2. Fragile sites
• Specific loci that show up during karyotyping as a gap on
a chromatid arm after culturing cells under specific
conditions. These sites are associated with an increase in
chromosome fragility
• In 1970 Frederick Hecht coined the term "fragile site".
• Based on their frequency, fragile sites are classified as
"common" or "rare.
• To date, more than 120 fragile sites have been identified in
the human genome.
3. Fragile sites appear as unstained or stretched
regions
• Aphidicolin – an inhibitor of DNA poly – induce fragile sites
• Camptothesin – an inhibitoor of topo I – enhance % of cells
• Def. folic acid or thymidine in culture
– FRAXA (Xq27.3) and FRAXE (Xq28)
• BrdU – rare fragile sites
• Distamycin A -----FRA16B—homozygous----no phenotypic
effect.
5. Common fragile sites (cFS)
• Common fragile sites are considered part of
normal chromosome structure and are present in
all (or nearly all) individuals in a population.
• These are of interest in cancer studies because they
are frequently affected in cancer and they can be
found in healthy individuals.
8. Common fragile sites
• FRA 3B……3p14.2….. FHIT……Lung cancer
• FRA 11B….11q23.3……Jacobsen syndrome
• FRA 8E…….8q24.11…..Langer-Giedion syndrome
• FRA3B was the first fragile site to be mapped and cloned and it was found to lie
within the Fragile Histidine Triad gene). FHIT spans ∼900 kb and includes two large
introns where FRA3B is centrally located, but encodes only a small 1.1 kb
transcript. There are no large di- or trinucleotide repeats within FRA3B, but the
region was found to contain human cervical cancer HPV-16 integration sites, which
led to other findings of HPV integrations at fragile sites in cervical tumors
• FRA16D lies within the large WWOX gene, which encodes a small 2.2 kb transcript
but extends >1 Mb due to the presence of two very large introns
11. FRAGILOME
• Human Cancer Genome endeavor and has its major
goal in
– (i)mapping and identifiying of the full repertoire of cFS
genes in the human genome,
– (ii) determining the genetic material damaged upon
activation and unfaithful repair of cFS, and
– (iii) defining the role that cFS genes may have in human
tumorigenesis
12. Heritable/ Rare fragile sites (rFS)
• Rare fragile sites are found in less than 5% of the
population, and are often composed of two- or
three-nucleotide repeats.
• They are often susceptible to spontaneous breakage
during replication, frequently affecting neighboring
genes.
• Example
– Fragile X syndrome (CGG repeats)
13. cFS Vs. rFS
• No abnormal phenotype has been definitely identified
with autosomal fragile sites, but some rare autosomal
recessive disorders may be due to homozygosity for
fragile sites.
• A fragile site on the X chromosome is associated with
Fragile X Syndrome.
• Fragile sites are designated by the letters "fra" followed
by the designation for the specific chromosome and locus.
41. How chromosome aberrations happens?
• When cells are irradiated, breaks are produced
in the chromosomes, broken ends appear to be
sticky and can rejoin with any other sticky end.
Once breaks are produced, different fragments
may behave in a variety of ways; breaks may
rejoin in their original configuration. Breaks
may fall to rejoin give rise to a deletion. And
broken ends resort and rejoin other broken
ends and give rise to chromosomes that appear
to be grossly distorted
• Study of chromosome aberrations through the effects of ionizing radiation
described in terms of their appearance at first metaphase after exposure to
radiation.
42.
43. Classification of Primary changes
Chromatid-type
where the breaks and re-joins
affect only one of the
sister-chromatids at any one
locus
Chromosome-type
where the breaks and re-joins
always
affect both sister-chromatids at
any one locus.
"breaks" followed by "re-joins"
EXCHANGES: 1) Interchanges, 2) Inter arm changes, 3) Intra arm changes
Complex exchanges
49. MN arise from a whole lagging chromosome
(aneugenic event leading to chromosome loss) or
an acentric chromosome fragment detaching from
a chromosome after breakage (clastogenic event)
which do not integrate in the daughter nuclei.
50.
51.
52.
53.
54.
