Abstract Gene mutation is responsible for the development of hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) infection; however, the characteristics and associated biological functions of highly mutated genes, in which the mutation frequencies are at least 5% in HCC patients with HBV infection, are not clearly evaluated. In the study, we analyzed the information regarding somatic mutation obtained by whole‐exome sequencing in 280 HBV‐related HCC tissues from public databases and published studies. Via integrative analysis, 78 genes, including TP53, TTN, MUC16, CTNNB1, and PCLO were summarized as highly mutated genes, and some of these mutated genes were further identified as cancer driver genes. Besides, we discovered that the highly mutated genes were enriched with various biological functions and pathways. The expression of many of highly mutated genes was found to be significantly altered in HBV‐related HCC, and several highly mutated genes were related to a variety of clinical factors and associated with the poor survival of the disease. Taken together, these results could enrich our understanding of highly mutated genes and their relationships with HBV‐related HCC. Some of the identified highly mutated genes might be used as novel biomarkers of disease prognosis, or as molecular targets for the treatment of HCC with HBV infection. Keywords: gene mutation, hepatitis B virus, hepatocellular carcinoma, integrative analysis __________________________________________________________________ The characteristics and associated biological functions of highly mutated genes, in which the mutation frequencies are at least 5% in HCC patients with HBV infection, are evaluated in the study, and these results could enrich our understanding of highly mutated genes and their relationships with HBV‐related HCC. graphic file with name CAM4-9-2462-g008.jpg 1. INTRODUCTION Hepatocellular carcinoma (HCC), the major pathological type of liver cancer, is still a leading cause of cancer‐related death worldwide.[40]1 In Asia and Africa, chronic hepatitis B virus (HBV) infection is the major cause of HCC development.[41]2, [42]3, [43]4 Although it has been suggested that several factors,[44]5, [45]6, [46]7 including the repeated inflammation mediated by immune reactions against the virus, the integration of HBV DNA into the host genome and the virus‐encoded oncoproteins (HBx and preS2/S proteins), are responsible for the development of HCC induced by HBV infection, the molecular mechanisms of hepatocarcinogenesis caused by HBV infection are still not well understanding, and no effective agents have been developed for the treatment of HBV‐related HCC. Hence, it is extremely important to discover novel molecules involved in the carcinogenesis of HBV‐associated HCC and offer the possibility of improving therapeutic approaches for HCC with HBV infection. Accumulation of genetic alterations is a hallmark of HCC,[47]8 and a great number of mutated genes, including CTNNB1, TSC1/2, PREX2, TP53, TERT, AXIN1, ARID1A, RET, and ARID2, have been identified in HCC, especially, in HBV‐related HCC.[48]3, [49]9, [50]10, [51]11, [52]12, [53]13, [54]14, [55]15 Current studies suggest that the mutations of CTNNB1 are associated with low stage HCC.[56]16 TERT promoter mutations are more frequently observed in HCC patients with low α‐fetoprotein (AFP) levels and advanced ages.[57]17 Ho et al report that TSC1/2 mutations contribute to the disruption of the downstream mTOR activity and lead to the more aggressive behavior of HCC cells.[58]15 In addition, special mutations in PREX2 gene are shown to have the capability of enhancing PREX2 protein stability and promoting cell proliferation.[59]18 TP53 mutations are observed to be related to the chemotherapy resistance, tumor recurrence, and decreased overall survival (OS) of HCC patients.[60]19 Besides these, the study from Ye et al shows that RET mutations are linked to poor disease free survival (DFS) and OS in patients with HCC.[61]20 Taken together, these findings indicate that the mutations of special genes are related to specific pathological characteristics and abnormal biological functions of HCC, and play important roles in promoting HCC progression. With the developing of sequencing technology, the mutations of several new genes, including APC, MYC, JAK1, COL11A1, LRP1B, FGF19, RB1, MUC16, and PCLO, in HCC tissues, especially in HBV‐related HCC tissues, have been discovered recently.[62]13, [63]21, [64]22, [65]23 However, to date, a comprehensive understanding of the highly mutated genes, which are responsible for the development of HBV‐related HCC, is lacking. In the present study, based on the public databases and published studies, we integrated and analyzed the data of somatic mutations that obtained from whole‐exome sequencing in HBV‐related HCC, assessed the amount of highly mutated genes, investigated their biological functions and associated pathways, and further evaluated the relationship between these mutated genes with clinical‐pathological features and prognosis of HCC with HBV infection. Our findings will help us better understand the molecular mechanisms associated with gene mutations in the development of HBV‐related HCC. 2. MATERIALS AND METHODS 2.1. Data acquisition and processing The somatic mutation information of HBV‐related HCC was obtained from cBioPortal database.[66]24, [67]25 The somatic mutation information from different contributors was checked and analyzed if met the following conditions: (a) the detail somatic mutation data and clinical information of HBV‐related HCC could be available from the cBioPortal database or the published studies; (b) the same sequence method was used to detect the somatic mutations of enrolled samples. Finally, we integrated the somatic mutation data of 280 HBV‐related HCC from three different studies for further analysis,[68]22, [69]26, [70]27 and the basic information of these three data is listed in Table [71]1. Table 1. The characteristics of three studies selected from the cBioPortal database Number of HBV‐related HCC Sequencing platform References