Literature DB >> 32650224

Genomic characterization of Chinese ovarian clear cell carcinoma identifies driver genes by whole exome sequencing.

Qin Yang1, Cancan Zhang2, Yuan Ren2, Huan Yi3, Tianjiao Luo4, Fangliang Xing5, Xuefeng Bai6, Lining Cui7, Linyan Zhu7, Jun Ouyang8, Pengcheng Jiang9, Weirong Fan2, Jianping Qiu10, Fengmian Wang2, Xin Xing2, Zhigang Zhang4, Xueli Zhang11, Rong Zhang12.   

Abstract

Little is known about the genetic alterations characteristic of ovarian clear cell carcinoma (OCCC). Our aim was to identify targetable genomic alterations in this type of cancer. Forty-two OCCC formalin-fixed, paraffin-embedded (FFPE) tissue samples were analyzed by whole-exome sequencing (WES), and 74 FFPE tissue samples underwent targeted sequencing (TS) to confirm the relevant driver mutations. Cell proliferation was assessed by cell counting kit-8 (CCK8) assays. In the 42 samples, ARID1A (64.3%) and PIK3CA (28.5%) were frequently mutated, as were PPP2R1A (11.9%), PTEN (7.1%) and KRAS (4.8%), which have been reported in previous OCCC studies. We also detected mutations in MUC4 (28.6%), MAGEE1 (19%), and ARID3A (16.7%); associations with these genes have not been previously reported. The functional protein-activated pathways were associated with proliferation and survival (including the PI3K/AKT, TP53, and ERBB2 pathways) in 83% of OCCCs and with chromatin remodeling in 71% of OCCCs. Patients with alterations in MAGEE1 (64% in the targeted sequencing cohort) had worse clinical outcomes (log-rank p < 0.05). A functional study revealed that two MAGEE1 mutants, one lacking two MAGE domains and the other containing two MAGE domains, significantly decreased the proliferative capacity of OCCC cells. We successfully identified novel genetic alterations in OCCC using whole-exome sequencing and targeted sequencing of OCCC patient samples and potential therapeutic targets for the treatment of this malignancy.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

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Keywords:  Driver mutation; MAGEE1; Ovarian clear cell carcinomas; Target sequencing; Whole exome sequencing

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Year:  2020        PMID： 32650224      PMCID： PMC7341065          DOI： 10.1016/j.neo.2020.06.002

Source DB:  PubMed          Journal:  Neoplasia        ISSN： 1476-5586            Impact factor:   5.715


Introduction

Among gynecologic malignancies, ovarian cancer is the second most common and the most deadly globally in 2020 cancer statistics [1]. Epithelial ovarian cancer accounts for over 80% of the malignant ovarian cancer cases [2] and consists of four major histological tumor subtypes, including serous, clear-cell, endometrioid, and mucinous [3]. Among the histological subtypes, ovarian clear cell carcinoma (OCCC) accounts for approximately 5% of all epithelial ovarian cancers, with an occurrence rate above 20% in certain Asian populations [4]. In addition, women with advanced OCCC have poorer survival outcomes. OCCC is a distinct subtype with a lower response rate than the serous subtype to platinum-based chemotherapy [5], [6], [7]. Although OCCC is the second leading cause of death from ovarian cancer, the etiology and pathogenesis of this devastating disease are poorly understood. The molecular characterization of solid tumors by whole genome or exome sequencing has provided important insights in cancer biology. Somatic mutations found in cancer may suggest personalized treatments options. OCCC-specific somatic mutations are clustered in AT-rich interactive domain 1A (SWI-like) (ARID1A), phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), protein phosphatase 2 scaffold subunit alpha (PPP2R1A) and K-ras (KRAS) [8], [9], [10], [11], [12]. Somatic copy number amplification of the ZNF217 gene in OCCC was also previously reported [13]. In this study, we obtained whole-exome sequencing (WES) data for Asian OCCC samples via next-generation sequencing (NGS) and integrated the independent single-nucleotide variant (SNV) and copy number variant (CNV) analyses to better elucidate the genomic architecture of our OCCC samples. To detect both high- and low-frequency pathogenic mutations, we performed targeted sequencing of a large cohort of 69 OCCC tumors and matched normal tissues. Finally, we validated recurrent mutants in several genes, including MAGEE1 in OCCC. Patients with alterations in MAGEE1 had worse clinical outcomes. The expression of mutant MAGEE1 significantly decreased the proliferative capacity of OCCC cells. These results indicate that MAGEE1-targeted drugs could guide the future development of therapeutic strategies for OCCC.

Materials and methods

Study population

Patients enrolled in this study were pathologically diagnosed with ovarian clear cell carcinoma between January 2008 and December 2016. Tissue samples were collected from formalin-fixed, paraffin-embedded (FFPE) blocks of tumor tissue from 69 ovarian clear cell cancer patients. The use of samples and medical records was approved by the research ethics committees of Shanghai University of Medicine & Health Sciences Affiliated with Sixth People’s Hospital South Campus (approval number: 2017-KY-01), Fujian Provincial Maternity and Children's Hospital (approval number: 2017049), Nanjing Medical University Affiliated with Changzhou Maternal and Child Health Care Hospital (approval number: 2017005), Nanjing Medical University Affiliated with Changzhou No. 2 People’s Hospital (approval number: 2016-017-01), and Nanjing Medical University Affiliated with Suzhou Municipal Hospital (approval number: L2017003). Genomic DNA was extracted from tumor areas of tissue sections from the FFPE blocks using the QIAGEN GeneRead DNA FFPE Kit (ID: 762174; this kit helps reduce errors due to DNA deamination caused by formalin fixation and aging).

Whole exome sequencing

Sequencing data were generated as detailed previously. In brief, whole-exome capture libraries were constructed from tumor and normal DNA after sample shearing, end repair, phosphorylation, and ligation to barcoded sequencing adaptors. DNA then underwent solution-phase hybrid capture with SureSelect v.2 Exome bait (Agilent Technologies), followed by sample multiplexing and sequencing on an Illumina HiSeq X Ten instrument. Raw sequencing reads were trimmed with Trimmomatic to filter low-quality reads. Clean reads were aligned to the reference human genome (UCSC, hg19) using Burrows-Wheeler Aligner (BWA). Duplicates were identified by Picard, and the remaining outputs were locally realigned using the Genome Analysis Toolkit (GATK). We detected somatic mutations with the MuTect algorithm and somatic indels based on concordant events identified by the Indelocator algorithm. To remove artifacts from the hydrolytic deamination of cytosine to uracil in FFPE samples, we filtered out C > T mutations consistent with a 20:1 single-strand bias based on read pair orientation. We used the eDriver, OncodriveFML, ActiveDriver, MutSigCV, Genome MuSic and OncodriveCLUST tools to infer significantly mutated genes. Hypermutated tumors are defined as those with a mutation count >1000. All somatic variants were annotated in dbSNP138, the 1000 Genomes Project and EXAC by ANNOVAR. Variants with allele frequencies greater than 0.5% of the allelic fraction in these databases were removed.

Validation with target sequencing

For targeted ultradeep sequencing of 56 genes, the Illumina HiSeq platform was used. After samples were library prepped and run on the HiSeq platform, reads were mapped to hg19 using BWA, and realigned using GATK. Variants were called using GATK. For targeted ultradeep sequencing, we required a depth ≥200 and a quality score ≥20.

Pathway enrichment analysis

Canonical cancer pathways were selected from MSigDB (including 1329 gene sets). The list of genes with recurrent mutations, including functional SNVs and CNVs, was analyzed. Pathway enrichment analyses of genes harboring somatic SNVs and CNVs were performed with KEGG or Gene Ontology by using the clusterProfiler package in R. P-values were calculated based on a hypergeometric distribution with FDR correction using the Benjamini method.

Cell culture

The OCCC cell lines OVISE and ES-2 were both preserved in Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. OVISE was cultured in RPMI 1640 medium, and ES-2 cells were cultured in McCoy’s 5A medium, containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 μg/ml streptomycin and 100 units/ml penicillin). All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

Plasmid construction and cell transfection

The MAGEE1 wild-type (MAGEE1-WT) sequence was obtained from Asia-vector Biotechnology Co., Ltd (Shanghai, China). Two mutants were constructed, mut1 lacking two MAGE domains and mut2 containing two MAGE domains. The hemagglutinin (HA) tag was added to the C terminus of the MAGEE1 mutants. All constructs were verified by sequencing. Cell transduction steps were performed according to the manufacturer’s protocols using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific).

Quantitative Real-Time PCR (qPCR)

Total RNA was extracted from OCCC cell lines by using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed as previously described [14]. β-Actin was used as internal control for quantification. The data were analyzed using the 2−ΔΔCt method. The primer sequences used in our study were as follows: MAGEE1-F, 5′-CCCAGAAGAGGTGACGG-3′; MAGEE1-R, 5′-GCAGCAGGAAGATG AGGA-3′; β-Actin-F, 5′-CTCTGGCTCCTAGCACCATGAAGA-3′; β-Actin-R, 5′-GTAAAACGCAGCTCAGTAACAGTCCG-3′.

Western blot

Western blot analysis was performed as described previously [14]. The membranes were incubated overnight at 4 °C with primary antibodies against HA (ab18181; Abcam) and β-actin (M1210-1, Huabio, Hangzhou, China), followed by incubation with species-specific secondary antibodies for 1 h. The signals were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE) and further quantified by ImageJ software.

Cell viability

To measure cell proliferation, cells were seeded at 2000 cells/well in a 96-well plate and detected by Cell Counting Kit-8 (CCK8, Dojindo, Japan) after 0, 1, 2, 3, and 4 days. The experiments were performed in triplicate and repeated twice.

Statistical analysis

Known prognostic factors for OCCC were recorded for each patient, as shown in Table 1. The characteristics of patients in different groups were compared using Fisher’s exact test (for categorical data) or the Mann-Whitney test (for continuous data). Only 57 patients with complete follow-up clinical data were included in the survival analysis. OS was calculated based from the date of OCCC diagnosis to the data of death from disease or the last follow-up. The Kaplan-Meier method was used to estimate the OS distribution, and differences in survival between groups were assessed using the log rank test. P values less than 0.05 indicated statistical significance. R software was used to perform all statistical analyses and generate all graphs.
Table 1

