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Mutation Screening
Dr. Derakhshandeh, PhD
TYPE OF MUTATIONS
WHICH TECHNIQUES DETECT
WHAT TYPE OF MUTATIONS
In classical genetics, three
types of mutations are
distinguished:
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Different types of mutations
 genome mutations: changes in chromosome
number
 chromosome mutations: changes in
chromosome structure
 gene or point mutations: mutations where
changes are at molecular level
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genome
mutations:
changes in
chromosome
number
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Examples (genome mutations)
Trinomials eg Trisomy 21
Monosomies eg Monosomy 14, 16
Sex Chromosome number changes eg 47,
XXY
haploidy, triploidy eg in leukemias,
abnormal fetuses
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Techniques
Karyotyping, conventional cytogenetics
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Down Syndrome (Trisomy 21(
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Trisomy 2(
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Down Syndrome
(Trisomy 21(
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Trisomy
18, 47 Ch
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Kleinefelter/47/XXY
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Kleinefelter
XXY
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chromosome
mutations:
changes in
chromosome
structure
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CHANGES IN CHROMOSOME
STRUCTURE
Translocations
Large Deletions/Insertions
Inversions
Duplications/Amplifications
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Techniques
Conventional cytogenetics
 molecular cytogenetics
FISH
Molecular:
 PFGE, Southern blotting,
Northern Blotting
Fluorescence Dosage
analysis
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Interphase FISH Examples
13 (green), and 21 (red)
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18 (aqua), X (green), and Y (red).
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female fetus with trisomy-21
• chromosomes 13
(green), and 21 (red)
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• chromosomes
18 (aqua), X
(green), and
Y (red).
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DiGeorge/Velo-Cardio-Facial/CATCH 22/Shprintzen
Syndrome which is caused by a microdeletion on
chromosome 22
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"painting" probe for chromosome 4 which causes the entire
chromosome to fluoresce. One chromosome 4 from this individual was
abnormal but it was difficult to determine from routine Cytogenetics if it
had a small terminal deletion at 4q or was the result of a more complex
rearrangement
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the same individual as the cell above has been hybridized with a probe for
the terminal part of chromosome 4q. Since there is only one green signal this
confirms that one chromosome 4 is missing material from the terminal end of
4q.
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probe for Steroid Sulfatase Deficiency which is caused by a
microdeletion on the X chromosome. The "Xp22.3" probe signal is
located at the Steroid Sulfatase region at Xp22.3. Since there are two X
chromosomes and only one has the Steroid Sulfatase gene signal
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Functional consequences:
depend on the extent and location of
change and the particular genes
involved
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gene or point
mutations:
mutations where changes
are at molecular level
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Methods for detection of
known mutations
Methods for detection of
unknown mutations
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Methods for detection of known
mutations
 PCR and size separation eg. DMD
 PCR and restriction enzyme digestion eg. SMN exon 7
&8
 Allele specific amplification (ASA)
 Allele refactory mutation system (ARMS) eg. CF
 Allele specific oligonucleotide hybridisation (ASO)
 Dot Blot eg. CF
 DNA chips eg. Brca1
 Genomic DNA sequencing
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ARMS
Amplification Refractory
Mutation System
Allele Specific PCR (ASPCR)
PCR Amplification of Specific Alleles
(PASA)
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ARMS
Two complementary reactions
one contains a primer specific for the normal
allele
the other contains one for the mutant allele
both have a common primer
one PCR primer perfectly matches one
allelic variant of the target but is
mismatched to the other
mismatch is located at/near 3' end of primer
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ARMS
genotyping is based on:
 whether there is amplification in one or
both reactions
band in normal reaction:
only indicates normal allele
band in mutant reaction:
only indicates mutant allele
bands in both reactions
 indicate a heterozygote
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Uses
Population screening
 rapid (1 working day)
 inexpensive
non-isotopic
Used for testing for
 B-thalassaemia
Cystic Fibrosis
alpha-1-antitrysin
sickle-cell anaemia
Phenylketonuria
Apolipoprotein E, etc
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Protocol f. ARMS
Primers- "Wallace temperature" ie [(A
+T)x2oC + (G + C)x4 oC] of 48-50 oC- GC
