Fanconi anemia

Fanconi anemia (FA) is a genetic disease with an incidence of 1 per 350,000 births, with a higher frequency in Ashkenazi Jews and Afrikaners in South Africa.

FA is the result of a genetic defect in a cluster of proteins responsible for DNA repair. As a result, the majority of FA patients develop cancer, most often acute myelogenous leukemia, and 90% develop bone marrow failure (the inability to produce blood cells) by age 40. About 60-75% of FA patients have congenital defects, commonly short stature, abnormalities of the skin, arms, head, eyes, kidneys, and ears, and developmental disabilities. Around 75% of FA patients have some form of endocrine problem, with varying degrees of severity. Median age of death was 30 years in 2000.

Treatment with androgens and hematopoietic (blood cell) growth factors can help bone marrow failure temporarily, but the long-term treatment is bone marrow transplant if a donor is available.

Because of the genetic defect in DNA repair, cells from people with FA are sensitive to drugs that treat cancer by DNA cross-linking, such as mitomycin C.

The disease is named after the Swiss pediatrician who originally described this disorder, Guido Fanconi. It should not be confused with Fanconi syndrome, a kidney disorder also named after Fanconi.

Genetic prevalence
FA is primarily an autosomal recessive genetic disorder. This means that two genes (one from each parent) are required to cause the disease. There is a 25% risk that each subsequent child will have FA. About 2% of FA cases are X-linked recessive, which means that if the mother has a Fanconi anemia gene there is a 50% chance that male offspring will present with Fanconi anemia.

Scientists have identified 15 FA or FA-like genes: FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP and RAD51C. FANCB is the one exception to FA being autosomal recessive, as this gene is on the X chromosome.

Approximately 1,000 persons worldwide currently suffer from the disease. The carrier frequency in the Ashkenazi Jewish population is about 1/90. Genetic counseling and genetic testing is recommended for families that may be carriers of Fanconi anemia.

Because of the failure of hematologic components to develop – leukocytes, red blood cells and platelets - the body's capabilities to fight infection, deliver oxygen, and form clots are all diminished.

Treatment
The first line of therapy is androgens and hematopoietic growth factors, but only 50-75% of patients respond. A more permanent cure is hematopoietic stem cell transplantation. If no potential donor exist, a savior sibling can be conceived by preimplantation genetic diagnosis (PGD) to match the recipients HLA type.

Prognosis
Many patients eventually develop acute myelogenous leukemia (AML). Older patients are extremely likely to develop head and neck, esophageal, gastrointestinal, vulvar and anal cancers. Patients who have had a successful bone marrow transplant and, thus, are cured of the blood problem associated with FA still must have regular examinations to watch for signs of cancer. Many patients do not reach adulthood.

The overarching medical challenge that Fanconi patients face is a failure of their bone marrow to produce blood cells. In addition, Fanconi patients normally are born with a variety of birth defects. For instance, 90% of the Ashkenazi children born with Fanconi's have no thumbs. A good number of Fanconi patients have kidney problems, trouble with their eyes, developmental retardation and other serious defects, such as microcephaly (small head).

Hematological abnormalities
Clinically, hematological abnormalities are the most serious symptoms in FA. By the age of 40, 98% of FA patients will have developed some type of hematological abnormality. It is interesting to note, however, the few cases in which older patients have died without ever developing them. Symptoms appear progressively, and often lead to complete bone marrow failure. While at birth, blood count is usually normal, macrocytosis/megaloblastic anemia, defined as unusually large red blood cells, is the first detected abnormality, often within the first decade of life (median age of onset is 7 years). Within the next 10 years, over 50% of patients presenting haematological abnormalities will have developed pancytopenia, defined as abnormalities in two or more blood cell lineages. Most commonly, a low platelet count (thrombocytopenia) precedes a low neutrophil count (neutropenia), with both appearing with relative equal frequencies. The deficiencies cause increased risk of hemorrhage and recurrent infections, respectively.

As FA is now known to affect the DNA repair, and given the current knowledge about dynamic cell division in the bone marrow, it is not surprising to find patients are more likely to develop bone marrow failure, myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The next sections will detail those pathologies.

Myelodysplastic syndromes
MDS, formerly known as preleukemia, are a group of bone marrow neoplastic diseases that share many of the morphologic features of AML, with some important differences. First, the percentage of undifferentiated progenitor cells, blasts cells, is always less than 20%, and there is considerably more dysplasia, defined as cytoplasmic and nuclear morphologic changes in erythroid, granulocytic and megakaryocytic precursors, than what is usually seen in cases of AML. These changes reflect delayed apoptosis or a failure of programmed cell death. When left untreated, MDS can lead to AML in about 30% of cases. Due the nature of the FA pathology, MDS diagnosis cannot be made solely through cytogenetic analysis of the marrow. Indeed, it is only when morphologic analysis of marrow cells is performed, that a diagnosis of MDS can be ascertained. Upon examination, MDS-afflicted FA patients will show many clonal variations, appearing either prior or subsequent to the MDS. Furthermore, cells will show chromosomal aberrations, the most frequent being monosomy 7 and partial trisomies of chromosome 3q 15. Observation of monosomy 7 within the marrow is well correlated with an increased risk of developing AML and with a very poor prognosis, death generally ensuing within 2 years.

Acute myeloid leukemia
FA patients are at elevated risk for the development of acute myeloid leukemia (AML), defined as presence of 20% or more of myeloid blasts in the marrow or 5 to 20% myeloid blasts in the blood. All of the subtypes of AML can occur in FA with the exception of promyelocytic. However, myelomonocytic and acute monocytic are the most common subtypes observed. It is also interesting to note that many MDS patients will evolve into AML given they survive long enough. Furthermore, the risk of developing AML increases with the onset of bone marrow failure.

