Genetic Validation Of The Causative Gene And Functional Mechanism Of Asparagine Synthetase Deficiency

Objective: Asparagine Synthetase Deficiency(ASNSD)(MIM 615574)is a rare neurometabolic disorder caused by mutations in the asparagine synthetase(ASNS)(MIM108370)gene,which is autosomal recessive.Clinical manifestations include progressive microcephaly,psychomotor retardation,progressive brain atrophy and drug-refractory epilepsy.In this study,we screened an ASNSD family for the causative gene,combined with Sanger sequencing for mutation site verification of this causative gene to determine the ASNS mutation type in patients,and also investigated the relationship between genotype and phenotype.Subsequently,cellular functional studies were performed on the three ASNS missense mutation loci identified to reveal their possible pathogenic mechanisms leading to ASNSD,providing a theoretical basis for early diagnosis,prenatal diagnosis,and effective treatment of children with ASNSD for the purpose of eugenic reproduction.Methods: In this study,after whole-exome resequencing to identify the pathogenic gene ASNS,Sanger sequencing was used for the validation of mutant loci.Pathogenicity prediction analysis of mutant loci was performed using bioinformatics software Poly Phen-2,Mutation Taster,PROVEAN and gnom AD,combined with multi-species homology comparison and 3D protein modeling to predict mutant protein alterations and initially identify pathogenic mutant.Then three missense mutations c.250A>T/p.M84 L,c.1211G>A/p.R404 H,c.1643C> T/p.S548 F for cell functional studies.Family co-dissociation analysis showed that c.250A>T/p.M84 L,c.1643C>T/p.S548 F from the mother and c.1211G>A/p.R404 H from the father.Firstly,the ASNS wild-type eukaryotic expression vector was constructed and the mutant eukaryotic expression vector was induced by point mutation kit.HFF-1 were transfected with non-specific control,wild-type and mutant ASNS eukaryotic expression vectors,and cultured in normal medium,histidine deficient medium and asparaginase supplemented medium.After 24 h and 48 h,total RNA and protein were extracted.The expression of HFF-1 ASNS m RNA was detected by fluorescence quantitative PCR(q PCR),and the expression of ASNS protein was detected by Western blotting.Cell Challenge experiments detect the growth of non-specific,wild-type and mutant cells cultured with asparaginase added medium and normal medium at 24 h,48h,and 72 h.Results: By using Whole exome sequencing and Sanger sequencing were used to verify that the proband carried three different missense mutations in ASNS,(c.250A>T/p.M84 L,c.1211G>A/p.R404 H,c.1643C>T/p.S548F).Patient 2 carried two missense mutations in ASNS(c.1211G>A/p.R404 H,c.1643C>T/p.S548F).Two mutations were predicted by bioinformatics analysis software(c.1211G>A/p.R404 H,c.1643C>T/p.S548F),c.250A>T/p.M84 L was a benign mutation.Multi-species homology comparison showed that R404 and S548 were located in highly conserved regions of the protein and M84 was located in a non-conserved region.Comprehensive analysis showed that there was no correlation between genotype and phenotype of ASNS mutation sites.3D protein model predictions show that mutant do not lead to structural changes in the protein.Cellular functional studies of the three missense mutations revealed that the expression and HFF-1 growth of M84 L,R404H,and S548 F in the WT and mutant groups were not statistically significant in normal medium.There was no statistical difference in M84 L,R404H and S548 F expression between WT and mutant groups in histidine deficient medium.In the medium supplemented with asparaginase,there was no statistically significant difference in the expression of M84 L,R404H,and S548 F in both WT and mutant groups,but there was a statistically significant difference in the growth of the WT and R404 H and S548 F groups.Conclusion: In this study,three ASNS missense mutations identified in ASNSD patients were reported,and perform cellular functional studies,confirming that the three variants had no effect on the regulation of the ASNS itself,nor on the basal m RNA and protein levels of ASNS.However,R404 H and S548 F hindered cell growth in the absence of extracellular asparagine.Therefore,we conclude that R404 H and S548 F are pathogenic mutation in ASNSD.At present,the pathogenesis of ASNSD is still unclear,and this study can provide more information for the pathogenesis and genetic intervention of ASNSD.