55. DNA damage – definition and causes
• DNA in the living cell is subjected to many chemical alterations, the
genetic information encoded in the DNA has to remain uncorrupted, if
any chemical change must be corrected. A failure to repair DNA
produces a mutation.
• Agents that damage DNA: 1) Radiation, 2) Ionizing rations such as
gamma and X rays 3) Ultraviolet rays 4) Highly reactive oxygen
radicals (produced during cellular respiration and other biochemical
pathways) 5) Chemicals in the environment: aromatic hydrocarbons
including cigarette smoke, plant and microbial products (Aflatoxins
from peanuts) chemicals used in chemotherapy.
56.
57. Types of DNA damages
• Although all cells possess a large number of
different types of repair system, each relatively
specific for a certain type of DNA damage, those
repair systems can be grouped into four broad
categories:
– Direct reversal of damage
– Excision of damaged region, followed by precise
replacement
– Double-strand break repair
– Damage bypass
73. Chromosomal breakage syndromes
• Syndrome is a group of symptoms that collectively indicate or
characterize a disease, psychological disorder, or other abnormal
condition. It may causes serious or even fatal conditions. It may curable
or incurable.
• Chromosomal breakage syndromes are conditions that due to improper
DNA repair
• How does CBS causes:
– Loss of DNA repair,
– Interstrand cross links,
– Increase exchange between sister chromatids,
– Change in nucleotide and double strand DNA breaks
74. Louis bar syndrome
(Ataxia Telangiectasia)
• In 1943 French Physician Denise Louse-Bar was described. It is AR
and is caused because of chromosome instability. It is a neuron
degenerative disease mainly affects cerebellum.
75.
76.
77.
78.
79. ATM gene (11q22.3)
• The ATM gene is a serine-protein kinase (eg. p53, BRCA1) provides
instructions for making a protein that is located primarily in the
nucleus of cells, where it helps control the rate at which cells grow
and divide. This protein also plays an important role in the normal
development and activity of several body systems, including the
nervous system and the immune system.
• Additionally, the ATM protein assists cells in recognizing damaged or
broken DNA strands. DNA can be damaged by agents such as toxic
chemicals or radiation. Breaks in DNA strands also occur naturally
when chromosomes exchange genetic material during cell division.
The ATM protein coordinates DNA repair by activating enzymes that
fix the broken strands. Efficient repair of damaged DNA strands helps
maintain the stability of the cell's genetic information.
• Because of its central role in cell division and DNA repair, the ATM
protein is of great interest in cancer research.
80.
81. Fanconi Anemia
• FA is one of the inherited (AR) anemias that cause bone marrow
failure and affects many parts of the body mainly thumb, forearm,
eys, ear (hearing loss), kidney and urinary tract abnormalities.
• There are many(>21) gene mutations are responsible. The normal
function of FA gene is to DNA repair, cell cycle control, oxygen
sensitivity, apoptosis, telomere maintenance and hematopoiesis.
• FA genes encoded proteins are involved in cell process known as FA
pathway.
• FA pathway is activated when the process making new (DNA
replication) copies. However, DNA damage usually blocks the
replication.
• FA pathway is particularly responsive to certain types damages
known as intercross links (ICLs).
• FA core complex activates FANC D2 and FANC I, then proteins
brings the DNA repair proteins to the area of ICL
86. Bloom Syndrome
• BS is inherited (AR) disorder, causes sun sensitive skin changes and an
increased risk of cancer, and other health problems include dilated
blood vessels, reddening in the skin, high pitched voice, increased risk
of diabetes, COPD and infections of upper respiratory tract including
ears and eyes.
• The BLM gene provides instructions for making a member of a protein
family called RecQ helicases (BLM, WRN, RecQ4, ReqQL, RecQ5B).
• BLM gene on 15q16.1 is known as “Caretaker of Genome” since it
maintenance of structural integrity of DNA by providing instructions
for making number of protein family helicases (enzymes that bind to
DNA and temporarily unwind the two spiral strand of the DNA
88. BLM gene on 15q26.1
• BLM mutation prevents BLM protein from performing its function in
maintain genomic instability. As a result of altering BLM protein
activity, the frequency of SCEs increase about 10 fold, and increased
cell death and resulting slow growth in affected individuals.
