Investigation of X-linked Noncoding Mutations in Autism

About This Grant

ABSTRACT Autism is a highly heritable neurodevelopmental condition, and males are over-represented in autism diagnoses. The X chromosome is enriched for genes associated with autism, suggesting it may contribute to the observed male bias. Loss-of-function coding mutations within these genes are often lethal in males, but females experience monogenic forms of autism. It is then possible that mild mutations within these genes are sufficient to cause autism in males but spare mosaic females. Males with idiopathic autism are enriched for maternally inherited noncoding mutations in cis-regulatory elements (CRE) proximal to these genes. The broad objective of this proposal is to characterize these X-linked CREs and autism-associated mutations found within them. MECP2 is a dosage-sensitive gene where loss- or gain-of-function mutations cause neurological disorders. Although MeCP2 levels are tightly controlled in typical individuals, the mechanisms by which its CREs, such as the promoter, control gene expression remain unclear. There are at least four mutations within the MECP2 promoter that are maternally inherited and segregate with autism in males. Using CRISPR-Cas9 technology, these mutations will be independently edited into the endogenous MECP2 promoter in human iPSCs (AIM 1). After differentiating these iPSCs into neurons, these mutations will be evaluated for their impact on MeCP2 levels and two representative target genes. For mutations that significantly alter MeCP2 levels, deep RNA sequencing will determine the effects of these mutations on the molecular phenotype of neurons. This dosage sensitivity could extend past MECP2. There are thousands of mutations in male autism probands that are inherited from the mother and localize to 197 different X-linked CREs in open chromatin in excitatory neurons. A majority of these CREs are proximal to 57 different X-linked genes known to cause neurological disease. Using a massively parallel reporter assay, these mutations will be functionally assessed in an unbiased, high-throughput screen (AIM 2). Downstream analyses will determine which genes and which specific regions are most impacted by autism-associated noncoding mutations. The top-ten autism-associated mutations that disrupt CRE activity will then be validated using a luciferase reporter assay. The overall impact of this proposal is to broaden the spectrum of known mutations that cause autism, addressing some of the missing heritability of autism. Additionally, by studying 197 X-linked CREs, this proposal will provide insight into the regulation of 57 separate X-linked genes known to cause neurological disorders, and it will provide a framework for investigating noncoding mutations. These results will enhance the current understanding of noncoding mutations and how they contribute to neurological disease.