Rajiv Gandhi University of Health Sciences s160

Rajiv Gandhi University of Health Sciences

Karnataka, Bangalore

Annexure 2

Proforma for registration of subjects for dissertation

1 . Name of the candidate and Address:

VIGNESWARAN.K

Postgraduate student, Department of Anatomy

St.John’s Medical College, Bangalore-560 034.

Residential address:

NO.9P, Santhosh Nagar

Agragaram Street,

Bahour -607402 ,

Puducherry

2. Name of the institution: St. John’s Medical College,

Bangalore - 560 034

3. Course of the study and subject: M.D (Anatomy)

4. Date of admission: 24th May, 2012

5. Title of the topic

“ Mitomycin-C induced stress-test for diagnostic differentiation of Fanconi anemia from idiopathic aplastic anemia”

1 6.1. Need for the study

Fanconi anaemia (FA) is an autosomal recessive condition which can be transmitted from parents to offsprings. FA has a variable clinical presentation and this leads to under diagnosis of true cases and also to differentiate from aplastic anemia. Sometimes patients presenting with typical symptoms of FA may not be actually suffering from FA. FA has more chance for developing malignancy because this is a chromosomal instability syndrome. The management of FA is also different from that of aplastic anaemia and hence a proper diagnosis is essential1,2,3.

Cytogenetic analysis not only helps in diagnostic evaluation, but also aids in genetic counselling for prevention genetic inheritance in future to their siblings. With this background, this study is conducted to identify the chromosomal damage in suspected cases of Fanconi anaemia (FA). This study also helps in diagnostic differentiation of FA from idiopathic aplastic anaemia. This study may be useful for early diagnosis and management which would improve the quality of life for patients

6.2. AIMS AND OBJECTIVES:

1. To estimate the prevalence of FA in patients suspected to have idiopathic aplastic anemia

2. To estimate the prevalence of the different clinical manifestation in patients with confirmed cases of FA ( phenotypic and genotypic correlation ).

3. To estimate the strength of association between number of breaks, number of gaps, number of triradii and number of quadriradii

6. REVIEW OF LITERATURE:

Fanconi anaemia (FA) is an autosomal recessive disease characterised by congenital abnormalities, defective haemopoiesis and high risk of developing acute myeloid leukaemia and certain solid tumors1. First description of fanconi anaemia dates back to 1927, where Fanconi described this condition2. Chromosomal instability, especially on exposure to alkylating agents, may be shown in affected subjects and is the basis for a diagnostic test4. Clinical features include skeletal abnormalities include radial ray, hip, vertebral scoliosis, rib 2 defects, skin pigmentation(Café au lait spots, hypopigmentation, hyperpigmentation), short stature, microphthalmia, renal, urinary tract abnormalities, hypogonadism, mental retardation, anorectal / duodenal atresia, cardiac anomalies, hearing defects, central nervous system anomalies 1 .

Few authors had studied chromosomal breaks in fanconi anemia. Fanconi anemia patients exhibit spontaneous chromosomal breaks3. Fanconi anemia patients cell are sensitive to DNA interstrand crosslink agents and expresses high frequency of chromosomal breakage.A detailed study was conducted in clinically diagnosed fanconi anemia patients in an Indian population. 33 patients were clinically diagnosed with fanconi anemia and had aplastic anemia with bleeding abnormalities. Genetic analysis revealed a significantly high frequency of parental consanguinity in FA patients compared to controls. Chromosomal analysis revealed spontaneous chromosomal breakage in 63.64% FA patients. Mitomycin-C induced cultures showed a significantly high frequency of chromosomal breakage and radial formation when compared to controls. Among thirty three patients, nine developed malignancies. At the highest concentration of mitomycin (MMC) 80ng/ml, 50 fold increase of chromosomal breaks and a 200 fold increase in radial figures were observed in FA anemia lymphocytes4. Spontaneous chromosomal aberrations were analysed in three FA anemia patients, eight family members and nine healthy individuals3. Peripheral blood lymphocytes obtained from each individual were cultured and cytogenetic analysis was performed on standard and sequential G-banded metaphases. The number of abnormal cells and breaks were found to be higher in FA patients compared to controls (P<0.001)3. Abnormally high levels of spontaneous and Mitomycin-C or diepoxybutane (DEB) induced chromosomal breakage were observed in lymphocytes of fanconi anemia patients. The results suggest that the combination of spontaneous and induced chromosomal breakage is a good aid in the differential diagnosis. Author suggested that increased chromosomal breakage is pathognomic for this recessive disorder5.

