Exome Sequencing Identifies Mutation Genes Related To Intrahepatic Cholangiocarcinoma And Validation Of The USP17 Mutation

Objective:To investigate candidate genes relevant to intrahepatic cholangiocarcinoma(ICC) by whole-exome deep sequencing and and validate mutation genes by Sanger sequencing, to provide scientific theoretical basis for ICC etiology and explore the ideal tumor biomarker and effective target in therapy.Methods:We sequenced the whole-exome of five ICCs and matched normal tissue with the method of Illumina deep sequencing to investigate potential variants related to this disease. After analyzed according to described method, we selected the genes which had underwent missense mutation. Then, we took 49 ICC samples, including 15 extrahepatic-metastasis samples and 34 non-extrahepatic-metastasis samples, in addition 49 matched normal liver tissues as control. After the experimental steps of polymerase chain reaction(PCR) amplification, Sanger sequencing was used to validate the result of the deep sequencing. We took 36 ICC paraffin specimens and matched paraffin specimens from the pathology department of the General hospital of PLA to observe the differences of USP17 protein expression between the tumor tissues and para-carcinoma tissues by immunohistochemistry.Results:The reads quality of deep sequencing was satisfactory, and we selected 12 somatic mutations in 8 genes, named USP17L10, SPDYE1, LRRC37A3, SIMC1, TPSD1, PRAMEF18, TBC1D3G, OBSCN, for next validation experiments. By Sanger sequencing, we found 7 having USP17L10 gene mutation in 49 specimens, the percentage was 14.3%, and all of them were point mutation, locating at the chromosome 4 g.9213473G>C (c.1091 G>C, AGT→ACT, Ser364Thr), and the mutation site was the same with deep sequencing. There were 5 mutations in 15 extrahepatic-metastasis specimens (33.3%),2 mutations in 34 non-extrahepatic-metastasis specimens(5.9%), and the difference was statistically significant (P<0.05). There were no mutations in matched para-carcinoma tissues. In 36 cases of ICC, USP17 protein expressed in 16 cases with positive rate of 44.4%, meanwhile in 5 cases of control group the expression of USP17 protein was positive(13.9%), the difference was statistically significant (P<0.05). The difference of USP17 positive rate between extrahepatic-metastasis group and non-extrahepatic-metastasis group was not statistically significant (P>0.05).Conclusions:USP17 may be a candidate gene involved in the development of ICC. The expression of USP 17 protein in tumor is higher than the control. The role of USP 17 gene mutation in ICC and whether the mutation can affect the expression and function of the protein remains to be further research.