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Chapter 29 Inherited Bone Marrow Failure Syndromes 353

investigation algorithms included FA complementation grouping to The activated core complex results in conversion of two downstream
determine the genetic group, followed by targeted gene analysis protein targets, FANCI and FANCD2 (called the ID2 complex),
(by Sanger sequencing and multiplex ligation-dependent probe from unubiquitinated isoforms to monoubiquitinated isoforms.
amplification). Monoubiquitination does not occur if the core complex upstream of
the ID2 complex is not intact, and therefore FA cells from patients
Genotype–Phenotype Correlations with upstream mutations do not show the monoubiquitinated
The clinical severity of FA is only partly determined by the specific FANCI/FANCD2. (3) Downstream effector complexes. In normal
FANC gene involved and by the type of mutation. Patients with cells after monoubiquitination of the ID2 complex, the wild-type
FANCA who are homozygous for null mutations and produce no core complex translocates the monoubiquitinated ID2 complex to
protein tend to have an earlier onset of anemia and a higher propen- chromatin and localizes the ID2 complex to nuclear foci, where it
sity to leukemic transformation compared with patients with hypo- probably forms a binding interface for single- and double-stranded
morphic FANCA who produce protein, albeit abnormal. Compared DNA and downstream effector complexes with additional FANC
with other FANCC mutations, FANCC IVS4+4A>T, commonly proteins (FANCD1/BRCA2, FANCJ/BACH1/BRIP1, FANCN/
found in Ashkenazi Jews, is particularly severe and is linked with PALB2, FANCO/Rad51C, FANCP/SLX4, FANCS/BRCA1) and
early-onset anemia, early BM failure, and severe physical anoma- other non-FA DNA repair proteins. There are three main DNA repair
lies, including some cases with physical features of VACTERL-H processes that the FA genes cooperate with: (1) excision repair that
syndrome (a well-known malformation association, including excises one DNA strand flanking the interstrand cross-link (via inter-
vertebral, anal, tracheoesophageal, renal, and limb abnormalities). action between SLX4 and MUS81, SLX1, and others), (2) translesion
Of genetic-ethnic interest, the identical IVS4+4A>T mutation in synthesis that extend the uncut strand (via recruitment of translesion
Japanese patients with FA manifests with a milder phenotype than in polymerase), (3) homologous recombination that is initiated after
Ashkenazi Jews. FANCD1/BRCA2 and FANCN/PALB2 mutations repair of the interstrand cross-link remnant possibly by ERCC4 and
are both associated with a significant predisposition to develop solid nucleotide excision repair protein, and involves interactions between
tumors, acute myeloid leukemia (AML) and with a severe physical FANCJ, FANCN, FANCO, FANCS/BRCA1, and RAD51. Other
anomaly phenotype. The VACTERL-H cluster of anomalies is closely DNA-repair proteins such as MRE11-RAD50-NBS1, replication
linked with biallelic mutations of FANCD1/BRCA2 and FANCB. protein A, PCNA, and BLM are also involved in the later stages of
Null mutations of FANCG correlate with very severe manifestations the DNA repair response.
compared with most other FANC mutations and correlate with early-
onset anemia and BM failure, a higher incidence of AML, and severe Cell Survival and Balancing Oxidative Stress. There are impor-
physical anomalies. FANCD2 and FANCI mutations also correlate tant protein–protein interactions between FA proteins and non-FA
with severe anomalies, and FANCI is associated with early-onset “binding partners” for cell survival. FANCC and FANCD2 form
anemia. complexes with members of the signal transducer and activator of
transcription (STAT) family of transcription factors in cytokine-
Murine Models mediated biologic responses. Secondly, heat shock proteins provide
Multiple FA mouse models have been generated in which targeted several cell survival functions, and FANCC protein specifically facili-
disruption of genes like Fanca, Fancc, Fancd1, Fancd2, Fancg, and tates the antiapoptotic role of Hsp 70. FANCC also interacts with
Fancn. Knock-out mouse models largely do not recapitulate the cdc2, PKR, and p53, suggesting that FANCC has other roles that
hypocellularity and cytopenia that characterizes FA, with few excep- are independent of DNA damage recognition and repair. GSTP1 is
−/−
−/−
−/−
tions (e.g., Slx or combined Fancc /Fancg ). However, these an enzyme that detoxifies byproducts of redox stress and xenobiotics
models provide insight into the various functions of the genes and and FANCC protein enhances GSTP1 activity in cells exposed to
the role of individual FA mutations. Consistent findings in some or apoptosis inducers.
