Gene-Environment interactions in Autism

摘要 孤独症谱系障碍（ASD）是一组神经发育疾病，其特征是社交功能受损， 互动，重复或限制性行为，以及沟通困难。ASD非常普遍， 在美国，每54名儿童中就有1名受到影响。全基因组和外显子组测序研究确定了192个高 相信ASD相关基因，其中许多在大脑发育过程中的各种细胞谱系中早期表达。 皮质发育，包括神经祖细胞、未成熟和成熟的神经元和神经胶质细胞。此外，本发明还提供了一种方法， GWAS研究表明，存在非编码基因组变异，有助于ASD表型。 小鼠暴露于环境中存在的化学物质，包括双酚A（BPA），导致ASD样 表型，大脑皮层细胞组成的改变，以及 转录因子（TF）参与ASD的基因。基于这些观察，我们假设， 存在于不同个体的非编码基因组中的序列变异，当改变调节基因时， 序列，可能会影响转录因子与其靶位点的相互作用，以响应环境化学品。 在携带特定序列变异的个体中，表型效应可能较弱或检测不到， 暴露于环境化学品可能会放大这些变异体与TF相互作用的影响， 随后的表型。为了验证这些假设，我们建议使用从以下来源获得的iPSC集合： 来自不同性别、年龄和种族背景的正常和ASD个体。这些iPSC将被用于生长 大脑皮质类器官，在分化过程中的不同时间暴露于BPA 来改变不同细胞类型的神经谱系中的基因表达。单核（sn）RNA-seq和snATAC- seq将被用于分析TF占有率和基因表达在特定的细胞群体中， 在存在或不存在BPA的情况下，脑类器官的分化。这将使我们能够监测 BPA对不同神经细胞谱系的分化途径和相对比例的影响。然后我们将确定 不同iPSC产生的脑类器官之间的差异BPA响应性ATAC-seq峰， 与ASD相关的细胞分化和基因表达表型。我们希望这些差异 ATAC-seq峰将对应于不同iPSC系的调控序列中存在的序列变体 影响ASD中特定基因的表达。这将使用大规模并行报告程序进行测试 在对应于受影响细胞类型的细胞系和脑类器官中进行MPRA。的作用 将使用单碱基无瘢痕基因组编辑进一步测试基因表达中的特定SNP。最后 这些BPA反应性SNP对自闭症表型的可能贡献将通过进行以下分析： snATAC-seq在来自ASD患者的死后脑样品中的应用。这些结果将填补我们的重要空白， 了解基因组变异体可以对存在于细胞中的化学物质作出反应的基本原理， 环境影响神经细胞的谱系定型并引发ASD症状。

ABSTRACT Autism spectrum disorder (ASD) is group of neurodevelopmental conditions characterized by impaired social interactions, repetitive or restrictive behaviors, and difficulties with communication. ASD is highly prevalent, affecting 1 in 54 children in the US. Whole genome and exome sequencing studies identified 192 high confidence ASD-associated genes, many of which are expressed early in various cell lineages during brain cortex development, including neural progenitors, immature and maturing neurons, and glial cells. In addition, GWAS studies suggest the existence of non-coding genome variants that contribute to ASD phenotypes. Exposure of mice to chemicals present in the environment, including bisphenol A (BPA), result in ASD-like phenotypes, alterations in the cellular composition of the brain cortex, and changes in the binding of transcription factors (TFs) in genes implicated in ASD. Based on these observations we hypothesize that sequence variants present in the non-coding genome of different individuals, when altering regulatory sequences, may influence the interaction of TFs with their target sites in response to environmental chemicals. Phenotypic effects may be weak or undetectable in individuals carrying specific sequence variants but exposure to environmental chemicals may amplify the effect of these variants on their interaction with TFs and the ensuing phenotypes. To test these hypotheses, we propose to use a collection of iPSCs obtained from normal and ASD individuals from different sex, age, and racial backgrounds. These iPSCs will be used to grow cerebral cortical organoids, which will be exposed to BPA at different times during the differentiation process to alter gene expression in different cell types of neural lineages. Single nucleus (sn) RNA-seq and snATAC- seq will be employed to analyze TF occupancy and gene expression in specific cell populations during the differentiation of brain organoids in the presence or absence of BPA. This will allow us to monitor the effect of BPA on differentiation pathways and relative ratios of different neural cell lineages. We will then identify differential BPA-responsive ATAC-seq peaks among brain organoids arising from different iPSCs that correlate with cellular differentiation and gene expression phenotypes related to ASD. We expect that these differential ATAC-seq peaks will correspond to sequence variants present in regulatory sequences of different iPSC lines that affect the expression of specific genes involved in ASD. This will be tested using massively parallel reporter assays (MPRAs) in cell lines corresponding to the affected cell type, and cerebral organoids. The role of specific SNPs in gene expression will be further tested using single-base scarless genome editing. Finally, the possible contribution of these BPA-responsive SNPs to autism phenotypes will be analyzed by performing snATAC-seq in post-mortem brain samples from ASD patients. These results will fill an important gap in our knowledge of the fundamental principles by which genome variants can respond to chemicals present in the environment to affect lineage commitment of neural cells and elicit ASD symptoms.