Authors :
Presenting Author: Azalea Lee, BA – Emory University
Junghwa Lim, BA – Lead Research Specialist, Emory University School of Medicine; Ying Zhou, PhD – Postdoctoral Researcher, Emory University School of Medicine; Michael Epstein, PhD – Professor, Emory University School of Medicine; David Cutler, PhD – Professor, Emory University School of Medicine; Emily Allen, PhD – Assistant Professor, Emory University School of Medicine; Zhexing Wen, PhD – Assistant Professor, Emory University School of Medicine; Peng Jin, PhD – Professor, Emory University School of Medicine
Rationale:
Fragile X syndrome (FXS), the most common inherited cause of intellectual disability (ID) and the leading monogenic cause of autism spectrum disorder (ASD), is caused by a CGG trinucleotide repeat expansion in the fragile X messenger ribonucleoprotein 1 (
FMR1) gene that leads to the functional loss of fragile X messenger ribonucleoprotein (FMRP). Twelve percent of children with FXS experience seizures and are more likely to have ASD, severe ID, and severe cognitive and language impairment compared to FXS patients without seizures (Berry-Kravis et al., 2021).
We utilized multiple analytical methods of whole genome sequencing data to identify potential genetic modifiers of seizures in FXS and conducted functional testing of those genes in Drosophila and human-induced pluripotent stem cell (hiPSC)-derived models.Methods:
We analyzed whole genome sequencing data in a cohort of 148 FXS patients (67 with seizure, 81 with no seizures) using multiple methodologies, including the detection of mutations in genes associated with epilepsy and rare-variant association testing (burden test and sequence kernel association test [SKAT]). The genes potentially modifying the seizure phenotype in FXS were then tested in Drosophila by RNA interference and in hiPSC-derived neurons. We used the vortex assay to measure seizure phenotypes in Drosophila and microelectrode array to measure the electrophysiological activity of neurons.
Results:
We identified mutations in several genes known to be associated with epilepsy, including CHD2, SLC9A6, KCNA1, DCX, DEPDC5, DNM1L, and NEXMIF, present in FXS patients with seizures. We also identified novel potential modifiers using rare-variant association testing, which included GK5, RGPD2, TTLL4, PDE10A, DNAJA1, EIF4B, and PXDN. In Drosophila, compared to controls, flies with Fmr1 knockdown (FXS flies) show a significantly increased rate of seizures. We observed that knocking down CHD2, SLC9A6, DEPDC5, GK5, RGPD2, and TTLL4 all independently ameliorated seizure phenotypes in FXS flies. Knocking down KCNA1 interestingly led to increased seizures. In hiPSC-derived neurons, FXS neurons displayed hyperexcitability measured by increased firing rate and average number of bursts compared to control neurons. The knockdown of CHD2 rescued the phenotype of increased firing rate in FXS neurons with no significant difference from those of control neurons, showing that knocking down CHD2 leads to the amelioration of seizure phenotypes in human neuronal models of FXS as was in flies.
Conclusions:
We have identified potential genetic modifiers of seizures in FXS by combining the findings of different methods of WGS analysis, followed by in vivo and in vitro functional modeling in Drosophila and hiPSC-derived neurons, respectively. Examining the unique modifiers of seizures in the setting of FXS can help better understand the underlying mechanisms as well as identify novel targeted treatments.
Funding:
NIH U54NS091859 and P50HD104463