Date of Award
1-11-2022
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Human genetic studies have identified a large number of patient-derived de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects since the impact of regulatory variants is highly dependent on the cellular context. In particular, current investigations have made significant investments in sequencing the genomes of autism spectrum disorder (ASD) families as a diagnostic method and to further understand the genetic architecture of this complex neurodevelopmental disease. These studies have revealed an enrichment of mutations in the untranslated regions (UTRs) of genes, which are noncoding regulatory regions, in ASD genomes. Assessment of these variations poses a challenge since these regions do not follow the triplet code and, even with prediction algorithms for RNA secondary structure or motif loss, the impact of these mutations must be defined experimentally. Furthermore, it is expected that only a subset fraction of these variants will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of thousands of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. To address these challenges, I generated a 3′UTR MPRA library with variants from ASD patients to functionally screen several hundred mutations in parallel. For this library, I implemented a Cre-dependent design to control expression and allow for cell type-specificity. I first validated the system in vitro then developed a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. Ultimately, I measured the effect of >500 3′UTR variants in excitatory neurons in the mouse brain and defined several ASD mutations that substantially altered transcript abundance. This new technique should enable robust functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.
Language
English (en)
Chair and Committee
Joseph Dougherty
Recommended Citation
Lagunas Jr, Tomas, "Massively Parallel Assessment of the Functional Effects of De Novo Autism Spectrum Disorder Patient-Derived Genetic Variants in 3’ Untranslated Regions" (2022). Arts & Sciences Electronic Theses and Dissertations. 3247.
https://openscholarship.wustl.edu/art_sci_etds/3247