The Research On Second-Generation Sequencing And Mutation Analysis Of 5 Patients With Gitelman's Syndrome Caused By Complex Heterozygous Mutation Of SLC12A3 Gene

Objective:To analyze the clinical and genetic features of 5 patients clinically diagnosed with Gitelman's Syndrome(GS),This research also included data of two patients clinically diagnosed with hypokalemia,with related genes sequenced with second generation sequencing.Materials and methods:This research enrolled 7 patients with hypokalemia,5 clinically diagnosed with Gitelman's Syndrome and 2 not,who have been admitted to the Department of Endocrinology the First Affiliated Hospital of Soochow University from 2017 to 2019.General clinical data and relevant laboratory results of the patients were collected and DNA samples from peripheral blood of the patients and their families were also collected to search for sequence genes associated with hereditary diseases by the second-generation sequencing.The allele frequency and related pathogenicity reports of the sequencing results were searched.Bioinformatic softwares SIFT,Polyphen2 and Mutation taster were used to predict gene function of missense mutations,and ScSNV,SPIDEX and MaxEntScan software were used to predict gene function of splice site mutations.The classification of gene variation was based on the ACMG classification standard.The physical and chemical properties of gene expression products were analyzed by ProtParam software,and the Swiss-pdbviewer software was used to compare and analyze the 3d structure model of protein.Results:Among the 5 patients clinically diagnosed with Gitelman's syndrome in this study,2 were male and 2 were female who were born at term,with clinical manifestations of fatigue,recurrent hypokalemia,hypomagnesia,increased inappropriate renal potassium excretion,metabolic alkalosis,low uric calcium,high plasma renin level,etc.Two other patients diagnosis with hypokalemia enrolled in this study were both males,who also had recurrent fatigue,without other typical features of Gitelman's syndrome.Mutations of SLC12A3 gene were found in all of the 5 patients diagnosed with Gitelman's syndrome,and a total of 8 mutation sites were found,all of which were complex heterozygous mutations;there were 7 missense mutation(Asn958Lys?Arg964Gln?Asp486Asn?Thr60Met?Leu891Val?Arg1018Gln?Arg334Trp)and 1 splice site mutation(c.2660+1G>A).There were 2 patients carried with Asp486Asn mutation and 2 patients carried with Leu891 Val mutation.In the other 2 patients with only hypokalemia,genetic test results were negative.Missense mutation Asn958Lys was not included in each database,while other mutations were very low frequency variation among the general population.Missense mutation Asn958Lys is a novel mutation,while the other mutations has been reported in the literature.Functional prediction of missense mutations showed that the mutation "Asn958Lys"was predicted as "Disease causing" only in Mutation taster;the mutation "Arg964Gln" was predicted as "Deleterious" in SIFT and as "Disease causing" in Mutation taster.Other mutations were all predicted as "Deleterious","Probably damaging" and“Diseases causing"respectively in SIFT,Polyphen2 and Mutation taster.The function prediction of the splice site mutation indicated that mutation c.2660+1G>A was predicted uniformly to affect correct gene expression in ScSNV,SPIDEX and MaxEntScan.According to the classification standard of ACMG variation,the variantion Asn958Lys,Arg964Gln,Arg1018Gln were all classified as likely pathogenic variants,and the variation Asp486Asn,c.2660+1G>A,Thr60Met,Arg334Trp,Leu891Val were all classified as pathogenic variation.The physical and chemical properties of the gene expression products were analyzed by ProtParam software,which indicated that the above mutations could lead to changes in the physical and chemical properties of the mutated NCC protein.The comparative analysis of the 3d structure model of the protein by Swiss-Pdb Viewer software shows that all the above mutations may lead to the change of NCC protein 3d structure.Conclusions:A novel Missense mutation Asn958Lys of SLC12A3 gene was found in one patient of Gitelman's syndrome.7 other reported mutations in this gene were also detected in other patients.This study confirmed the clinical importance of second-generation sequencing in molecular diagnosis of Gitelman's syndrome,a rare endocrine system disease,suggesting that inclusion of SLC12A3 in the genetic testing group may contribute to the early diagnosis and high diagnostic success rate of Gitelman's syndrome,providing a basis for clinical diagnosis,clinical treatment,genetic counseling and prenatal diagnosis.