Functional Investigation of Somatic Variants in Pediatric Epilepsy Using Single-Cell DNA/RNA Assays from Patient Derived Single Cells (P3-9.009)

Abstract

<h3>Objective:</h3> Genetic diagnosis for epilepsy and other neurological disorders are not optimized to detect somatic mutations, representing a significant unsolved challenge in neurology. Our group identified candidate somatic variants from the Epi4K consortium, a large cohort of epilepsy trio exomes, using MosaicHunter, a sensitive framework to identify candidate somatic variants. Here, we analyze a specific loss-of-function somatic single nucleotide variant (sSNV) in ARHGAP31 to determine its functional effect as a potential novel epilepsy gene. <h3>Background:</h3> Once somatic variants have been identified and characterized in-silico, their functional and biological consequences must be experimentally validated. Here, we describe a clinical case with somatic candidate stop-gain variant in ARHGAP31, and use a patient cell line to investigate ARHGAP31, a gene that plays a key role in cellular signaling by regulating proteins Cdc42 and Rac1 (Lamarche-Vane and Hall, 1998). <h3>Design/Methods:</h3> To determine the effect of our ARHGAP31 variant on ARHGAP31 gene expression, concurrent genotyping and RNA expression analysis of individual patient-derived lymphoblasts was performed using a single-cell workflow with primers for Sanger sequencing and probes for digital droplet PCR (ddPCR). Additionally, we analyzed Epi25K, a cohort of exome sequencing data from epilepsy cases and controls, to determine whether ARHGAP31 somatic variants are preferentially present in epilepsy cohorts. <h3>Results:</h3> We hypothesized that wild-type cells express ARHGAP31 at a higher level than cells carrying the sSNV and report that in a population of 13 patient single-cells expressing ARHGAP31 at the highest level, 100% (13/13) were wild-type genotype (p-value = 0.00005). Additionally, we report several loss-of-function somatic variants in ARHGAP31 in large cohort analysis from Epi25K. <h3>Conclusions:</h3> Based upon single-cell gene expression data and large epilepsy cohort data, we present evidence for ARHGAP31 as a new epilepsy candidate gene. Clinically, this work is foundational to future efforts to determine additional somatic mutations which contribute to pediatric epilepsy. <b>Disclosure:</b> Mr. Cheng has nothing to disclose. An immediate family member of Mr. Chen has received personal compensation for serving as an employee of Lathrop &amp; Gage. Dr. Huang has nothing to disclose. Junseok Park has nothing to disclose. The institution of Dr. Kirsch has received research support from Ricoh, Inc. Dr. Walsh has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Maze therapeutics. Dr. Walsh has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Third Rock Ventures. Dr. Walsh has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Flagship Pioneering. Dr. Walsh has received personal compensation in the range of $10,000-$49,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Annals of Neurology. Dr. Shao has nothing to disclose.