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theses

Tourette Disorder (TD) is a complex neurodevelopmental disorder characterized by the presence of both chronic motor and vocal tics. Psychiatric disorders, particularly Attention Deficit Hyperactivity Disorder (ADHD) and Obsessive Compulsive Disorder (OCD) are often comorbid. Despite the fact that TD is heritable, a specific genetic cause of TD has not been discovered. This is likely because multiple genes are involved, but common molecular pathways are affected. Whole exome sequencing analysis of an extended TD pedigree revealed a heterozygous segregating nonsense mutation in the PNKD gene (c.415C>T) in affected individuals. The PNKD gene has at least three alternatively spliced isoforms (short, medium, and long) with differential tissue expression and cellular localization patterns. Previously we reported that human neurons derived from induced pluripotent stem cells (iPSC) of two members of the TD pedigree heterozygous for the PNKDc.415C>T nonsense mutation had both reduced levels of PNKD and reduced levels of the synaptic active zone protein, RIMS1. RIMS1 regulates Ca2+ induced neurotransmission. Ptacek et. al. 2014 previously reported that PNKD stabilizes RIMS1 protein at the presynaptic terminal. Because RIMS1 is critical in the regulation of neurotransmission and PNKD-long stabilizes RIMS1, we hypothesized that haploinsufficiency of the PNKD-long isoform causes reduction of RIMS1 in subjects heterozygous for the PNKDc.415C>T nonsense mutation resulting in TD symptoms within the studied TD pedigree. To further investigate the role of the PNKDc.415C>T nonsense mutation in the etiology of TD, we created mice which harbored the same nonsense mutation using the CRISPR/CAS9 genome modification system. Additionally, we studied Pnkd knockout mice which were readily available through Jackson labs. We performed behavior and molecular studies on both groups of mice. We hypothesized that both mutant Pnkd-long and -medium alternatively spliced mRNA transcripts with the early translation termination codon would be rapidly degraded through nonsense mediated decay. Because of this, we anticipated mice harboring the Pnkdc.415C>T nonsense mutation would have the same phenotype as Pnkd knockout mice. However, we report here that the mutant Pnkd-medium mRNA transcript is stable while the mutant Pnkd-long transcript is rapidly degraded. Additionally, distinct behavioral differences were discovered between homozygous Pnkdc.415C>T mice and homozygous Pnkd Knockout mice. Due to these findings, we propose an alternative hypothesis postulating that a stable mutant Pnkd-medium isoform may be the cause, or a contributing factor to the TD phenotype in the family studied. Alternatively, haploinsufficiency of Pnkd-long, expressed only in neurons, as well as an aberrantly expressed mutant -medium isoform may each play a separate role in the etiology of TD and possibly OCD and/or ADHD within this multigenerational pedigree.

ETD_8647

Ph.D.

Includes bibliographical references

by Cara Marie Nasello

doi:10.7282/T3H70K06

ETD doctoral

The author owns the copyright to this work.