To calculate the number of chromosomal aberration induced by MMC or DEB, the total number of spontaneous aberration scored in 50 cells from each person was substracted from the total number of aberration scored in 50 treated cells. Treatment with 0.1, 0.25, 0.5 microliter per ml MMC resulted in the induction of 0 to 4 (mean 0.58), 0 to 5 (mean 2.0), and 0 to 17 (mean 5.2) chromosomal aberrations per 50 cells of normal persons. Majority of the induced chromosomal aberrations were chromatid gaps and breaks with occasional

3 quadriradials. Similar results were obtained with lymphocytes from three fanconi anemia heterozygotes. Treatment of FA lymphocytes with 0.1 , 0.25 , 0.5 microgram per ml MMC resulted in induction of 19 to 133 , 47 to 100 , 112 to 265 aberrations per 50 cells. This is much higher than levels in normal subjects 5. Variations were observed between the levels of MMC induced chromosome aberrations in repeated blood samples from individual patient5. Drug therapy received by patients may affect chromosomal instability. Six of the patients included in this study had received steroid therapy at some time although only three were receiving such treatment at the time the blood samples were taken. High levels of breakage were seen in cells from untreated patients6. According to another author a family was described in which two of seven sibs have fanconi aplastic anemia. Chromosomal aberrations including endoreduplication were found in the peripheral blood of the two patients, as well as in a sister who was not anemic but had several of the congenital abnormalities usually associated with this syndrome. Remaining sibs and both parents were free of clinical, haematological and chromosomal abnormalities7.

The chromosome of bone marrow cells from a patient with fanconi anemia showed a lower aberration frequency than that of either lymphocyte or fibroblast cultures from the same patient. This difference between the aberration frequency in vivo and in vitro may provide a clue to nature of the inherited metabolic defect underlying fanconi anemia8. According to another author, DEB is used in a widely accepted alternative protocol9. As the management of patients with fanconi anemia differs from that of idiopathic aplastic anemia, it is essential to differentiate these disorders as early as possible10. Patients with FA sometimes have normal cytogenetic results11. Some authors did susceptibility test using caffeine, chloramphenicol, actinomycin D, nitrogen mustard, MMC, UV irradiation12,13,14. Patients with FA have a high risk of squamous cell carcinomas of head, neck, and esophagus15. Age of onset occurs between the ages of four and seven16.

7. MATERIAL AND METHODS

7.1. SOURCE OF DATA: Suspected patients with clinical features of Fanconi Anemia referred to Division of Human Genetics, St John’s Medical College, Bangalore for cytogenetic analysis.

SAMPLE SIZE: 25 patients suspected to be having FA/Idiopathic aplastic anemia

4 Justification for Sample size: On an average per year, the number of patients referred to the Division of Human Genetics, St John’s Medical College, Bangalore, is 500. Of which 10 patients are referred with suspected Fanconi Anaemia. Hence a sample size of 25 was selected for a period of 3 years.

7.2. INCLUSION CRITERIA:

SUBJECTS

1. Patients with suspected aplastic anemia or with a clinical diagnosis of FA.

7.3. EXCLUSION CRITERIA:

SUBJECTS

1. Patients who received blood transfusion recently.

2. Patients with a h/o any malignancy and on treatment with alkylating agents.

7.4. STUDY DESIGN: Descriptive cross-sectional study

7.5. Protocol:

1. 4ml of venous blood will be aseptically collected from subjects in heparinized vaccutainer.

2. Informed written consent will be taken from the guardians of the subjects.

3. Culture media will be prepared. 5ml of RPMI media with 18% NBCS+300 microliter (µl ) of phytohemagglutinin (PHA) with 300µl of sample will be added to each culture vial. For each case 3 cultures with different concentrations of MMC and 3 controls will be setup. Cultures will be incubated at 37ºC for 72 hours. After 24 hours, MMC will be added to the culture vials in concentrations of 25µl, 6.5µl, 0µl and again incubated for 48hours to complete 72 hours.

4. At 69th hour of incubation, 50 microliter of colcemid (10 microliter per ml) will be added and incubated again for 50 minutes. Harvesting is done as per the standard protocols.

5. The pellets are then fixed and after ageing, the slides are prepared, 2 each from each of the different concentrations of MMC from the subject and control sample . The slides are then 5 scored for chromosomal breakage, under oil immersion microscopy(100X). The slides of the subjects are compared with that of the controls to evaluate the effect of MMC

Patient Blood Sample

72hours PHA stimulated culture

After 24hours: Add MMC

25µl 6.5µl 0µl (nil)

Harvesting at 69th hour

Termination (Hypotonic Treatment & Fixation)

Slide Preparation

Slides are stained (Giemsa Stain)

Microscopic Analysis

7.6. STATISTICAL ANALYSIS

6 The proportion of patients with fanconi anemia will be calculated. In patients diagnosed with FA, the proportion of each clinical manifestation will be calculated. The strength of association between different indicators of chromosomal damage will be estimated using pearson correlation coefficient(parametric) or spearmanns correlation coefficient (non-parametric). P value of <0.05 will be considered to be statistically significant.

