Fanconi anemia is a rare autosomal recessive syndrome comprised of progressive bone marrow failure, congenital anomalies and a predisposition to malignancy. The heterozygote rate in the United States may be as high as 1 in 300. The mean age for the onset of aplastic anemia is approximately eight years. Although improved supportive care has prolonged the survival of these patients from only a few years from the diagnosis of bone marrow failure, the mean age of death is still approximately 24 years of age. Most patients die from complications of bone marrow failure including bleeding, or infection, or from malignancy or complications of stem cell transplantation. In a recent 20 year review of patients in the Fanconi anemia registry, the actual risk of developing leukemia or other cancers was approximately 30%.
The diagnosis of Fanconi anemia initially rested upon finding the combination of bone marrow failure with congenital anomalies. These anomalies include cafe au lait spots and/or hypo pigmentation of the skin, short stature, upper limb malformations (often involving the thumb or radius), renal and gastrointestinal abnormalities, microcephaly, and characteristic facies with a broad nasal base, epicanthal folds, narrow set and small eyes and micrognathia. The bone marrow failure is characterized by slow progression to severe bone marrow aplasia and pancytopenia, stress erythropoiesis with fetal features including macrocytosis, elevated hemoglobin F, and i antigen expression. Attempts to culture bone marrow progenitors in vitro from patients with Fanconi anemia demonstrates decreased numbers of myeloid and erythroid colonies (CFU) consistent with clinical bone marrow failure.
Fanconi anemia cells appear to have a defect in DNA repair that leads to increased spontaneous chromosomal breakage. This feature increases the susceptibility of Fanconi anemia cells to DNA bifunctional cross-linking agents such as mitomycin C and diepoxybutane (DEB). The diagnosis of Fanconi anemia now relies upon detecting increased chromosomal breakage after in vitro treatment with DEB. 11 Similarly, cells cultured from patients with Fanconi anemia display increased susceptibility to the cytotoxicity of mitomycin C. More recently, cells from patients with Fanconi anemia have been demonstrated to display G2 phase prolongation/arrest, increased sensitivity to toxicity by oxygen, defective p53 induction and increased apoptosis.
Fanconi anemia can be classified into at least thirteen complementation groups by somatic cell hybrids. The complementation is based upon correction of the chromosomal sensitivity to cross-linking agents in hybrid cells. Twelve independent genes have been cloned and characterized within these 13 complementation groups. A loss of function in any of these genes including FANC A, B, C, D2, E, F, G, J, L, M, N, and FANC D1 (which is BRCA2) can cause Fanconi anemia. However, complementation groups A, C, and G account for approximately 80-85% of patients with Fanconi anemia in the United States. Discrete mutations in these genes have been identified in families with the disorder. Expression of the complementary cDNA gene in the respective Fanconi anemia cells in vitro corrects the increased chromosomal breakage from DEB and the increased sensitivity to mitomycin C. Expression of gene products in bone marrow progenitors from patients with Fanconi anemia increases survival in in vitro assays.
The current treatment for Fanconi anemia relies upon hematological support in the form of red blood cell and platelet transfusions. Aplastic anemia will transiently respond to androgen therapy in 50% of children. G-CSF has also been utilized in published studies from our own group to improve the number of myeloid cells in the peripheral circulation. Bone marrow transplantation has cured some patients of their bone marrow failure; however, there appears to be more toxicity to the conditioning regimens and there may be increased numbers of solid tumors post transplant compared to patients without the disorder. Survival five years after a matched sibling transplant now exceeds 65% and after an unrelated donor transplant 30%. More recent studies in unrelated donor transplant for Fanconi anemia at Cincinnati Children's Hospital and the University of Minnesota have reported survival rates approaching those observed in matched sibling donor transplants. However, graft failure resulting in death remains a major obstacle. The availability of sufficient numbers of (previously purified and cryopreserved) autologous HSC for re-infusion after graft failure may prevent this complication.
Finally, gene therapy approaches are being pursued, but to date, there is no evidence for cure with this approach in humans, although correction has been reported in murine models. These studies are hampered by the fact that mouse knockouts of FA genes do not develop spontaneous aplastic anemia and thus are not phenocopies of the human disease. Thus in previously reported mouse-studies, gene therapy approaches required ablative total body irradiation of the recipient mice to ensure engraftment of the gene corrected stem cells.
An obvious limitation of Fanconi anemia hematopoietic stem cell gene transfer is that the necessary target for genetic manipulation, the hematopoietic stem cell (or its surrogate, CD34+ cell) is progressively lost during the development of aplastic anemia. Thus at the time of severe aplasia and the greatest need for treatment, target stem cells for genetic modification are deficient. Collection of a meaningful number of HSC prior to the onset of aplastic anemia for eventual use in a therapeutic gene therapy trial will be explored in the study outlined here. Key questions remaining are whether corrected HSC from Fanconi anemia patients will engraft after autologous re-infusion without any cyto-reductive treatment of the recipient and, if engrafted, whether the corrected cells will demonstrate a proliferative advantage over uncorrected stem cells.
