Whole-genome sequencing (WGS) now enables a survey of most of the genome and has resulted in the discovery of two orders of magnitude more de novo variants than exome studies. This has resulted in the identification of several million inherited rare variants per genome. However, identifying ASD-causing mutations from WGS data outside of the ~1 percent of protein coding sequences is challenging because 1) the ‘search space’ is so much larger, and thus, many more mutations occur by chance, and 2) unlike in coding regions, there is no simple code to distinguish deleterious mutations in noncoding sequences; thus, functional mutations must be defined experimentally. In addition, the consequences of mutations and variations in noncoding sequences are often highly dependent on the specific cell type in which nearby genes are expressed. To address these challenges, Joseph Dougherty’s laboratory plans to functionally analyze mutations found in noncoding regions within the Simons Simplex Collection (SSC) WGS data set using a massively parallel reporter assay in ASD-relevant cell-types both in vitro and in vivo.

The primary goal of this project is to identify de novo mutations in noncoding untranslated regions (UTRs). As each UTR is associated with a defined gene, this proposal avoids the complication of predicting the affected target gene, a problem inherent to the study of distal noncoding elements (e.g., enhancers). To identify likely causal UTR mutations, Dougherty will utilize post-transcriptional regulatory element (PTRE) sequencing and a massively parallel reporter assay (MPRA) to measure the impact of UTR variation on mRNA stability and protein translation, using methods developed in Barak Cohen’s and Sergei Djuranovic’s laboratories at Washington University in St. Louis^1-3. These protocols will be combined with additional methods developed by the Dougherty and Cohen labs that enable assessment of these UTR variants in specific cell types in the mouse brain using viral delivery and translating ribosome affinity purification (TRAP)^{4, 5}.

By screening thousands of ASD variants within the SSC, this project will provide a functionally driven interpretation of UTR noncoding ASD variants. UTR mutations that result in a functional haploinsufficiency of the target gene should produce effects similar to those observed in loss-of-function mutations within the affected gene’s coding region, allowing the identification of likely causal UTR ASD mutations. By combining the expertise of the Dougherty, Cohen and Diuranovic laboratories, this project will provide insight into the extent to which UTR mutations contribute to ASD risk.

1. Cottrell K.A. et al. Nat. Commun. 9, 301 (2018) PubMed
2. Maricque B.B. et al. Nucleic Acids Res. 45, e16 (2017) PubMed
3. Kwasnieski J.C. et al. Proc. Natl. Acad. Sci. USA. 109, 19498-19503 (2012) PubMed
4. Heiman M. et al. Cell 135, 738-748 (2008) PubMed
5. Doyle J.P. et al. Cell 135, 749-762 (2008) PubMed