University of Leicester
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Studies on the basis of the inherited bone marrow failure syndromes: Fanconi's anaemia and dyskeratosis congenita.

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posted on 2015-11-19, 08:52 authored by Inderjeet Singh. Dokal
A number of inherited disorders are associated with bone marrow failure. Amongst them Fanconi's anaemia (FA) is the most common and, together with dyskeratosis congenita (DC), the best characterized. The aim of this study was twofold: A. To devise a strategy which may prermit "expression cloning" of the FA genes. B: To characterise cytogenetic (and molecular) features of cells from FA and DC patients which may provide a better understanding of the basis of these disorders. As a first step towards cloning the gene (s) for Fanconi's anaemia (FA) I devised a selection system [based on the DNA cross-linking agents mitomycin-c (MMC) and diepoxybutane (DEB)] that discriminates between FA and normal cells. 'Mixing experiments' (where approximately 10 normal cells were co-plated with 106 FA cells) demonstrated that it is possible to kill FA cells at high density without significantly affecting the cloning efficiency of normal cells. Transfection of FA fibroblasts with normal DNA (mouse genomic, human genomic, and human cDNA) either by calcium phosphate precipitation or by electroporation yielded 11 DEB and MMC resistant colonies. However, southern analysis of the DNA from these colonies with the appropriate probes gave no positive signal, and thus no "handle" to recover the FA gene (s). Experiments addressing the effect of the specific DNA topoisomerase I inhibitor, camptothecin, on FA and normal cells showed: 1. The FA lymphocytes have increased chromosomal breakage compared to normal lymphocytes after incubation with camptothecin (p=0.006). 2. Incubation of peripheral blood lymphocytes (pbl) from normal subjects with camptothecin, produced the same type of chromosomal breakage as that seen in FA lymphocytes. 3. FA fibroblasts were more sensitive to camptothecin than normal fibroblasts. These data are compatible with either a defect which makes topoisomerase I more crucial, or its function being abnormal in some FA patients. In patients with DC, primary skin fibroblast cultures were abnormal in both morphology and growth rate. Survival studies using 4 clastogens and gamma-irradiation showed no significant difference between DC and normal fibroblasts. Cytogenetic studies performed on pbl showed no difference between DC and normal lymphocytes with or without prior incubation with clastogens. However, bone marrow from 1 out of 3 patients and fibroblasts from 2 out of 4 patients showed numerous unbalanced chromosomal rearrangements in the absence of clastogenic agents. Although patients with FA and DC share some features in common they appear to differ in two fundamental ways: Firstly, unlike Fanconi cells, DC cells are not hypersensitive to clastogens. Secondly the primary defect in DC appears to predipose cells to developing chromosomal rearrangements rather than to chromosomal gaps and breaks seen in FA.


Date of award


Author affiliation

College of Medicine, Biological Sciences and Psychology

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • MD



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