7.7. Does the study require any investigations or interventions to be conducted on patients or ther human or animals? If so please describe briefly

No interventions will be done

7.8. Has ethical clearance obtained from your institution in case of 7.3?

Yes it has been submitted for Institutional ethical clearance

8. REFERENCES:

7 1.MD Tischkowitz, SV Hodgson-Fanconi anemia -Journal of medical genetics 2003,40,1- 108.

2.Tushar B.Parikh, Rekha H.udani ,Ruchi N.Nanavathi, Babu Rao V-Fanconis anemia in newborn-Indian paediatrics-vol.42 2005

3.Seema korgaonkar, kanjaksha Ghosh and Babu Rao vundinti-Clinical ,genetic and cytogenetic study of fanconi anemia in an indian population – accepted on 18 july 2009 , hematology 2010 vol 15

4.Jaroslav cervenka, MD,CSc, Diane Arthur, MD and Carmen Yasis-mitomycin –c test for diagnostic differentiation of idiopathic aplastic anemia and fanconi anemia –peadiatrics vol . 67 ,January 1981

5.A.Fundia, N.Gorla and I.Larripa- spontaneous chromosomal aberrations in fanconi anemia patients are located at fragile sites and acute myeloid leukemia breakpoints –Hereditas 120;47-50(1992.

6.G Duckworth –Rysiecki, Hulten, J Mann and A M R Taylor-clinical and cytogenetic diversity in fanconi anemia -Journal of medical genetics ,1984 ,21,197-2035

7.John Perkins, John Timson and Alan E. H. Emery-clinical and chromosome studies in fanconis aplastic anemia –Journal of medical genetics, 1969 6,28

8.MJ Shahid , F P Khouri and S K Ballas-fanconi anemia :report of a patient with significant chromosomal abnormalities in bone marrow cells –Journal of medical genetics-1972 9:474- 478

9.Anneka B.Oostra, Aggie W.M.Nieuwint, Hans Joenje, and Johan P.de Winter. Diagnosis of fanconi anemia:chromosomal breakage analysis.

10.Rashmi Talwar, V.P.Choudhry, Kiran Kucheria. Differentiation of fanconi anemia from Idiopathic Aplastic Anemia by induced chromosomal breakage study using Mitomycin- c(MMC). Indian paediatrics vol41,may17,2004.

11.Daniel G.Kuffel, B.S., Noralane M.Lindor, M.D., Mark R.Litzow,M.D., Alian R.Zinsmeister,Ph.D., and Gordon W.Dewald,Ph.D. Mitomycin C chromosome stress test to 8 identify hypersensitivity to bifunctional alkalyating agents in patients with Fanconi anemia or aplastic anemia. Mayo foundation for medical education and research 1997.

12.Masao S .Sasaki and Akira Tonomura.A High susceptibility of Fanconi anemia to chromosome breakage by DNA cross –linking agents.Cancer Research 1973;33:1829-1836

13.Makoto Higurashi and Patrick E.Conen. In Vitro chromosomal radiosensitivity in Fanconi anemia. Blood 1971 38 :336-342.

14.Helga seyschab, Richard Friedl, Yujie Sun ,Detlev Schindler,Holger Hoehn,Sabine Hentze, and Traute Schroeder-kurth. Comparative evaluation of diepoxybutane sensitivity and cell cycle blockage in the diagnosis of Fanconi anemia. Blood vol85,No 8,1995

15.Philip S.Rosenberg ,Gerard Socie, Blanche P.Alter and Eliance Gluckman. Risk of head and neck squamous cell cancer and death in patients with Fanconi anemia who did and did not receive transplants.American society of hematology 2005 105;67-73

16.Anthony Glanz and F Clarke Fraser. Spectrum of anomalies in Fanconi anemia . Journal of Medical Genetics ,1982,19,412-416.

9. Signature of candidate

10. Remarks of Guide:

11. Name and designation

Dr. REMA DEVI

Prof and Head,

Division of Human Genetics

Department of Anatomy,

St. John’s Medical College

Bangalore-560 034.

11.2 Signature of the guide:

11.3 Co-guide:

11.4 Signature:

11.5 Head of the Department: Dr. Roopa Ravindranath

11.6 Signature:

12.1 Remarks of the chairman & Principal:

10 12.2 Signature