Transcript Chapter 21: Molecular Basis of Cancer

Tecniche per l’analisi di
mutazioni
Vincenzo Nigro
Dipartimento di Patologia
Generale, Seconda Università
degli Studi di Napoli
Telethon Institute of Genetics and
Medicine (TIGEM)
What is a mutation?
a variation of the DNA sequence….

..that is only found in affected individuals

..that is never found in non affected individuals

..that accounts for the pathological process/status

..that, when corrected in time, disease is rescued
..that is only found in affected and that is
never found in non affected
incomplete penetrance
that is more often found in affected
than in non affected...
50.000 private variants = innocuous
differences belonging to one family
CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC
CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT
GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG
GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG
GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA
AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC
TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA
AGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT
CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA
TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT
TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG
GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA
GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG
TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT
TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA
GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC
CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG
CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT
GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT
AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG
CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA
TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT
AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG
TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA
CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA
TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT
GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG
TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG
AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA
TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTT
GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG
GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG
AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC
TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA
CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT
CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC
CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACC
CCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTT
GCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCG
GCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAG
GCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAA
AAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTC
TAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAA
AGAATGTTAACTATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCT
CAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAA
TGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCT
TCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGG
GGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCA
GGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTG
TTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCAT
TTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAA
GGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATC
CCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTG
CTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTT
GTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAAT
AAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGG
CAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACA
TCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTT
AAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATG
TGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAA
CACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAA
TTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCT
GTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAG
TGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAG
AAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTA
TAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGCTCCAAAGCTCTTGCTTT
GTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAG
GGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGG
AGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCC
TGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAA
CTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGAT
CTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC
1-allele diseases


monoallelic mutations may be responsible for
dominant or X-linked disorders
new random mutations are the rule with an
unpredictable pattern of distribution
gender effect in mutations




For mutations other than point mutations, sex biases in
the mutation rate are very variable
Small deletions are more frequent in females
Germline base substitution mutations occur more
frequently in males than in females, especially in older
males
Point mutations at some loci occur almost exclusively in
males, whereas others occur ten times more than in
females
relative frequency of de novo
achondroplasia for different paternal ages
Relative frequency of de novo
neurofibromatosis for different paternal ages
the number of male germ-cell divisions
2-allele diseases




novel mutations are rare, usually mutations have
a long history (100-1000 generations)
mutations have an ethnical signature with a
predictable pattern of distribution and frequency
biallelic mutations may be responsible for
autosomal recessive disorders
polymorphisms and private variants are more
easily discriminated vs true mutations
2-allele diseases

consanguineity is a risk factor for homozygosity

high carrier frequency is a risk factor for
compound heterozygosity
The effect of an allele

null or amorph = no product

hypomorph = reduced amount / activity

hypermorph = increased amount / activity

neomorph = novel product / activity

antimorph = antagonistic product /
activity
Mutation detection

mutation scanning


or resequencing methods for identifying
previously unknown mutations
genotyping

methods for scoring previously known mutations
or single nucleotide polymorphisms (SNPs)
Key questions for mutation detection
strategy

expected mutations are monoallelic or biallelic?

is the gene well recognized for that disease?

is the mutation pattern known? (deletion, dup, small
mutations, etc.)

which is the complexity of the gene?

how many patients must be examined?

how many controls should be examined?


