Comprehensive Study On Clinical Phenotype,functional Influence And Targeted Therapy Of De Novo GRIN Variants In NMDA Receptor M2 Channel Pore-forming Loop

Background: N-methyl-D-aspartate(NMDA)receptors bind synapticallyreleased glutamate and mediate a slow component of excitatory synaptic transmission in the brain,thereby exerting a key role in brain development and function.It is intolerant to missense variation in the M2 reentrant loop.Genetic variation in the GRIN genes encoding NMDA receptor subunits are associated with various neurological diseases.Methods: Here we summarize the clinical information for 18 patients(from Center for Functional Evaluation of Rare Variants,Emory University School of Medicine,USA and Pub Med Databases by Dec.31,2018)harboring 13 de novo missense variants in GRIN1,GRIN2 A,GRIN2B that alter residues in the M2 reentrant loop,a region that lines the pore and is intolerant to missense variation.And we evaluate the functional effects on NMDA receptors of these variants by molecular biology and electrophysiology experiments.Subsequently,we evaluate the potency of several FDA-approved NMDA receptor-targeted drugs to GRIN mutations located in M2 reentrant loop.Results: All patients in this study including 6 males and 5 females present onset age from 3 days to 7 months.These de novo variants were identified in patients with various neurological disorders including epilepsy,developmental delay,intellectual disability,hypotonia/hypertonia,autism,speech disorder and dysmorphic features.And neurological developmental delay with epilepsy is the dominant disorder.Evaluation of the pharmacological properties and biophysical characteristics show that these variants can have modest changes in agonist potency,proton inhibition,current amplitude,response time course,mean open time of single channel,single channel conductance and open probability.However,voltage-dependent magnesium inhibition was significantly reduced in all variants.Incorporation of a single copy of a mutant subunit into the NMDA receptor produced a dominant reduction in magnesium inhibition for some variants.Additionally,these M2 variants-containing NMDA receptors show differential responses to NMDA receptor-targeted drugs.Conclusions:(1)Patients perform epilepsy and neurological developmental disorders during childhood,without obvious causes,should be considered as genetic variants in NMDA receptors probably.Relevant gene test is necessary.(2)These variants encoding M2 reentrant loop obviously alter many functions of NMDA receptor and produce complex and varied effects on NMDA receptor properties,which may underlie the patients' phenotypes.(3)Actually,it is important and necessary to study the effect of mutation in NMDAreceptor from different aspects.(4)Specific drug screening for genetic variants in NMDA receptors has an important clinical guiding role,especially in targeted therapy.