Lectures 1-3 Chromosomal DNA
Fragility Syndromes
• There exist numerous genetic disorders, marked
by chromosome instability
• Chromosomal instability is associated with cancer
development
• Both the chromosomal instability and neoplastic
outcome are related to abnormalities of:
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DNA metabolisms
DNA repair
Cell cycle control
Apoptosis control
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Cellular DNA damage causes
• Endogenous
– Oxidative free radicals
– Inappropriate DNA methylation
– Aberrant telomeres
• Exogenous:
– Chemicals
– Radiations
Continued genetic integrity requires appropriate responses
to exogenous and endogenous alterations. Otherwise,
mutations eventually result, which can lead to cancer.
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Chromosome breakage syndromes
The question of the number of genes responsible for the defects of ataxia
telangectasia was resolved by the positional cloning of ATM. All cases of
the disease are due to one of the 200 different mutations (usually null
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mutations) in this disease.
Chromosome breakage syndromes
and cancer
• Diseases of defective DNA recombination
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Ataxia telangiectasia
Nijmegen breakage syndrome
Breast cancer
Fanconi anemia
ATM
NBS
BRCA1/BRCA2
11 genes
• Diseases of mutant DNA helicases
– Bloom Syndrome
– Werner syndrome
– Rothmund-Thompson syndrome
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Ataxia Telangiectasia
• Defect in G1/S transition: p53 mediated
• Defect in G2/M, mitotic spindle, and S phase checkpoints
• All cases of the diseases are due to one of the 200 different
mutations.
• ATM protein expressed in many tissues (thymus, spleen,
developing CNS).
• ATM is able to phosphorylate several proteins (To date,
more than 30 ATM-dependent substrates have been
identified in multiple pathways that maintain genome
stability and reduce the risk of disease) :
– p53 (p21, GADD45 pathway)
– cAbl: Rad-51
– RAD50/MRE11/NBS complex
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ATM and DNA repair
• The recognition and repair of DNA double-strand breaks (DSBs) is a
complex process that draws upon a multitude of proteins.
• This is not surprising since this is a lethal lesion if left unrepaired and
also contributes to genome instability and the consequential risk of
cancer and other pathologies.
• Some of the key proteins that recognize these breaks in DNA are
mutated in distinct genetic disorders that predispose to agent
sensitivity, genome instability, cancer predisposition and/or
neurodegeneration.
• These include members of the Mre11 complex (Mre11/Rad50/Nbs1)
and ataxia-telangiectasia (A-T) mutated (ATM), mutated in the human
genetic disorder A-T. The mre11 (MRN) complex appears to be the
major sensor of the breaks and subsequently recruits ATM where it is
activated to phosphorylate in turn members of that complex and a
variety of other proteins involved in cell-cycle control and DNA
repair.
Oncogene. 2007 Dec 10;26(56):7749-58.
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Acta Pharmacologica Sinica 2005 Aug; 26 (8): 897–907
Fanconi Anemia
• Inheritance: Autosomal recessive; frequency is
about 2.5/105 newborns
• Fanconi anemia is a chromosome instability
syndrome with progressive bone marrow failure
and an increased risk of cancers
• OMIM # FANCA (607139), FANCB (300515),
FANCC (227645), FANCD1 (605724), FANCD2
(227646), FANCE (600901), FANCF (603467),
FANCG (602956), FANCJ (605882), FANCL
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(608111), or FANCM (609644).
Fanconi Anemia
• FA is a chromosome instability syndrome characterized by
childhood-onset aplastic anemia, cancer/leukemia susceptibility,
and cellular hypersensitivity to DNA crosslinking agents.
• Cultured FA cells are unusually sensitive to DNA crosslinking
agents such as mitomycin C whereas their sensitivity to radiation
is close to normal.
• Heterogeneous responses of various cell lines to DNA
crosslinking treatments suggest genetic heterogeneity
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Phenotype and clinics
• The male to female ratio is 1.24. The median survival age
has improved to 30 years in patients reported between
1991 and 2000.
• Survival was 19 years in those reported between 19811990.
• The common congenital defects seen in FA patients
includes short statue (51%), abnormalities of the skin
(55%), upper extremities (43%), head (26%), eyes (23%),
kidneys (21%), ears (11%) and developmental disability
(11%).
• Thirty two percent of male FA patients show abnormal
gonads, although abnormal gonads has been described in
only 3% of female FA patients. A significant percentage
(25%-40%) of the FA patients were reported to be
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physically normal.
Neoplastic risk
• Myelodysplasia (MDS) and acute non
lymphocytic leukaemia (ANLL): 15% of cases;
i.e. a 15000 fold increased risk of MDS and
ANLL has been evaluated in FA, and it has
been assumed that it is reasonable to regard the
Fanconi anemia genotype as "preleukemia";
mean age at diagnosis: 13-15 yrs
• Hepatocarcinoma (androgen-therapy induced)
in 10%; mean age at diagnosis: 16 yrs
• Other cancers in 2-5%: in particular squamous
cell carcinoma
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Treatment
• Androgens and steroids to improve
hematopoietic functions; bone marrow
transplantation prevents from terminal
pancytopenia (decrease in all types of
blood cell), and from ANLL as well.