The clinical pathological characteristics in OCCC patients

SampleAgeGradeStatusOSChemotherapyEndometriosisWESTarget
OCCC_0164IIAlive2124YNYY
OCCC_0235IAlive1946YNYY
OCCC_0350ICAlive933YNYY
OCCC_0452IAAlive455YYYY
OCCC_0559IIIADead902YYYY
OCCC_0645IAAlive455YNYY
OCCC_0743IICDead908YNYY
OCCC_0856IAAlive455YYYY
OCCC_0959ICAlive1978YNYY
OCCC_1060ICAlive360YNYY
OCCC_1146IICDead1234YNYY
OCCC_1250ICAlive392YNYY
OCCC_1343ICAlive850NYYY
OCCC_1448ICAlive265YYYY
OCCC_1553IAlive758YYYY
OCCC_1649IIDead545YYYY
OCCC_1750ICAlive708YYYY
OCCC_1850IIAlive568NNYY
OCCC_1964IAAlive1551YNYY
OCCC_2053IIAlive600YYYY
OCCC_2153IAlive517YYYY
OCCC_2240IAAlive600YNYY
OCCC_2333IAAlive517YNYY
OCCC_2463IAAlive517YNYY
OCCC_2537IIICDead336YYYY
OCCC_2644ICAlive1267YNYY
OCCC_2756ICDead631YNYY
OCCC_2852IBAlive423NNYY
OCCC_2970IIBDead301NNYY
OCCC_3061ICAlive455YNYY
OCCC_3138IAAlive1946YNYY
OCCC_3251IAAlive933YNYY
OCCC_3365IIIAlive1580YNYY
OCCC_3447IAAlive789YNYY
OCCC_3540ICAlive1393YYYY
OCCC_3654ICAlive423YYYY
OCCC_3732ICDead268YYYY
OCCC_3858IIICAlive1293YNYY
OCCC_3954IIICAlive1632YYYY
OCCC_4041IIIBAlive392YYYY
OCCC_4174IIICDead63NNYY
OCCC_4251IIICDead533YYYY
OCCC_4356IIIBDead122NNNY
OCCC_4466IIAlive392YNNY
OCCC_4581IBDead2854NNNY
OCCC_4654IIICAlive2584YNNY
OCCC_4742IAAlive1821YNNY
OCCC_4845IAlive3282YYNY
OCCC_4968IIICDead300YNNY
OCCC_5054IAAlive1361YNNY
OCCC_5154IAAlive1361YNNY
OCCC_5243IICAlive1519YYNY
OCCC_5355ICAlive1393YNNY
OCCC_5458IAAlive1884YNNY
OCCC_5534IAAlive2708YNNY
OCCC_5653IAlive2394YYNY
OCCC_5753IIAlive758YYNY
OCCC_5857IIBAlive2310YNNY
OCCC_5945IAlive2311YYNY
OCCC_6046ICAlive1695YNNY
OCCC_6160IAAlive600YNNY
OCCC_6257ICAlive663YNNY
OCCC_6350IIIAlive1425YNNY
OCCC_6445ICDead724YYNY
OCCC_6554ICAlive600YNNY
OCCC_6630IBAlive663YNNY
OCCC_6751IAAlive2886YYNY
OCCC_6847IIAlive2332YNNY
OCCC_6964IAlive120YYNY
The clinical pathological characteristics in OCCC patients

Results

Clinical cohort

A cohort of 69 patients diagnosed with ovarian clear cell cancer and treated surgically from 2008 to 2016 was recruited. Ovarian tumor tissue samples and matched solid normal tissue samples were obtained. This cohort was included in the WES cohort (n = 42) and the targeted sequencing cohort (n = 69 including the WES cohort). The detailed clinical pathological characteristics of the patients in the cohorts, including age, tumor stage, chemotherapy, the presence of endometriosis and survival, are presented in Table 1. The median age at diagnosis was 52 years, but a notable percentage of the patients (10%) were younger than 40 years at diagnosis. The tumor was stage I in 45 patients, stage II in 12 subjects and stage III/IV in 12 individuals.

Mutation detection analysis

To detect somatic mutations in tumor samples, whole-exome sequencing was performed on 84 samples (42 ovarian tissues, including both cancer and adjacent normal tissues). For the tumor and matched normal DNA samples, each targeted base was sequenced by a mean of 180 independent reads. A total of 5868 somatic exonic mutations were identified in all tumor samples, including nonsynonymous variants, in_frameshift variants, frameshift variants, nonsense variants and splice site variants, with a median of 153 alterations per tumor (range, 44–794). The number of somatic mutations was higher in patients with stage III/IV disease than in patients with stage I/II disease (P = 0.039; Wilcoxon test) (1). However, no correlation was observed between the number of somatic mutations and patient age (group 1 ≤52 years; group 2 >52 years; P = 0.31) or the presence of endometriosis (P = 0.50) (Fig. S1). The previously reported hotspot genes with somatic mutations in OCCC included ARID1A, PIK3CA, KRAS, PPP2R1A, PTEN, MLL3, ARID1B and PIK3R1 (Fig. S2). In total, 63 mutations were discovered across these 8 genes in 42 OCCCs (Table S1). Of these, ARID1A and PIK3CA were the two most frequently mutated genes in all patients. ARID1A accounted for 34 mutations in 27 patients, which was consistent with a previous report, but these mutations were scattered along the entire length of the genes. PIK3CA accounted for 14 mutations in 12 patients, and 4 of these patients had PIK3CA hotspot mutations (c.1624G > A [p. Glu542Lys] and c.1633G > A [p. Glu545Lys]). Genes predicted by more than one method may be more likely to be drivers. By applying six independent mutation prediction algorithms, eDriver (Table S2), OncodriveFML (Table S3), ActiveDriver (Table S4), MutSigCV (Table S5), Genome MuSic (Table S6) and OncodriveCLUST (Table S7), we computed the significance levels of the frequently mutated genes, accounting for the gene size and the background mutation rate. Fig. 1a shows the most significantly mutated genes identified by more than two cancer driver detection methods in our panel of 43 cases, with their mutation frequencies derived from whole-exome sequencing; these genes included ARID1A, MAGEE1, PIK3CA, MUC4, ARID3A, FLG2, TCHH, GRM3, MUC17, ZNF208 and GAGE12J. In addition to ARID1A and PIK3CA, other genes were newly identified as novel SMGs in our study. The frequencies of significant somatic mutations in the oncogenes MUC4 and ARID3A were 28.6% and 16.7%, respectively. MAGEE1, FLG2, TCHH, GRM3, MUC17, ZNF208, and GAGE12J were mutated in 19.0%, 26.2%, 23.8%, 4.8%, 23.8%, 9.5% and 9.5% of samples, respectively (Fig. 1b).
Fig. 1

Mutations identified by whole-exome sequencing in OCCC patients. (a) Eleven SMGs identified in 42 OCCC samples. Venn diagram of the overlap of the significantly mutated genes as assessed by eDriver, OncodriveFML, ActiveDriver, MutSigCV, Genome MuSic and OncodriveCLUST. (b) Mutation oncoprint of the 11 SMGs identified by at least two of the three methods. (c) Distribution of specific nucleotide changes among somatic variations by whole exome sequencing in cancer tissues compared to adjacent tissues. (d) The bars show the percentage of somatic single nucleotide variations identified in each sample. (e) Two mutational signatures were observed in the genomes of OCCC samples: a defective DNA mismatch repair signature (signature 6) and an age-associated signature (signature 1). These data were adapted from COSMIC database (http://cancer.sanger.ac.uk/cosmic/signatures).

Mutations identified by whole-exome sequencing in OCCC patients. (a) Eleven SMGs identified in 42 OCCC samples. Venn diagram of the overlap of the significantly mutated genes as assessed by eDriver, OncodriveFML, ActiveDriver, MutSigCV, Genome MuSic and OncodriveCLUST. (b) Mutation oncoprint of the 11 SMGs identified by at least two of the three methods. (c) Distribution of specific nucleotide changes among somatic variations by whole exome sequencing in cancer tissues compared to adjacent tissues. (d) The bars show the percentage of somatic single nucleotide variations identified in each sample. (e) Two mutational signatures were observed in the genomes of OCCC samples: a defective DNA mismatch repair signature (signature 6) and an age-associated signature (signature 1). These data were adapted from COSMIC database (http://cancer.sanger.ac.uk/cosmic/signatures). The mutation spectrum revealed C: G > T: A transitions (69.41%), as the most abundant alteration, and other transversions, including C: G > G: C (6.91%), T: A > C: G (7.83%), C: G > A: T (1.02%), T: A > G: C (2.25%) and T: A > A: T (3.37%) (Fig. 1c and d). Using known mutational signatures reported in the COSMIC database, the most frequent mutation signature was signature 6 (likely with defective DNA mismatch repair), which is most frequently found in colorectal and uterine cancers. The second most common signature observed was signature 1, a pattern associated with spontaneous deamination of 5-methylcytosine that accounts for a significant percentage of the critical somatic driver mutations observed in most cancers, including OVs (Fig. 1e). To further validate the frequently mutated genes found in the WES cohort, we performed targeted sequencing of all 69 tumor samples. The targeted gene panel included over 56 previously identified genes that are prone to mutation and involved in OCCC (Table S8). This panel is composed of genes from four categories: MutSigCV-positive cancer driver genes (6 genes), genes with a high variant frequency (19 genes), previously reported genes (9 genes) and other candidate genes (22 genes). We analyzed tumor DNA from a total of 138 OCCC samples (ovarian tissues including both cancer and adjacent normal tissues). After quality control for library preparation, 124 tumors (62 tumor samples) were suitable for analysis. On average, 96% of the reads mapped uniquely to the targeted sequences, and we obtained an average read depth of 800 reads per base in the targeted region. After filtering out changes in intronic regions and polymorphisms (present in dbSNP134), we identified a total of 1816 variants in all sequenced exons of the 62 OCCC patients. After discarding alterations in noncoding RNAs or the 5′ or 3′ untranslated regions, and synonymous mutations, only 1096 single nucleotide variants and indels were identified that resulted in missense mutations, frameshift mutations, inframe mutations or nonsense mutations, with an average of 17.62 mutations per case (Table 2). AHNAK2 was the most frequently mutated gene (60.3%), followed by OBSCN (55.5%), ARID1A (54.0%), and AHNAK (49.2%), MUC19 (46.0%), DSPP (42.9%), PIK3CA (41.3%), FLG (41.3%) and MUC17 (41.3%) (Fig. S2). In ARID1A, we identified 45 heterozygous variants (21 frameshift, 11 nonsense, 7 missense changes, 3 in frame variants and 3 splice site variants) spread across the coding exons of the gene in 33 patients. Heterozygous mutations were also identified in PIK3CA, and these mutations mostly clustered in exons 10 and 21. The majority of the PIK3CA mutations were missense mutations (n = 26). In MAGEE1, we identified 13 heterozygous variants including 7 inFrameDel mutations, 4 missense mutations, 1 frameshiftIns mutation and 1 inFrameIn mutation (Fig. S3).
Table 2

Gene mutations identified by targeted sequencing in 62 patients with OCCC.

