Understanding Fanconi Anemia Genetics and Implications for Therapy

1. In Memory of Brian Alan Frith

2. Utililzation of Geneticsin 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. • 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? • Why are FA genes important? • What tests are used to screen for Fanconi Anemia? • How do you diagnose Fanconi Anemia? • How can you treat Fanconi Anemia? • Implications

5. What is Fanconi Anemia? • Fanconi Anemia is an autosomal recessive disorder. • 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). 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).

7. Function of FA genes • DNA repair • Cell cycle control • Oxygen sensitivity • Apoptosis and telomere maintenance • 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. • Mutated FA genes result in a prolonged G2/M checkpoint and reduced efficacy of the DNA repair mechanism. G0 G1 http://en.wikipedia.org/wiki/Mitosis Tischkowitz, M., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10.

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

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

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

13. S/Sx • 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 Horseshoe Kidney http://www.md.huji.ac.il/mirror/webpath/RENAL004.jpg

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

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

17. Diagnosis • DEB and MMC tests • 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. • 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 • Lentivirus mediated gene transfer http://www.kfdunn.com/vector/gene-therapy.gif

19. Risks of Gene Therapy • 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. 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. • 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.

21. Sensitivity to Treatment • 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. 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.

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

23. Is this ethical? • A genetic disorder that predisposes an individual to cancer—why not screen the population at large? • Cost/benefit • Wait until Fanconi Anemia shows symptoms before finding out you carry mutated FA genes? • Early intervention • Carriers • 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. • 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. • 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). • Fanconi Anemia Research Fund, Inc., Diagnosis. 2006, Available at: http://www.fanconi.org/aboutfa/Diagnosis.htm, Accessed on 2006 Nov 26. • 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. • 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., Hodgson, S. Fanconi anaemia. J. Med. Genet. 2003; 40:1-10.