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Clinical Course of FA
• FA is an autosomal (all complementation groups except for FA-B
group) or X-linked (FA-B group) recessive disease, clinically
characterized by multiple congenital abnormalities, bone marrow
(BM) failure, and cancer susceptibility.
• The prevalence of FA is estimated to be 1-5 per million and
heterozygous carrier frequency is estimated to be 1 in 300, although
the true frequency is probably higher.
• FA patients show extreme clinical heterogeneity
• The median age at diagnosis is 6.5 years for male patients, and 8
years for female patients, although the age at diagnosis ranges from
0 to 48 years.
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FA Population Genetics
• On the basis of complementation analysis of 47 FA patients
from Europe and U.S./Canada, the following frequencies of
the various subtypes were identified by Buchwald (1995): 31
were group A (66%), 2 were group B (4.3%), 6 were group
C (12.7%), 2 were group D (4.3%), and 6 were group E
(12.7%).
• Reporting for the European Fanconi Anaemia Research
Group, Joenje (1996) found that among ethnically and
clinically unselected FA patients from Germany and the
Netherlands, FA-A was most prevalent in Germany (13/22,
59%), whereas in the Netherlands a majority of patients were
FA-C (4/6, 67%).
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Clinical heterogeneity of FA
• Mean age at death: 16 years; most patients die from marrow aplasia (haemorrhage,
sepsis), and others from malignancies; MDS and ANLL in FA bear a very poor
prognosis (median survival of about 6 mths); survival is also poor in the case of a
squamous cell carcinoma.
• It has recently been shown that significant phenotypic differences were found
between the various complementation groups. In FA group A, patients homozygous
for null mutations had an earlier onset of anemia and a higher incidence of leukemia
than those with mutations producing an altered protein. FA group G patients had
more severe cytopenia (decrease in blood cells) and a higher incidence of leukemia.
FA group C patients had less somatic abnormalities, which, in reverse, were more
frequent in the rare groups FA-D, FA-E, and FA-F. FA group G patients patients
and patients homozygous for null mutations in FANCA are high-risk groups with a
poor hematologic outcome and should be considered as candidates both for frequent
monitoring and early therapeutic intervention.
• There may also be a certain degree of clinical heterogeneity according to the degree
of mosaicism. Therefore, clinical manifestations may be variable within a given
family, according to the stage of embryonic development at which the somatic
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reverse mutation occurred.
Cytogenetics: Inborn conditions
• Spontaneous chromatid/chromosome
breaks, triradials, quadriradials
• Hypersensitivity to the clastogenic effect
of DNA cross-linking agents (increased
rate of breaks and radial figures);
diepoxybutane (DEB), mitomycin C, or
mechlorethamine hydrochlorid are used
for diagnosis
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A: gaps; B: breaks; C: deletion; D: triradials; E: quadriradials; F:
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complex figures; G: dicentric. Giemsa staining
Fanconi anemia genes and cancer
• Inactivation of FA genes has been observed in a
wide variety of human cancers in the general
population (non-FA patients)
• Defects of DNA repair and cell cycle checkpoints,
such as the defects of the FA pathway, are possible
mechanisms of genomic instability in cancer and
may also be responsible for the hypersensitivity of
cancer cells to certain types of chemotherapeutic
drugs and radiation.
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Other findings
• Slowing of the cell cycle (G2/M
transition, with accumulating of cells in
G2)
• Impaired oxygen metabolism
• Defective P53 induction
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Genes involved and Proteins
• The most prevalent complementation groups
are: group A (65-70% of cases), groups C and G
(10-15% each)
Rare complementation groups are groups B, D,
E, and F (<1 to 3 % each).
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Molecular Defects in FA
• Identification of eleven genes for FA has led to
progress in the molecular understanding of this
disease. FA proteins, including a ubiquitin ligase
(FANCL), a monoubiquitinated protein
(FANCD2), a helicase (FANCJ/BACH1/BRIP1)
and a breast/ovarian cancer susceptibility protein
(FANCD1/BRCA2), appear to cooperate in a
pathway leading to the recognition and repair of
damaged DNA. Molecular interactions among FA
proteins and responsible proteins for other
chromosome instability syndromes (BLM, NBS1,
MRE11, ATM, and ATR) have also been found.
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Figure. Schematic representation of the eleven human Fanconi Anemia
proteins. The relative sizes of the FA proteins are shown to scale. The
FANCF and FANCL proteins are the smallest, and the FANCM and
FANCD1/BRCA2 proteins are the largest. The only FA
proteins with known enzymatic activity are FANCJ (helicase), FANCM
(DNA translocase), and FANCL (E3 ubiquitin ligase). FANCD2 and
FANCD1/BRCA2 have been shown to have
direct DNA binding activity. dsDNA; double strand DNA, HD; helical
domain, NES; nuclear export sequences, NLS; nuclear localization
signals, OB; oligonucleotide/oligosaccharide binding folds, ssDNA;
single strand DNA, TD; tower domain, TPR; tetratricopeptide repeat.
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References
• Taniguchi T, D'Andrea AD: The molecular
pathogenesis of fanconi anemia: recent
progress. Blood. 2006 Feb 21; [Epub ahead of
print]
• Yamashita T, Nakahata T: Current knowledge
on the pathophysiology of Fanconi anemia:
from genes to phenotypes. Int J Hematol 2001;
74(1): 33-41
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