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ABSTRACT: Targeted resequencing using next-generation sequencing technology is being rapidly applied to the molecular diagnosis of human genetic diseases. The group of muscular dystrophies may be an appropriate candidate for this approach because these diseases exhibit genotype-phenotype heterogeneity. To perform a proof-of-concept study, we selected four patients with congenital muscular dystrophies with defective glycosylation of alpha-dystroglycan. A custom-solution-based target enrichment kit was designed to capture whole-genic regions of the 26 muscular-dystrophy-related genes, including six genes implicated in alpha-dystroglycanopathies. Although approximately 95% of both coding and noncoding regions were covered with at least 15-read depth, parts of the coding exons of FKRP and POMT2 were insufficiently covered. Homozygous and compound heterozygous POMGnT1 mutations were found in two patients. Two novel noncoding variants of FKTN were identified in one patient who had a retrotransposon insertion mutation of FKTN in only one allele. The current targeted resequencing strategy yielded promising results for the extension of this method to other muscular dystrophies. As suboptimal coverage in a small subset of coding regions may affect the sensitivity of the method, complementary Sanger sequencing may be required.
Neuromuscular Disorders 02/2013; · 2.80 Impact Factor
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Hansoo Park,
Seungbok Lee,
Hyun-Jin Kim,
Young Seok Ju, Jong-Yeon Shin,
Dongwan Hong,
Marcin von Grotthuss,
Dong-Sung Lee,
Changho Park,
Jennifer Hayeon Kim,
Boram Kim,
Yun Joo Yoo,
Sung-Il Cho,
Joohon Sung,
Charles Lee,
Jong-Il Kim,
Jeong-Sun Seo
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ABSTRACT: BACKGROUND: Musical abilities such as recognising music and singing performance serve as means for communication and are instruments in sexual selection. Specific regions of the brain have been found to be activated by musical stimuli, but these have rarely been extended to the discovery of genes and molecules associated with musical ability. METHODS: A total of 1008 individuals from 73 families were enrolled and a pitch-production accuracy test was applied to determine musical ability. To identify genetic loci and variants that contribute to musical ability, we conducted family-based linkage and association analyses, and incorporated the results with data from exome sequencing and array comparative genomic hybridisation analyses. RESULTS: We found significant evidence of linkage at 4q23 with the nearest marker D4S2986 (LOD=3.1), whose supporting interval overlaps a previous study in Finnish families, and identified an intergenic single nucleotide polymorphism (SNP) (rs1251078, p=8.4×10(-17)) near UGT8, a gene highly expressed in the central nervous system and known to act in brain organisation. In addition, a non-synonymous SNP in UGT8 was revealed to be highly associated with musical ability (rs4148254, p=8.0×10(-17)), and a 6.2 kb copy number loss near UGT8 showed a plausible association with musical ability (p=2.9×10(-6)). CONCLUSIONS: This study provides new insight into the genetics of musical ability, exemplifying a methodology to assign functional significance to synonymous and non-coding alleles by integrating multiple experimental methods.
Journal of Medical Genetics 11/2012; · 6.36 Impact Factor
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Jeong-Sun Seo,
Young Seok Ju,
Won-Chul Lee, Jong-Yeon Shin,
June Koo Lee,
Thomas Bleazard,
Junho Lee,
Yoo Jin Jung,
Jung-Oh Kim,
Jung-Young Shin,
Saet-Byeol Yu,
Jihye Kim,
Eung-Ryoung Lee,
Chang-Hyun Kang,
In-Kyu Park,
Hwanseok Rhee,
Se-Hoon Lee,
Jong-Il Kim,
Jin-Hyoung Kang,
Young Tae Kim
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ABSTRACT: All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.
Genome Research 09/2012; · 13.61 Impact Factor
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Jee-In Heo,
Soo-Jin Oh,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim, Jong-Yeon Shin,
Min-Ju Kim,
Minju Kim,
Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: DNA damage in eukaryotic cells induces signaling pathways mediated by the ATM, p53 and ERK proteins, but the interactions between these pathways are not completely known. To address this issue, we performed a time course analysis in human embryonic fibroblast cells treated with DNA-damaging agents. DNA damage induced the phosphorylation of p53 at Ser 15 (p-p53) and the phosphorylation of ERK (p-ERK). Inhibition of p53 by a dominant negative mutant or in p53(-/-) fibroblast cells abolished ERK phosphorylation. ERK inhibitor prevented p53 phosphorylation, indicating that phosphorylations of p53 and p-ERK are interdependent each other. A time course analysis showed that ATM interacted with p-p53 and p-ERK in early time (0.5 h) and interaction between ATM-bound p-p53 and p-ERK or ATM-bound p-ERK and p-p53 occurred in late time (3 h) of DNA damage. These results indicate that ATM mediates interdependent activation of p53 and ERK through formation of a ternary complex between p-p53 and p-ERK in response to DNA damage to cause growth arrest.