• SCEs is hallmark of BS and is an indicator of chromosome instability.
It also associated with gaps and breaks that impair cell activities and
cause the health problems.
89. Xeroderma Pigmentosum
• Xeroderma pigmentosum, which is commonly known as
XP, is an inherited condition characterized by an
extreme sensitivity to ultraviolet (UV) rays from
sunlight.
• This condition mostly affects the eyes and areas of skin
exposed to the sun.
– The signs usually appear in infancy or early
childhood. Many affected children develop a severe
sunburn after spending just a few minutes in the sun.
• Some affected individuals also have problems involving
the nervous system.
96. Chromosomal Breakage Syndromes
• Chromosomal breakage syndromes are a group of genetic disorders. They are
– Ataxia telangiectasia (AT)
– Bloom syndrome (BS)
– Fanconi anemia (FA)
– Xeroderma pigmentosum (XP)
all these typically transmitted in an AR mode of inheritance
• In culture, cells from affected individuals exhibit elevated rates of chromosomal
breakage or instability, leading to chromosomal rearrangements.
• The disorders are characterized by a defect in DNA repair mechanisms or genomic
instability, and patients with these disorders show increased predisposition to cancer.
• Patients with ataxia telangiectasia are hypersensitive to ionizing radiation, while
patients with Bloom syndrome, Fanconi anemia, and xeroderma pigmentosum are
sensitive to UV radiation. The ataxia telangiectasia Rad3–related (ATR) protein
responds to UV damage, whereas the ataxia telangiectasia mutated (ATM) protein
responds to double-strand breaks (DSBs) caused by ionizing radiation and
radiomimetic compounds.
97. Ataxia telangiectasia
Telangiectasia
Mutated (ATM)
gene is
responsible for
AT.
AT patients
strongly
radiosensitive
Pleiotropic defects
• Ataxia refers to poor coordination and telangiectasia to small dilated
blood vessels, both of which are hallmarks of the disease.
• A-T affects many parts of the body:
– It impairs certain areas of the brain including the cerebellum, causing
difficulty with movement and coordination.
– It weakens the immune system causing a predisposition to infection.
– It prevents repair of broken DNA, increasing the risk of cancer.
• Symptoms
– Ataxia, Occulomotor apraxia, Telangectesia, Problems with infections
(especially ears, sinusitis), slow growth rate/ pubertal development,
Increased incidence of cancer,
progressive
neurondegenera
tion
Chromosome instability
AR disorder,
1 out of 40,000...
also known as Louis-Bar syndrome
98. ATM gene on 11q22.3
• Function
– The protein encoded by this gene belongs to the PI3/PI4-kinase family.
This protein is an important cell cycle checkpoint kinase that
phosphorylates; thus, it functions as a regulator of a wide variety of
downstream proteins, including tumor suppressor proteins p53 and
BRCA1, checkpoint kinase CHK2, checkpoint proteins RAD17 and
RAD9, and DNA repair protein NBS1.
– This protein and the closely related kinase ATR are thought to be master
controllers of cell cycle checkpoint signaling pathways that are required
for cell response to DNA damage and for genome stability.
100. Bloom syndrome
• Bloom syndrome is a rare human autosomal recessive disorder belonging to
a group of chromosomal breakage syndromes.
• BS is characterized by marked genetic instability, including a high level of
sister chromatid exchanges, associated with a greatly increased
predisposition to a wide range of cancers commonly affecting the general
population.
• The constant clinical features of BS are proportionate pre- and postnatal
growth retardation and cancer predisposition.
• Additional clinical features include dolichocephaly (longhead), facial
sun-sensitive telangectic erythema, patchy areas of hyper – and
hypopigmentation of the skin and moderate immunodeficiency manifested
by recurrent respiratory tract and gastrointestinal infections.
101. Bloom syndrome
• A 10 fold increase in the rate of SCEs in BS cells compared to
control cells is the only objective criteria for BS diagnosis.
102. Fanconi anemia
• Fanconi anaemia is a type of aplastic
anemia. inherited blood disorder that leads to bone
marrow failure i.e., prevents BM from making
enough new blood cells for body to work
normally.