all of the mice include impaired proliferation of BM hematopoietic Previous studies suggested a role of oxidative stress in the evolu-
progenitors, hypogonadism, impaired fertility, growth retardation, tion of BM failure and leukemia in FA. Reactive oxygen species
microphthalmia, development of cancers, hypersensitivity of BM (ROS) were shown to be elevated in FA cells, and high oxidative
progenitor cells to administered MMC, as well as to interferon-γ stress caused increased DNA damage, increased hematopoietic stem
(INF-γ) or tumor necrosis factor-α (TNF-α) in vitro and in vivo. The cell (HSC) senescence, and a decreased HSC pool, thereby leading
phenotype of these mutant mice shows abnormal G 2 /M progression to BM failure. Further, in vivo and in vitro studies demonstrated the
of the cell cycle similar to patients with FA. Interestingly, double ability of the antioxidant N-acetylcysteine to reduce DNA damage,
knockout of several Fanc genes together with genes that play a role in reduce HSC senescence, and improve HSC reconstitution ability.
−/−
balancing oxidative stress and other genotoxic agents (e.g., Fancd2 / Therefore it is possible that patients with FA are particularly sensitive
−/−
−/−
−/−
−/−
Foxo3a, Fancc /Sod1 , Fancd2 /Aldh2 ) leads to a phenotype to ROS-induced DNA damage because of impaired DNA repair
−/−
−/−
that is closer to the human FA disease. For example, Fancc /Sod1 mechanisms. This increased sensitivity may be caused, at least in
−/−
−/−
mice develop hypocellular BM; Fancd2 /Foxo3a mice feature an part, by impaired detoxification of ROS and naturally produced
initial expansion followed by a progressive decline of BM stem and aldehydes.
−/−
−/−
progenitor cells, and Fancd2 /Aldh2 develop low progenitors and In the skin fibroblasts of patients with FA, N-acetylcysteine was
leukemia. able to reduce ROS levels and apoptosis as measured by activation
−/−
of caspase-3 and PARP cleavage. In fancc mice, N-acetylcysteine
Functions of FANC Proteins rescues hematopoietic colony formation that is impaired by spon-
DNA damage repair. Cells and cell lines from patients with FA are taneous secretion of TNF-α. It also reduces TNF-α-mediated
phenotypically similar regardless of the complementation group that hematopoietic colony formation and HSC senescence and HSC
−/−
they represent. A hypothesis was therefore formulated and subse- reconstitution potential. Using a fancd2 mouse model, treatment
quently substantiated that the various wild-type FANC proteins with the antioxidant drug, resveratrol, has also been shown to pre-
function in a common response pathway to repair DNA damage serve HSC quiescence, partially correct the abnormal cell cycle status
incurred during DNA replication. and significantly improve the spleen colony-forming capacity of BM
There are three general steps in the FA DNA damage response cells. Importantly, treatment of FA mice with N-acetylcysteine has
pathway: (1) Core complex. Eight wild-type FANC proteins been shown to reduce the accumulation of cytogenetic abnormalities
(FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, (that are commonly seen in patients with FA who transform to
and FANCM) and four additional proteins (FAAP16, FAAP 20, MDS/AML). In one study the antioxidant, tempol, delayed cancer
−/−
+/−
FAAP24, and FAAP100) form a single large nuclear protein core in tumor-prone fancd2 /Trp53 mice. However, in another study
complex as the first step. The core complex functions as a ubiquitin neither N-acetylcysteine nor the antioxidant resveratrol had this
ligase of which FANCL is the catalytic subunit. (2) ID2 complex. property.