Two Gene Mutations In MEN1 Founded By Exome Gene Sequencing Causing Sporadic Insulinomas

Objective: Insulinoma,also known as islet β-cell neoplasia,is a disease characterized by seizure-induced hypoglycemia caused by the secretion of large amounts of insulin,and is a more common cause of organic hypoglycemia.By using exome sequencing technology,exome sequencing of tumor tissue and blood DNA of patients with sporadic insulinoma was carried out to screen for pathogenic gene mutations of sporadic insulinoma.Our present study is aimed to identify gene mutations responsible for tumorigenesis of sporadic insulinoma.Methods:In our present study,3 patients(INS1,INS2 and INS3)with primary insulinoma were enrolled and the paired tumor and peripheral blood of each sample for DNA preparations were then collected from patients after surgical removal.First,the paired tumor and peripheral blood of each sample were extracted with DNA.Then,Illumina HiSeq 2500 gene sequencing was performed.After sequencing,the sequencing fragment was compared with the reference genome using BWA software.The reference genome used in this experiment was hg19.To obtain the important candidate gene,the MuTect software were used to detect single nucleotide variants(SNVs).Variants were filtered for minimum genotype quality of 50 and minimum coverage depths of 10.Then,the software ANNOVAR was applied to annotate the qualified variants.Finally,the variants were obtained and the deleteriousness of variants was subsequently predicted by various bioinformatics programs(e.g.SIFT,Polyphen2,LRT,Mutation Taster,Mutation Assessor,FATHMM,RadialSVM,LR).Thereby finding the effect of gene mutations on the pathogenesis of insulinoma.Results:1.Six DNA samples that passed the test were sequenced using Illumina HiSeq 2500 Sequencer(Illumina,San Diego,CA,USA).Tumor exomes were sequenced,which led to 104472186,107751402 and 112709600 reads for INS1,INS2 and INS3,respectively.The matched blood exomes were sequenced,which led to 110592066,98905302 and 109660740 reads for INS1,INS2 and INS3,respectively.2.Exome sequencing analysis overall identified 40210,40272 and 41910 SNVs for tumor tissue,whereas 41106,40050 and 41451 SNVs for the matched blood samples.We identified 55 rare somatic mutations among the 3 patients,including 39 nonsynonymous,4 were stopgain and 12 were synonymous.A c.C681G/p.Y227 X mutation was found in exon 4 of the MEN1 gene in INS1,while c.G346T:p.E116 X mutation was found in exon 2 of the MEN1 gene in INS2.The mutations were not present in the corresponding leukocyte DNA.3.After using SIFT and polyphen analysis the effect of protein translation of the two somatic termination mutations(c.C681G/p.Y227 X and c.G346T/p.E116X)found in the study,the two stopgain mutations locate in the functional domain of MEN1,indicating that it may affect the binding of MLL1.Conclusions:Y227X mutation and E116 X mutation all results in a significant truncation of MEN1 protein,which may destroy the function structure domain and affect the functions of MEN1.