In Vitro Splicing Assay Of NMDAR Subunit Mutations With Idiopathic Epilepsy

Background and PurposeIdiopathic epilepsy(IE),including idiopathic generalized epilepsy(IGE)and idiopathic partial epilepsy(IPE),are caused by genetic causes accounting for 40-60% of all epilepsies.more then 40% of the IE pathogenic genes are unknown.In the pathogenesis of epilepsy,genes encoding ion channels are the most important.Nmethyl-D-aspartate receptor(NMDAR)is a ligand-gated ion channel,which is not only an important regulatory structure of excitatory synaptic transmission,plasticity and excitotoxicity in the central nervous system,but also acts as a target of many antiepileptic drugs.The mutations of NMDAR subunits are associated with a variety of neuropsychiatric disorders,including epilepsy,Alzheimer disease,Huntington disease,depression,schizophrenia,mental retardation,autism,etc.,among which epilepsy is the most important phenotype.The mutations in NMDAR subunits contain missense mutations,truncated mutations,splicing mutations,deletions/insertions,chromosome microdeletions,chromosome translocations and copy number variations et al,among which missense mutations are in the majority.Missense mutations affect the structure of protein throuth amino acid replacement.However,at present,the splcing sites of the coding genes of NMDAR subunits,especially exon mutations,lead to few splcing of IE.The molecular mechanism of pathogenesis is not completely clear and needs further study.In this study,the molecular mechanism of mutations in the coding genes of NMDAR subunits was explored by in vitro splcinganalysis.MethodTwo hundred and eighty-seven patients with idiopathic epilepsy were enrolled and were screened by whole exome sequencing(WES).Then,mutant genes of the NMDAR subunits were analyzed according clinical features of patients and bioinformatics predictions of mutations.Five mutations(GRIN1 c.394-1G>C;GRIN2A c.702C>T;GRIN2B c.1287C>T;GRIN2D c.690C>G and GRIN3 B c.556G>A),which probably affected RNA splicing,were selected in this study.PTarget-GRIN1-E2-E4,pTarget-GRIN2A-E3-E5,pTarget-GRIN2B-E4-E7,pTarget-GRIN2D-E2-E4 and pTarget-GRIN3B-E1-E3 were constructed in the wild type The mutant plasmids were constructed by using SY5 Y cells in vitro and RT-PCR.The mutations of NMDAR subunits were analyzed by Q-PCR.The relative expression of this.Result1.RT-PCR showed that GRIN1 c.394-1G>C disrupted the 3 'receptor site and led to deletion of 2 bases on exon 3,which resulted in early termination of translation and had truncated NR1 protein(p.Lys131fsX21);other mutants(GRIN2A c.702C>T,GRIN2 B c.1287C>T,GRIN2 D c.690C>G and GRIN3 B c.556G>A)did not have significantly abnormal transcriptional bands,and then sequencing results of these mutants showed no effect on the splcing.2.Real-time quantitative PCR showed that the relative quantification of GRIN1 c.394-1G> C mutant was: the relative amount of c.394-1G> C abnormal mRNA was 86.45 ± 2.7% and the relative amount of normal mRNA was 10.64 ± 1.7% Abnormal and normal mRNA relative amount of contrast,the difference was statistically significant.Conclusion1.GRIN1 c.394-1G> C mutation is likely to affect the shear by destroying the original shear receptor site,resulting in truncated NR1 protein(p.Lys131fsX21),which leads to the occurrence of the disease.2.The mutations(GRIN2A c.702C> T,GRIN2 B c.1287C> T,GRIN2 D c.690C> G and GRIN3 B c.556G> A)had no effect on the splicing.