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Characterization of a New Fusion Gene in Acute Myeloid Leukemia
Jocelyn ReaderPh.D. Candidate in Human
GeneticsUniversity of Maryland Baltimore
Advisor: Dr. Yi NingFeb. 28th, 2007
Why a Ph.D. is for Me
Educational and Scientific Research Experiences
Professoriate Applied vs. Basic Research
Basic research – research driven by scientist’s curiosity or interest in a scientific question
Applied research – research designed to solve practical problems rather than acquire knowledge for knowledge’s sake
Career Goals and Options
Introduction
Cancer genetics Cytogenetics
Studying the structure of chromosome material
Cancer Leukemia
Acute Myeloid Leukemia
http://en.wikipedia.org/wiki/Image:Bcrablmet.jpg
Societal Impact of Cancer
In 2007 ~1.44 billion new cases of cancer
expected ~560,000 deaths due to cancer in the
U.S. > 1,500 people a day
Leukemia New cases expected : 44,240 Expected number of deaths : 21,790
American Cancer Society; National Cancer Institute
Societal Impact of Cancer Cancer is responsible for more estimated
years of life lost than any other cause of death
Person-years of Life Lost The difference between the actual age of death due to a
cancer and the expected age of death
Background: DNA & Chromosomes
http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/chromosome.cfm?key=chromosomehttp://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/gene.cfm?key=gene
Background: The Central Dogma of Molecular Biology
Concept of how genes lead to protein formation
Protein domains are sequences that give the protein a specific function
http://fig.cox.miami.edu/~cmallery/150/gene/c7.17.12.domains.jpg
Background: Etiology of Cancer
Campisi J. 2003. Nat Rev Cancer. 3:339-49. Chen Z, Sandberg AA. 2002. Am J Med Genet. 115:130-41.
Cancer Uncontrolled growth of abnormal
cells Mutations in DNA sequence or gross
chromosomal changes Usually occurring in tumor
suppressor genes or proto-oncogenes
Deletions or translocations
Background: Tumor Suppressor Genes & Proto-oncogenes
Gatekeepers Tumor
Suppressors Proto-oncogene
Typically involved in cellular growth
Can become an oncogene through several mechanisms
Campisi et al. 2003
Background: Chromosomal Alterations
Deletion
Types Deletions Translocations
Why study? Role in disease diagnosis,
prognosis and treatment Identification of new
tumor suppressor genes or proto-oncogenes
Outcome Loss of an important
gene(s) Create a new gene with a
new function
Background: Hematological Malignancies
Hematological Malignancies Cancers that affect the blood, bone marrow
and lymph node Lymphoma and Leukemia
Lymphoma Hodgkin’s disease Non-Hodgkin lymphoma
Leukemia
Leukemia Speed of cell growth
Acute / Chronic Cell lineage
Lymphoid / Myeloid Acute lymphoblastic Acute myeloid Chronic
lymphoblastic Chronic myeloid
Acute Myeloid Leukemia (AML) Arrests cells in early
stage of development
Most common acute leukemia in adults
Background: Hematological Malignancies
http://www.bloodlines.stemcells.com/img/Metcalf_Fig3_2.gif
Patient History
42-year-old male patient with recurrent AML
Presented with a possible abnormality on 17p
4 months after last treatment – patient relapsed with same abnormality
Objectives & Experimental Design
Identify/ verify the chromosomal anomaly in the patient Fluorescence in situ hybridization (FISH)
Identify and characterize the genes involved in the chromosomal anomaly Molecular Genetic Techniques
RT-PCR Cloning and Sequencing
Molecular Cytogenetics & Genetics Techniques
Fluorescence in situ hybridization (FISH)
http://en.wikipedia.org/wiki/Image:FISH_%28technique%29.gif
PCR & RT-PCR Reverse transcriptase
– polymerase chain reaction (RT-PCR) Start with RNA
instead of DNA First reaction use a
RNA-dependent DNA polymerase
Make cDNA (complementary DNA)
PCR products are visualized by agarose gel electrophoresis
Polymerase Chain Reaction (PCR)
http://en.wikipedia.org/wiki/Image:Pcr.png#file
Cloning of DNA sequences
http://employees.csbsju.edu/hjakubowski/classes/SrSemMedEthics/Human%20Genome%20Project/plasmid.gif
Identification and Characterization of a New Oncogenic Fusion Gene in Acute Myeloid Leukemia
FISH Analysis on 17p
FISH Analysis TP53 not
affected Sub-telomeric
probe shows translocation to 11p TP53 (Orange)
17p13.2 (Green)
Identification of a Candidate Gene for the 11;17 Translocation
NUP98 11p15.5 Protein that forms part of nuclear pore complex
Promiscuous fusion partner gene plays a role in hematological malignancies
http://www.infobiogen.fr/services/chromcancer/Genes/NUP98.html
Identification of NUP98s Involvement in 11;17
Translocation
Created two probes that contained sequence from the 5’ and 3’ end of the NUP98 gene
No involvement Fused signal
Involvement Split signal
Nup98cen (Green)Nup98tel (Orange)
RP1-4G17
RP11-542C16
Translocation Breakpoint Mapping on 17p
RP11-186B7 (Green)RP1-4G17 (Orange)
RP11-542C16 (Green)
NUP98 Fusion Gene Partner Candidate Gene
Candidate Gene
PHF23
Component Map
RP11-542C16
RP1-4G17
Modified from NCBI Map Viewer http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?taxid=9606&query=542c16&qchr=&strain=All&advsrch=off
RT-PCR Amplification and Sequence Analysis of NUP98-PHF23 Fusion
Transcript
Create DNA primer for NUP98 and PHF23 gene
RT-PCR Amplification and visualization
Clone PCR product Perform sequence
analysis
1. NUP98-PHF23 Gene Fragment
2. Full-length NUP98-PHF23 Fusion Gene
NUP98-PHF23
5’-CCGATGTCAGACCCTAAGAAGAAGGAAGAGGCCCCCGACAGTGCTACCTTGCTTGAGAAG-3’
P M S D P K K K E E A P D S A T L L E K
Schematic of Normal and Predicted Proteins
COOHNH2FG FGGLEBS
NUP98
920 aaNUP98-PHF23
FG GLEBS FGNH2 COOHPHD
797 aa
403 aa
NH2 COOH
PHF23
PHD
Normal and Fusion ProteinsRNP binding domain
NUP98-PHF23 fusion protein combination of NUP98 and PHF23 proteins Predicted to have a function different from the
normal proteins
PHF23
PHD Finger Protein 23
PHD motif Plant
Homeodomain Finger
Involved in regulating transcription
Additional fusion genes leading to AML contain PHD domains NUP98-NSD1 NUP98-NSD3
NUP98/NSD3
Jaju RJ, et al. 2001. Blood 98:1264-7. Rosati R, et al. 2002 Blood 99:3857-60.
NH2 COOHPHD
Summary and Conclusions
Summary Types and causes of leukemia Identified a novel fusion gene
Conclusion Why study this fusion gene
Possible common mechanism of action for these cancer causing genes
New therapeutic drug targets Application to other cancers
Future Directions Verify oncogenicity of fusion gene
Acknowledgements
Dr. Yi Ning – Advisor Clinical Cytogenetics Laboratory –
University of Maryland Baltimore PROMISE Program in Human Genetics Marlene and Stewart Greenebaum
Cancer Center