1. In Memory of Brian Alan Frith

2. Utililzation of Genetics in Screening for Fanconi Anemia and Subsequent Implications for Therapy Cheryl Vanderford, PA-S Bettye Hollins, PhD, Advisor

3. Why am I interested in this topic? At the age of seventeen months, my brother was diagnosed with megakaryoblastic leukemia. He fought a short four month battle. At the age of seventeen months, my brother was diagnosed with megakaryoblastic leukemia. He fought a short four month battle. In 2003, doctors informed us that Brian’s cell-line (CHRF- 288) was being used in research for Fanconi Anemia. In 2003, doctors informed us that Brian’s cell-line (CHRF- 288) was being used in research for Fanconi Anemia. http://www.affinityonline.net/local/1999-05/mcd-002.asp

4. Overview What is Fanconi Anemia? What is Fanconi Anemia? Why are FA genes important? Why are FA genes important? What tests are used to screen for Fanconi Anemia? What tests are used to screen for Fanconi Anemia? How do you diagnose Fanconi Anemia? How do you diagnose Fanconi Anemia? How can you treat Fanconi Anemia? How can you treat Fanconi Anemia? Implications Implications

5. What is Fanconi Anemia? Fanconi Anemia is an autosomal recessive disorder. Fanconi Anemia is an autosomal recessive disorder. We all have FA genes. We all have FA genes. Known mutations involved: FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, and FANCM (Kennedy and D’Andrea, 2006). Known mutations involved: FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, and FANCM (Kennedy and D’Andrea, 2006). http://www.wellspring.in/images/centers_of_excellence/common.jpg

6. What is Fanconi Anemia? Fanconi Anemia predisposes an individual to numerous types of cancer, including acute myeloid leukemia, breast cancer, squamous cell carcinoma of the head and neck, and cancers of the gynecological system, skin, esophagus, liver, and kidney (Kennedy and D’Andrea, 2006). Fanconi Anemia predisposes an individual to numerous types of cancer, including acute myeloid leukemia, breast cancer, squamous cell carcinoma of the head and neck, and cancers of the gynecological system, skin, esophagus, liver, and kidney (Kennedy and D’Andrea, 2006).

7. Function of FA genes DNA repair DNA repair Cell cycle control Cell cycle control Oxygen sensitivity Oxygen sensitivity Apoptosis and telomere maintenance Apoptosis and telomere maintenance Haemopoiesis Haemopoiesis Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10.

8. Fanconi Anemia Pathway: DNA Repair Mirchandani, K., D’Andrea, A. The fanconi anemia/BRCA pathway: a coordinator of cross-link repair. Experimental Cell Research. 2006; 312: 2647-2653.

9. FA Role in Cell Cycle Regulation Checkpoint exists at G2/M stage of mitosis to prevent progression of damaged DNA. Checkpoint exists at G2/M stage of mitosis to prevent progression of damaged DNA. Mutated FA genes result in a prolonged G2/M checkpoint and reduced efficacy of the DNA repair mechanism. Mutated FA genes result in a prolonged G2/M checkpoint and reduced efficacy of the DNA repair mechanism. http://en.wikipedia.org/wiki/Mitosis Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10. G0G0 G1G1

10. FA Role in Haemopoiesis Common presentations of patients with Fanconi Anemia: Common presentations of patients with Fanconi Anemia: Anemia Anemia Pancytopenia Pancytopenia Bone marrow hypoplasia Bone marrow hypoplasia Thrombocytopenia Thrombocytopenia Gene therapy to target this role of FA genes in individuals with FA gene mutations. Gene therapy to target this role of FA genes in individuals with FA gene mutations.

11. S/Sx Low Birth Weight Low Birth Weight http://www.successby6ottawa.ca/lbwfpn/images/pink.jpg

12. S/Sx This is a three year old boy with Fanconi Anemia. This is a three year old boy with Fanconi Anemia. http://www.emedicine.com/ped/ images/19911991PED3022- 01A.JPG  Short stature

13. S/Sx Absence of or malformity in hands and arms, for example the absence of a thumb or the presence of polydactyly Absence of or malformity in hands and arms, for example the absence of a thumb or the presence of polydactyly http://www.fanconi.org/aboutfa/Diagnosis.htm Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10.

14. S/Sx Presence of only one kidney or of a horseshoe kidney Presence of only one kidney or of a horseshoe kidney Horseshoe Kidney http://www.md.huji.ac.il/mirror/webpath/RENAL004.jpg

15. S/Sx Café-au-lait spots Café-au-lait spots http://www.dental.mu.edu/oralpath/lesions/cafe/cafe1.JPG

16. Screening DEB test DEB test MMC test MMC test Prenatal screening Prenatal screening Carriers of Fanconi Anemia Carriers of Fanconi Anemia Blood test Blood test Rapid means of screening population at large Rapid means of screening population at large Immunoblotting and immunofluorescence Immunoblotting and immunofluorescence Subtyping Subtyping Retroviral vectors Retroviral vectors

17. Diagnosis DEB and MMC tests DEB and MMC tests Diagnosis typically occurs before the age of twelve (Fanconi Anemia Research Fund, Inc., 2006). Diagnosis typically occurs before the age of twelve (Fanconi Anemia Research Fund, Inc., 2006). MMC test is used to diagnose Fanconi Anemia at the University of Kentucky. MMC test is used to diagnose Fanconi Anemia at the University of Kentucky. Subtyping via use of retroviruses needs to be incorporated into standard protocol when diagnosing a patient with Fanconi Anemia. Subtyping via use of retroviruses needs to be incorporated into standard protocol when diagnosing a patient with Fanconi Anemia.

