Impacts Of CASP9 Missense Mutations On Protein Function And Apoptosis In Human Neural Tube Defects

BackgroundNeural tube defects(NTDs)are complex multifactorial diseases caused by failure of neural tube closure.The genetic factor mainly include gene null or variations associated with biological processes such as differentiation,proliferation,apoptosis,and cell polarity,while environmental factors include folic acid,blood glucose,and retinoic acid,etc.The folic acid deficiency in maternal serum increases the incidence of NTDs.Apoptosis is an indispensable biological process during development.In vitro culture experiments found that closure of neural tube in the cranium of mice requires normal apoptosis,especially at the junction of closure.In addition,there were also studies that reported that genetic defects in the apoptosis pathways resulted in mouse NTDs.These genes include the anti-apoptotic gene Bel10,and the pro-apoptotic genes Apaf1,Casp9,and Casp3.Based on the contribution of apoptosis genes to mouse NTDs,this study intends to investigate genetic contribution of apoptosis gene in human NTDs and interaction between genetic factor and low folic acid environmental factor.Objective1.To explore the genetic contribution of apoptosis associated genes to human NTDs.2.To verify the apoptosis functional changes of apoptosis gene missense mutations detected in human NTDs.MethodsSecond-generation sequencing of target genetic sequences was carried out.355 NTD patients and 225 ethnicity-and region-matched controls were selected.Sequencing was carried out on the exons and highly conserved regions in 14 apoptosis-associated genes.ClustalX,SIFT,and PolyPhen2 were used to carry out bioinformatics analysis of conservation,changes in properties of amino acids,and prediction of functional disruption in the missense mutations that were detected from screening,in order to predict the possible functional changes due to missense mutations.After the construction of wild type and mutant plasmids,NE-4C and HEK293T cells were used as study objects to establish a transient CASP9 mutant cell model.Real-time PCR,western blot,quantitation of caspase activity,and Annexin V-FITC/PI double staining were used to carry out apoptosis functional verification,at the molecular and cellular level,of missense mutations that were predicted through biological analysis to be deleterious.At the same time,the effects of missense mutations on apoptosis under low folic acid levels were examined.Results1.In NTD patients,we detected 28 missense mutations in apoptosis-associated genes,compared with 4 missense mutations i revealed in the control group,and this difference was statistically significant(P<0.05).Therefore,missense mutations were enriched in patients with NTDs.Among these missense mutations,the proportion of missense mutations in the CASP9 gene was 25%(7/28).2.We found that 4 missense mutations in the CASP9 gene(p.G66A,p.R173C,p.R191G,and p.Y251C)occurred in 7 patients with NTDs,and 4 of these patients had p.R191G mutations.The position of the p.G66A and p.Y251C missense mutations was at a highly conserved amino acid,while p.R173C and p.R191G was located at a poorly conserved amino acid.In these 4 missense mutations,the p.G66A mutation was located in the CARD domain of CASP9,while p.R173C,p.R191G,and p.Y251C were located in the catalytic domain,and p.Y251C was located near the catalytic cysteine residue.SIFT and Polyphen2 were used for protein predictions arising from the missense mutations.Results showed that every mutation could show functional disruption by at least one prediction method.According to the recurrence frequency,amino acid position,conservation,and bioinformatics analysis in patients with NTDs,we predicted that the CASP9 missense mutations(p.G66A,p.R191G,and p.Y251C)could affect biological function.3.In the NE-4C and HEK293T transient transfection cell models constructed with exogenous overexpression of wild type or mutant CASP9 plasmids,the protein expression levels of pro-CASP9 and cleaved-CASP9 in CASP9 p.Y251C was significantly decreased,and the activity of the apoptosis enzyme CASP9 was also significantly decreased with this mutation.4.In molecular assays,we found that the CASP9 p.Y251C mutation decreases the levels of cleaved-CASP3 and cleaved-PARP in the intrinsic apoptosis pathway,and decreased the activity of the apoptosis enzyme,CASP3.In cellular experiments,we also found that the CASP9 p.Y251C mutation decreased the number of apoptotic cells.5.Under low folic acid culture conditions,the CASP9 p.R191G and p.Y251C mutations were found to decrease the activity of the apoptosis enzymes CASP9 and CASP3 and decreased the number of apoptotic cells,compared with wild type CASP9.ConclusionWe found that missense mutations in apoptosis-associated genes were enriched in NTD patients and carried out functional verification of CASP9 missense mutations.We found that missense mutations significantly affect protein function and its downstream events,inducing significant apoptosis defects in cells,and it is influenced by the low folic acid environment,which could probably play an important role in the pathological mechanisms of NTDs.