Project III - Comprehensive Genomic Evaluation of Structural Birth Defects

项目摘要 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 国际吧大多数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。基于阵列的 技术鉴定了导致SBD的拷贝数变异，并使基因组范围内的关联成为可能。 问题研究大规模并行的“下一代”（NGS）方法对大量短（50-300个碱基）序列进行测序， 对）DNA片段，允许对所有编码区（外显子组）或整个 基因组一次。血液来源DNA的外显子组和基因组测序确定了 ~50%的儿童为多系统综合征型SBD。然而，大多数SBD的病因-无论是孤立的， 综合征-仍然未知。 其潜在原因是基于短读段的血液来源的DNA测序不提供对血液来源的DNA的测序。 对个体基因组的全面了解。 该提案的目标是在SBD儿童的前瞻性队列中使用新技术， 目前的方法无法识别的遗传变异。这些“隐藏的”变体包括重复、倒置、 重复扩增/收缩，以及标准的基于短读段的方法不能进行的表观遗传修饰 确定（目标1）。隐藏的变异还包括合子后出现的DNA突变（“嵌合体”）， 存在于血液来源的DNA中（目的2）。 我们将对患者使用基于长读段的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）为非诊断性。患者 对于临床检查为非诊断性的孤立性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鉴定嵌合变体。 这项建议符合NICHD的既定目标，因为我们的研究结果将成为新战略的基础， 诊断，治疗和预防人类结构性出生缺陷。我们的工作通过以下方式推进这一目标： 在患有SBD的儿童中识别先前未检测到的遗传变异。这是关键的第一步， 建立在临床诊断竞技场中利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.