18. Treatment Retrovirus mediated gene transfer Retrovirus mediated gene transfer Lentivirus mediated gene transfer Lentivirus mediated gene transfer http://www.kfdunn.com/vector/gene-therapy.gif

19. Risks of Gene Therapy Retrovirus potential to stimulate oncogenes. Retrovirus potential to stimulate oncogenes. Lentivirus association with arthritis and encephalitis in goats, leukemia in cattle, anemia in horses, and immunodeficiency in cats, cattle, primates, and humans. Lentivirus association with arthritis and encephalitis in goats, leukemia in cattle, anemia in horses, and immunodeficiency in cats, cattle, primates, and humans. Anson, D. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genetic Vaccines and Therapy. 2004; 2(1): 9. (Published online).

20. Somatic Mosaicism Reversal of mutated FA genes has been successfully accomplished by nature alone. Reversal of mutated FA genes has been successfully accomplished by nature alone. How? How? Better understanding of nature’s own gene therapy for Fanconi Anemia may allow us to someday mimic the same mechanism to eliminate Fanconi Anemia. Better understanding of nature’s own gene therapy for Fanconi Anemia may allow us to someday mimic the same mechanism to eliminate Fanconi Anemia.

21. Sensitivity to Treatment Tischkowitz, M., Ameziane, N., Waisfisz, Q., De Winter, J., Harris, R., Taniguchi, T., D’Andrea, A., Hodgson, S., Matthew, C., Joenje, H. Bi-allelic silencing of the fanconi anaemia gene FANCF in acute myeloid leukaemia. Br J Haematol. 2003; 123: 469-471. CHRF-288 is Brian’s cell line. CHRF-288 is Brian’s cell line. Hypermethylation of FANCF is hypothesized to “be an early step in sporadic carcinogenesis” Recent studies have shown that the degree of methylation to FANCF can indicate the responsiveness of the individual to a specific treatment.

22. Genetic Counseling Provide knowledge about: Provide knowledge about: What Fanconi Anemia is What Fanconi Anemia is What symptoms are associated What symptoms are associated Who can be affected Who can be affected What the disorder means for the future of the individual What the disorder means for the future of the individual What treatments are available and their risks and benefits What treatments are available and their risks and benefits Importance of screening other family members at risk Importance of screening other family members at risk http://www.childbirthsolutions. com/articles/preconception/gen eticcounseling/Gen-1.jpg

23. Is this ethical? A genetic disorder that predisposes an individual to cancer—why not screen the population at large? A genetic disorder that predisposes an individual to cancer—why not screen the population at large? Cost/benefit Cost/benefit Wait until Fanconi Anemia shows symptoms before finding out you carry mutated FA genes? Wait until Fanconi Anemia shows symptoms before finding out you carry mutated FA genes? Early intervention Early intervention Carriers Carriers Guilt if not aware of the risk of possibly passing mutated FA genes to your child. Guilt if not aware of the risk of possibly passing mutated FA genes to your child.

24. Summary Fanconi Anemia is an autosomal recessive disorder that predisposes individuals to a variety of cancers. Fanconi Anemia is an autosomal recessive disorder that predisposes individuals to a variety of cancers. Screening techniques exist, such as the DEB and MMC tests, that should be used to screen the population at large. Screening techniques exist, such as the DEB and MMC tests, that should be used to screen the population at large. Subtyping of which FA gene(s) is (are) mutated should become standard protocol with diagnosis. Subtyping of which FA gene(s) is (are) mutated should become standard protocol with diagnosis. Gene therapy may someday eliminate Fanconi Anemia. Gene therapy may someday eliminate Fanconi Anemia.

25. References Anson, D. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genetic Vaccines and Therapy. 2004; 2(1): 9. (Published online). Anson, D. The use of retroviral vectors for gene therapy-what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genetic Vaccines and Therapy. 2004; 2(1): 9. (Published online). Fanconi Anemia Research Fund, Inc., Diagnosis. 2006, Available at: http://www.fanconi.org/aboutfa/Diagnosis.htm, Accessed on 2006 Nov 26. Fanconi Anemia Research Fund, Inc., Diagnosis. 2006, Available at: http://www.fanconi.org/aboutfa/Diagnosis.htm, Accessed on 2006 Nov 26. http://www.fanconi.org/aboutfa/Diagnosis.htm Kennedy, R., D’Andrea, A. DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes. Journal of Clinical Oncology. 2006; 24: 3799-3808. Kennedy, R., D’Andrea, A. DNA repair pathways in clinical practice: lessons from pediatric cancer susceptibility syndromes. Journal of Clinical Oncology. 2006; 24: 3799-3808. Mirchandani, K., D’Andrea, A. The fanconi anemia/BRCA pathway: a coordinator of cross-link repair. Experimental Cell Research. 2006; 312: 2647-2653. Mirchandani, K., D’Andrea, A. The fanconi anemia/BRCA pathway: a coordinator of cross-link repair. Experimental Cell Research. 2006; 312: 2647-2653. Pettigrew, A. Personal Interview. January 2007. Pettigrew, A. Personal Interview. January 2007. Tischkowitz, M., Ameziane, N., Waisfisz, Q., De Winter, J., Harris, R., Taniguchi, T., D’Andrea, A., Hodgson, S., Matthew, C., Joenje, H. Bi-allelic silencing of the fanconi anaemia gene FANCF in acute myeloid leukaemia. Br J Haematol. 2003; 123: 469-471. Tischkowitz, M., Ameziane, N., Waisfisz, Q., De Winter, J., Harris, R., Taniguchi, T., D’Andrea, A., Hodgson, S., Matthew, C., Joenje, H. Bi-allelic silencing of the fanconi anaemia gene FANCF in acute myeloid leukaemia. Br J Haematol. 2003; 123: 469-471. Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10. Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10.