• FA is a chromosome instability syndrome with
progressive bone marrow failure and an increased
risk of cancers
• autosomal recessive; frequency is about
2.5/105
newborns
• growth retardation (70% of cases)skin
abnormalities: hyperpigmentation and/or café au
lait spots in 80%squeletal malformations (60%),
particularly radius axis defects (absent or
hypoplastic thumb or radius...)
103. Core FA pathway
• The defect in FA is due to a failure to
respond to and repair DNA damage during
replication, but perhaps the most striking
aspect concerns genetic heterogeneity: the
FA phenotype can result from recessive
mutations in up to 13 distinct genes.
• DNA damage activates the complex of
Fanconi anemia−associated proteins FANCA,
FANCB, FANCE, FANCF, FANCG and
FANCL. FANCL is a ubiquitin ligase that
probably causes the ubiquitination of
FANCD2. This protein, together with another
FA associated protein, BRCA2 and RAD51, is
involved in the repair of DNA damage
106. Xeroderma pigmentosum
• Chromosomal breakage syndromes are relatively
rare; most practicing physicians may never see a
patient with a chromosomal breakage syndrome.
• Inability to repair damage of UV rays to skin,
usually found at a very young age(1-2 years) Six
times more common in Japanese people than any
other group. Diagnosis is complicated because the
symptoms may be varied and complex. These
disorders are often lethal.
• Symptoms: 1) severe sunburn/blistering after short
sun exposure, 2) many freckles at a young age, 3)
thin and excessively dry skin, 4) dark spots on the
skin, 5) early aging of the lips, skin, eyes, mouth,
and tongue and 6) spiderly blood vessels
Seven XP repair genes XPA through
XPG genes (role in NER), have
been identified
XPA: 9q22.3
1.XPB/ERCC3: 2q21
2.XPC: 3p25
3.XPD/ERCC2: 19q13.2-q13.3
4.XPE/DDB2: 11p12-p11
5.XPF/ERCC4: 16p13.3-p13.13
6.XPG/ERCC5: 13q33
7.XPV/POLH: 6p21.1-p12
Increased gaps and breaks,
increased SCE after UV
108. XP and Ocular problems
Ocular characteristics of patients with XP
• Structural eyelids abnormalities
• Neoplasms of the ocular surface and eyelids
• Ocular surface disease and inflammation
• Corneal abnormalities
Test/ Diagnosis
Eye exam may show Clouding of
cornea, Keratitis,
Lid tumor,
Blepharitiss
Treatment of XP
1. Cryotherapy: The removal of heat from the body
2. Fluorouracil: Pyrimidine analogue used to treat cancer
3. Reduced exposure to sun’s UV rays
109. ICF
1. ICF syndrome (or Immunodeficiency, Centromere instability
and Facial anomalies syndrome) is a very rare AR immune disorder.
2. Characterized by variable reductions in serum immunoglobulin levels
which cause most ICF patients to succumb to infectious
diseases before adulthood.
3. ICF syndrome patients exhibit facial anomalies which
include hypertelorism, low-set ears, epicanthal
folds and macroglossia.
4. ICF syndrome can be caused by a mutation in the
DNA-methyltransferase-3b (Dnmt3b) gene, located on chromosome
20q11.2. The disease is inherited in an autosomal recessive manner
110. Aging is a process that accumulates changes in organisms or objects
overtime. Human aging process involves multidimensional changes on
physical, psychological, cultural and social levels. This comprehensive
guide provides information on various health and lifestyle subjects
associated with aging, longevity and old age.
111. Cell Division Cell Cycle Aging
What mechanisms control the proliferation of cells?
What governs the life span of an organism?
Cell death as a necessary and important part of development:
Apoptosis (programmed cell death)
Aging
112. George Martin, 1978
‘Genetic Syndromes in Man with Potential Relevance to the Pathology of
Aging’
< 7000 genes: involved in degenerative processes associated with aging
Between 70 and 7 genes: control processes having large impact on
senescence
What is cell senescence?