Hugo_SymbolchromosomeStart_PositionEnd_PositionStrandVariant_ClassificationTumor_Seq_Allele1Tumor_Seq_Allele2Tumor_Samle_Barcodeprotein_change
ARID1A12708734627087346+Splice_SiteGTOCCC_22
OBSCN1228528833228528833+Missense_MutationGAOCCC_22p.R5912H
MSH357995072479950724+Missense_MutationGCOCCC_22p.A60P
MUC177100683482100683482+Missense_MutationCTOCCC_22p.P2929S
AHNAK116229605962296059+Missense_MutationCAOCCC_22p.V1944L
AHNAK214105413318105413318+Missense_MutationCTOCCC_22p.G2824R
AHNAK214105413471105413471+Missense_MutationTCOCCC_22p.K2773E
TCHH1152083777152083777+Missense_MutationTGOCCC_37p.Q639P
FLG1152277704152277704+Missense_MutationCGOCCC_37p.D3220H
FLG1152281635152281635+Missense_MutationCAOCCC_37p.R1909S
OBSCN1228462377228462377+Missense_MutationGAOCCC_37p.V1930M
OBSCN1228468087228468087+Missense_MutationCTOCCC_37p.A2624V
DSPP48853741288537412+Missense_MutationGAOCCC_37p.D1200N
PTPRN27157959931157959931+Missense_MutationGAOCCC_37p.A201V
PLEC8144998243144998243+Missense_MutationGAOCCC_37p.R2089C
PLEC8145024703145024703+Missense_MutationGAOCCC_37p.R58W
AHNAK116229396862293968+Missense_MutationCTOCCC_37p.V2641M
MUC19124087378140873781+Missense_MutationTCOCCC_37p.S1776P
MUC19124087398940873989+Missense_MutationCTOCCC_37p.S1845L
AHNAK214105407327105407327+Missense_MutationGAOCCC_37p.P4821S
AHNAK214105407525105407525+Missense_MutationGAOCCC_37p.P4755S
AHNAK214105413516105413516+Missense_MutationCGOCCC_37p.V2758L
AHNAK214105413517105413517+Missense_MutationGCOCCC_37p.N2757K
AHNAK214105413791105413791+Missense_MutationGAOCCC_37p.S2666F
AHNAK214105419549105419549+Missense_MutationGTOCCC_37p.P747T
AHNAK214105419610105419610+Missense_MutationCGOCCC_37p.Q726H
CASKIN11622311272231127+Missense_MutationCTOCCC_37p.G748S
ERBB2173786328737863287+Missense_MutationGAOCCC_37p.E40K
SPTBN4194106299241062992+Missense_MutationGAOCCC_37p.A1785T
PPP2R1A195271598252715982+Missense_MutationCTOCCC_37p.R183W
LAMA5206090957960909579+Splice_SiteCAOCCC_37p.E861*
HRNR1152191230152191230+Missense_MutationGTOCCC_50p.Q959K
OBSCN1228474615228474615+Missense_MutationGAOCCC_50p.R3140Q
PIK3CA3178952085178952085+Missense_MutationAGOCCC_50p.H1047R
DSPP48853673788536737+Missense_MutationGAOCCC_50p.D975N
PLEC8145009181145009181+Missense_MutationTAOCCC_50p.N412Y
EVPL177401054574010545+Missense_MutationCAOCCC_50p.G779C
HELZ2206219437562194375+Missense_MutationGAOCCC_50p.R1934W
ARID1A12710556527105565+Nonsense_MutationGTOCCC_04p.E1726*
ARID1A12710615927106159+Missense_MutationGAOCCC_04p.E1924K
DST65635778156357781+Missense_MutationCTOCCC_04p.R6514Q
AHNAK214105413284105413284+Missense_MutationGAOCCC_04p.S2835L
SPTA11158592867158592867+Missense_MutationCTOCCC_47p.R2009H
SPTA11158617396158617396+Missense_MutationGAOCCC_47p.R1277C
OBSCN1228462332228462332+Missense_MutationGAOCCC_47p.E1915K
OBSCN1228464316228464316+Missense_MutationCGOCCC_47p.P2129R
OBSCN1228468069228468069+Missense_MutationGAOCCC_47p.S2618N
OBSCN1228475581228475581+Missense_MutationGAOCCC_47p.R3244Q
OBSCN1228503679228503679+Missense_MutationAGOCCC_47p.T4382A
OBSCN1228506756228506756+Missense_MutationCTOCCC_47p.S4768L
OBSCN1228509733228509733+Missense_MutationCTOCCC_47p.A5064V
OBSCN1228555619228555619+Missense_MutationATOCCC_47p.Y6554F
OBSCN1228559933228559933+Missense_MutationATOCCC_47p.T7152S
OBSCN1228562312228562312+Missense_MutationCTOCCC_47p.R7508C
OBSCN1228564758228564758+Missense_MutationGAOCCC_47p.R7682H
OBSCN1228566387228566387+Missense_MutationGAOCCC_47p.R7933Q
PIK3CA3178916623178916623+Nonsense_MutationCTOCCC_47p.R4*
MUC43195511070195511070+Missense_MutationCGOCCC_47p.D2461H
MUC43195511208195511208+Missense_MutationTGOCCC_47p.T2415P
MUC43195513530195513530+Missense_MutationACOCCC_47p.L1641V
SHROOM347766240877662408+Missense_MutationGAOCCC_47p.E1028K
DST65635774056357740+Missense_MutationCTOCCC_47p.E6528K
MUC177100683993100683993+Missense_MutationCAOCCC_47p.T3099N
AHNAK116229932562299325+Missense_MutationTGOCCC_47p.K855T
SPTBN2116645757166457571+Missense_MutationGAOCCC_47p.R1917W
SPTBN2116647818266478182+Missense_MutationGAOCCC_47p.S315L
SPTBN2116647841166478411+Missense_MutationCTOCCC_47p.V288M
KRAS122539828425398284+Missense_MutationCTOCCC_47p.G12D
MUC19124083801740838017+Missense_MutationGAOCCC_47p.A867T
SPTB146526054365260543+Missense_MutationCAOCCC_47p.S613I
AHNAK214105413318105413318+Missense_MutationCTOCCC_47p.G2824R
AHNAK214105415607105415607+Missense_MutationCTOCCC_47p.V2061M
AHNAK214105417358105417358+Missense_MutationTCOCCC_47p.E1477G
CASKIN11622319182231918+Missense_MutationCTOCCC_47p.V548M
XPO6162816740228167402+Missense_MutationCTOCCC_47p.D364N
SPTBN4194106013441060134+Missense_MutationACOCCC_47p.S1586R
SPTBN4194106298741062987+Missense_MutationGAOCCC_47p.R1783Q
VASP194602126646021266+Missense_MutationGAOCCC_47p.R86H
PPP2R1A195271632352716323+Missense_MutationCAOCCC_47p.S256Y
LAMA5206088998560889985+Missense_MutationGAOCCC_47p.T2689M
LAMA5206089952560899525+Missense_MutationCTOCCC_47p.R1872H
HELZ2206219063062190630+Missense_MutationCTOCCC_47p.R2640H
MAPK1222212357922123579+Missense_MutationTCOCCC_47p.M333V
MAPK1222215339622153396+Missense_MutationGAOCCC_47p.R172C
ARID1A12705778827057788+Missense_MutationCTOCCC_25p.S499L
ARID1A12710664827106648+Missense_MutationGAOCCC_25p.G2087R
RPTN1152128065152128065+Missense_MutationCTOCCC_25p.G504R
HRNR1152191050152191050+Missense_MutationCTOCCC_25p.G1019R
FLG1152281145152281145+Missense_MutationTGOCCC_25p.K2073Q
FLG1152282178152282178+Missense_MutationCGOCCC_25p.E1728D
FLG1152283430152283430+Missense_MutationAGOCCC_25p.F1311S
FLG21152324114152324114+Missense_MutationCTOCCC_25p.A2050T
OBSCN1228464337228464337+Missense_MutationCTOCCC_25p.S2136L
LRP1B2141625794141625794+Missense_MutationCTOCCC_25p.R1403H
MUC43195508586195508586+Missense_MutationACOCCC_25p.S3289A
DSP675840727584072+Missense_MutationGAOCCC_25p.E2193K
DST65648054756480547+Missense_MutationCTOCCC_25p.R2573Q
MUC177100679633100679633+Missense_MutationCTOCCC_25p.P1646S
MUC177100682351100682351+Missense_MutationAGOCCC_25p.I2552V
AHNAK116229587062295870+Missense_MutationTCOCCC_25p.M2007V
AHNAK116229633562296335+Missense_MutationCTOCCC_25p.A1852T
SPTB146525105065251050+Missense_MutationCTOCCC_25p.R1306Q
SPTB146526049565260495+Missense_MutationCTOCCC_25p.R629Q
SPTB146526337065263370+Missense_MutationGAOCCC_25p.R416W
AHNAK214105405284105405284+Missense_MutationGAOCCC_25p.R5502W
AHNAK214105408638105408638+Missense_MutationGCOCCC_25p.L4384V
AHNAK214105410804105410804+Missense_MutationCGOCCC_25p.D3662H
CASKIN11622314472231447+Missense_MutationGAOCCC_25p.P641L
CASKIN11622390892239089+Missense_MutationCTOCCC_25p.D186N
EVPL177401849474018494+Splice_SiteCTOCCC_25p.P202P
PPP2R1A195271598252715982+Missense_MutationCTOCCC_25p.R183W
ARID1A12709994727099947+Nonsense_MutationCTOCCC_01p.R1276*
HRNR1152191019152191019+Missense_MutationGTOCCC_01p.S1029Y
HRNR1152191565152191565+Missense_MutationGAOCCC_01p.T847M
SPTA11158581062158581062+Missense_MutationCAOCCC_01p.G2418C
OBSCN1228520965228520965+Missense_MutationTAOCCC_01p.L5266Q
MUC43195515435195515435+Missense_MutationCTOCCC_01p.A1006T
SHROOM347763140477631404+Missense_MutationCTOCCC_01p.A140V
SHROOM347767621977676219+Missense_MutationCAOCCC_01p.P1528Q
SHROOM347770004777700047+Missense_MutationGAOCCC_01p.R1903Q
ANK3106196555261965552+Missense_MutationCTOCCC_01p.E431K
AHNAK116229281662292816+Missense_MutationCTOCCC_01p.V3025M
AHNAK214105407965105407965+Missense_MutationGAOCCC_01p.A4608V
CASKIN11622372362237236+Missense_MutationTAOCCC_01p.T256S
EVPL177400507274005072+Missense_MutationACOCCC_01p.L1405R
EVPL177400507374005073+Missense_MutationGTOCCC_01p.L1405I
EYA2204580146545801465+Missense_MutationGAOCCC_01p.R383H
ARID1A12705634927056349+Nonsense_MutationCTOCCC_32p.Q449*
PIK3CA3178936091178936091+Missense_MutationGAOCCC_32p.E545K
AHNAK214105415160105415160+Missense_MutationCGOCCC_32p.V2210L
KRT10173897837238978372+Missense_MutationGAOCCC_32p.R156C
AKT2194074596240745962+Missense_MutationGAOCCC_32p.P210L
ARID1A12710655827106558+Missense_MutationCTOCCC_34p.R2057W
MUC177100680354100680354+Missense_MutationCGOCCC_34p.T1886S
TCHH1152081230152081230+Missense_MutationACOCCC_35p.L1488R
FLG1152280731152280731+Missense_MutationGCOCCC_35p.H2211D
PIK3CA3178936082178936082+Missense_MutationGAOCCC_35p.E542K
MUC43195489015195489015+Missense_MutationGAOCCC_35p.L583F
DST65641721156417211+Missense_MutationATOCCC_35p.V5249D
SPTB146528974565289745+Missense_MutationCAOCCC_35p.W23L
DST65650540556505405+Splice_SiteT-OCCC_35
HRNR1152188862152188862+Missense_MutationGAOCCC_61p.S1748L
FLG1152276886152276886+Missense_MutationGCOCCC_61p.D3492E
FLG1152279527152279527+Missense_MutationTCOCCC_61p.D2612G
FLG1152280002152280002+Missense_MutationTCOCCC_61p.T2454A
FLG21152325817152325817+Missense_MutationTCOCCC_61p.H1482R
FLG21152325818152325818+Missense_MutationGCOCCC_61p.H1482D
FLG21152325820152325820+Missense_MutationTGOCCC_61p.Y1481S
MUC43195510896195510896+Missense_MutationGAOCCC_61p.P2519S
MUC177100680017100680017+Missense_MutationAGOCCC_61p.I1774V
MUC177100682427100682427+Missense_MutationGCOCCC_61p.R2577T
MUC177100682967100682967+Missense_MutationTCOCCC_61p.L2757P
PLEC8145003304145003304+Missense_MutationTCOCCC_61p.Q1213R
AHNAK214105407327105407327+Missense_MutationGAOCCC_61p.P4821S
AHNAK214105413791105413791+Missense_MutationGAOCCC_61p.S2666F
AHNAK214105414923105414923+Missense_MutationTCOCCC_61p.K2289E
PPP2R1A195272295652722956+Missense_MutationCTOCCC_61p.R381W
LAMA5206090967160909671+Missense_MutationCTOCCC_61p.R830Q
DST65651583056515831+Splice_SiteTC-OCCC_61p.G232fs
FLG1152276490152276490+Missense_MutationCGOCCC_03p.E3624D
