Author(s): Kristin C. Bussey, Clemson University; Kristin C. Bussey, Clemson University; Robert R. H. Anholt, Clemson University; Trudy F. C. Mackay, Clemson University //

ABSTRACT: Mediator subunit 12 (MED12) is a component of the mediator complex, which acts as a co-activator/co-repressor of RNA polymerase II. Mutations in different domains of the MED12 protein are associated with rare genetic disorders, including FG Syndrome, Ohdo Syndrome, and Lujan Syndrome, which are characterized by a range of neurodevelopmental, physical, and behavioral anomalies. It is increasingly recognized that these disorders overlap in symptoms forming a spectrum of MED12-related phenotypes with variable penetrance. However, identifying genetic modifiers of rare diseases is impossible in human populations. To investigate the pathogenicity of MED12 mutations and to identify possible epistatic modifiers, we generated a Drosophila model centered on the fly ortholog of MED12, kohtalo (kto). First, we used RNAi-mediated knockdown of kto and measured locomotion and sleep parameters and startle response as a proxy for sensory motor integration in 3-5 day old flies and 14-16 day old flies, sexes separately.   Using two independent RNAi lines we found that knockdown of kto resulted in reduced nighttime sleep at both ages and in both sexes. Reduction in expression of kto also resulted in increased startle behavior in young flies and a reduction in startle response in 14-16 day old females. After we verified that impairment of kto expression results in phenotypic effects that are analogous to symptoms of MED12 disorders, we generated a CRISPR/Cas9 mediated excision line, which contains an 8kb deletion removing the entire kto locus, to identify epistatic modifiers of these phenotypes.  We will cross this line and its co-isogenic control to lines of the Drosophila Genetic Reference Panel (DGRP), a population of sequenced wild-derived inbred lines and measure startle behavior and sleep phenotypes in F1s. To identify epistatic modifiers of the phenotypic effects of the kto knockout we will perform a genome-wide association analysis across the DGRP on the differences between hybrids from the CRISPR deletion line and its control. Human orthologs of epistatic modifiers of kto will represent candidate genes that may cause variation in the manifestation of MED12-related disorders. This comparative genomics approach provides a general blueprint for similar studies on modifiers of other rare disease genes that have conserved orthologs across phyla.

Source of Funding: Supported by NIH grant GM128974 to TFCM and RRHA