Molecular Biology Reports 05/2012; 39(8):8007-14. · 2.93 Impact Factor
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ABSTRACT: The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.
Genome Research 12/2011; 22(3):436-45. · 13.61 Impact Factor
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ABSTRACT: Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology.
A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25
16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication.
Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction.
The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.
Journal of Medical Genetics 11/2011; 48(11):731-6. · 6.36 Impact Factor
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ABSTRACT: Progesterone has a potential protective effect against ovarian carcinoma induced by estrogen. Progesterone is also known to cause apoptosis while tamoxifen induces growth arrest. Therefore, we attempted to determine whether combined treatment with progesterone and tamoxifen has a synergistic effect on anti-cancer activity. Although progesterone is known to cause apoptosis while tamoxifen induces growth arrest in many cancer cells, the detailed action of progesterone and tamoxifen and the anticancer effect of combined treatment have not been tested in ovarian cancer cells. Therefore, we tested the growth and apoptosis activity of progesterone and tamoxifen and the anticancer effect of combined treatment of progesterone and tamoxifen in ovarian cancer cells. Ovarian cancer cells, PA-1, were treated with progesterone, tamoxifen, or a combination of progesterone and tamoxifen. The anti-cancer effects were investigated by use of flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, DNA fragmentation analysis, and Western blot analysis. We found that 100 µM progesterone induced typical apoptosis in PA-1 cells. Treatment of PA-1 cells with 10 µM tamoxifen resulted in an increase in the levels of p21, p27, p16 and phospho-pRb, indicating typical G1 arrest. Co-treatment of PA-1 cells with 100 µM progesterone and 10 µM tamoxifen resulted in typical apoptosis, similar to that induced by treatment with 100 µM progesterone alone. These results indicate that progesterone caused apoptosis and tamoxifen induced G1 arrest. Combined treatment with tamoxifen and progesterone caused apoptosis similar to that induced by treatment with progesterone alone and had no additional anti-cancer effect in ovarian cancer cells.
Oncology Reports 09/2011; 27(1):87-93. · 1.84 Impact Factor
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Young Seok Ju,
Jong-Il Kim,
Sheehyun Kim,
Dongwan Hong,
Hansoo Park, Jong-Yeon Shin,
Seungbok Lee,
Won-Chul Lee,
Sujung Kim,
Saet-Byeol Yu, [......],
Maryam Yavartanoo,
Hyunseok Peter Kang,
Omer Gokcumen,
Diddahally R Govindaraju,
Jung Hee Jung,
Hyonyong Chong,
Kap-Seok Yang,
Hyungtae Kim,
Charles Lee,
Jeong-Sun Seo
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ABSTRACT: Massively parallel sequencing technologies have identified a broad spectrum of human genome diversity. Here we deep sequenced and correlated 18 genomes and 17 transcriptomes of unrelated Korean individuals. This has allowed us to construct a genome-wide map of common and rare variants and also identify variants formed during DNA-RNA transcription. We identified 9.56 million genomic variants, 23.2% of which appear to be previously unidentified. From transcriptome sequencing, we discovered 4,414 transcripts not previously annotated. Finally, we revealed 1,809 sites of transcriptional base modification, where the transcriptional landscape is different from the corresponding genomic sequences, and 580 sites of allele-specific expression. Our findings suggest that a considerable number of unexplored genomic variants still remain to be identified in the human genome, and that the integrated analysis of genome and transcriptome sequencing is powerful for understanding the diversity and functional aspects of human genomic variants.
Nature Genetics 07/2011; 43(8):745-52. · 35.53 Impact Factor
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Jee-In Heo,
Soo-Jin Oh,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim, Jong-Yeon Shin,
Min-Ju Kim,
Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.