FLG1152282852152282852+Missense_MutationAGOCCC_03p.Y1504H
FLG1152283256152283256+Missense_MutationCGOCCC_03p.R1369T
ZNF71737578802875788028+Missense_MutationGAOCCC_03p.S199L
MUC177100678724100678724+Missense_MutationCAOCCC_03p.P1343T
MUC177100682922100682922+Missense_MutationGCOCCC_03p.R2742P
KRAS122539828425398284+Missense_MutationCGOCCC_03p.G12A
MUC19124082039640820396+Missense_MutationGAOCCC_03p.R125Q
AHNAK214105410804105410804+Missense_MutationCGOCCC_03p.D3662H
ARID1A12710691527106915+Nonsense_MutationCTOCCC_30p.Q2176*
PIK3CA3178952077178952077+Missense_MutationTGOCCC_30p.N1044K
FLG1152277137152277137+Missense_MutationGCOCCC_69p.R3409G
FLG1152281635152281635+Missense_MutationCAOCCC_69p.R1909S
CELSR334868942348689423+Missense_MutationCAOCCC_69p.R1937L
MUC43195513010195513010+Missense_MutationGAOCCC_69p.P1814L
MUC177100679169100679169+Missense_MutationCAOCCC_69p.A1491E
MUC177100679388100679388+Missense_MutationACOCCC_69p.Q1564P
MUC177100682261100682261+Missense_MutationGCOCCC_69p.V2522L
PLEC8144995266144995266+Missense_MutationGTOCCC_69p.A3045D
PLEC8144998339144998339+Missense_MutationGAOCCC_69p.R2057W
AHNAK116229507162295071+Missense_MutationTAOCCC_69p.D2273V
SPTBN4194099372340993723+Missense_MutationCTOCCC_69p.R97W
UNC13B93539763235397632+Splice_Site-CCATCGGGAAGGTGCTGATGATGTCTGCATACTGCATCAGCACCTTOCCC_69p.T1144fs
ARID1A12710655927106559+Missense_MutationGAOCCC_63p.R2057Q
OBSCN1228456294228456294+Missense_MutationCTOCCC_63p.A1642V
SHROOM347766246877662468+Missense_MutationCTOCCC_63p.R1048C
DST65648536656485366+Nonsense_MutationGAOCCC_63p.R1156*
AHNAK214105412913105412913+Missense_MutationGAOCCC_63p.R2959W
SPTBN4194101938841019388+Missense_MutationGAOCCC_63p.G898S
MAPK1222215339522153395+Missense_MutationCTOCCC_63p.R172H
KRT5125291352452913524+Splice_SiteA-OCCC_63
HELZ2206219754562197545+Missense_MutationGTOCCC_67p.T877N
MAPK8IP2225104298051042980+Missense_MutationCTOCCC_67p.P152L
OBSCN1228412271228412271+Missense_MutationCTOCCC_41p.T922M
SHROOM347767800577678005+Nonsense_MutationCTOCCC_41p.R1705*
DST65641770656417706+Missense_MutationTCOCCC_41p.Q5084R
PLEC8144992259144992259+Missense_MutationCAOCCC_41p.Q4047H
PLEC8145003701145003701+Missense_MutationCTOCCC_41p.E1125K
AHNAK116228536462285364+Missense_MutationCTOCCC_41p.V5509M
KRT5125291147452911474+Missense_MutationCTOCCC_41p.R331H
AHNAK214105412598105412598+Missense_MutationACOCCC_41p.L3064V
AHNAK214105412633105412633+Missense_MutationGAOCCC_41p.P3052L
MAGEE1X7565021175650211+Missense_MutationGAOCCC_41p.V630M
ARID1A12710096127100961+Nonsense_MutationCTOCCC_17p.Q1415*
FLG1152280568152280568+Missense_MutationCAOCCC_17p.R2265I
CTNNB134127564841275648+Nonsense_MutationCTOCCC_17p.R515*
ZNF71737578808875788088+Missense_MutationCTOCCC_17p.R179K
ZNF71737579080075790800+Missense_MutationCTOCCC_17p.D49N
PIK3CA3178936095178936095+Missense_MutationATOCCC_17p.Q546L
MUC43195508955195508955+Missense_MutationCTOCCC_17p.A3166T
SHROOM347767552877675528+Missense_MutationCTOCCC_17p.R1298C
SHROOM347767773077677730+Missense_MutationCTOCCC_17p.T1613I
KRT5125291280552912805+Missense_MutationCTOCCC_17p.S232N
AHNAK214105408107105408107+Missense_MutationGTOCCC_17p.L4561I
AHNAK214105412871105412871+Missense_MutationCTOCCC_17p.A2973T
ARID1A12702381827023818+Nonsense_MutationCGOCCC_18p.Y308*
HRNR1152186877152186877+Missense_MutationCGOCCC_18p.G2410R
FLG1152281113152281113+Missense_MutationGCOCCC_18p.S2083R
FLG1152283589152283589+Nonsense_MutationGTOCCC_18p.S1258*
FLG1152283590152283590+Missense_MutationACOCCC_18p.S1258A
FLG1152284318152284318+Missense_MutationCTOCCC_18p.G1015D
FLG1152284319152284319+Missense_MutationCGOCCC_18p.G1015R
FLG21152328326152328326+Missense_MutationCTOCCC_18p.G646R
ZNF71737578826075788260+Missense_MutationCAOCCC_18p.G122W
MUC177100677793100677793+Missense_MutationGAOCCC_18p.M1032I
MUC177100680228100680228+Missense_MutationCAOCCC_18p.A1844E
MUC177100682045100682045+Missense_MutationCAOCCC_18p.P2450T
MUC177100682552100682552+Missense_MutationAGOCCC_18p.K2619E
MUC177100682556100682556+Missense_MutationAGOCCC_18p.D2620G
UNC13B93539822735398227+Missense_MutationATOCCC_18p.N1176I
AHNAK116229807462298074+Missense_MutationCGOCCC_18p.R1272P
AHNAK214105413471105413471+Missense_MutationTCOCCC_18p.K2773E
SPTB146525843665258452+Splice_SiteCCTGGTGTTCAGATGGT-OCCC_18p.DHLNTR930fs
FLG1152276045152276045+Missense_MutationAGOCCC_48p.Y3773H
FLG1152284263152284263+Missense_MutationGCOCCC_48p.H1033Q
FLG1152284673152284673+Missense_MutationGAOCCC_48p.R897C
FLG1152284791152284791+Missense_MutationGTOCCC_48p.H857Q
FLG21152329942152329942+Missense_MutationCTOCCC_48p.R107Q
OBSCN1228476577228476577+Missense_MutationCGOCCC_48p.L3443V
ZNF71737578692175786921+Missense_MutationGAOCCC_48p.P568L
MUC43195477841195477841+Missense_MutationCTOCCC_48p.V1028I
MUC43195513605195513605+Missense_MutationGAOCCC_48p.P1616S
MUC177100681211100681211+Missense_MutationGCOCCC_48p.V2172L
MUC177100681533100681533+Missense_MutationCAOCCC_48p.T2279N
MUC177100681985100681985+Missense_MutationATOCCC_48p.T2430S
PLEC8144991243144991243+Missense_MutationGAOCCC_48p.S4386L
PLEC8144998614144998614+Missense_MutationCTOCCC_48p.R1965Q
ANK3106182948361829483+Missense_MutationCTOCCC_48p.R3719H
ANK3106183165161831651+Missense_MutationCGOCCC_48p.Q2996H
AHNAK116229565662295656+Missense_MutationATOCCC_48p.V2078D
MUC19124087869540878695+Missense_MutationAGOCCC_48p.I3414V
MUC19124087869740878697+Missense_MutationAGOCCC_48p.I3414M
MUC19124092407440924074+Splice_SiteGTOCCC_48
KRT5125290879452908794+Missense_MutationCTOCCC_48p.G569R
AHNAK214105409546105409546+Missense_MutationTGOCCC_48p.K4081T
AHNAK214105413266105413266+Missense_MutationAGOCCC_48p.L2841P
AHNAK214105416046105416046+Missense_MutationGCOCCC_48p.F1914L
AHNAK214105419446105419446+Missense_MutationTGOCCC_48p.K781T
CASKIN11622367592236759+Missense_MutationGAOCCC_48p.R333W
CDC27174521936445219364+Missense_MutationATOCCC_48p.M469K
ARID1A12710614227106142+Missense_MutationGAOCCC_66p.R1918Q
HRNR1152191206152191206+Missense_MutationAGOCCC_66p.S967P
OBSCN1228432236228432236+Missense_MutationGAOCCC_66p.G1149R
PIK3CA3178952085178952085+Missense_MutationAGOCCC_66p.H1047R
MUC43195506411195506411+Missense_MutationCTOCCC_66p.A4014T
MUC43195508937195508937+Missense_MutationTAOCCC_66p.T3172S
SHROOM347766144677661446+Missense_MutationAGOCCC_66p.K707R
MSH358008864380088643+Missense_MutationCAOCCC_66p.P879T
MSH358008864480088644+Missense_MutationCAOCCC_66p.P879Q
ANK3106182993661829936+Missense_MutationCTOCCC_66p.G3568E
KRAS122539828425398284+Missense_MutationCAOCCC_66p.G12V
AHNAK214105413284105413284+Missense_MutationGAOCCC_66p.S2835L
CASKIN11622308142230814+Missense_MutationGAOCCC_66p.A852V
CDC27174523559845235598+Missense_MutationGTOCCC_66p.S150Y
OBSCN1228522798228522798+Missense_MutationGTOCCC_19p.V5402L
LRP1B2141625780141625780+Missense_MutationATOCCC_19p.S1408T
LRP1B2141625782141625782+Missense_MutationGTOCCC_19p.A1407D
MUC43195477909195477909+Missense_MutationCTOCCC_19p.R1005Q
MUC177100683723100683723+Missense_MutationGCOCCC_19p.R3009T
AHNAK116229554962295549+Missense_MutationCTOCCC_19p.A2114T
EVPL177400379474003794+Missense_MutationTCOCCC_19p.Y1831C
VASP194602101246021012+Missense_MutationTAOCCC_19p.F33I
VASP194602101346021013+Missense_MutationTGOCCC_19p.F33C
LAMA5206090599260905992+Missense_MutationCAOCCC_19p.C1220F
FLG1152280430152280430+Missense_MutationGCOCCC_51p.S2311C
FLG21152324093152324093+Missense_MutationGCOCCC_51p.Q2057E
FLG21152324146152324146+Missense_MutationTGOCCC_51p.H2039P
FLG21152328192152328192+Missense_MutationACOCCC_51p.H690Q
SPTA11158604407158604407+Missense_MutationCGOCCC_51p.E1831Q
OBSCN1228463635228463635+Missense_MutationGAOCCC_51p.R2043H
OBSCN1228475464228475464+Missense_MutationGAOCCC_51p.R3205H
CELSR334868945648689456+Missense_MutationGCOCCC_51p.S1926C
MUC43195512117195512117+Missense_MutationCGOCCC_51p.A2112P
DSP675858467585846+Missense_MutationCTOCCC_51p.S2784F
PLEC8144999110144999110+Missense_MutationCGOCCC_51p.E1800Q
AHNAK116228425162284251+Missense_MutationCTOCCC_51p.E5880K
MUC19124087671040876710+Missense_MutationCTOCCC_51p.P2752L
AHNAK214105412801105412801+Missense_MutationGCOCCC_51p.S2996C
CASKIN11622302362230236+Missense_MutationCGOCCC_51p.A1045P
KRT10173897523238975232+Missense_MutationTCOCCC_51p.S519G
EVPL177401054574010545+Missense_MutationCAOCCC_51p.G779C
ARID3A19932572932572+Nonsense_MutationCTOCCC_51p.R175*
LAMA5206088991160889911+Missense_MutationCTOCCC_51p.A2714T
FLG21152324440152324440+Missense_MutationAGOCCC_33p.I1941T
AHNAK214105412100105412100+Missense_MutationGCOCCC_33p.Q3230E
LAMA5206090967260909672+Missense_MutationGAOCCC_33p.R830W
SPTBN2116646064466460666+Splice_SiteTCCTGGCCCTCACCTTGGCCTTC-OCCC_33p.EKAKV1615fs
PIK3CA3178952085178952085+Missense_MutationAGOCCC_06p.H1047R
DMRTB115392541253925412+Missense_MutationCTOCCC_59p.R96C
TCHH1152084066152084066+Missense_MutationCTOCCC_59p.E543K
HRNR1152189067152189067+Missense_MutationCTOCCC_59p.G1680R
FLG1152282178152282178+Missense_MutationCGOCCC_59p.E1728D
CELSR334868534948685349+Nonsense_MutationGAOCCC_59p.R2352*
MUC43195515449195515449+Missense_MutationAGOCCC_59p.V1001A
DSP675799317579931+Missense_MutationGAOCCC_59p.E1170K
MUC177100683644100683644+Missense_MutationGAOCCC_59p.G2983S
PLEC8144995227144995227+Missense_MutationGAOCCC_59p.T3058M
SPTBN2116646162766461627+Missense_MutationCTOCCC_59p.D1496N
KRT5125291280552912805+Missense_MutationCTOCCC_59p.S232N
AHNAK214105408172105408172+Missense_MutationACOCCC_59p.V4539G
AHNAK214105412720105412720+Missense_MutationGTOCCC_59p.T3023N
AHNAK214105419313105419313+Missense_MutationCGOCCC_59p.E825D