should not have self complementary seqs
of 4bp or more
Amplification- (94 oC 1min, 50 oC 2min,
72 oC 3min)x3025ul reaction: 250ng
genomic DNA
10mM Tris-HCl pH 8.3
50mM KCl
200uM each dNTP
0.5 U Taq Pol
0.05-1uM of each oligonucleotide
1.5-4.5 mM MgCl2
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Optimization of ARMS
Magnesium titration (1.5, 2.5, 3.5, 4.5mM)
 oligo titration (1.0, 0.25, 0.1, 0.05uM)
 adjust MgCl2 & oligo: to give spurious
bands that do not interfere with specific
detection but act as an internal control for
successful PCR in absence of the specific
band
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Other Optimization Tips
• decreasing oligo conc to 0.05uM may
increase specificity (but < 0.025uM may
give weak signal)
- MgCl2 conc can be lowered below
1.5mM to achieve specificity
- Adding EDTA can decrease the
effectively MgCl2
- MgCl2 conc >4.5mM can give useful
spurious bands as internal controls
- Dilution of template DNA to improve
specificity & avoid problems of
contaminating PCR inhibitors
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Modifications/Adaptions to the
original ARMS methodology
Multiplex ARMS
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Multiplex ARMS
Many genetic diseases
have more than one
mutation
often closely spaced eg CF
(over 900 mutations)
 now known - majority are
rare but some a relatively
common eg F508, G551D
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To set up multiplex ARMS
Determine commonest mutations in the
respective population
develop the muliplex ARMS for the
commonest mutations
validate the results of the multiplex test
on samples with known mutations
determined via another methology
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Agarose gels showing the
feasibility of the ARMS concept
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Thalassemia Minor
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Thalassemia
 Onset: Childhood
 Hypo chromic / Microcrystal anemia
 Low level of MCV / MCH
 -Thal: Elevated HbA2 (α22)
HbF (α2γ2)
 α-Thal: Normal HbA2, HbF
Thalassemia Minor
• Thalassemia minor is an inherited form of
hemolytic anemia that is less severe than
thalassemia major.
• This blood smear from an individual with
thalassemia shows small (microcytic), pale
(hypochromic), variously-shaped
(poikilocytosis) red blood cells.
• These small red blood cells (RBCs) are able to
carry less oxygen than normal RBCs .
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Thalassemia Major
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Thalassemia major
an inherited form of hemolytic anemia
characterized by red blood cell
(hemoglobin) production
abnormalities.
the most severe form of anemia
the oxygen depletion in the body
becomes apparent within the first 6
months of life.
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untreated
death usually results within a few
years.
Note the small, pale (hypochromic),
abnormally-shaped red blood cells
associated with thalassemia major.
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 globin mutations
RNA-Processing (º)
 Splice junction




IVSI-1
IVSI-2
IVSI-3’ end del 25bp
IvsI-130
 Consensus splice sites (º/ +)
 IVSI-5
 IVSI-6
 IVSII-844
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 globin mutations
 Cryptic splice sites in Introns (+)
 IVSI-110
 IVSII-745
 Cryptic splice sites in exons
 Cd 26 (HbE)
 Cd 121 (HbD panjab/O Arab)
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Prenatal diagnosis
 I. ARMS-PCR (22 common mut.)
 II. PCR-RFLP (9 inf. RFLPs)
 III. RDB (60 mut.)
 IV. Sequencing
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ARMS-PCR
1
N
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2
M
3
N
4
M
5
N
6
M
7
N
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M
9
N
10
M
11
N
12
M
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PCR-RFLP
1
2
UD -/+
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3
M
4
-/-
LSV
UD
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5
+/+
6
7
+/+
-/-
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Sickle Cell disorder
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Sickle Cell disorder
 Stuck the red cell in the vessels
 In children: Spleen, chest, wrists,ankles
 In adults: hips and shoulders
 Anemia (Hb 7-8 g/dl)
 Infections (take antibiotics)
 Painful crises (6-18 months)
 Swollen and inflamed (hand/food syndrome)
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Methods for
detection of
unknown
mutations
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detection of unknown mutations
Small Mutations
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Physical methods
Denaturing gradient gel
electrophoresis (DGGE)
eg. DMD, Thal
Single stranded
conformation
polymorphism analysis
(SSCP)
Heteroduplex analysis
(HA)
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Methods for unknown mutations
(diagnostic methods)
These methods are relatively
simple, but still require:
experience and skill to perform.
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DGGE
Denaturing gradient gel
electrophoresis
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DGGE
is often used in diagnostic
laboratories
non-radioactive tracers and
detects almost all mutations
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