Browsing by Author "Yeh, Kun-Tu"

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Evaluation of whole exome sequencing by targeted gene sequencing and Sanger sequencing
(2017-08-01)
Chang, Ya-Sian
;
Huang, Hsien-Da
;
Yeh, Kun-Tu
;
Chang, Jan-Gowth
;
生物科技學系
Background: Targeted gene sequencing (TGS) and whole exome sequencing (WES) are being used in clinical testing in laboratories. We compared the performances of TGS and WES using the same DNA samples. Methods: DNA was extracted from 10 endometrial tumor tissue specimens. Sequencing were performed with an Illumina HiSeq 2000. We randomly selected variants to confirm through Sanger sequencing or mutant-enriched PCR with Sanger sequencing. Results: We found that the variants identified in both TGS and WES were true positives (47/47), regardless of the sequencing depth. Most variants found in TGS only were true positives (34/40), and most of the variants found by WES only were false positives (8/18). From these results, we suggest that the sequencing depth may not play important role in the accuracy of NGS-based methods. After analysis, we found that WES had a sensitivity of 72.70%, specificity of 96.27%, precision of 99.44%, and accuracy of 75.03%. Conclusions: The results of NGS-based methods must currently be validated, especially for important reported variants regardless of the methods used, and for the use of WES in cancers a higher false negative rate must be considered. More sensitive methods should be used to confirm the NGS results in uneven cancer tissues.
Genetic alterations in endometrial cancer by targeted next-generation sequencing
(2016-02)
Chang, Ya-Sian
;
Huang, Hsien-Da
;
Yeh, Kun-Tu
;
Chang, Jan-Gowth
;
生物科技學系
Many genetic factors play important roles in the development of endometrial cancer. The aim of this study was to investigate genetic alterations in the Taiwanese population with endometrial cancer. DNA was extracted from 10 cases of fresh-frozen endometrial cancer tissue. The exomes of cancer-related genes were captured using the NimbleGen Comprehensive Cancer Panel (578 cancer-related genes) and sequenced using the Illumina Genomic Sequencing Platform. Our results revealed 120 variants in 99 genes, 21 of which were included in the Oncomine Cancer Research Panel used in the National Cancer Institute Match Trial. The 21 genes comprised 8 tumor suppressor candidates (AIM, MSH2, PIK3R1, PTCH1, PTEN, TET2, TP53, and TSC1) and 13 oncogene candidates (ALK, BCL9, CTNNB1, ERBB2, FGFR2, FLT3, HNF1A, KIT, MTOR, PDGFRA, PPP2R1A, PTPN11, and SF3B1). We identified a high frequency of mutations in PTEN (50%) and genes involved in the endometrial cancer-related molecular pathway, which involves the IL-7 signaling pathway (PIK3R1, n = 1; AKT2, n = 1; FOXO1, n = 1). We report the mutational landscape of endometrial cancer in the Taiwanese population. We believe that this study will shed new light on fundamental aspects for understanding the molecular pathogenesis of endometrial cancer and may aid in the development of new targeted therapies. (C) 2015 Elsevier Inc. All rights reserved.
Identification of novel mutations in endometrial cancer patients by whole-exome sequencing
(2017-05-01)
Chang, Ya-Sian
;
Huang, Hsien-Da
;
Yeh, Kun-Tu
;
Chang, Jan-Gowth
;
生物科技學系
The aim of the present study was to identify genomic alterations in Taiwanese endometrial cancer patients. This information is vitally important in Taiwan, where endometrial cancer is the second most common gynecological cancer. We performed whole-exome sequencing on DNA from 14 tumor tissue samples from Taiwanese endometrial cancer patients. We used the Genome Analysis Tool kit software package for data analysis, and the dbSNP, Catalogue of Somatic Mutations in Cancer (COSMIC) and The Cancer Genome Atlas (TCGA) databases for comparisons. Variants were validated via Sanger sequencing. We identified 143 non-synonymous mutations in 756 canonical cancer-related genes and 1,271 non-synonymous mutations in non-canonical cancer-related genes in 14 endometrial samples. PTEN, KRAS and PIK3R1 were the most frequently mutated canonical cancer-related genes. Our results revealed nine potential driver genes (MAPT, IL24, MCM6,TSC1,BIRC2, CIITA, DST, CASP8 and NOTCH2) and 21 potential passenger genes (ARMCX4, IGSF10, VPS13C, DCT,DNAH14,TLN1,ZNF605,ZSCAN29, MOCOS, CMYA5, PCDH17, UGT1A8, CYFIP2, MACF1, NUDT5, JAKMIP1, PCDHGB4, FAM178A, SNX6, IMP4 and PCMTD1). The detected molecular aberrations led to putative activation of the mTOR, Wnt, MAPK, VEGF and ErbB pathways, as well as aberrant DNA repair, cell cycle control and apoptosis pathways. We characterized the mutational landscape and genetic alterations in multiple cellular pathways of endometrial cancer in the Taiwanese population.
Transcribed pseudogene psi PPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma
(2013-04-01)
Chan, Wen-Ling
;
Yuo, Chung-Yee
;
Yang, Wen-Kuang
;
Hung, Shih-Ya
;
Chang, Ya-Sian
Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene psi PPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted psi PPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected psi PPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of psi PPM1K. Furthermore, expression of NEK8 in psi PPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of psi PPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.