Functional Investigation of Somatic Variants in Pediatric Epilepsy Using Single-Cell DNA/RNA Assays from Patient Derived Single Cells
Drew Cheng1, Michael Zhu Chen2, August Yue Huang3, Junseok Park3, Heidi Kirsch4, Christopher Walsh5, Diane Shao6
1Harvard University, 2Harvard-MIT Medical Scientist Training Program & Harvard Bioinformatics and Integrative Genomics PhD Program, 3Department of Genetics and Genomics, Boston Children’s Hospital & Department of Pediatrics, Harvard Medical School, 4UCSF Epilepsy Center, UCSF Department of Neurology, 5Department of Pediatrics, Division of Genetics, Boston Children’s Hospital & Howard Hughes Medical Institute, 6Department of Neurology, Boston Children’s Hospital
Objective:
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.
Background:
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).
Design/Methods:
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.
Results:
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.
Conclusions:
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.