how many mutations and how many variations have already
been identified in this gene?
are there more members of the same gene family (or
pseudogenes) in the genome?
Dimension of the mutation detection
study
Number of
patients
Gene size
X
Number of
controls
General strategy for mutation
detection
screening
of recurrent
mutations
YES
frequent
mutations
are known?
NO
mutations
are identified?
NO
mutation
scanning
YES
SEQUENCING
Log-PCR = 4 multiplex-PCR (2x20+2x18)
with uniform spacing and gel position
according to chromosomal position
A
DMD
B
C
BMD
D
1: del ex 43
2: del ex 11, 17, 19, 21
3: del ex 17, 19, 21
4: del ex 50, 52
5: del ex 7, 11, 17, 19
6: del ex 61
1
2
3
4
5
6
1: no del
2: del ex 8, 12, 18, 20, 22
3: del ex 12, 18, 20, 22
4: del ex 46, 51
5: del ex 6, 8, 12, 18
6: del ex 62
MLPA ligation
Probes are ligated by a thermostable ligase
PCR amplification
A universal primer pair is used to amplify all ligated probes
The PCR product of each probe has a unique length (130 480 bp)
Separation and quantification by
capillary electrophoresis
Each peak is the
amplification product
of a specific probe.
Samples are
compared to a control
sample.
A difference in relative
peak height or peak
area indicates a copy
number change of the
probe target sequence
MLPA can be used to detect known mutations
Mismatch
Mismatch at the probe ligation site 
No ligation, no amplification product
Perfect match
Ligation of the two probe oligonucleotides
 Amplification product
MS-MLPA
M
M
Methylated Target
Denaturation and
Multiplex probe
hybridization
Ligation and
Digestion with
methylation
sensitive
endonucleases
Unmethylated Target
M
M
Only undigested (methylated) and ligated probes are exponentially amplified
MRC-Holland b.v.
Molecular inversion probe (MIP)
genotyping
•MIP genotyping uses circularizable probes with 5′ and
3′ ends that anneal upstream and downstream of the
SNP site leaving a 1 bp gap
•Polymerase extension with dNTPs and a non-stranddisplacing polymerase is used to fill in the gap
Ligation seals the nick, and exonuclease I is used to remove
excess unannealed and unligated circular probes
•The resultant product is PCR-amplified and the orientation of
the primers ensures that only circularized probes will be
amplified
•The resultant product is hybridized and read out on an array of
universal-capture probes
GoldenGate genotyping assay

GoldenGate uses extension ligation between annealed locusspecific oligos (LSOs) and allele-specific oligos (ASOs)

An allele-specific primer extension step is used to preferentially
extend the correctly matched ASO (at the 3′ end) up to the 5′ end
of the LSO primer

Ligation then closes the nick
GoldenGate genotyping assay


A subsequent PCR amplification step is used to amplify the
appropriate product using common primers to ‘built-in’
universal PCR sites in the ASO and LSO sequences
The resultant PCR products are hybridized and read out on an
array of universal-capture probes
PTT
protein truncation test



Sensitivity
1000-bp fragment > 85%
Detects only nonsense mutations
Post PCR time: 48-72 hours
(translation/trascription, gel preparation, loading
and run, analysis of results)



Use of 35S radioactivity
No special equipment required
mRNA as starting template
Applications of PTT
(% of truncating mutations)

Polycystic Kidney Disease PKD1
95%

Familial Adenomatous Polyposis APC
95%

Ataxia telangiectasia ATM
90%

Hereditary breast and ovarian cancer BRCA1-2
90%

Duchenne Muscular Dystrophy DMD
90%

Fanconi anemia FAA
80%

Hereditary non-polyposis colorectal cancer hMSH1-2

Neurofibromatosis type 2 NF2
65%

Hunter Syndrome IDS
50%

Neurofibromatosis type 1 NF1
50%

Cystic Fibrosis CFTR
15%
70%-80%
SSCP
Mutation detection by
heteroduplex analysis: the
mutant DNA must first be
hybridized with the wild-type
DNA
to form a mixture of two
homoduplexes and two
heteroduplexes
Heteroduplex analysis
DHPLC
denaturing HPLC from Transgenomic
DHPLC analysis
at different
temperatures of
the column
DHPLC analysis of the CAPN3 gene
(exon 11)
UV
2
0
1:2
FLUO
100
0
1:4
1:6
1:8
1:10
Sequencing artifacts
FALSE POSITIVE (specificity)
when
searching for heterozygous DNA differences there
are a number of potential mutations, together with
sequence artifacts, compressions and differences in peak
intensities that must be re-checked with additional primers
and costs
FALSE NEGATIVE (sensitivity)
loss
of information farther away or closer to the primer
does
not detect a minority of mutant molecules in a wildtype environment
Sanger DNA sequencing
Massive parallel DNA sequencing
454 technology:
a water-in-oil emulsion is created:
a single molecule of DNA with a
single bead
454 technology:
Beads with clones are selected and
assembled onto a planar substrate
454 technology:
Sequencing by synthesis
pyrosequencing
Up to 100 Million bp
in 8 hours can be
read
Ambiguities arise for
homopolymeric
tracts
Emulsion PCR or
Bridge PCR?
7.4 x coverage
234 runs
24.5 billions bp
NimbleGen sequence
capture
11 genetic diseases !!