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Mapping mobile DNA elements: Sources of human genetic diversity and disease. Kathleen H. Burns, M.D., Ph.D. Johns Hopkins Department of.

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Presentation on theme: "Mapping mobile DNA elements: Sources of human genetic diversity and disease. Kathleen H. Burns, M.D., Ph.D. Johns Hopkins Department of."— Presentation transcript:

1 Mapping mobile DNA elements: Sources of human genetic diversity and disease. Kathleen H. Burns, M.D., Ph.D. Johns Hopkins Department of Pathology

2 Overview I. The Mobile Genome: Transposable elements in human diversity & disease. II. Finding a needle in a haystack - one new mobile element insertion in a million. III. Novel microarray-based transposon mapping technology and data analysis IV. Future directions

3 The (junk) Genome Data from Jasinska & Krzyzosiak, FEBS Letters (2004). 55% of the human genome is repetitive 45% of the human genome is comprised of interspersed repeats non-repetitive tandem repeats 21% of the human genome is comprised of long interspersed LINEs LTRs (HERVs) SINEs (Alu)

4 LINEs are dynamic genetic sequences. Multiplication by “copy-and-paste” AAAAA5’ UTRORF1ORF2 3’ UTR ASP

5 Major epigenetic remodeling takes place in germ cell and embryo development. PGCsBlastocystFertilization extraembryonic tissue epiblast From Burns & Matzuk, “Rewriting and its Risks” in Preimplantation Embryo Development, Knobil and Neill’s Physiology of Reproduction, 3 rd Ed., Total Genome Methylation Zygote

6 Retrotransposons can act as insertional mutagens “Accidental Discoveries” of LINEs as Agents of Disease LINE compromise transcriptional elongation splicing disruption “gene breaking”/pre-mature polyA LINE regulatory/epigenetic effectsdisrupted ORF Disrupted GenesDisorderInsertion SiteInserted ElementsReference CYBB CGDExonL1 TaMeischl et al. (2000) IntronBrouha et al. (2002) F8 Haemophilia AExonL1 TaKazazian et al. (1988) L1 preTaKazazian et al. (1988) F9 Haemophilia BExonL1 TaLi et al. (2001) Mukherjee et al. (2004) HBB  thalassemia ExonL1 TaDivoky et al. (1996) IntronKimberland et al. (1999)

7 Tumor suppressors are down-regulated in neoplasia in the context of broad genome hypomethylation Modified from Melki & Clark (2002). Normal Cell Cancer Cell Hypermethylation of tumor suppressors Overall hypomethylation TumorEvidence for LINE hypomethylationReference breast cancer5’ flanking sequences of hypomethylated L1Hs elements isolated by MSP iPCRAlves, et al chronic myeloid leukemia (blast phase) methylation-specific PCR of primary samples; hypomethylation associated with ↑ BCR-ABL mRNA, resistance to tyrosine kinase inhibitors Roman-Gomez, et al chronic lymphoid leukemia primary specimens analyzed by HpaII digest and Southern blot Dante, et al colorectal adenocarcinomacompared to neighboring normal colon ; alternate MSI progression pathway Estecio, et al hepatocellular carcinoma hepatocellular carcinomas compared to surrounding, cirrhotic liver; HpaII restriction enzyme digest Takai, et al pancreatic endocrine and carcinoid tumors compared to surrounding tissue; LINE hypomethylation correlates with lymph node metastasis, cytogenetic aberrations Choi, et al prostate cancercompared to surrounding tissue; hypomethylation associated with Gleason grade, clinical stage, and cytogenetic abnormalities Santourlidis, et al. 1999; Schultz, et al. 2002; Cho, et al urothelial carcinomaappreciated by Southern blot or MSP-PCR in most specimensNeuhausen, et al

8 Miki, et al. Cancer Research BglIIPstIMspIHindIIIEcoRI Chr5: A known case of colon cancer associated with APC mutation by a somatic LINE insertion

