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.