Genomic Instability in EBV-Transformed Lymphoblastoid Cell Lines
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Genomic instability in EBV-transformed lymphoblastoid cell lines Ji Hee Oh The Graduate school Yonsei University Graduate Program in Science for Aging Genomic instability in EBV-transformed lymphoblastoid cell lines Dissertation Submmitted to Graduate Program in Science for Aging And Graduated school Yonsei University In partial fulfillment of the requirements For the degree of Doctor of Philosophy Ji Hee Oh December 2013 감사의 글 길고도 짧았던 박사 학위 과정을 미흡하나마 논문으로 결실을 맺게 되었습 니다. 그 동안 많은 가르침과 격려 해주신 모든 분들께 이 글을 통하여 감사의 마음을 전합니다. 무엇보다도 부족한 저에게 배움의 기회를 주시고 많은 가르침을 주신 이종 호 교수님께 고개 숙여 깊이 감사 드립니다. 바쁘신 가운데 변함없이 관심 을 가져주신 이수복 교수님, 이승민 교수님께 감사의 인사를 드립니다. 석 사 학위과정 때부터 꾸준히 학문적으로 많은 가르침을 주신 채지숙 교수님, 김오연 교수님, 정지형 교수님께 감사의 인사를 드립니다. 대학원 전 과정 동안 가장 오랜시간동안 도와주고 걱정해 준 진태원군 너 무 고마웠습니다. 박사 학위과정 입학과 졸업을 같이 할 수 있어서 너무 기쁘며 축하합니다. 김윤경 선생님, 박사되고 승급하고 결혼도하고 너무 축하해요. 같이 졸업 준비하면서 고생도 많이 하고 고맙고 고생 많았습니 다. 오랜 시간 동안 학위과정을 할 수 있도록 도와주신 형질연구 과 선생님들 에게 고마운 마음을 전합니다. 김봉조 과장님, 김흥태 연구사님, 이주영 연 구사님 감사합니다. 너무 많은 도움을 항상 받으면서 논문 준비도 같이 해 준 김영진 선생님 진심으로 고맙습니다. 불광동에서부터 지금까지 변치 않 고 항상 많은 도움을 주신 문상훈 박사님 감사합니다. 두 분의 도움으로 논문을 완성할 수 있었습니다. 지금은 떨어져있어 그리운 김남희 선생님, 이은주 박사님, 박미의 박사님, 김동준 선생님, 김광중 박사님, 복정 선생 님의 진심 어린 충고 감사하고 보고 싶습니다. 많은 시간을 같이 보내진 않았지만 항상 옆에 있어준 김연정 선생님, 황미영 선생님, 윤준호 선생님, 허룡 선생님 앞으로도 더욱 많이 서로 도와주고 서로 힘이 되어주는 사이 가 계속 되었으면 좋겠습니다. 국립보건연구원 형질연구과의 황주연 선생 님, 고민진 선생님, 김정민 선생님, 이헌식 선생님, 배재범 선생님, 박수연 선생님, 이영 선생님, 박인규 선생님, 박태준 선생님, 유호영 선생님, 송영 웅 선생님, 최지영 선생님, 한소희 선생님 항상 도와주셔서 감사합니다. 이세상 누구보다 사랑하는 나의 가족에게 고마움과 감사함을 전합니다. 언 제나 부모님의 기대에 미치지는 못하지만 사랑과 믿음으로 묵묵하게 저의 뒷바라지를 해주셨기에 오늘의 제가 있을 수 있었습니다. 크나큰 은혜에 이 결실 조금이나마 부모님께 기쁨을 드리고 싶습니다. 지금은 홀로 해외 에 있는 하나밖에 없는 내 동생, 항상 걱정하고 고맙고 사랑 하다고 전하 고 싶습니다. 항상 내 의견을 존중해주고 양보해주고 언제나 큰 힘이 되 어준 지민기군에게 사랑하고 고마운 마음을 전하고 싶습니다. 저의 이 모든 결실인 부모님께 이 논문을 바칩니다. 2014년 1월 오지희 드림 CONTENTS List of Figures ································································· v List of Tables ····························································· vi List of Appendices ···················································· viii ABSTRACT ······························································· ix Ⅰ. BACKGROUND ······················································· 1 1. LYMPHOBLSTOID CELL LINES (LCLs) ····························· 1 1.1. Epstein-Barr Virus (EBV) ············································ 1 1.2. EBV immortalization ················································· 2 1.3. Subculture ······························································· 3 2. GENOMIC ABERRATION ··············································· 3 2.1. Copy number variation (CNV) ···································· 3 2.2. Loss of heterozygosity (LOH) ····································· 4 2.3. Silent LOH ······························································ 4 3. MICROARRAY TECHNOLOGY ······································· 5 i Ⅱ. GENOTYPE INSTABILITY DURING LONG- TERM SUBCULTURE OF LYMPHOBLASTOID CELL LINES ·································································· 6 1. INTRODUCTION ························································· 6 2. SUBJECTS AND METHODS ··········································· 8 2.1. Samples ································································· 8 2.2. Subculture of LCLs ··················································· 9 2.3. Genotyping ··························································· 12 2.4. Examination of genotype concordance ······················· 12 3. RESULTS ··································································· 13 3.1. Estimated genotype instability in LCLs and PBMCs across sample pairs ·················································· 13 3.1.1. Investigation of concordance of SNP genotypes between LCLs and PBMCs ································· 13 3.1.2. Test of the concordance of GWAS SNPs ·············· 17 3.1.3. Calculation of genotype concordance between LCLs and PBMCs ··········································· 19 3.1.4. Identification of the chromosomal regions vulnerable to genotyping errors ························ 22 ii 3.1.5. Detection of loss of heterozygosity regions ············ 30 4. DISCUSSION ······························································· 36 Ⅲ. INTEGRATED GENOMIC ABERRATION, GENE EXPRESSION AND miRNA EXPRESSION ANALYSIS IN LYMPHOBLASTOID CELL LINE DURING LONG- TERM CELL CULTURE ·········································· 39 1. INTRODUCTION ······················································· 39 2. MATERIALS AND METHODS ······························· 41 2.1. Samples ····································································· 41 2.2. Genotyping ································································· 43 2.3. Gene expression microarray ············································ 43 2.4. miRNA microarray ······················································· 44 2.5. Gene ontology analysis ·················································· 44 3. RESULTS ··································································· 45 3.1. Exome array genotyping ··········································· 45 3.2. CNV and LOH detection ··········································· 45 3.3. Identification of differentially expressed genes ·············· 48 3.4. Functional correlation of DEGs ·································· 51 3.5. Identification of DEGs overlapped with CNVs ·············· 58 iii 3.6. Functional correlation of DEGs overlapped with CNVs··· 61 4. DISCUSSION ······························································· 66 REFERENCES ···························································· 69 ABSTRACT IN KOREAN ·············································· 75 APPENDICES ···························································· 77 iv List of Figures Figure 1. Boxplot: Genotype concordance of between PBMC and LCLs at 6 different propagation stages ·························· 18 Figure 2. Comparison of genotype concordance of LCLs among SNP based on the chromosomal location. ······················ 23 Figure 3. The LOH observed in chromosome ······························ 25 Figure 4. The LOH identified at different chromosomal regions ···· 31 v List of Tables Table 1. Description of samples ··············································· 11 Table 2. Genotype concordance between PBMCs and LCLs from SNP filtered call rate ················································· 15 Table 3. Estimation of the concordance rate between duplicated6 experiments from the same samples ······························ 16 Table 4. Comparison of genotype concordance of LCLs among SNP groupings based on missing rate, HWE p-value, and MAF ······················································ 21 Table 5. LOH with increased numbers of LCL passages through culture ····································································· 24 Table 6. Description of samples ··············································· 42 Table 7. Detecting LOH regions ··············································· 47 Table 8. Integration of gene expression data in ≥5 samples with 10 top signals ordered by M-value ································· 49 Table 9. Integration of miRNA expression data in ≥5 samples with 10top signals ordered by M-value··························· 50 vi Table 10. Functional annotation clusters of significant DEGs from gene expression data with top clusters ordered by FDR ··································································· 52 Table 11. Functional annotation clusters of significant DEGs from miRNA expression data with top clusters ordered by FDR ··································································· 55 Table 12. Integration of CNVs with gene expression data ·············· 59 Table 13. Integration of CNVs with miRNA expression data·········· 60 Table 14. Functional annotation clusters of significant DEGs with CNVs from gene expression data ·························· 62 Table 15. Functional annotation clusters of significant DEGs with CNVs from miRNA expression data ······················ 63 vii List of Appendices Appendix Ⅰ. Detection of CNVs by exome chip ························· 78 Appendix Ⅱ. Functional annotation clusters of significant DEGs from gene expression data ···································· 89 Appendix Ⅲ. Functional annotation clusters of significant differentially expression genes from miRNA expression data ················································· 92 Appendix Ⅳ. Functional annotation clusters of significant DEGs with CNVs from miRNA expression data ··············· 105 viii ABSTRACT Genotype instability in EBV-transformed lymphoblastoid cell lines Ji Hee Oh Graduate Program Science for Aging The Graduate School Yonsei University Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) promise to address the challenge posed by the limited availability of primary cells needed as a source of genomic DNA for genetic studies. However, the genetic stability of LCLs following prolonged culture has never been rigorously investigated. The impact of genomic aberration on the biological experiments such as gene ix expression and miRNA expression level has not been explored. Purposes of current study are to investigate whether genetic instability of LCLs might cause the accumulation of genetic modifications following their long-term subculture and to examine a correlation between genomic aberration and expression level in genes and miRNAs. To accomplish our goals, we isolated genomic DNA from human peripheral blood mononuclear cells and LCLs collected from 20 individuals and genotyped