Project III - Comprehensive Genomic Evaluation of Structural Birth Defects

项目总结 结构性出生缺陷(SBD)影响3%-6%的活产，是婴儿发病率和死亡率的主要原因 全世界。大多数SBD是孤立的，发生在人类发育的前10周，被认为是 由遗传、表观遗传和环境因素驱动。 DNA测序技术的进步推动了我们对SBD遗传学的理解。基于数组的 技术确定了导致SBD的拷贝数变异，并使全基因组关联成为可能 学习。大规模并行的下一代(NGS)方法对大量短(50-300个碱基)进行排序 对)DNA片段，允许对所有编码区(外显子组)或整个 一次测得基因组。外显子组和基因组测序确定了血液来源DNA的分子病因学 ~50%的儿童多系统、综合征性SBD。然而，大多数SBD的病因-既有孤立的，也有 综合征--仍不清楚。 一个潜在的原因是，基于短读的血液来源DNA测序并不提供 对个体基因组的全面了解。 这项建议的目标是在SBD儿童的预期队列中使用新技术来识别 遗传变异不能被目前的方法识别。这些“隐藏的”变体包括复制、倒置、 重复扩展/收缩和表观遗传修饰，这是标准的短读方法所不能做到的 确定(目标1)。隐藏的变异还包括合子后产生的DNA突变(“马赛克”)，而不是 存在于血液来源的DNA中(目标2)。 我们将在患者身上使用基于长阅读的DNA和RNA测序方法(PacBio和Oxford Nanopore) 患有综合征性SBD，其临床检查(包括外显子组测序)一直未被诊断。在病人中 对于临床检查尚未确诊的分离的SBD，我们将应用基于深度短读的DNA 对多种非血液来源的组织进行测序，以识别马赛克变体。 这项建议与《消除一切形式种族歧视国际公约》的既定目标相一致，因为我们的研究结果将构成以下新战略的基础 人类结构性出生缺陷的诊断、治疗和预防。我们的工作通过以下方式进一步实现了这一目标 在SBD儿童中识别以前未检测到的基因变异。这是迈向 建立在临床诊断领域使用这些新技术的框架。

PROJECT SUMMARY Structural birth defects (SBD) affect 3-6% of live births and are a leading cause of infant morbidity and mortality worldwide. Most SBD are isolated, occur in the first 10 weeks of human development, and are thought to be driven by genetic, epigenetic, and environmental factors. Technical advances in DNA sequencing have propelled our understanding of the genetics of SBD. Array based technologies identified copy number variants that contribute to SBD and enabled genome wide association studies. Massively parallel “Next Generation” (NGS) methods sequence large numbers of short (50-300 base pairs) pieces of DNA simultaneously, permitting sequencing of all coding regions (the exome) or the entire genome at once. Exome and genome sequencing of blood-derived DNA identifies the molecular etiology of ~50% of children multisystemic, syndromic SBD. However, the etiology of most SBD- both isolated and syndromic- remains unknown. A potential reason for this is that short-read based sequencing of blood-derived DNA does not provide a comprehensive view of an individual’s genome. The goal of this proposal is to use novel technologies in prospective cohorts of children with SBD to identify genetic variation not identified by current methods. These “hidden” variants include duplications, inversions, repeat expansions/contractions, and epigenetic modifications that standard short-read based methods cannot identify (Aim 1). Hidden variants also include DNA mutations that arise post-zygotically (“mosaic”) and are not present in blood-derived DNA (Aim 2). We will use long-read based DNA and RNA sequencing methods (PacBio and Oxford Nanopore) on patients with syndromic SBD whose clinical workup (including exome sequencing) has been non-diagnostic. In patients with isolated SBD whose clinical workup has been non-diagnostic, we will apply deep short-read based DNA sequencing of multiple, non-blood derived tissues to identify mosaic variants. This proposal aligns with the stated goals of the NICHD, as our findings will form the basis of new strategies for the diagnosis, treatment, and prevention of human structural birth defects. Our work furthers that goal by identifying previously undetected genetic variation in children with SBD. This is a key first step towards establishing a framework for utilizing these novel technologies in the clinical diagnostic arena.