XPO6162812871228128712+Missense_MutationCTOCCC_59p.R644Q
EVPL177400574274005742+Missense_MutationCTOCCC_59p.V1182M
DST65645715756457158+Splice_SiteCT-OCCC_59
MUC19124085822040858228+Splice_SiteCAGGAAGTA-OCCC_59p.QEV1199del
HRNR1152186844152186844+Missense_MutationGAOCCC_44p.R2421C
HRNR1152187048152187048+Missense_MutationCTOCCC_44p.G2353S
HRNR1152187194152187194+Missense_MutationCAOCCC_44p.R2304L
HRNR1152192259152192259+Missense_MutationTCOCCC_44p.T616A
OBSCN1228432198228432198+Missense_MutationCTOCCC_44p.A1136V
ZNF71737578603675786036+Missense_MutationAGOCCC_44p.F863S
ZNF71737578821775788217+Missense_MutationGAOCCC_44p.T136I
MSH357995072479950724+Missense_MutationGCOCCC_44p.A60P
MUC177100679782100679782+Missense_MutationAGOCCC_44p.I1695M
MUC177100679783100679783+Missense_MutationACOCCC_44p.T1696P
MUC177100683099100683099+Missense_MutationCGOCCC_44p.P2801R
AHNAK214105413588105413588+Missense_MutationGCOCCC_44p.L2734V
EVPL177401968074019680+Missense_MutationCTOCCC_44p.R85H
ARID1A12709912227099122+Splice_Site-GOCCC_44p.S1180fs
HRNR1152185695152185695+Missense_MutationCAOCCC_08p.G2804C
HRNR1152187879152187879+Missense_MutationTCOCCC_08p.R2076G
OBSCN1228400030228400030+Nonsense_MutationCAOCCC_08p.Y182*
OBSCN1228505767228505767+Missense_MutationGTOCCC_08p.G4675V
MUC177100680579100680579+Missense_MutationCAOCCC_08p.S1961Y
HELZ2206219527762195277+Missense_MutationGAOCCC_08p.P1633L
LRP1B2141267574141267574+Missense_MutationCAOCCC_15p.C2774F
EYA2204580143745801437+Missense_MutationGAOCCC_15p.V374M
OBSCN1228463548228463548+Missense_MutationCAOCCC_49p.P2014Q
FLG1152286344152286344+Missense_MutationCGOCCC_39p.D340H
ANK3106183236761832367+Missense_MutationCTOCCC_39p.V2758I
SPTBN2116648110566481105+Missense_MutationCTOCCC_39p.E257K
MUC19124087649440876494+Missense_MutationTCOCCC_39p.I2680T
EYA2204581200445812004+Missense_MutationGAOCCC_39p.G501S
MAPK1222216018822160188+Missense_MutationCTOCCC_39p.R148H
PIK3CA3178952007178952007+Missense_MutationAGOCCC_62p.Y1021C
UNC13B93537607435376074+Missense_MutationCTOCCC_62p.S473L
KRT1125307120053071200+Missense_MutationCTOCCC_62p.R343H
AHNAK214105418508105418508+Missense_MutationCAOCCC_62p.V1094L
LAMA5206090825860908258+Missense_MutationGAOCCC_62p.S1057L
PIK3CA3178936067178936067+Nonsense_MutationCTOCCC_31p.R537*
MUC43195515006195515006+Missense_MutationGCOCCC_31p.H1149D
MUC177100683472100683472+Missense_MutationATOCCC_31p.E2925D
MUC177100683474100683474+Missense_MutationTGOCCC_31p.V2926G
UNC13B93538246435382464+Missense_MutationACOCCC_31p.N840T
AHNAK116228998162289981+Missense_MutationCTOCCC_31p.V3970I
OBSCN1228566387228566387+Missense_MutationGAOCCC_11p.R7933Q
CTNNB134126609741266097+Missense_MutationGTOCCC_11p.D32Y
PLEC8144997792144997792+Missense_MutationCTOCCC_11p.R2239H
AHNAK214105412466105412466+Missense_MutationCTOCCC_11p.G3108S
CDC27174524943245249432+Splice_SiteTAOCCC_11
CDC27174524943145249431+Splice_SiteC-OCCC_11
EVPL177401937274019372+Splice_Site-CTGTTTCTGCTCCAGCACGCGTGCCCAGAAACAGGTCAGGAAOCCC_11
ARID1A12710007027100070+Splice_SiteGAOCCC_07
FLG21152331330152331330+Missense_MutationCTOCCC_07p.V11I
PIK3CA3178952085178952085+Missense_MutationAGOCCC_07p.H1047R
PPP2R1A195271597052715970+Missense_MutationCAOCCC_07p.P179T
PLEC8145004373145004373+Missense_MutationCTOCCC_20p.V988M
PLEC8145005812145005812+Missense_MutationGAOCCC_20p.R869W
KRT10173897526138975261+Missense_MutationGAOCCC_20p.S509F
LAMA5206091022960910295+Splice_SiteCCAGTGTGCCCCCAAATCCCCCACACCTTGGTCCTCAGACTCACCGGCTGGCACTCAGCAACTCCAC-OCCC_20p.GGVAECQP785fs
PIK3CA3178919287178919287+Missense_MutationGTOCCC_14p.D258Y
MUC43195489009195489009+Missense_MutationCGOCCC_14p.A585P
MUC19124087807840878078+Missense_MutationCTOCCC_14p.T3208I
AHNAK214105420865105420865+Missense_MutationCTOCCC_14p.R308H
TCHH1152083846152083846+Missense_MutationCAOCCC_43p.R616L
FLG1152284450152284450+Missense_MutationCTOCCC_43p.R971H
OBSCN1228399923228399923+Missense_MutationGAOCCC_43p.G147R
CELSR334869698848696988+Missense_MutationCTOCCC_43p.R1027Q
DST65634687056346870+Missense_MutationCTOCCC_43p.R6850Q
PLEC8144999694144999694+Missense_MutationCTOCCC_43p.R1605H
PLEC8145001886145001886+Missense_MutationGAOCCC_43p.R1287W
UNC13B93540394135403941+Missense_MutationCTOCCC_43p.R1563W
ANK3106183463161834631+Missense_MutationTAOCCC_43p.Q2003L
SPTB146524008065240080+Missense_MutationGAOCCC_43p.A1679V
AHNAK214105418067105418067+Missense_MutationCTOCCC_43p.G1241S
EYA2204572580245725802+Missense_MutationGAOCCC_43p.G295R
LAMA5206088586160885861+Missense_MutationGAOCCC_43p.R3436W
MAPK8IP2225104259251042592+Missense_MutationCTOCCC_43p.R23C
MAGEE1X7564837475648374+Missense_MutationGTOCCC_43p.K17N
RPTN1152128196152128196+Missense_MutationCTOCCC_46p.S460N
DSPP48853698088536980+Missense_MutationAGOCCC_46p.N1056D
MUC177100683179100683179+Missense_MutationGAOCCC_46p.G2828S
PLEC8144991180144991180+Nonsense_MutationCTOCCC_46p.W4407*
AHNAK214105413066105413066+Missense_MutationTCOCCC_46p.K2908E
AHNAK214105418918105418918+Missense_MutationCAOCCC_46p.G957V
ARID1A12710109927101099+Nonsense_MutationCTOCCC_21p.R1461*
FLG1152282672152282672+Missense_MutationGCOCCC_21p.P1564A
LRP1B2141459361141459361+Missense_MutationGAOCCC_21p.T2119M
CELSR334868170448681704+Missense_MutationCTOCCC_21p.A2704T
CELSR334869783848697838+Missense_MutationCTOCCC_21p.V744M
PIK3CA3178936083178936083+Missense_MutationAGOCCC_21p.E542G
PIK3CA3178952013178952013+Missense_MutationGAOCCC_21p.R1023Q
MUC177100685327100685327+Missense_MutationTAOCCC_21p.S3544T
MUC19124087395640873956+Missense_MutationGTOCCC_21p.G1834V
MAPK8IP2225104426251044262+Missense_MutationGAOCCC_21p.R409Q
HRNR1152187606152187606+Missense_MutationGAOCCC_53p.R2167C
LOR1153233513153233513+Missense_MutationGAOCCC_53p.G30S
LRP1B2141660736141660736+Splice_SiteTAOCCC_53
PIK3CA3178936082178936082+Missense_MutationGCOCCC_53p.E542Q
PLEC8144990707144990707+Missense_MutationCTOCCC_53p.V4565M
ANK3106195638561956385+Splice_SiteTAOCCC_53
AHNAK214105411020105411020+Missense_MutationCTOCCC_53p.A3590T
AHNAK214105418481105418481+Missense_MutationCTOCCC_53p.V1103I
AKT2194074194840741948+Missense_MutationCTOCCC_53p.E342K
MAPK1222215339622153396+Missense_MutationGAOCCC_53p.R172C
OBSCN1228494303228494342+Splice_SiteGGCTCCCAGGCCACCAGTGCCACCCTCACTGTCACAGGTG-OCCC_53p.GSQATSATLTVTG3964fs
CASKIN11622395672239567+Splice_Site-GOCCC_53
PIK3CA3178936082178936082+Missense_MutationGAOCCC_26p.E542K
UNC13B93540387335403873+Missense_MutationGAOCCC_26p.G1540D
CDK12173764686637646866+Missense_MutationGAOCCC_26p.R663H
ARID1A12705802927058029+Nonsense_MutationTGOCCC_27p.Y579*
ARID1A12710662127106621+Nonsense_MutationGTOCCC_27p.E2078*
FLG1152285252152285252+Missense_MutationCGOCCC_27p.A704P
OBSCN1228564757228564757+Missense_MutationCTOCCC_27p.R7682C
CTNNB134126609741266097+Missense_MutationGTOCCC_27p.D32Y
CTNNB134127523941275239+Missense_MutationCTOCCC_27p.R469C
PIK3CA3178951955178951955+Missense_MutationAGOCCC_27p.M1004V
PLEC8145007424145007424+Missense_MutationGTOCCC_27p.F590L
KRT1125307020853070208+Missense_MutationGCOCCC_27p.N442K
AHNAK214105412500105412500+Missense_MutationGCOCCC_27p.D3096E
AHNAK214105413588105413588+Missense_MutationGCOCCC_27p.L2734V
AHNAK214105416455105416455+Missense_MutationAGOCCC_27p.M1778T
PPP2R1A195271598352715983+Missense_MutationGAOCCC_27p.R183Q
MAPK8IP2225104192551041925+Nonsense_MutationCTOCCC_27p.Q149*
ARID1A12702390427023904+Nonsense_MutationGAOCCC_05p.W337*
FLG21152328546152328546+Missense_MutationCAOCCC_05p.L572F
FLG21152328547152328547+Missense_MutationAGOCCC_05p.L572S
PIK3CA3178928226178928226+Missense_MutationCTOCCC_05p.P471L
UNC13B93540390335403903+Missense_MutationGAOCCC_05p.R1550Q
ANK3106182991961829919+Missense_MutationGAOCCC_05p.R3574C
AHNAK116228881062288810+Missense_MutationCTOCCC_05p.S4360N
HRNR1152190900152190900+Missense_MutationTAOCCC_29p.S1069C
HRNR1152191862152191862+Missense_MutationAGOCCC_29p.L748S
HRNR1152191964152191964+Missense_MutationCGOCCC_29p.S714T
HRNR1152193378152193378+Missense_MutationCTOCCC_29p.G243S
FLG1152277315152277315+Missense_MutationCGOCCC_29p.E3349D
OBSCN1228494989228494989+Missense_MutationCAOCCC_29p.Q4075K
MUC177100679059100679059+Missense_MutationGCOCCC_29p.K1454N
MUC177100679919100679919+Missense_MutationACOCCC_29p.N1741T
AHNAK116229047062290470+Missense_MutationCTOCCC_29p.V3807M
CASKIN11622289042228904+Splice_SiteGAOCCC_29p.R1400C
OBSCN1228504548228504548+Missense_MutationCTOCCC_64p.P4475L
OBSCN1228526694228526694+Missense_MutationGAOCCC_64p.R5742H
LRP1B2141457870141457870+Missense_MutationTCOCCC_64p.N2250D
PIK3CA3178952085178952085+Missense_MutationAGOCCC_64p.H1047R
PLEC8145008841145008841+Missense_MutationCTOCCC_64p.D472N
CDK12173768730637687306+Missense_MutationCTOCCC_64p.R1404C
LAMA5206089246460892464+Missense_MutationCTOCCC_64p.R2483H
HELZ2206219187962191879+Missense_MutationCTOCCC_64p.E2485K
TCHH1152083325152083325+Missense_MutationATOCCC_60p.L790M
OBSCN1228559450228559450+Missense_MutationGAOCCC_60p.G6991R
PIK3CA3178936094178936094+Missense_MutationCGOCCC_60p.Q546E
PLEC8144999935144999935+Missense_MutationGAOCCC_60p.R1525W
AHNAK116229130062291300+Missense_MutationTCOCCC_60p.K3530R
KRAS122539828425398284+Missense_MutationCTOCCC_60p.G12D
CASKIN11622393052239305+Missense_MutationGTOCCC_60p.N140K
MINK11747949864794986+Missense_MutationGAOCCC_60p.R659H
SPTBN4194097864040978640+Missense_MutationGAOCCC_60p.A38T
PPP2R1A195271598352715983+Missense_MutationGTOCCC_60p.R183L
HELZ2206219141062191410+Missense_MutationTCOCCC_60p.T2566A
HELZ2206219471362194713+Missense_MutationATOCCC_60p.L1821Q
FLG1152278643152278643+Missense_MutationAGOCCC_57p.W2907R
