Deep Sequencing Identifies Mutation Genes Related To TCCB And Validation Of The HECW1Mutation

Objective: To investigate candidate genes related to TCCB by whole-exome deepsequencing and validate mutation genes by Sanger sequencing,to provide scientifictheoretical basis for TCCB etiology and explore the ideal tumor biomarker and effectivetarget in therapy.Methods:In the first part,we sequenced the whole-exome of two individuals who werediagnosed as non-muscle-invasive TCCB(non-muscle-invasive group) and two individualswho were diagnosed as muscle-invasive TCCB(muscle-invasive group) with the method ofSOLID deep sequencing to investigate potential variants related to this disease.Afteranalyzed according to described method,we selected the genes which had underwentmissense mutation.In the second part,we took84TCCB paraffin specimens from the pathologydepartment of the First Affiliated Hospital of Soochow University,including54muscle-invasive specimens and30non-muscle-invasive specimens;and15cases of post-operativetumor tissues,including7muscle-invasive cases and8non-muscle-invasive cases,inaddition10para-carcinoma tissues as control,then made them into paraffin specimens.Using OMEGA DNA extraction kit to extract DNA from these specimens,after theexperimental steps of polymerase chain reaction (PCR) amplification,Sanger sequencingwas used to validate the result of the deep sequencing.Observed the differences of HECW1protein expression between the tumor tissues and para-carcinoma tissues by immunohisto-chemistry.Results:In the first part:The dates quality of deep sequencing were satisfaction,we identifiedmissense mutation in10genes,including4genes in non-muscle-invasive group,namedIVL,RGPD5,ZNF79,SRRM5;3genes in muscle-invasive group,named CPNE7,RBMXL 3,ACSM2A;3genes in both group,named HECW1,ZNF273,TCHH.In the second part:we found6having HECW1gene mutation in99specimens,thepercentage was6.06%,and all of them were point mutation,locating at the exon-11and themutation sites were the same with deep sequencing or nearby areas.There was onesynonymous mutation in38non-muscle-invasive specimens(2.63%),four missensemutations and one nonsense mutation in muscle-invasive specimens(8.20%),the differencewas not statistically significant (P>0.05),there were no mutations in10para-carcinomatissues.In15cases of TCCB,HECW1protein expressed in9cases,with positive rate of60%,meanwhile only in one case of control group the expression of HECW1protein waspositive(10%),the difference was statistically significant (P<0.05).Conclusion:(1)Deep sequencing technology, especially the whole-exome sequencing,would be a efficient experiment in the study of urogenital neoplasms etiology.(2)Sangersequencing is the gold standard for verifying the result of deep sequencing.(3)HECW1may be a candidate gene involved in the development of bladder cancer.(4)The expressionof HECW1protein in tumor is higher than the control.(5)The role of HECW1genemutation in bladder cancer and whether the mutation can affect the expression and functionof the protein remains to be further research.