Molecular Biology Reports 11/2010; 38(4):2785-91. · 2.93 Impact Factor
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Dongwan Hong,
Sung-Soo Park,
Young Seok Ju,
Sheehyun Kim, Jong-Yeon Shin,
Sujung Kim,
Saet-Byeol Yu,
Won-Chul Lee,
Seungbok Lee,
Hansoo Park,
Jong-Il Kim,
Jeong-Sun Seo
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ABSTRACT: High-throughput genomic technologies have been used to explore personal human genomes for the past few years. Although the integration of technologies is important for high-accuracy detection of personal genomic variations, no databases have been prepared to systematically archive genomes and to facilitate the comparison of personal genomic data sets prepared using a variety of experimental platforms. We describe here the Total Integrated Archive of Short-Read and Array (TIARA; http://tiara.gmi.ac.kr) database, which contains personal genomic information obtained from next generation sequencing (NGS) techniques and ultra-high-resolution comparative genomic hybridization (CGH) arrays. This database improves the accuracy of detecting personal genomic variations, such as SNPs, short indels and structural variants (SVs). At present, 36 individual genomes have been archived and may be displayed in the database. TIARA supports a user-friendly genome browser, which retrieves read-depths (RDs) and log2 ratios from NGS and CGH arrays, respectively. In addition, this database provides information on all genomic variants and the raw data, including short reads and feature-level CGH data, through anonymous file transfer protocol. More personal genomes will be archived as more individuals are analyzed by NGS or CGH array. TIARA provides a new approach to the accurate interpretation of personal genomes for genome research.
Nucleic Acids Research 11/2010; 39(Database issue):D883-8. · 8.03 Impact Factor
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Soo-Jin Oh,
Jee-In Heo,
Yoon-Jung Kho,
Jeong-Hyeon Kim,
Hong-Joon Kang,
Seong-Hoon Park,
Hyun-Seok Kim, Jong-Yeon Shin,
Min-Ju Kim,
Sung Chan Kim,
Jae-Bong Park,
Jaebong Kim,
Jae-Yong Lee
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ABSTRACT: Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.
Experimental neurobiology. 09/2010; 19(2):83-9.
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Hansoo Park,
Jong-Il Kim,
Young Seok Ju,
Omer Gokcumen,
Ryan E Mills,
Sheehyun Kim,
Seungbok Lee,
Dongwhan Suh,
Dongwan Hong,
Hyunseok Peter Kang, [......],
Hyeran Kim,
Song Ju Yang,
Kap-Seok Yang,
Hyungtae Kim,
Matthew E Hurles,
Stephen W Scherer,
Nigel P Carter,
Chris Tyler-Smith,
Charles Lee,
Jeong-Sun Seo
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ABSTRACT: Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.
Nature Genetics 04/2010; 42(5):400-5. · 35.53 Impact Factor
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Hyun-Seok Kim,
Eui-ju Yeo,
Seong-Hoon Park,
Joo-In Park,
Sang-Chul Park, Jong-Yeon Shin,
Min-Ju Kim,
Soo-Jin Oh,
Moo-Ho Won,
Tae-Chun Kang,
Jae-Bong Park,
Jaebong Kim,
Jong-Il Kim,
Hyun-Yong Lee,
Jae-Yong Lee
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ABSTRACT: Hydoxyurea induces senescence-like growth arrest in normal human fibroblasts. p21(WAF/CIP1/SDI1), a cyclin dependent kinase inhibitor, was found to be upregulated during this growth arrest. Levels of p21(WAF/CIP1/SDI1) protein and mRNA were increased nine-fold by hydroxyurea in these cells. In order to determine whether p21(WAF/CIP1/SDI1) mRNA is increased by hydroxyurea at the transcriptional level, human fibroblast cells were transfected with reporter constructs containing a p21(WAF/CIP1/SDI1) promoter fragment and then treated with hydroxyurea. The luciferase activities in the reporter-transfected fibroblast cells were not increased by hydroxyurea, indicating that p21(WAF/CIP1/SDI1) transcription was not elevated by hydroxyurea. The half-life of the p21(WAF/CIP1/SDI1) mRNA was increased by 2.5-fold but that of p21(WAF/CIP1/SDI1) protein was not. Our results suggest that increased mRNA stability is the major mechanism of p21(WAF/CIP1/SDI1) elevation in the hydroxyurea-induced growth arrest of human fibroblasts.
Mechanisms of Ageing and Development 01/2006; 126(12):1255-61. · 3.44 Impact Factor