FLG1152282506152282506+Missense_MutationTAOCCC_57p.Y1619F
FLG1152282507152282507+Missense_MutationAGOCCC_57p.Y1619H
FLG1152284081152284081+Missense_MutationGTOCCC_57p.P1094H
FLG21152326837152326837+Missense_MutationGCOCCC_57p.A1142G
MUC43195506887195506887+Missense_MutationAGOCCC_57p.M3855T
MSH358006477080064770+Missense_MutationGAOCCC_57p.R734Q
AHNAK116229280162292801+Missense_MutationATOCCC_57p.F3030I
AHNAK116229521662295216+Missense_MutationCTOCCC_57p.V2225M
KRAS122539828125398281+Missense_MutationCTOCCC_57p.G13D
AHNAK214105411448105411448+Missense_MutationCTOCCC_57p.R3447K
AHNAK214105413093105413093+Missense_MutationCAOCCC_57p.V2899L
CDC27174521468945214689+Missense_MutationCTOCCC_57p.R581Q
HRNR1152191577152191577+Missense_MutationCTOCCC_13p.R843Q
FLG21152326978152326978+Missense_MutationTCOCCC_13p.N1095S
PIK3CA3178936091178936091+Missense_MutationGAOCCC_13p.E545K
DST65648083856480838+Missense_MutationGAOCCC_13p.A2476V
MUC177100681363100681363+Missense_MutationCAOCCC_13p.F2222L
AHNAK116229605962296059+Missense_MutationCAOCCC_13p.V1944L
KRAS122539828125398281+Missense_MutationCTOCCC_13p.G13D
MUC19124083576840835768+Nonsense_MutationATOCCC_13p.K706*
MUC19124087884340878843+Missense_MutationAGOCCC_13p.E3463G
AHNAK214105412294105412294+Missense_MutationAGOCCC_13p.L3165S
PPP2R1A195271632652716326+Missense_MutationGTOCCC_13p.W257L
EVPL177400662574006625+Splice_Site-CTGACTTCTGGGCCCTTCCTTCCTTTTTTTTTTTTTTTTTTTTOCCC_13
ARID1A12710036927100369+Missense_MutationAGOCCC_56p.M1361V
FLG1152286281152286281+Missense_MutationCTOCCC_56p.A361T
PIK3CA3178952085178952085+Missense_MutationAGOCCC_56p.H1047R
PTPRN27157449185157449185+Missense_MutationGAOCCC_56p.T687M
MUC19124087825840878258+Missense_MutationCTOCCC_56p.T3268I
MUC19124087826040878260+Missense_MutationAGOCCC_56p.T3269A
KRT10173897529838975298+Missense_MutationCAOCCC_56p.G497C
TCHH1152084549152084549+Missense_MutationGCOCCC_09p.Q382E
HRNR1152191783152191783+Missense_MutationGCOCCC_09p.H774Q
FLG1152277106152277106+Missense_MutationCTOCCC_09p.R3419Q
FLG1152279642152279642+Missense_MutationGCOCCC_09p.Q2574E
FLG1152279952152279952+Missense_MutationATOCCC_09p.S2470R
OBSCN1228432152228432152+Missense_MutationGCOCCC_09p.G1121R
OBSCN1228467542228467542+Missense_MutationCTOCCC_09p.R2473W
OBSCN1228476583228476583+Missense_MutationGAOCCC_09p.V3445M
OBSCN1228509289228509289+Missense_MutationGAOCCC_09p.R4916Q
CELSR334867934948679349+Missense_MutationCTOCCC_09p.R2920Q
SHROOM347766251677662516+Missense_MutationGAOCCC_09p.D1064N
SHROOM347766266477662664+Missense_MutationGAOCCC_09p.R1113H
MUC177100683102100683102+Missense_MutationCTOCCC_09p.T2802I
MUC177100683800100683800+Missense_MutationGAOCCC_09p.G3035S
MUC177100684875100684875+Missense_MutationGCOCCC_09p.S3393T
PTPRN27157370768157370768+Missense_MutationCTOCCC_09p.R854Q
UNC13B93539691835396918+Missense_MutationGAOCCC_09p.R1090Q
UNC13B93540397835403978+Missense_MutationGAOCCC_09p.R1575Q
AHNAK116230347762303477+Missense_MutationCTOCCC_09p.V32I
AHNAK214105410716105410716+Missense_MutationGTOCCC_09p.P3691H
AHNAK214105414686105414686+Missense_MutationCTOCCC_09p.V2368I
AHNAK214105414938105414938+Missense_MutationCTOCCC_09p.V2284M
VASP194602121146021211+Missense_MutationCTOCCC_09p.R68W
HELZ2206219508562195085+Missense_MutationGAOCCC_09p.A1697V
MAPK8IP2225104263751042637+Missense_MutationGAOCCC_09p.A38T
MAPK8IP2225104515651045156+Missense_MutationGAOCCC_09p.R466Q
SPTA11158637741158637741+Nonsense_MutationCAOCCC_36p.E649*
PIK3CA3178916876178916876+Missense_MutationGAOCCC_36p.R88Q
PIK3CA3178952072178952072+Missense_MutationAGOCCC_36p.M1043V
MUC177100683008100683008+Missense_MutationGCOCCC_36p.V2771L
KRT5125291051752910517+Missense_MutationCTOCCC_36p.R448Q
HRNR1152187900152187900+Missense_MutationCTOCCC_02p.G2069S
HRNR1152191425152191425+Missense_MutationCTOCCC_02p.G894S
FLG1152282147152282147+Missense_MutationGTOCCC_02p.Q1739K
FLG1152282753152282753+Missense_MutationGAOCCC_02p.P1537S
FLG1152283598152283598+Missense_MutationTCOCCC_02p.E1255G
FLG1152285686152285686+Missense_MutationTCOCCC_02p.H559R
PIK3CA3178936082178936082+Missense_MutationGAOCCC_02p.E542K
MUC43195506438195506438+Missense_MutationTCOCCC_02p.S4005G
AHNAK214105416839105416839+Missense_MutationGTOCCC_02p.A1650E
CASKIN11622291882229188+Missense_MutationCTOCCC_02p.R1305Q
MINK11747888084788808+Missense_MutationGAOCCC_02p.R180H
HELZ2206220357862203578+Missense_MutationGCOCCC_02p.A54G
FLG21152327408152327408+Missense_MutationTCOCCC_24p.R952G
FLG21152328778152328778+Missense_MutationCTOCCC_24p.C495Y
SPTA11158605725158605725+Missense_MutationGAOCCC_24p.L1804F
OBSCN1228469825228469825+Missense_MutationCTOCCC_24p.R2797W
SHROOM347767761877677618+Missense_MutationAGOCCC_24p.K1576E
MUC177100675451100675451+Missense_MutationCGOCCC_24p.Q252E
MUC177100676667100676667+Missense_MutationAGOCCC_24p.N657S
MUC177100677995100677995+Missense_MutationAGOCCC_24p.S1100G
MUC177100678029100678029+Missense_MutationGCOCCC_24p.R1111T
PTPRN27157929370157929370+Missense_MutationCTOCCC_24p.D384N
KRT5125291280552912805+Missense_MutationCTOCCC_24p.S232N
PPP2R1A195271632352716323+Missense_MutationCTOCCC_24p.S256F
CDC27174523215445232154+Splice_Site-TGGGGTTAATGOCCC_24
DSP675806587580658+Missense_MutationTAOCCC_28p.L1412Q
DSP675854327585432+Missense_MutationCTOCCC_28p.T2646M
LAMA5206090906260909062+Missense_MutationGAOCCC_28p.P925S
MUC43195506386195506386+Missense_MutationGAOCCC_65p.P4022L
AHNAK116229825262298252+Missense_MutationCTOCCC_65p.V1213M
KRAS122539828525398285+Missense_MutationCAOCCC_65p.G12C
DST65641768856417688+Missense_MutationGAOCCC_10p.S5090L
MUC177100677582100677582+Missense_MutationCGOCCC_10p.T962S
FLG21152327579152327579+Missense_MutationTAOCCC_58p.S895C
SPTA11158592886158592886+Missense_MutationATOCCC_58p.S2003T
OBSCN1228433270228433270+Missense_MutationGAOCCC_58p.R1213H
OBSCN1228506756228506756+Missense_MutationCTOCCC_58p.S4768L
PIK3CA3178936082178936082+Missense_MutationGAOCCC_58p.E542K
MUC43195506555195506555+Missense_MutationCTOCCC_58p.A3966T
SHROOM347767630877676308+Missense_MutationCGOCCC_58p.Q1558E
MUC177100680105100680105+Missense_MutationTCOCCC_58p.L1803P
AHNAK116229557662295576+Missense_MutationGAOCCC_58p.L2105F
AHNAK214105414686105414686+Missense_MutationCTOCCC_58p.V2368I
AHNAK214105419180105419180+Missense_MutationAGOCCC_58p.S870P
HELZ2206219138362191383+Missense_MutationGAOCCC_58p.R2575W
NEURL1B5172113300172113301+Splice_SiteGT-OCCC_58
FLG1152278594152278594+Missense_MutationATOCCC_54p.L2923Q
FLG21152323277152323277+Nonsense_MutationGAOCCC_54p.Q2329*
MUC19124087612640876126+Missense_MutationTGOCCC_54p.H2557Q
CDC27174523559845235598+Missense_MutationGTOCCC_54p.S150Y
DSP675707747570804+Splice_SiteTCAGGGCCATGACAATCGCCAAGGTATGTCC-OCCC_54p.IRAMTIAK560fs
FLG21152326057152326057+Missense_MutationCGOCCC_12p.G1402A
FLG21152326058152326058+Missense_MutationCAOCCC_12p.G1402C
SPTA11158606445158606445+Missense_MutationCTOCCC_12p.E1766K
MUC19124087830640878306+Missense_MutationGAOCCC_12p.G3284E
ARID1A12709995827099958+Nonsense_MutationTAOCCC_40p.Y1279*
FLG1152280083152280083+Missense_MutationCTOCCC_40p.A2427T
FLG21152325292152325292+Missense_MutationCGOCCC_40p.S1657T
FLG21152328192152328192+Missense_MutationACOCCC_40p.H690Q
CELSR334869886448698864+Missense_MutationGAOCCC_40p.R402C
PIK3CA3178936082178936082+Missense_MutationGAOCCC_40p.E542K
PIK3CA3178938935178938935+Missense_MutationACOCCC_40p.E726A
MSH357995072479950724+Missense_MutationGCOCCC_40p.A60P
DSP675584617558461+Missense_MutationGAOCCC_40p.R129Q
DST65648511656485116+Missense_MutationCTOCCC_40p.R1239H
MUC177100676360100676360+Missense_MutationCGOCCC_40p.P555A
PLEC8144997144144997144+Missense_MutationGAOCCC_40p.A2455V
AHNAK214105411236105411236+Missense_MutationCTOCCC_40p.A3518T
AHNAK214105413372105413372+Missense_MutationTCOCCC_40p.M2806V
AHNAK214105414134105414134+Missense_MutationGAOCCC_40p.P2552S
AHNAK214105414343105414343+Missense_MutationCTOCCC_40p.R2482K
AHNAK214105418818105418818+Missense_MutationGCOCCC_40p.D990E
XPO6162816785028167850+Splice_SiteTAOCCC_40
EVPL177400518774005187+Missense_MutationCTOCCC_40p.E1367K
EYA2204580139045801390+Missense_MutationCTOCCC_40p.S358L
HELZ2206219132162191321+Missense_MutationATOCCC_40p.N2595K
MAGEE1X7564899375648993+Missense_MutationGAOCCC_40p.V224M
MAGEE1X7565006875650068+Missense_MutationGTOCCC_40p.G582V
RPTN1152129136152129136+Missense_MutationCTOCCC_23p.G147S
FLG1152282120152282120+Missense_MutationGTOCCC_23p.Q1748K
FLG21152327858152327858+Missense_MutationTCOCCC_23p.S802G
MUC177100676109100676109+Missense_MutationACOCCC_23p.N471T
AHNAK116229580162295801+Missense_MutationTCOCCC_23p.M2030V
AHNAK214105410322105410322+Missense_MutationACOCCC_23p.I3822M
AHNAK214105419653105419653+Missense_MutationAGOCCC_23p.L712P
AHNAK214105419654105419654+Missense_MutationGAOCCC_23p.L712F
CDC27174523567045235670+Splice_Site-AATATACAOCCC_23
FLG1152284655152284655+Missense_MutationTCOCCC_38p.R903G
OBSCN1228464942228464942+Missense_MutationGAOCCC_38p.E2228K
SHROOM347766195277661952+Missense_MutationCTOCCC_38p.R876C
DST65632952856329528+Missense_MutationGAOCCC_38p.R7252C
DST65648186456481864+Missense_MutationGAOCCC_38p.T2134M
PLEC8144996830144996830+Missense_MutationCTOCCC_38p.A2560T
AHNAK116229276662292766+Missense_MutationATOCCC_38p.D3041E
AHNAK116229438162294381+Missense_MutationGCOCCC_38p.A2503G
SPTBN2116646171766461717+Missense_MutationCTOCCC_38p.V1466M
XPO6162818730528187305+Missense_MutationGAOCCC_38p.R107W
PPP2R1A195271630152716301+Missense_MutationCTOCCC_38p.R249C
Gene mutations identified by targeted sequencing in 62 patients with OCCC.

