源于患有由单个基因中有害的双等位基因突变引起的尚未描述的隐性遗传疾病的个体。我们之前通过候选基因方法鉴定这些细胞系中突变的尝试已经产生了与该模型一致的结果;我们鉴定了两个具有双等位基因LIG 4突变的兄弟姐妹和第二个尚未报道的具有RNF 168双等位基因突变的患者。这些结果表明，RS面板是丰富的DNA损伤反应的遗传缺陷的患者，但在面板中的细胞系之间的辐射敏感性的原因是多种多样的，候选基因的研究将是一个低效的方法来确定在大多数这些细胞系的辐射敏感性的遗传病变。幸运的是，现在有可能通过新开发的外显子组测序方法鉴定由一个或几个受影响个体代表的单基因疾病中的突变。我们建议在此应用外显子组测序以分阶段方法鉴定我们的RS-LCL组中的致病突变;对RS-LCL的代表性样品进行测序，通过筛选该组剩余部分中已鉴定的基因来寻求确认，最后通过检测突变基因的野生型拷贝来补充单个细胞系的放射敏感性。

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.