Characterization of a de novo mutation in the GRIN2D gene

NMDA receptors are ligand-gated cation channels. Following binding of glutamate and glycine, they
open and mediate excitatory synaptic transmission in the brain. In our work, we study the case of a
proband, diagnosed with epileptic encephalopathy, severe developmental delay and motor deficits.
Whole exome sequencing identified a de novo mutation in the GRIN2D gene, one of the four genes
that encode the GluN2 subunit. The identified pathogenic variant is GRIN2D c.1999 G>A, p.
Val667Ile; localized to the transmembrane domain of the receptor. A previous report showed that this
mutation leads to profound gain-of-function. Specifically, the channel is 6-times more likely to open
and its current deactivation is 2-fold slower (Li et al, AJHG 2016). We hypothesize that this gain-of-
function leads to neuronal dysfunction. Our goal is to find an allosteric inhibitor of the mutated
protein, in order to correct the aberrant brain activity. Our first step was to create a CRISPR/Cas9
mouse model that replicates the proband’s genomic variant (Grin2d-mice). The mouse phenotypes
will be validated by behavioral, electrophysiological and morphological tests. With the goal of drug
repurposing, we will search for a known drug that is able to correct the activity of the channel. To this
end, we will produce stable cell lines, using viral infection, to express the wild-type NMDAR and the
proband variant. Both groups will be exposed to a small-molecule library consisting of thousands of
substances and analyzed using fluorescent high-throughput screening (HTS). Positive candidates will
be further studied in Grin2d-mice and their therapeutic potential will be examined.