Pathogenicity Analysis Of GPRASP2 Gene Mutations In Epilepsy Patients

Background and PurposeEpilepsy is a common neurological disorder that affects about 5‰of the world’s population.The etiology of epilepsy is complex,and current studies believe that it is closely related to genetics.So far,more than 1000 genes have been found to be related to epilepsy and we found that gene GPRASP2 is a new candidate gene of epilepsy.The GPRASP2 gene encodes G protein-coupled receptor-associated sorting protein 2,a member of a family that regulates the activity of G protein-coupled receptors（GPCRs）.Its encoded protein has been shown to interact with several GPCRs,including the M1muscarinic acetylcholine receptor and calcitonin receptor.GPRASP2 gene is widely expressed in multiple human tissues,with the highest expression in brain.Male mice hemizygous for a knock-out allele showed increased body weight,autism-like behavior,alterations hippocampal pyramidal neuron dendrite complexity,impaired glutamate transmission,impaired spatial learning,and enhanced long-term hippocampal depression.However,the relationship between GPRASP2 gene variants and epilepsy was not determined.By analyzing the pathogenicity of GPRASP2 gene mutations,the clinical characteristics of patients with GPRASP2 gene mutations and the relationship between GPRASP2 gene and epilepsy,this study suggested that GPRASP2 gene might be a potential pathogenic gene for epilepsy.MethodsIn this study,clinical data of patients with epilepsy were collected from multiple medical centers,and epileptic patients with GPRASP2 gene mutation were screened by whole exome sequencing.All samples with GPRASP2 gene mutation were verified by Sanger sequencing.The clinical characteristics of epileptic patients with GPRASP2gene mutation were analyzed and summarized.The secondary protein structure map was made to analyze the location characteristics of the mutation sites.The conservation of the sites was analyzed by the sequence alignment between different species and software were also used to analyze the pathogenicity and conservation the sites.Hydrogen bond changes of different missense mutations were predicted by Py MOL.Fisher’s exact test was used to compare the frequency of GPRASP2 variation in different populations in the study group and gnom AD database,and statistical analysis was performed.Those methods are used to explore the relationship between GPRASP2gene mutation and epilepsy.ResultsFive novel variants in GPRASP2 were identified in five unrelated cases by conducting trio-based whole exome sequencing in 347 patients with epilepsy.All the five patients had missense mutations,and the age of onset ranged from 1 year to 10years.Four patients were diagnosed with partial epilepsy,and one patient was diagnosed with febrile seizure,with Case 5 having the most severe clinical phenotype.Among the variants,Ser657Arg was located in the armadillo-like repeat domain,Ala260val was located in the GASP motif,and the other variants were located between the domain and motif or between GASP motifs.The sequence comparison among different species suggested that each point was conservative.In terms of software prediction,Ala260Val,Arg322Gly,Ser485Pro and Gln556Arg had at least one software prediction for its harmful effects.And Ala260Val,Gln556Arg,Ser485Pro had software prediction conservatism.Py MOL predicted that Ser657Arg had hydrogen bonds changes and protein structural stability decreased at all sites,among which Arg322Gly protein stability decreased the most（DDG=-1.73Kcal/mol）.In the gnom AD database,the frequencies of these mutations were all less than 0.005,among which,Ala260val and Ser657Arg had no frequency in the population,Arg322Gly,Ser485Pro,Gln556Arg mutations had a very low frequency in the population(5.46×10^-6,1.64×10^-5,2.18×10^-5).Statistical analysis showed that the GPRASP2variants identified in this study was statistically significant between our cohort and the two controls for gnom AD.Conclusions1.GPRASP2 gene is potentially a new candidate pathogenic gene of epilepsy.2.Patients with GPRASP2 variants presented partial epilepsy or febrile seizure.3.Most patients had a good response to antiepileptic drugs.4.Variants leading to important functional region sequence change or obvious protein damage may cause a severer phenotype.