Screening Of Pathogenic Mutant Genes And Study Of Mutant Proteins In CHARGE Syndrome

Objectives: CHARGE syndrome is a rare congenital anomaly with extremely complex clinical phenotype.The incidence of CHARGE syndrome is about 1/8,500 to1/15,000.The pathogenesis is still unclear and there is no effective cure.The main objective of this study is to screen the pathogenic gene in a family with CHARGE syndrome,identify the pathogenic gene,and explore the effect of mutation on the protein structure and function of related pathogenic gene,so as to clarify its pathogenic mechanism and provide key evidence for clinical prenatal screening,diagnosis and development of reliable therapeutic drugs for CHARGE syndrome.Methods:(1)A family with CHARGE syndrome was collected.According to the clinical diagnostic criteria and treatment guidelines of CHARGE syndrome,the patient was diagnosed with CHARGE syndrome,and the family map was drawn.(2)After whole exome sequencing,pathogenic genes were screened based on mutation harmfulness,recessive inheritance and de novo mutation.Significant enrichment analysis was used to determine the most important biochemical metabolic pathways and signal transduction pathways involved in the mutated pathogenic genes,and Sanger sequencing was used to verify the candidate pathogenic gene mutation sites.(3)The physical and chemical properties,hydrophobic properties,subcellular localization,signal peptide,transmembrane helix structure and secondary structure of the mutant protein were analyzed at the protein level and their differences with the wild type.(4)The three-dimensional protein model of the core region of the candidate pathogenic gene was constructed,and the three-dimensional protein structure model of the candidate pathogenic gene mutation protein was modeled using the established three-dimensional protein structure model to analyze the effect of mutation on the structure and function of the protein.(5)According to the predicted results of proteinprotein interaction,protein-protein docking was performed,and the changes and effects of protein-protein interaction before and after mutation were compared to speculate the pathogenic mechanism in the pathogenesis of CHARGE syndrome.Results:(1)Four pathogenic genes CUBN(c.1952G>C),AMOT(c.1412G>A),TRPM4(c.1169A>T)and CHD7(c.3089A>G)were screened and verified by Sanger sequencing.(2)The physical and chemical properties of the mutant proteins were changed in different degrees.(3)CUBN(Arg651Pro),AMOT(Arg471His),TRPM4(Lys390Met)and CHD7(Asn1030Ser)mutant proteins had increased hydrophobicity,among which TRPM4(Lys390Met)had the most significant increase.(4)TRPM4(Lys390Met)mutation caused changes in 5 of its 8 transmembrane helices.(5)The mutant proteins CUBN(Arg651Pro),AMOT(Arg471His),TRPM4(Lys390Met)and CHD7(Asn1030Ser)had different effects on the secondary structure of the protein.(6)The mutation site of the pathogenic gene caused a significant change in the threedimensional conformation of the protein.(7)The mutant sites caused changes in the interaction between CUBN-AMOT,CHD7-AMOT and CUBN-Amot-CHD7 proteins.Conclusions:(1)CUBN(c.1952G>C),AMOT(c.1412G>A),TRPM4(c.1169A>T)and CHD7(c.3089A>G)gene missense mutations were screened by whole exome sequencing.(2)The physicochemical properties of the mutant proteins Arg651 Pro,Arg471His,Lys390 Met and Asn1030 Ser changed in different degrees.(3)Protein hydrophobicity analysis showed that CUBN(Arg651Pro),AMOT(Arg471His),TRPM4(Lys390Met)and CHD7(Asn1030Ser)mutant proteins had increased hydrophobicity,among which TRPM4(Lys390Met)had the most significant increase.The change of hydrophobicity may affect the 3D structure of the protein.(4)TRPM4(Lys390Met)mutation caused conformational changes in 5 out of 8 transmembrane helices of TRPM4.A change in the position of the transmembrane helical region due to a change in the hydrophobic nature of the mutant protein was considered.(5)Protein secondary structure prediction showed that CUBN(Arg651Pro),AMOT(Arg471His),TRPM4(Lys390Met)and CHD7(Asn1030Ser)mutant proteins changed its secondary structure.(6)Three-dimensional models of wild type and mutant CUBN,AMOT,TRPM4 and CHD7 proteins were successfully constructed by homology modeling software.After superposition and comparative analysis of the three-dimensional structures of the respective wild type and mutant proteins,it was found that there were important domain changes in CUBN and CHD7 mutant proteins.AMOT,TRPM4 and CHD7 mutants also caused significant changes in protein structure.(7)Protein-protein interaction analysis showed that CUBN-AMOT,CHD7-AMOT and CUBN-AMOT-CHD7 proteins could interact with each other,and the protein interaction contact area,hydrophobic interaction,hydrogen bond,salt bridge and cation-π interaction changed greatly after mutation.These results suggest that the interaction pattern of the mutant proteins may be one of the key pathogenic mechanisms of CHARGE syndrome.