A Novel sequencing-based approach to discovering rare genetic factors for ALL.

描述(申请人提供)：急性淋巴细胞性白血病(ALL)是儿童最常见的癌症，尽管有很多研究，但这种复杂、致命和变异很大的疾病的遗传病因仍不清楚。这种知识的缺乏是预测个体结果和设计更有针对性、毒性更低的治疗剂方面取得进展的关键障碍。越来越多的证据表明，在复杂疾病中观察到的变异性在很大程度上是由于个体间罕见的遗传变异。识别罕见的因果遗传变异需要对一群受影响的个人的多个遗传基因座进行深度重新测序，与来自未受影响的队列的类似测序相比，最具信息量。对于如此大规模的分析，传统的双脱氧测序方法成本和时间都太高。然而，通过应用我们设计的定向的、基于人群的混合DNA测序方法来利用下一代测序的大规模并行、高通量能力，我们建议从96名未受影响的儿童的种系DNA和96名匹配的来自儿童高危ALL患者的非肿瘤和肿瘤DNA中对涉及儿童高危白血病发生的56个基因进行深度重新排序。虽然特定原因的罕见变异是个别罕见的，但在基于群体的规模上询问与疾病相关的遗传位点时，可能会显示出各种不同的罕见变异，所有这些变异都会导致相似的表型。我们希望在我们的儿科ALL队列中确定这样的基因座。我们将在250名儿科高危ALL患者的验证队列中证实这些结果，随后对验证的基因座进行个体基因分型，以确定潜在的个体间基因-基因相互作用。我们期望这些结果能够确定与儿童白血病发生有关的各种基因和生化途径，然后形成基于细胞的长期功能研究的基础，最终目标是提供额外的数据，可用于风险评估、个体化治疗和新的靶向治疗药物设计的预期个体患者基因分型。 公共卫生相关性：越来越多的证据表明，罕见基因变化的独特个体组合可能在很大程度上解释了复杂疾病，特别是儿童白血病的易感性和治疗反应的变异性。我们设计了一种新的高通量、靶向性、低成本的DNA测序方法。通过应用这种方法并对数十个在儿童白血病中重要的基因进行测序，我希望能迅速识别出各种新的基因变化和生化途径，当这些变化发生时，可能会使儿童在这么小的年龄就容易患上白血病。

DESCRIPTION (provided by applicant): Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, and despite much research, the genetic etiology of this complex, deadly, and widely variable disease remains unknown. This lack of knowledge is a critical obstacle to advances in predicting individual outcomes and designing more targeted, less toxic therapeutic agents. There is mounting evidence suggesting that a large degree of the variability observed in complex disease is due to inter-individual rare genetic variants. Identifying rare causal genetic variants requires deep resequencing of multiple genetic loci from a population of affected individuals, and is most informative when compared to similar sequencing from an unaffected cohort. Traditional dideoxy sequencing methods are too cost and time prohibitive for such large scale analyses. However, by applying a targeted, population-based pooled DNA sequencing method that we have designed to leverage the massively parallel, high-throughput capacity of next-generation sequencing, we propose to deeply resequence 56 genes implicated in pediatric high-risk leukemogenesis from the germline DNA of 96 unaffected children and 96 matched non-tumor and tumor DNA from pediatric high-risk ALL patients. While a specific causal rare variant is individually rare, a disease-associated genetic locus, when interrogated on a population-based scale, might demonstrate a variety of different rare variants all resulting in a similar phenotype. We expect to identify such loci in our pediatric ALL cohort. We will confirm these results in a validation cohort of 250 pediatric high-risk ALL patients followed by individual genotyping of validated loci to identify potential inter-individual gene-gene interactions. We expect these results to identify a variety of genes and biochemical pathways involved in pediatric leukemogenesis which would then form the basis for long-term cell based functional study, with the ultimate goal of providing additional data that could be used for prospective individual patient genotyping for risk assessment, individualized therapy, and design of new targeted therapeutic agents. PUBLIC HEALTH RELEVANCE: There is increasing evidence that unique individual combinations of rare genetic changes may account for a significant degree of variability in the susceptibility and treatment response of complex disease, particularly pediatric leukemia. We have designed a new high-throughput, targeted, cost-effective method for DNA sequencing. By applying this method and sequencing dozens of genes important in pediatric leukemia, I expect to rapidly identify a variety of new genetic changes and biochemical pathways that, when altered, may predispose a child to developing leukemia at such a young age.