Authors :
Presenting Author: Hongjie Yuan, MD, PhD – Emory University School of Medicine
Scott Myers, PhD – Emory University School of Medicine; Riley Perszyk, PhD – Emory University School of Medicine; Jing Zhang, Ms – Emory University School of Medicine; Sukhan Kim, Ms – Emory University School of Medicine; Kelsey Nocilla, Ms – Emory University School of Medicine; James Allen, Ms – Emory University School of Medicine; Jennifer Bain, PhD – Columbia University Irving Medical Center; Johannes Lemke, MD – University of Leipzig Medical Center; Dennis Lal, PhD – Cleveland Clinic; Timothy Benke, MD, PhD – University of Colorado School of Medicine; Stephen Traynelis, PhD – Emory University School of Medicine
Rationale:
Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions, including epilepsy. The data have provided diagnosis of rare diseases, including pathogenic missense variants in
GRIN genes encoding N-methyl-D-aspartate receptors (NMDARs). Functional analysis is needed to assess multiple properties to understand how
GRIN variants impact receptor function in neurons. One can use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here we describe an analytical and comprehensive framework by which to categorize
GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to
GRIN variants identified in patients and the general population.
Methods:
Six assays were performed that assess glutamate potency, glycine potency, Mg
2+ potency, open probability, deactivation time course and surface expression. The first four assays were generated
via two-electrode voltage clamp (TEVC) recordings from
Xenopus oocytes (PMID27839871). Two assays were from whole cell voltage clamp recordings and
beta-lactamase reporter assay performed on transfected HEK cells (PMID27839871). The effects of a GRIN variant on the relative non-synaptic and synaptic charge transfer were estimated as previously described (PMID34227748, 37000222).
Results:
We functionally assessed patient-derived GRIN2B missense variants and benign GRIN2B variants from healthy individuals (gnomAD). Analysis of patient-derived variants yielded a wide range of results, with single or multiple parameters changing in a manner that should increase or decrease overall synaptic function, while NMDARs hosting benign variants showed properties like WT receptors. We proposed thresholds for each assay for discrete determination of GoF and LoF. We classify GRIN variants into six categories: Likely GoF, Possible GoF, Likely LoF, Possible LoF, Indeterminant, and No Detectable Effect. We use a discrete classification method, which is similar to the approach used by ACMG to assess pathogenicity, as the primary means for determining GoF and LoF, with net synaptic and non-synaptic charge transfer as supporting information used to classify variants that are less clear. To further illustrate the utility of this approach, we have applied it to published and new variants for which a full data set (all six assays) is available. Conclusions:
We have developed a suite of
in vitro assays in heterologous expression systems that assess si features that are determinants of NMDAR function and applied this to over 100 variants. These results have revealed a wealth of information about
GRIN disorders, patient characteristics and prognosis, and potential treatment strategies. Based on our approach, we expect that the methodological framework established should facilitate advances in clinical development of targeted approaches to treating
GRIN disorders.
Funding:
Simon’s, GRIN2B, CureGRIN Foundations; NIHNS111619, HD082373, AG075444, AG072142, NS108874;
GRIN Therapeutics