Clinical and Molecular Characterization of Fanconi Anemia: An Indian Perspective

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by a defect in one of the DNA repair pathways. FA exhibits a high degree of clinical heterogeneity and the exact molecular basis of the difference in the phenotypes has not been well understood. Study of this disease in geographic regions with high consanguinity will provide valuable insights in genotype-phenotype correlation and the genetic and environmental modifiers of this disease. We carried out a comprehensive clinical and molecular analysis in 101 patients with pancytopenia, with age <20 years, seen at the department of Haematology, Christian Medical College, India between 2009 and 2014. Seventy six patients had characteristic physical abnormalities of FA, of which perioral hyperpigmentation (42%) was the most common in these patients. We also included 25 patients with aplastic anemia as controls. The median age at diagnosis was 11 years (range 4-30) and sex ratio between males to females was 3:2. Chromosome breakage analysis (CBA) was performed on peripheral blood samples at diagnosis. Forty metaphases each from patient and normal control were assessed for sensitivity to Mitomycin C as per the protocol published in Mayo Clinic Proceedings (1997). CBA score greater than the cut off value of 40 was seen in 63% (n=48/76) of the patients. However, 23% (n=18/76) had ambiguous or borderline score (score 30-40; n= 12, score 20-30; n=4, score <10-20; n=2) and 13% (n=10/76) of patients were not sensitive to Mitomycin C. The control group did not display sensitivity or had mild degree of breakage (CBA score: 4.1-20). FANCD2 ubiquitination analysis of these patients with physical abnormalities in peripheral blood and fibroblasts showed absence of ubiquitination in 69 (90%) of the patients while the control group had normal ubiquitination pattern. To evaluate mosaicism, both peripheral blood and fibroblast samples were analyzed in 59 patients and FANCD2 ubiquitination was defective in both tissues in 55 (93%) patients. In 3 patients, FANCD2 ubiquitination was defective in fibroblasts, but not in peripheral blood indicative of mosaicism of this disease in these patients. Three patients had a normal ubiquitination pattern but had high CBA score and this might be due to mutations in any of the genes that function in the pathway downstream of FANCD2 ubiquitination and requires further evaluation. Four patients who had physical features suggestive of FA were negative by both CBA and FANCD2 western blot. In the control group, both peripheral blood and fibroblast samples were available in 19 patients all of which showed normal FANCD2 ubiquitination. In order to further characterize the FA subtypes, we generated lentiviral vectors to express FANCA, FANCC and FANCG genes for complementation assay. Fibroblasts from 20 patients were transduced with lentiviral FANCA and in 17 (85%) FANCD2 ubiquitination could be restored suggesting a high frequency of FANCA defects in Indian population. Mutation screening of FANCA in a subset of patients showed large deletions in five of which three are novel (ex 10_37 del,ex 6_14 del and ex 1_4 del). There was a deletion of two nucleotides resulting in a frameshift mutation in one patient (c.3760_3761delGA) and a missense mutation (c.2786A>C) in another. Our data suggests that FANCD2 ubiquitination analysis in conjunction with CBA is useful for the diagnosis of FA and detection of mosaicism and the complementation analysis shows high frequency of FANCA defects in patients with FA in Indian population.