Copy number variant analysis

We next applied the software tool Control-FREEC to detect unique CNVs in 42 ovarian clear cell tumors. The merged copy number pattern of the 42 patients showed arm-level and focal SCNAs across all chromosomes (Fig. 2). While many small amplified/deleted regions were detected across the genome, there were five large blocks of amplifications (spanning >1 Mb) involving chr8q, which includes MYC; chr20q, which includes ZNF217; and chr17q, which includes ERBB2, PPP1R1B, and TBC1D3. Other loci, including the PIK3CA, EIF3E and CDH17 loci, were also amplified (11.9%, 47.6%, and 35.7% of samples, respectively). On the other hand, frequent large blocks of deletions were observed on chr9q in the region including NOTCH1 and PAEP (19% and 16.6%).
Fig. 2

Somatic copy number aberrations (SCNA) landscape in 42 OCCC samples. Deletions and amplifications are indicated by boxes in different shades of blue and red, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Somatic copy number aberrations (SCNA) landscape in 42 OCCC samples. Deletions and amplifications are indicated by boxes in different shades of blue and red, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) At the chromosome arm level, we examined focal somatic copy number alterations by performing GISTIC 2.0 analysis and found that the amplified loci in OCCC samples were at 19q, 2q, 8q, 17q, 1p, 1q, 5q, 12q, and 20q, and the deletions common to the data set were at 19p, 11p, 16p, 9q, 21q, 1p, 7q, 17q, 11q, 16q, 19q, 4p and 2q (cutoff q-value <0.01) (Fig. S4). All focal genes identified by GISTIC 2.0 are shown in Table S9 (amplification cutoff = 0.1, deletion cutoff = -0.1, cutoff q value <0.05).

Pathways in ovarian clear cell carcinoma

To discover the important altered pathways in OCCC, we performed an integrative analysis of single nucleotide variant (SNV) data and copy number variant (CNV) data and selected recurrently mutated genes in two or more individuals that mapped to canonical cancer pathways using MSigDB and frequent alterations across multiple pathways, including p53 effectors, NGF signaling, focal adhesion, and PTEN-dependent cell cycle arrest and apoptosis (Table 3 and Fig. 5S). KEGG pathway enrichment analysis further identified significant overlaps (q value <0.001) with KEGG cancer, platinum drug resistance and the AMPK signaling pathway (Table S10).
Table 3

MSigDB canonical pathway database enrichment for recurrent mutant genes in OCCC samples. This table presents the top 20 modules. Module: name of the module.

Gene Set NameGenes in Gene Set (K)DescriptionGenes in Overlap (k)k/Kp-valueFDR q-value
KEGG_PATHWAYS_IN_CANCER328Pathways in cancer250.07625.43E−187.22E−15
PID_P53_DOWNSTREAM_PATHWAY137Direct p53 effectors130.09493.58E−112.18E−08
KEGG_TYPE_II_DIABETES_MELLITUS47Type II diabetes mellitus90.19156.19E−112.18E−08
KEGG_PROSTATE_CANCER89Prostate cancer110.12366.55E−112.18E−08
REACTOME_DEVELOPMENTAL_BIOLOGY396Genes involved in Developmental Biology190.0481.82E−104.84E−08
REACTOME_SIGNALLING_BY_NGF217Genes involved in Signalling by NGF140.06451.08E−092.39E−07
KEGG_THYROID_CANCER29Thyroid cancer70.24141.52E−092.84E−07
SIG_PIP3_SIGNALING_IN_CARDIAC_MYOCTES67Genes related to PIP3 signaling in cardiac myocytes90.13431.71E−092.84E−07
KEGG_FOCAL_ADHESION201Focal adhesion130.06474.15E−096.13E−07
REACTOME_IMMUNE_SYSTEM933Genes involved in Immune System260.02799.84E−091.31E−06
KEGG_COLORECTAL_CANCER62Colorectal cancer80.1291.93E−082.33E−06
REACTOME_ADAPTIVE_IMMUNE_SYSTEM539Genes involved in Adaptive Immune System190.03532.77E−083.07E−06
KEGG_NOTCH_SIGNALING_PATHWAY47Notch signaling pathway70.14895.45E−085.58E−06
PID_HES_HEY_PATHWAY48Notch−mediated HES/HEY network70.14586.34E−086.02E−06
SIG_INSULIN_RECEPTOR_PATHWAY_IN_CARDIAC_MYOCYTES51Genes related to the insulin receptor pathway70.13739.79E−088.67E−06
KEGG_ENDOMETRIAL_CANCER52Endometrial cancer70.13461.12E−079.34E−06
REACTOME_SIGNALING_BY_FGFR112Genes involved in Signaling by FGFR90.08041.64E−071.12E−05
PID_FGF_PATHWAY55FGF signaling pathway70.12731.67E−071.12E−05
BIOCARTA_ERK5_PATHWAY18Role of Erk5 in Neuronal Survival50.27781.68E−071.12E−05
BIOCARTA_PTEN_PATHWAY18PTEN dependent cell cycle arrest and apoptosis50.27781.68E−071.12E−05
MSigDB canonical pathway database enrichment for recurrent mutant genes in OCCC samples. This table presents the top 20 modules. Module: name of the module. Given that the four functional protein activating pathways, including the (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, TP53 pathway, ERBB2 pathway, and chromatin remolding pathways were more frequently activated in OCCCs, these pathways represent potential therapeutic pathways for targeted treatment approaches (Fig. 3). Of the 42 samples, 35 (83%) contained at least one mutation in one of the four pathways. The PI3K/AKT, TP53, ERBB2, and chromatin remolding pathways were mutated in 83%, 67%, 40% and 71% of cases, respectively (Fig. S6).
Fig. 3

