Deep mutational scanning of CHD2 for variant interpretation in neurodevelopmental disorders

项目摘要 CHD2中的新生致病性变体是神经发育障碍的最常见原因之一。 这些疾病包括难治性癫痫、智力残疾和自闭症谱系障碍。在 此外，在生命后期获得的体细胞CHD2变体在某些类型的白血病中高度复发。为 这两种类型的条件，损失的CHD2功能是可能的致病机制，大多数变异是 截断。然而，也有证据表明，错义变体可以破坏或废除CHD2功能， 导致功能丧失，尽管它们更难解释，而且许多被归类为不确定性的变体。 显著性（VUS）。VUS是人类遗传学家面临的最大挑战之一， 临床基因检测报告中返回的一类变异，部分原因是我们对如何 错义变体影响蛋白质功能。我们的研究通过设计一个双峰来解决这些挑战。 使用下游蛋白确定VUS对CHD2功能的影响的高通量测定 丰度（Aim 1）和CHD2附加特征（Aim 2）。我们将利用CHD2是一种染色质的事实， 重塑，从而影响下游蛋白质表达和DNA甲基化景观， 开发一种高通量、多模式的方法来鉴定改变CHD2功能的变体。我们还将 使用pegRNA池实施引物编辑以产生30种CHD2变体，并使用 我们希望在未来的工作中扩展到数百个CHD2变体。我们的工作和方法具有广泛的吸引力，因为它可以 在未来扩展到包括其他NDD和癌症。最后，本研究的目标是开发一个 实验范例，可以扩展到解决所有CHD2 VUS，为患者提供答案， 家庭，并确定那些有资格在未来接受精确治疗的人。

Project summary De novo pathogenic variants in CHD2 are one of the most common causes of the neurodevelopmental disorders (NDDs) that include refractory epilepsy, intellectual disability and autism spectrum disorders. In addition, somatic CHD2 variants, acquired later in life, are highly recurrent in certain types of leukemias. For both types of conditions, loss-of-CHD2 function is the likely pathogenic mechanism, and most variants are truncations. However, there is also evidence that missense variants can disrupt or abrogate CHD2 function, leading to loss-of-function, though they are harder to interpret, and many are classified as variants of uncertain significance (VUS). VUS are one of the biggest challenges for human geneticists, and represent the biggest class of variants returned on clinical genetic test reports, in part because we have a poor understanding of how missense variants impact protein function. Our research addresses these challenges by designing a bimodal high-throughput assay to determine the impact of VUS on CHD2 function using downstream protein abundance (Aim 1) and the CHD2 episignature (Aim 2). We will leverage the fact that CHD2 is a chromatin remodeler and thus affects both downstream protein expression and the DNA methylation landscape to develop a high-throughput, multimodal approach to identify variants that alter CHD2 function. We will also implement prime-editing using pools of pegRNAs to create 30 CHD2 variants and multiplex this assays, with a view to scale to hundreds of CHD2 variants in future work. Our work and approach has broad appeal as it can be scaled to include other NDDs and cancers in the future. Finally, the goal of this research is to develop an experimental paradigm that can be scaled to resolve all CHD2 VUS, providing answers for patients and families, and identifying those individuals that will be eligible for precision therapies in the future.