9 chr4; human genome sequencing project Reducing the haystack: Seeing the transcriptionally active T(a)LINE subset Boissinot & Furano, AAAAAA ACA L1 fossil L1 elements polymorphic L1 elements

10 Where are the transposons? TIP-chip strategy Tiling Array: masked feature microarray slide

11 L1 T(a) LINE mapping strategy: Vectorette PCR

12 R1 R2 R3 R1 R2 R3 2kb LINE mapping strategy: Vectorette PCR

13 EnzymeTotal Coverage Covering 3 billion base pairs of the human genome Total Length =

14 Comparison to the reference genome location of a T(a)LINE in the reference Partial overlap of mapped T(a)LINEs with the human genome reference

15 6,932,527 11,641,350 11,863,056 33,335,616 34,746,985 43,049,918 45,334,456 49,615,819 54,160,944 56,745,095 63,148,056 65,272,709 67,180,024 72,523,645 73,829,561 75,459,361 75,866,879 76,328,405 76,340,938 77,583,298 80,989,514 85,496,585 94,744,098 98,150, ,115, ,453, ,776, ,225, ,498, ,905, ,910, ,342, ,399, ,237, ,255, ,398, ,583,236 P MSD P MSD PM SD Insertion carrier Inferred heterozygote No insertion The T(a)LINE TIP-Chip is a robust genotyping tool P M S D

16 Gaussian Distribution of Background and Foreground Values log 2 scale Reference peak Other peaks Noise estimate Frequency LN scale

17 Modeling Peak Shape Strong signal Weak signal Absent signal Kilobases states x 2 peak directions = 42 peak states + 1 background state = 43 states

18 LISA LINE Insertion Signal Analysis Ranking of 34 known L1 insertion signals on the X chromosome

19 Reference (Ta)L1are polymorphic Absent Present

20 P MSD P MSD Finding novel T(a)LINE insertions 6,932,527 11,641,350 11,863,056 33,335,616 34,746,985 43,049,918 45,334,456 49,615,819 54,160,944 56,745,095 63,148,056 65,272,709 67,180,024 72,523,645 73,829,561 75,459,361 75,866,879 76,328,405 76,340,938 77,583,298 80,989,514 85,496,585 94,744,098 98,150, ,115, ,453, ,776, ,225, ,498, ,905, ,910, ,342, ,399, ,237, ,255, ,398, ,583,236 Up Dn Up H H A P P P P P A P A P P

21 LINE methylation is relaxed in germ cells and preimplantation embryos as well as in some types of cancer. LINE insertions arising in the former can cause heritable disease. Relaxed LINE silencing may have an underappreciated roles in tumor progression. We have developed a comprehensive mapping method for the most active LINEs in the modern genome. This is a robust method for genotyping known insertions and identifying novel insertions. This has immediate pertinence to understanding common polymorphisms and heritable disease. The approach is expected to have special utility in comparing tumor and normal DNA. Summary

22 Applications of total genome transposon profiling in cancer research. Comprehensive T(a)LINE mapping to compare tumor and normal tissue. Correlate findings with markers of epigenetic T(a)LINE silencing. Development of HERV-K mapping strategies. Transposon mapping for identifying causes of familial cancer susceptibility syndromes. Nimblegen HD2 platform Total probes 2.1 million Probe length mer (ChIP-chip whole genome tiling) Feature size 13μm x 13μm Array size 62mm x 14mm Slide size 1” x 3” (25mm x 76mm) glass Future Directions

23 Center for High Throughput Biology Jef Boeke, Ph.D. & the Boeke Lab Cheng Ran Huang Tejas Niranjan Department of Oncology Curt Civin, M.D. & the Civin Lab Institute of Genetic Medicine Dave Valle, M.D. Acknowledgements Funding: Burroughs Wellcome Foundation National Cancer Institute Sol Goldman Pancreatic Cancer Research Center Goldhirsh Brain Tumor Research Foundation


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