VIMS Journal: December 2016

Original Article

Incidence of Fanconi Anaemia in MorphologicallyNormal Aplastic Anaemia Patients

Ms. Atreyee Dutta, Dr. Rajib De, Dr. Tuphan Kanti Dolai, Ms. Shanoli Ghosh,Sri Akshay Ramesh Gore, Dr. Pradip Kumar Mitra, Dr. Ajanta Halder

Key words:
Fanconi Anaemia, Aplastic Anaemia, StressCytogenetics, Radial Formation.

Keywords :
Type 1 Diabetes, IDDM Epidemiology


Introduction :
Fanconi anaemia (FA) is an autosomal recessivedisease, characterized by pancytopenia, bonemarrow failure, congenital malformation (skeletalmalformation, hyperpigmentation, urogenital,renal and cardiac anomalies). FA patients aresusceptible to DNA cross linking agents. Positivestress cytogenetics has implication for thetreatment decision of FA.

Aims and Objective :
The exact incidence of FA in West Bengal isunknown due to lack of proper designed trials.Our study was aimed to find out the FA cases inmorphologically normal Aplastic Anaemia (AA)patients.

Materials and Methods:
Clinical history of 70 AA patients was taken andtheir physical examination was done. Bonemarrow aspiration and biopsy were done toconfirm the diagnosis of AA. Peripheral venousblood was collected with consent from 49 AApatients (< 50 years). Stress Cytogenetics usingMytomycin C was done to exclude thepossibilities of FA.

Among 49, 36(73.46%) were male and13(26.63%) female. Median age was 15.5 years(age range 3-50 years). Four (8.16%) patientsshowed positive stress cytogenetics and in eachcase breaks, gap, radial and tri radial were found.Breaks and radial structure were not found inage and sex matched control samples.

Around 8.16% patients had FA even if they aremorphologically normal. Stress cytogeneticanalysis will influence initiation of treatment.Further studies on a large sample are required.

The Fanconi anaemia is defined by geneselucidated a novel DNA repair mechanismrequired for maintaining genomic stability andpreventing cancer.[1] If mutation occurs in thesegenes, it can cause a specific clinicallyrecognizable entity with a unifying cellularfeature of hypersensitivity to crosslinking agentslike Mytomycin C, di-epoxy butane.[2] FanconiAnemia (FA) is a rare disorder with incidenceof 1in 350,000 births. They have an increasedrisk of solid tumours and leukaemia. Thirteencomplementation groups, defined by somaticcell hybridization,are associated with thedevelopment of Fanconi anemia.[3] Thecomplementation groups have been designated FANCA, B, C, D1, D2, E, F, G, I, J, L, M, and N.The most frequent mutations are found in FANCA,FANCC, or FANCG.[4] It has been proposed thatthe A and C gene produces cytoplasmic proteinsform an "FA core complex" whereas the productsof genes B, E, F, G,L, and M, forms adaptors orphosphorylators.[4,5] The complex translocatesto the nucleus and ubiquitylates FANCD2 andprotects the cell from DNA cross-linking thereby participating in DNA repair mechanism. NowDNA damage triggers the activation of theFA/BRCA pathway and ubiquitylation ofFANCD2 which in turn activates the DNA repairmechanism by the help of DNA repair proteinslike BRCA1, FANCD1/BRCA2, FANCN/PALB2, and RAD51.[6] In presence of a mutantprotein the normal function is hampered, leadingto the damage of hematopoietic stem cells andbone marrow failure. Usually Fanconi anaemiapatients face some typical abnormalities like,developmental delay, short stature, microcephaly,abnormal skin pigmentation, malformations ofskeletal system, limbs, eyes, ears, kidneys andurinary tract, heart, genitalia, gastrointestinalsystem and central nervous system, absence ofthumb and radius. So far known the carrierfrequency of Fanconi anaemia is 1:181 in USand the carrier frequency is 1:93 in Israel. Butthe exact incidence of Fanconi anaemia in Indiais not known due to lack of properly designedtrials. Few uncontrolled studies are there but nowell design population based study depicting theincidence of Fanconi anaemia has been done tilldate. It might be the first study in its kind. Ourstudy was aimed to find out the Fanconi anaemiacases in morphologically normal AplasticAnaemia (AA) patients.