Somatically altered pathways in OCCC patients. (a) Somatically altered genes in the PI3K/AKT, (b) TP53, (c) ERBB2 and (d) chromatin remolding pathways. Non-synonymous somatic mutations and copy number deletions were considered as inactivating mutations (shades of blue), while copy number amplifications were considered as activating mutations (shades of red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Somatically altered pathways in OCCC patients. (a) Somatically altered genes in the PI3K/AKT, (b) TP53, (c) ERBB2 and (d) chromatin remolding pathways. Non-synonymous somatic mutations and copy number deletions were considered as inactivating mutations (shades of blue), while copy number amplifications were considered as activating mutations (shades of red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Clinical relevance of aberrant genes mutations

For the WES cohort of 42 OCCC cases, the median follow-up time was 27.6 months, and 78% of patients were alive at the time of last follow-up. Kaplan-Meier analysis with the log-rank test revealed a shorter survival period for the 42 OCCC patients with PLEC mutations (HR 0.27, 95% confidence interval 0.76 to 0.04, P = 0.004) or CDC27 mutations (HR 3.6, 95% confidence interval 1.2 to 10.7, P = 0.035) than for those without the corresponding mutation (Fig. S7). In addition, based on the targeted sequencing data, the OS was not significantly different between the PLEC or CDC27 mutation-positive patients and the wild-type patients (P = 0.227 or P = 0.954) (Fig. S7). However, the OS analysis of OCCC patients grouped by MAGEE1 mutation revealed that OCCC patients with MAGEE1 mutations had a shorter survival time than those without MAGEE1 mutations (log-rank p < 0.05) (Fig. 4). The OBSCN mutation was found to have a significant effect on overall survival in all OCCC patients (Fig. 4). The human OBSCN gene on chromosome 1q42.13 region is comprised of over 80 exons and encodes a ∼720 kDa protein. The OBSCN gene is frequently and consistently mutated in various cancers with a strong correlation with breast, colorectal and other female related cancers [15]. For known OCCC driver genes, such as ARID1A, PIK3CA and PPP2R1A, no survival difference was observed based on the presence of genetic abnormalities. These data suggest that mutations in these genes may represent a poor prognostic factor and are likely involved in the pathogenesis of OCCC.
Fig. 4

Kaplan-Meier estimate of overall survival by OBSCN mutational status (a) and MAGEE1 mutational status (b). Patients with tumors harboring mutations had significantly worse overall survival than those with corresponding WT genes in their tumors (log-rank test, OBSCN P = 0.042; MAGEE1 P = 0.043).

Kaplan-Meier estimate of overall survival by OBSCN mutational status (a) and MAGEE1 mutational status (b). Patients with tumors harboring mutations had significantly worse overall survival than those with corresponding WT genes in their tumors (log-rank test, OBSCN P = 0.042; MAGEE1 P = 0.043).

MAGEE1 mutants affects cell growth in OCCC cells

To further elucidate the relevance of MAGEE1 to OCCC progression, we first analyzed MAGEE1 levels in OCCC cell lines (Fig. 5a). Then, we performed a functional assay and found that MAGEE1 overexpression significantly decreased the viability of OCCC cells (Fig. 5c). MAGEE1 contains a nuclear localization signal in the N-terminal region and two MAGE domains in the C-terminal region. To determine whether the two domains of the MAGEE1 gene are associated with proliferation, OVISIE and ES-2 cells expressing the MAGEE1 mutants (MAGEE1-mut1 and MAGEE1-mut2) were constructed and analyzed (Fig. 5b). CCK8 assays revealed that both OVISIE and ES-2 cells transfected with MAGEE1-mut1 or MAGEE1-mut2 exhibited a significant decrease in cell proliferation (Fig. 5c), suggesting that both domains of the MAGEE1 gene are closely associated with OCCC cell proliferation. However, the viability of OCCC cells transfected with the MAGEE1-mut2 was lower than that of cells transfected with the MAGEE1-mut1, revealing that the second domain of MAGEE1 has a greater contribution to OCCC cell proliferation.
Fig. 5

MAGEE1 overexpression inhibits cell viability in vitro. (a) The expression of MAGEE1 in the ES-2 and OVISE cell lines transfected with empty vector control (NC), MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2 was determined by RT-PCR. (b) Western blotting of MAGEE1 expression levels in ES-2 and OVISE cells transfected with empty vector control (NC), MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2. (c) CCK8 assay showed a significant reduction in the viability of cells transfected with MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2 compared to cells transdfected with empty vector control (NC).

MAGEE1 overexpression inhibits cell viability in vitro. (a) The expression of MAGEE1 in the ES-2 and OVISE cell lines transfected with empty vector control (NC), MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2 was determined by RT-PCR. (b) Western blotting of MAGEE1 expression levels in ES-2 and OVISE cells transfected with empty vector control (NC), MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2. (c) CCK8 assay showed a significant reduction in the viability of cells transfected with MAGEE1-WT, MAGEE1-Mut1 or Magee1-Mut2 compared to cells transdfected with empty vector control (NC).

Discussion

OCCC is one of the most malignant subtypes of epithelial ovarian cancer and is more prevalent in Asians (11.1% of total EOC cases) than in Caucasians or Africans in the USA (4.8 and 3.1%, respectively) [16], [17]. OCCC is a rare form or subtype of epithelial ovarian cancer that has a unique structure (morphology), unfavorable prognosis, and chemotherapeutic resistance. Most notably, ARID1A and PIK3CA were the most frequently mutated genes in OCCC patients. Yasuda et al. indicated that ARID1A and PIK3CA was the most frequently mutated gene, occurring in approximately 66.7% and 50% of patients with OCCC (n = 48) in Japanese women using whole exome sequencing [12]. In addition, Seo et al. revealed that PIK3CA mutations and ARID1A were found in 40% and 40% in the 15 Korean OCCCs using whole exome sequencing [11]. In another study, ARID1A mutations and PIK3CA mutations were detected in 77.8% and 66.7% of OCCC (n = 16) in the Taiwanese population using targeted sequencing [18]. In the present study, we successfully characterized the genomic landscape of 69 Chinese patients with OCCC. To our knowledge, this is the first report of an NGS WES study in Chinese patients with OCCC. The most frequent mutated gene identified in this study was the ARID1A gene (66.7%, 32/48), which encodes a key component of the SWI/SNF chromatin-remodeling complex that is conserved in all eukaryotes, plays an important role in controlling gene expression and is critical in development, differentiation, and tumor suppression. Although the frequency of somatic ARID1A mutations in OCCC was recently published to be 46% and 57% based on whole-exome and transcriptome sequencing analyses, respectively, the frequency reported in this study was 66.7%. The frequency of ARID1A mutations in this study was slightly higher than that in a previous report, but the difference was not statistically significant. Mutation of the SWI/SNF-related gene SMARCA2 was identified in six cases; hence other genes in the ARID1 pathway may be mutated in the remaining cases. Other known OCCC-related genes were also frequently mutated, including PIK3CA (50%, 24/48), PPP2R1A (18.8%, 9/48) and KRAS (16.7%, 8/48) (Fig. 3). MAGEE1 was also predicted to be a cancer driver gene by three independent mutation prediction algorithms. MAGEE1 is a member of the melanoma antigen gene (MAGE) family and is encoded on the X chromosome, spanning one exon, containing 957 amino acids. The MAGE family has garnered growing interest as cancer biomarkers and immunotherapy targets because a subset of these human proteins has been classified as cancer-testis antigens (CTAs), which have restricted expression in the testis (and occasionally in the ovary and placenta) and are aberrantly re-expressed in cancer and can be immunogenic. Collectively, MAGE genes have been found to be broadly expressed in many tumor types, including colon, melanoma, brain, lung, prostate, and breast cancer, among others. Furthermore, MAGEE1 is mutated frequently enough to be classified as a candidate cancer gene (CAN-gene) in breast cancer and thus potentially a driver of tumorigenesis. In this study, mutations in MAGEE1 were found in eight patients in the WES cohort and fifth patients in the targeted sequencing cohort. The other genes, including MUC4, ARID3A, FLG2, TCHH, GRM3, MUC17, ZNF208 and GAGE12J, were identified as novel SMGs in our study. MUC4 and MUC17 are related to cell apoptosis/anti-adhesive [19] and cell restitution processes [20], respectively, and we identified somatic mutations in these genes in 28.6% and 23.8% of OCCC samples, respectively. ARID3A, FLG2, TCHH, GRM3, ZNF208, and GAGE12J were mutated in 4.8% to 28.6% of samples, respectively. ARID3A is a member of the human AT-rich interaction domain (ARID) family, is located at 19p13.3, and is a nuclear matrix-associated transcription factor that blocks cell differentiation and promotes cell proliferation [21]. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa belonging to the fibrinogen-related protein superfamily that has apoptotic effects on effector T-cells and prevents the maturation of dendritic cells [22]. TCHH (trichohyalin) is a member of the S100-fused type proteins (SFTP) family and probably contributes to tumorigenic processes such as cell proliferation, metastasis, angiogenesis and immune evasion [23]. GRM3 is a group II metabotropic glutamate receptors that activates the AKT signaling pathway [24]. Additionally, mutations in GRM3 that lead to constitutive receptor activation have been shown to provide cell proliferation and survival signals in melanoma [25]. ZNF208 is a member of the zinc finger family of proteins that bind to DNA through a series of zinc finger motifs and regulate gene transcription [26]. Mutations in ZNF208 have been observed in gastric cancer [27]. The GAGE12J gene is located on the X chromosome and encodes a cancer-testis antigens that promotes gastric cancer growth and metastasis by modulating the expression of gastric cancer metastasis-related genes [28]. This current study has several limitations. First, the study population of this retrospective case-control study was small (n = 69). Second, the median follow-up time was 58 months from the SEER database for patients with white and Asian. And early-stage OCCC confined to ovary has favorable prognosis. Regarding the stage of disease (n = 69) in our cohort, it was stage I in 45(65.2%), stage II in 12 (17.4%), stage III in 12(17.4%). The median followup of 27.6 months in our cohort is relatively short and 78% of patients were alive at last followup. Therefore, this is a limit regarding the impact of reported mutations on survival outcomes. Third, the expression of the genes with somatic mutations was not investigated. Paired tumor/normal whole transcriptome sequencing or microarray analyses to discover the effects of genetic alterations would improve the accuracy and completeness of the genomic profiling results. Despite these limitations, this study is the first to use whole exome sequencing to genetically characterize OCCC in an Asian population. By analyzing cancer tissue samples and matched normal samples from individual OCCC patients, integrative somatic analyses were completed.

Conclusions

In conclusion, the present study successfully characterized the genomic landscape of 69 patients with OCCC. We identified potential therapeutic targets for the treatment of OCCC. Additional larger studies including whole transcriptome sequencing to determine the effects of genetic alterations are warranted.

Conflict of interest

No potential conflicts of interest were disclosed.
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