Investigation of mutations in Fanconi anemia genes and malignancy predisposition in Brazilian patients.

INTRODUCTION: This study proposed to identify Fanconi anemia (FA) mutations in Brazilian patients and to investigate their impact on clinical manifestations and malignancies onset. METHODS: A total of 116 patients were screened for nine mutations in FANCA, FANCC, FANCG. Those with no mutations were investigated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing for FANCA, FANCC, FANCE, FANCF, FANCG, FANCD1/BRCA2. RESULTS: Genetic subtype was identified in 107/116 (78 FA-A, 8 FA-C, 13 FA-G, 8 FA-E), with only one mutation in 1/116, and no mutations in 9/116 patients. Before hematopoietic cell transplantation (HCT), malignancies were detected in 16/116 patients (14/78 FA-A, 01/08 FA-C, 01/08 FA-E), and 12 of them were hematological. Observed to expected ratio (O/E) of hematologic malignancy was 303.7 (95% CI = 148.6-458.7). CONCLUSION: This study allowed the identification of biallelic mutations in 91.4% of patients. FANCG and FANCC mutations had significantly earlier bone marrow failure onset, and FANCG severe cytopenia at diagnosis. Despite the inherent limitations of the small number of malignancy events in each genetic subtype, the hematologic malignancies O/E ratio was very high. Cumulative incidence of malignancy before HCT was higher in the third and fourth decades of life, considering HCT and death as competing risks. The cumulative incidence of HCT increased during the first decade, competing with malignancy development.

Somatic mosaicism in patients with Fanconi anaemia: Proposal of alternative tissue for inconclusive diagnoses.

INTRODUCTION: Fanconi anaemia (FA) is a rare genetic disorder marked by progressive bone marrow failure, chromosomal fragility, and increased cancer susceptibility. Laboratory diagnosis includes chromosomal instability test and mutation investigation. A total of 15%-25% of all patients may have somatic mosaicism, characterized by two distinct haematopoietic cell populations, one resistant and one sensitive to agents that induce chromosomal breakage, which complicates the diagnosis by a high incidence of reverted cells leading to inconclusive or false-negative results. The study aimed to evaluate the use of bone marrow stromal mesenchymal cells (BM-MSCs) as an alternative, non-haematopoietic tissue for diagnosis. METHODS: Bone marrow mesenchymal stromal cells from 12 patients with positive diepoxybutane (DEB) tests were cultivated and analysed by cytogenetics and mutation investigation. RESULTS: The DEB test was performed at 0.1 and 0.01 µg/ml concentrations, with an index ranging from 0.24 to 1.00. At higher concentration, the metaphases number was lower, probably due to toxicity. Regarding the molecular investigation, all the mutations previously found in peripheral blood were identified on BM-MSC. CONCLUSION: This study demonstrated the possibility of using BM-MSCs as an alternative tissue for cytogenetic and molecular investigation. Future tests using an intermediate DEB concentration may lead to an optimal protocol that could be non-toxic to cells but provides conclusive results.

Transplantation for Fanconi anaemia: lessons learned from Brazil.

Fanconi anaemia is a challenging disease to manage, and haematopoietic stem-cell transplantation (HSCT) is the treatment of choice for the haematological complications related to this disease. Over these past two decades, we have observed a substantial improvement in survival outcomes after matched related and unrelated donor HSCT, even for patients living in low-income and middle-income countries. Long-term overall survival is still suboptimal because of the risk of malignancies and other disease-related complications. For patients without well matched donors, alternative donor transplantation using mismatched related donors is an option but is historically associated with a high incidence of graft failure and graft-versus-host disease (GVHD). Herein we discuss the development of a HSCT programme for Fanconi anaemia in our centre in Curitiba, Brazil. Because ex vivo, T-cell depletion is unavailable in our country, we adapted the haploidentical donor transplantation platform using post-HSCT cyclophosphamide to overcome graft failure and GVHD associated with HLA-mismatched donor transplantation. The withdrawal of pre-HSCT cyclophosphamide reduced the severity of mucositis and did not interfere with engraftment. The addition of serotherapy improved overall survival by decreasing the incidence of severe acute and chronic GVHD. Although we have improved overall survival and expanded access to HSCT for Fanconi anaemia, our patients face many challenges, especially viral reactivation and GVHD disease, that merit attention. We acknowledge that there is a learning curve to adopt the haploidentical approach for Fanconi anaemia to low-resourced settings, and this Brazilian experience might require further modifications along with national and international collaborations to be implemented in other countries.