Materials and Methods:
Patient screening:
Peripheral venous bloodsamples were collected with proper consentfrom 70 diagnosed aplastic anaemia patients forlast one year from the department of Hematologyof N.R.S Medical College & Hospital, Kolkata,West Bengal. Diagnosis was establishedaccording to the guidelines of InternationalAgranulocytosis and Aplastic Anaemia StudyGroup, 1987. At least two of the three criteriawith hypocellular bone marrow must be presentto define aplastic anaemia which are, (1)haemoglobin < 100 g/L, (2) platelet count< 50*109/L, (3) neutrophil count < 1.5 * 109/L.Patients were treated according to standardprotocol based on British Committee forStandards in Hematology (BCSH) Guidelines.Among 70 morphologically normal aplasticanaemia patients, peripheral venous bloodsamples were collected from 49 patients (< 50years) for stress cytogenetics and chromosomalbreakage test with Mitomycin C was done.Controlled Samples were collected from thehealthy age and sex matched persons.

Methodology :
Peripheral venous blood samples were collectedin heparinized vacutainer. Media compositionfor peripheral venous blood sample was RPMI1640 Gibco, USA (4ml) and Foetal bovine serum(1ml). Phytohemagglutinin (200ul) was addedbecause it is necessary for the selectivestimulation of T-lymphocyte in the culture.Peripheral venous blood samples were incubatedat 37°C for 72 hours. For stress cytogeneticsDNA cross-linking agents (Mitomycin C) wasintroduced to the culture after 24 hours ofinoculation. Three cultures for patient's sample(0ng/ml MMC, 50ng/ml MMC, 100ng/mlMMC) and three cultures for control sample(0ng/ml MMC, 50ng/ml MMC, 100ng/mlMMC) were established for every cases. For each case age and sex matched control samplewere set. Colcimid Solution was added to theculture and harvesting was done according tostandard protocol (modified moorhead et al,1960). The control peripheral blood sampleswere harvested in same manner. Metaphases areobserved under microscope 100X magnification.Presence of breaks, radial, bi-radial, tri-radialdenotes the characteristics sensitivity ofchromosome to DNA cross-linking agentssuggestive for Fanconi anaemia.

Results & Discussion:
As inherited aplastic anaemia is constitutionaldisease, bone marrow is not the preferable samplefor stress cytogenetics. Between them 73.46%(n=49) were male and 26.53 % (n=49) werefemale.
The median age of the patients was 15.5 years(n=49). The age range of the patient was (3-50years). Four (8.16%) patients showed positivefor stress cytogenetics which is suggestive forFanconi anaemia. In every cases break, gap,radial, and tri radial were observed under themicroscope (100X magnification). Breaks andradial structure were not found in controlsamples.

Showing tri radials (Red arrow) and breaks (Green arrow)

Table 1: Showing the results of Stress cytogenetics analysis:

Median age 15.5 years
Median age of stress cytogenetics (+Ve) 6 years
Age range (3-50)years
Sex Male =36(73.46%), Female=13(26.53%)
VSAA 31(63.26%)
SAA 14(28.57%)
NSAA 4(8.16%)
(+)Ve Stress cytogenetics 4(8.16%)[7Y/F,9Y/M,5Y/F,5Y/M]

Very severe Aplastic Anaemia (VSAA), Severe Aplastic Anaemia (SAA), Non Severe Aplastic Anaemia (NSAA)

In 8.16% cases were found to be positive forstress cytogenetics which is suggestive forFanconi anaemia. Therefore, it can be concludedthat, in morphologically normal aplastic anaemiapatients, stress cytogenetics can be positive.Consequently stress cytogenetic study as aroutine examination in aplastic anaemia isessential. Nonetheless study on larger samplesize is required for the establishment of thefindings.

We are indebted to, Department of Genetics,Vivekananda Institute of Medical Sciences,Kolkata, and DST-WB, for giving us all kind offinancial and inventory support. We also obligedto Department of Haematology, N.R.S MedicalCollege & Hospital, Kolkata for providingsamples and all kind of assistances.

Declaration of interest:
The authors report no conflicts of interest. Theauthors alone are responsible for the contentand writing of this paper.

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  4. Jacquemont C, Taniguchi T: The Fanconi anemiapathway and ubiquitin. BMC Biochemistry, 8 (Suppl1):S10 , 22 November 2007, DOI: 10.1186/1471-2091-8-S1-S10.

  5. Alter BP, Giri N, Sharon A. Savage and Philip S.,Cancer in dyskeratosis congenital, Blood, 2009,113:6549-6557; doi:10.1182/blood-2008-12-192880.

  6. Shukla P, Ghosh K, Vundinti BR, Current and emergingtherapeutic strategies for Fanconi anemia, The HUGOJournal, 2012, 6:1.


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