Divide certain # of times then enter G0 and eventually die
113. Aging a multigene process
10 genetic diseases that mimic aging process—but only in part
Chromosomal aneuploides🡪 Down’s
syndrome
Known single mutant gene🡪 Werner’s syndrome
Unknown but thought to be single gene🡪 Hutchinson-Gilford’s Progeria
Dementia, cataracts, diabetes, hair graying, cancer
Skin ‘thinning’, Hair graying and loss, atherosclerosis,
Cataracts, cancer diabetes, osteoporosis
Skin ‘thinning’ hair loss, atherosclerosis, osteoporosis, hypertension
In 20’s Helicase
Birth/1yr ??
Appears Birth
114. Progeroid syndromes
• Progeroid means "resembling premature aging",
• PS are a group of rare genetic disorders that mimic physiological
aging, making affected individuals appear to be older than they are.
All disorders within this group are thought to be monogenic.
• Familial Alzheimer's disease and familial Parkinson's disease are two
accelerated aging diseases associated with aged individuals; they
affect only one tissue and can be classified as unimodal progeroid
syndromes.
• However, PS usually refers to segmental progeria, which tends to
affect multiple or all tissues and causes affected individuals to exhibit
only some of the features associated with aging.
119. Progeroid syndromes
• Individuals with these disorders tend to have a
reduced lifespan. PS have been widely studied in the
fields of aging, regeneration, stem cells and cancer
• The most widely studied of the PS are
– Werner syndrome and
– Hutchinson-Gilford progeria,
as they are seen to most resemble natural aging.
139. Werner’s history
• Named for C. W. Otto Werner (1879-1936)
• Rare autosomal recessive disease
• Approx 1 in 200 people carriers for defective gene
• Approx 3 in 1,000,000 people have the disease
• (Slightly higher percentage in Japan)
• Onset of symptoms early to mid 20’s,
• Major cause of death—heart attack in mid 40s
140. Cells of Progeria and WS
Cell Culture
What do cells need to proliferate?
Progeric Fibroblasts:
Rarely ever double
Few cell generations before death
When compare fibroblasts of child with Progeria and their parent
Child’s cells are ‘older’ in terms of replication
Fibroblasts
‘normal cells’ divide ~12 to 24 hours
Divide approx 50times in culture
Note: Often ‘Progeric’ used to describe any premature aging as well as
the disease Hutchinson Gilford Progeria
141. Why do we age?/How how do we age?
Short answer: Don’t know
Hypotheses for Aging:
The 3 R’s: Mutation effecting DNA reading, replicating or repair
Free Radical Theory: Aging due to accumulation of
damage from free radicals
Helicase defect: Mutation Chromosome 8 in WRN gene
•all 35 known mutations result in truncated protein
•all ‘remove’ nuclear targeting sequence
•different mutations associated with different cancers
Telomere Theory: Chromosome ends shorten with divisions
Cause of Werner’s syndrome
142. Werner syndrome - Cause
• Genetic mutations, which lead to defects in the cellular machinery
that repairs DNA. The DNA damage theory of aging proposes that
aging is a consequence of the accumulation of naturally occurring
DNA damages. These damages may arise from reactive oxygen
species (ROS), chemical reactions (e.g. with intercalating agents),
radiation, depurination, and deamination.
• Mutations in two classes of DNA repair proteins, RecQ protein-like
helicases (RECQLs) and nucleotide excision repair (NER)
proteins.
143. Werner syndrome - Cause
• RecQ is a family of conserved ATP-dependent helicases required for
repairing DNA and preventing deleterious recombination and
genomic instability. DNA helicases are enzymes that bind to
double-stranded DNA and temporarily separate them. This
unwinding is required in replication of the genome during mitosis,
but in the context of PS, it is a required step in repairing damaged
DNA. Thus, DNA helicases, such as RecQ, maintain the integrity of
a cell, and defects in these helicases are linked to an increased
predisposition to cancer and aging phenotypes.
144. Werner syndrome - Cause
• There are five genes encoding RecQ in humans
(RECQ1-5),
– RECQL2/WRN,
– RECQL3/BLM and
– RECQL4RTS
• On the cellular level, cells of affected individuals
exhibit chromosomal abnormalities, genomic
instability, and sensitivity to mutagens
