Germline Structural Variant Identification and Functional Determination in Childhood Cancer

摘要 已在癌症易感综合征中发现了一些种系致病结构变异(SV)， 例如，林奇综合征中的MSH2倒置。短读全基因组测序(WGS)的出现 促进了SVS的检测。然而，这种测序方法的一个缺陷是无法捕获所有 SVS，假设读取不能很好地映射到低复杂性区域，并且当前用于识别SVS的算法 短读数据的敏感度很低，假阳性率很高。这一奖学金的目标之一是 是优化并实现了一个SV调用流水线，该流水线使用多种算法来提高敏感度 以及从胚系短读WGS三联体中识别变异体的特异性。这条管道目前正在 在美国国立卫生研究院资助的贝勒先进测序项目中登记的儿童癌症患者上进行开发和测试 儿童癌症护理(基础3)外显组研究，包括种族和种族多样化的儿科 实体(中枢和非中枢)肿瘤患者。这个基本的子集包括63个先证者/父母三人组，他们有 随后进行了胚系短读WGS。运行五个SV调用方，然后使用 百分比倒数重叠滤波法或基于人工智能的邻近度图滤波法 Novo和继承的SVS。这个队列最近被选为长读测序试点，数据 作为心动过速检测的黄金标准。短读WGS生成的SV呼叫与长读WGS生成的SV呼叫的比较 阅读数据将有助于确定不同调用的敏感性和特异性，并进一步提高短读 管道(目标1A)。优化后，这一短读WGS SV流水线将应用于儿童优先遗传 胚胎性和肺泡型横纹肌肉瘤队列(n=900)，作为一种独立的评估方法和 此外，还为了鉴定这种癌症中尚未得到很好特征的复发生殖系SVS(目标1B)。这个 第二个训练和科学目标是探索一种新的新生SV的功能效应，通过 初步分析。前列腺素还原酶2(PTGR2)增强子的新生殖系复制和 在1例儿童后颅窝A型(PF-A)室管膜瘤中发现了启动子区域。转录组 来自患者肿瘤的数据显示PTGR2表达增加。PTGR2蛋白可将15-酮基-2-甲氧基苯乙酸乙酯 前列腺素E_2至15-酮-13，14-二氢前列腺素E_2。对成人恶性肿瘤的研究表明，增加的15-酮- 13，14-二氢前列腺素E_2可能通过STAT3信号通路与癌症风险相关。增加了 据报道，STAT3信号是PF-A室管膜瘤的一个明显特征。已识别的部分复制 PTGR2将被工程化为室管膜瘤前体细胞，以功能评估对STAT3的影响 信号、细胞增殖和DNA合成。同时，进一步分析PTGR2的表达和结构 将对儿科室管膜瘤队列中的变异进行分析(目标2)。这项试验计划的完成和 在Plon博士和Milosavljevic博士的指导下所描述的培训活动将为Owen Hirschi提供高度跨学科的 关于基因组数据分析、结构变异及其对儿科癌症生物学下游影响的培训。

Abstract A few germline pathogenic structural variants (SV) have been identified in cancer predisposition syndromes, e.g., MSH2 inversion in Lynch syndrome. The advent of short-read whole-genome sequencing (WGS) has facilitated the detection of SVs. However, a pitfall of this sequencing methodology is the inability to capture all SVs, given the reads do not map well to low complexity regions, and current algorithms used to identify SVs from short-read data have very low sensitivity and very high false-positive rates. One of the objectives of this fellowship is to optimize and implement an SV calling pipeline that utilizes multiple algorithms to increase the sensitivity and specificity of variant identification from germline short-read WGS trios. This pipeline is currently being developed and tested on pediatric cancer patients enrolled in the NIH-funded Baylor Advancing Sequencing into Childhood Cancer Care (BASIC3) exome study, which consists of ethnically and racially diverse pediatric patients with solid (CNS and non-CNS) tumors. This BASIC3 subset includes 63 proband/parent trios who have subsequently undergone germline short-read WGS. Five SV callers are run and then filtered using either a percent reciprocal overlap filter or a proposed Artificial Intelligence-based proximity graph filter to identify de novo and inherited SVs. This cohort has recently been selected for a long-read sequencing pilot, data which serves as the gold standard for SV detection. Comparison of short-read WGS generated SV calls with the long- read data will help determine the sensitivity and specificity of variant calls and further improve the short read pipeline (Aim 1A). Once optimized this short-read WGS SV pipeline will be applied to the Kids First Genetics of Embryonal and Alveolar Rhabdomyosarcoma cohort (n=900), as an independent assessment of the method and also to identify recurrent germline SVs in this cancer which has not yet been well characterized (Aim 1B). The second training and scientific objective is to explore the functional effect of a novel de novo SV identified through the initial analysis. A de novo germline duplication of the Prostaglandin Reductase 2 (PTGR2) enhancer and promoter region was found in a pediatric Posterior Fossa subtype A (PF–A) ependymoma patient. Transcriptome data from the patient’s tumor revealed increased PTGR2 expression. The PTGR2 protein converts 15-keto- prostaglandin E2 to 15-keto-13,14-dihydro-PGE2. Studies in adult malignancies suggest that increased 15-keto- 13,14-dihydro-PGE2 is associated with cancer risk potentially through the STAT3 signaling pathway. Increased STAT3 signaling is reported as a distinct feature of PF-A ependymoma. The identified partial duplication of PTGR2 will be engineered into ependymoma progenitor cells, to functionally assess the effects on STAT3 signaling, cell proliferation, and DNA synthesis. In parallel, further analysis of PTGR2 expression and structural variants will be analyzed in pediatric ependymoma cohorts (Aim 2). Completion of this experimental plan and described training activities under Drs. Plon and Milosavljevic will provide Owen Hirschi highly interdisciplinary training in analysis of genomic data, structural variation, and the downstream effects on pediatric cancer biology.