Tecniche per l

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Tecniche per lanalisi di mutazioni

• Vincenzo Nigro
• Dipartimento di Patologia Generale, Seconda
  Università degli Studi di Napoli

Telethon Institute of Genetics and Medicine
(TIGEM)
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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

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..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...
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50.000 private variants innocuous differences
belonging to one family
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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

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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

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relative frequency of de novo achondroplasia for
different paternal ages
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 Relative frequency of de novo neurofibromatosis
for different paternal ages
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the number of male germ-cell divisions
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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

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2-allele diseases

• consanguineity is a risk factor for homozygosity
• high carrier frequency is a risk factor for
  compound heterozygosity

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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

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Mutation detection

• mutation scanning
• or resequencing methods for identifying
  previously unknown mutations
• genotyping
• methods for scoring previously known mutations or
  single nucleotide polymorphisms (SNPs)

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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?

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Dimension of the mutation detection study
Number of patients
Gene size
X
Number of controls
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General strategy for mutation detection
frequent mutations are known?
screening of recurrent mutations
YES
NO
mutation scanning
SEQUENCING
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Log-PCR 4 multiplex-PCR (2x202x18) with
uniform spacing and gel position according to
chromosomal position
1 2 3 4 5 6
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MLPA ligation

• Probes are ligated by a thermostable ligase

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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)

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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
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MLPA can be used to detect known mutations
Mismatch
Perfect match
Ligation of the two probe oligonucleotides ?
Amplification product
Mismatch at the probe ligation site ? No
ligation, no amplification product
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MS-MLPA
Only undigested (methylated) and ligated probes
are exponentially amplified
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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-strand-displacing polymerase is used to fill
  in the gap

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• 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

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GoldenGate genotyping assay

• GoldenGate uses extension ligation between
  annealed locus-specific 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

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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

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PTTprotein truncation test

• Sensitivity 1000-bp fragment gt 85
• Detects only nonsense mutations
• Post PCR time 48-72 hours (translation/trascript
  ion, gel preparation, loading and run, analysis
  of results)
• Use of 35S radioactivity
• No special equipment required
• mRNA as starting template

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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 70-80
• Neurofibromatosis type 2 NF2 65
• Hunter Syndrome IDS 50
• Neurofibromatosis type 1 NF1 50
• Cystic Fibrosis CFTR 15

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SSCP
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Mutation detection by heteroduplex analysis the
mutant DNA must first be hybridized with the
wild-type DNAto form a mixture of two
homoduplexes and two heteroduplexes
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Heteroduplex analysis
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DHPLCdenaturing HPLC from Transgenomic
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DHPLC analysis at different temperatures of the
column
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DHPLC analysis of the CAPN3 gene (exon 11)
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110
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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 wild-type environment

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Sanger DNA sequencing
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• Massive parallel DNA sequencing

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454 technology a water-in-oil emulsion is
created a single molecule of DNA with a single
bead
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454 technology Beads with clones are selected
and assembled onto a planar substrate
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454 technology Sequencing by synthesis pyrosequen
cing
Up to 100 Million bp in 8 hours can be
read Ambiguities arise for homopolymeric tracts
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Emulsion PCR or Bridge PCR?
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7.4 x coverage 234 runs 24.5 billions bp
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NimbleGen sequence capture
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11 genetic diseases !!