Frequency of FANCA and FANCG G enes Mutation in Sudanese Fanconi Anemia Patients

1. Frequency of FANCA andFANCG Genes Mutation in Sudanese Fanconi Anemia Patients By Ream Elzain Abdelgadir Supervisor: Imad Fadl-Elmula

2. Historicalbackground Guido Fanconi (1927)

3. Incidence (Worldwide) Uroepithelial cancer Uroepithelial cancer Rarely occur before the age of 40 years E 54,000 new cases E 12,500 deaths E €  Three times more frequently in than in E € 4th most common cancer in  7th most common cancer in E I n areas where Schistosoma haematobium is endemic E € The most common cancer (25%) in E 25% up to 30% of pediatric aplastic patients

5. Fanconi genes

6. Clinical Presentation

7. Diagnosis Chromosomal breakage test Fibroblasts cultures Cell cycle arrest Determination of complementation groups

8. Treatment Supportive treatment Bone marrow transplantation

9. Present StudyDate of mission 2011

10. To identify the prevalence of Fanconi anemia among patients in aplastic anemia Sudanese patients • To characterize FA genetic profile in Sudanese • To assess the influence of the WBCs in chromosomal breakage test

11. Specific Objective 1- To detect the frequency of Fanconi anemia patients among aplastic anemia using chromosomal breakage test. 2- To determine the prevalence of FANCA gene among Sudanese patients with FA. 3- To identify the molecular genetics profile of FANCA gene mutation (exon 10, 11, 12 and31) in Sudanese patients with Fanconi anemia. 4- To detect FANCG 637-643 deletion mutation among Sudanese AA patients. 5- To determine the relationship between total white blood cell count and successful of cytogenetic cell culture among aplastic anemia patients. 6- To correlate between the TWBC count and amount of blood to obtain a successful cytogenetic cell culture among aplastic anemia patients.

12. working planning From 2011-2014

13. Material and methods

14. Sample collection 69 child with aplastic anemia Gaffer Ibnaof Hospital Hematology unit During 2011-2012 5 ml Venous blood

15. Clinical Presentation Café au lait Short stature Abnormal thumbs Microcephaly

16. Complete Homogram Using Sysmex

17. Chromosomal breakage test Culture of blood in RPM1 media after adding MM-C. Incubate 37C° for 72 hours.

18. Chromosomal breakage test Harvesting steps: Adding Colcemid, hypotonic solution, Fixative.

19. Chromosomal breakage test Slide preparation: Metaphase dropping, Ageing of cells, SSC treatment, staining.

20. Analysis of chromosomal breakage test Analysis of 5-25 metaphase.

21. PCR Technique PCR mixture DNA Amplification Salting out method Sequencing in Korea company

22. Data Analysis Frequency, percentage and correlation carried out by SPSS 13 software program.

23. Cons +ve 77% Demographic and hematological result M:65% F: 35%

24. Cytogenetic Results Prevalence of FA among AA patients

25. Analysis of chromosomal breakage test Breakage positive: Breakage, gaps, redial, ring.

26. Transformation to Leukemia 4 patients transform to AML AML M4 AML M5 AML M6

27. Sex distribution Bone marrow Failure 19 (90.5%) 18(85.7%) Female 5 (23.8%) Male 16 ( 76.2%) 18 (85.7%)

28. Clinical presentation

29. BMF in Fanconi anemia

30. Molecular results # 16 /21 was +ve for FANCA gene  Prevalence of FANCA gene was 76.1%. #A total of 47 mutations were detected of which 34 were intronic mutation whereas 13 were exonic mutation. Only one was reported. Frame shift mutation 3 Nonsense mutation 9 Missense mutation 18 Silent mutation 17

31. Molecular results

32. Correlation between WBC count and the success of cell culture A total of 60/69 (87%) showed an adequate number of metaphase for chromosomal breakage analysis. Of the 60 cases 8 (11.6%) cases showed excellent metaphase appearance, 25 (36.2%) showed good metaphase appearances, and 27 (39.2%) revealed bad metaphase appearances. The remaining 9 (13 %) showed failure in culture.

33. Correlation between WBC count and the success of cell culture Direct correlation between : WBC count  Metaphase Number WBC count  Metaphase quality WBC count  amount of needed blood

34. Correlation between WBC count and the success of cell culture

35. Conclusion and recommendation *In conclusion , the prevalence of FA among Sudanese patients are higher than the other countries although the disease is consider as a rare syndrome. This make the importance of screening FA among aplastic anemia patients to use the appropriate treatment and prevent the appearance of the disease complication.

36. Conclusion and recommendation * The equation developed by the present study improved the out come of chromosomal breakage test and thus should be used to calculate the needed amount of blood to perform successful chromosomal breakage test.

37. Acknowledgment Our acknowledge to the patients and their family who agreed to participate in this study. Our thanks extend to the staff of department of Pediatrics, Hematology unit, Gaffer Ibnaof hospital for children and Elite for Health & Medical Services center for genetic study at Khartoum-Sudan.

38. Acknowledgment ليت البيان مطاوعا لمرادى .... حتي أصوغ تحيه لعماد عقل تفجر فى مرابع قطرنا .... أزرى علاه بشامخ الاطواد علم الوراثه كان رائد بحثه .... فاعجب بني بسابق الرواد ليت القريض يمدنى ببيانه .... حتى أريك روائع الانشاد فأنهض الى العلياء دون تردد .... لا تستجيب لمقاله الحساد الله يحفظكم بحسن عناية ... يكفيك شر قوائل الاحقاد ريم الزين ريم