Although risk of developing either MDS or AML before the age of 20 is only 27%, this risk increases to 43% by the age of 30 and 52% by the age of 40. Even with a marrow transplant, about 1/4 of FA patients diagnosed with MDS/ALS will die from MDS/ALS-related causes within 2 years. (Butturini, A et al 1994. Blood. 84:1650-4)

Bone marrow failure
The last major haematological complication associated with FA is bone marrow failure, defined as inadequate blood cell production. Several types of failure are observed in FA patients, and generally precede MDS and AML. Detection of decreasing blood count is generally the first sign used to assess necessity of treatment and possible transplant. While most FA patients are initially responsive to androgen therapy and haemopoietic growth factors, these have been shown to promote leukemia, especially in patients with clonal cytogenetic abnormalities, and have severe side effects, including hepatic adenomas and adenocarcinomas. The only treatment left would be bone marrow transplant; however, such an operation has a relatively low success rate in FA patients when the donor is unrelated (30% 5-year survival). It is therefore imperative to transplant from an HLA-identical sibling. Furthermore, due to the increased susceptibility of FA patients to chromosomal damage, pretransplant conditioning cannot include high doses of radiations or immunosuppressants, and thus increase chances of patients developing graft-versus-host disease. If all precautions are taken, and the marrow transplant is performed within the first decade of life, 2-year probability of survival can be as high as 89%. However, if the transplant is performed at ages older than 10, 2-year survival rates drop to 54%.

A recent report by Zhang et al. investigates the mechanism of bone marrow failure in FANCC-/- cells. They hypothesize and successfully demonstrate that continuous cycles of hypoxia-reoxygenation, such as those seen by haemopoietic and progenitor cells as they migrate between hyperoxic blood and hypoxic marrow tissues, leads to premature cellular senescence and therefore inhibition of haemopoietic function. Senescence, together with apoptosis, may constitute a major mechanism of haemopoietic cell depletion occurred in bone marrow failure.

Molecular basis of FA
There are 13 genes responsible for FA, and one of them is identical to the breast-cancer susceptibility gene BRCA2. They are involved in the recognition and repair of damaged DNA; genetic defects leave them unable to repair DNA. The FA core complex of 8 proteins is normally activated when DNA stops replicating because of damage.The core complex adds ubiquitin, a small protein that combines with BRCA2 in another cluster to repair DNA. At the end of the process, ubiquitin is removed.

Recent studies have shown that eight of these proteins, FANCA, -B, -C, -E, -F, -G, -L and –M assemble to form a core protein complex in the nucleus. According to current models, the complex moves from the cytoplasm into the nucleus following nuclear localization signals on FANCA and FANCE. Assembly is activated by replicative stress, particularly DNA damage caused by cross-linking agents(mitomycin C or cisplatin) or reactive oxygen species (ROS). Indeed, FANCA and FANCG have been observed to multimerize when a cell is faced with oxidative stress-induced damage.

Following assembly, the protein core complex activates FANCL protein which acts as an E3 ubiquitin-ligase and monoubiquitinates FANCD2.

Monoubiquitinated FANCD2, also known as FANCD2-L, then goes on to interact with a BRCA1/BRCA2 complex. Details are not known, but similar complexes are involved in genome surveillance and associated with a variety of proteins implicated in DNA repair and chromosomal stability. With a crippling mutation in any FA protein in the complex, DNA repair is much less effective, as shown by its response to damage caused by cross-linking agents such as cisplatin, diepoxybutane and Mitomycin C. Bone marrow is particularly sensitive to this defect.

In another pathway responding to ionizing radiation, FANCD2 is thought to be phosphorylated by protein complex ATM/ATR activated by double-strand DNA breaks, and takes part in S-phase checkpoint control. This pathway was proven by the presence of radioresistant DNA synthesis, the hallmark of a defect in the S phase checkpoint, in patients with FA-D1 or FA-D2. Such a defect readily leads to uncontrollable replication of cells and might also explain the increase frequency of AML in these patients.

Other FA protein interactions
Although the above described pathway seems to be the most integral part of the DNA damage response in cells and explains the pathology of FA, novel approaches have determined that most FA proteins have an alternate role. Indeed, recent investigations on FANCC, one of the intensively studied proteins, have shown that it plays an important role in cellular responses to oxidative stress. For example, it has been found to interact with NADPH cytochrome P450 reductase, associated with increased production of ROS, and glutathione S-transferase, responsible for production of the anti-oxidant glutathione. These two enzymes are both involved in either triggering or detoxifying ROS. Not surprisingly, mice with Cu/Zn superoxide dismutase and FANCC mutations demonstrate defective haemopoiesis. FANCC was also shown to bind STAT1 and help receptor docking and phosphorylation of STAT135, which helps in tumor suppression. This leads to the conclusion that FANCC participates in cell growth arrest and cell cycle progression, inhibiting apoptosis, a possible cause of bone marrow failure due to depletion of haemopoietic progenitors. Another FA protein linked to protection against oxidative damage is FANCG. Indeed, this protein interacts with cytochrome P450 2E1 suggesting a possible role in detoxifying cytochrome ROS, produced readily by the members of this superfamily36. Furthermore, FANCG is identical to post-replication repair protein XRCC9, hinting at the possibility that FANCG also interacts directly with DNA by means of its internal leucine zipper. Thus it is readily seen that FA proteins also act outside of the Fanconi pathway, either by helping neutralize ROS or by taking part in DNA repair. Such mechanisms help understand the causes behind bone marrow failure, where reoxygenation-induced oxidative stress is very common. Furthermore, it is known that cross-linking agents produce ROS and it is possible that FA cell hypersensitivity to cross-linkers is not due directly to them, but rather to the cell’s impaired ability to cope with increased ROS production.