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Transforming growth factor beta (TGF-beta) secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-beta in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-beta isoforms. In ten FA patients, mutations were detected in FANCA (n = 7), FANCG (n = 1) and FANCD2 (n = 2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. beta-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-beta 1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-beta was absent in FA-D2 BM-MSCs. Absence of TGF-beta secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-beta 1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-beta 1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.

Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, ‘FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility., Fanconi anaemia is an inherited disorder characterised by developmental abnormalities, bone marrow failure and predisposition to cancer. Here the authors report a de novo mutation in the DNA repair gene Rad51 in an atypical subtype of Fanconi anaemia.

Serous type Fallopian tube carcinoma (FTC) and ovarian carcinoma (OVCA) are gynaecological malignancies with a poor prognosis. Although most of the encountered tumours are sporadic, a positive family history is an important risk factor. Both tumour types have been linked to mutations in the BRCA1 and BRCA2 genes and share several cellular, molecular and clinical features. However, a detailed and unbiased comparison at the molecular level had been lacking. In this thesis the molecular pathogenesis of serous FTC and serous OVCA was further studied. After an introduction in Chapter 1, we describe a genome-wide array comparative genomic hybridization (array CGH) study of 14 serous FTCs (Chapter 2). All FTCs showed a high frequency of copy number aberrations, while these were remarkably homogeneous. Recurrent regions of amplification suggested known oncogenes MYC, CCNE1 and AKT2 to be driver genes. In Chapter 3 we describe the immunohistochemical analysis of p53, HER-2/neu and p27Kip1 on 28 serous FTCs and we correlated expression levels with DNA copy number. The studies suggested that p53 accumulation and p27Kip1 down-regulation are early events, whereas HER-2/neu overexpression may be involved in progression. Furthermore, overexpression of HER-2/neu may be caused partly by amplification. In Chapter 4 we were the first to report on a direct comparison with array CGH of serous FTCs and OVCAs. The analysis suggested that these display (quantitative) differences in their genomic profiles, besides shared features. Multiplex ligation-dependent probe amplification (MLPA) directly identified EIF2C2 as a new possible driver gene. Gains/amplifications of CCNE1 and MYC, often in conjunction with changes of EVI1 and PTK2, seemed to be common in earlier stages, whereas changes of HER2 were associated with advanced stages (see Chapter 3). In Chapter 5 we report on the development of dedicated MLPA gain-probe-sets, specifically tailored at high-grade serous FTC and OVCA and primarily based on our previous array CGH (see Chapter 4). We identified, besides known genes PIK3CA, AKT2, FGFR1 and PDGFB, new putative genes MYCBP, LIMK1, SOCS1, SMARCA4, DPF1, BCL2L1, NCOA3, PTPN1, NFATC2 and KCNQ2. As an application, we performed logistic regression, resulting in a model that could discriminate 76% of the FTCs and 80% of the OVCAs on basis of 3 markers. Since serous endometrial carcinomas (ECs) share their aggressive clinical behaviour and their histological appearance with serous OVCA and FTC, it could be hypothesized that these serous cancers share common denominators. In Chapter 6, we examined 9 serous ECs with the same array as before and compared the profiles with those described in Chapter 4. We could show that serous ECs can be discriminated from serous OVCAs. In Chapter 7 the results of the studies are discussed. We conclude that despite their histomorphological and clinical resemblance, high-grade serous FTC and OVCA show differences in their genomic profiles, besides also shared features, and should be regarded as distinct entities. We have identified new candidate genes for carcinogenesis and validated certain known genes, which has resulted in a more detailed knowledge on the pathogenesis of this class of tumours.