Tecniche per l’analisi di mutazioni

Vincenzo Nigro

Dipartimento di Patologia Generale, Seconda Università

degli Studi di Napoli

Telethon Institute of Genetics and Telethon Institute of Genetics and Medicine (TIGEM)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 affectedthan in non affected...

50.000 private variants = innocuous differences belonging to one family

CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTTGCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAAAAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAAAGAATGTTAACAATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATCCCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAGTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGATCCAAAGCTCTTGCTTTGTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGGAGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCCTGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAACTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGATCTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC

CCCCAGCCTCCTTGCCAACGCCCCCTTTCCCTCTCCCCCTCCCGCTCGGCGCTGACCCCCCATCCCCACCCCCGTGGGAACACTGGGAGCCTGCACTCCACAGACCCTCTCCTTGCCTCTTCCCTCACCTCAGCCTCCGCTCCCCGCCCTCTTCCCGGCCCAGGGCGCCGGCCCACCCTTCCCTCCGCCGCCCCCCGGCCGCGGGGAGGACATGGCCGCGCACAGGCCGGTGGAATGGGTCCAGGCCGTGGTCAGCCGCTTCGACGAGCAGCTTCCAATAAAAACAGGACAGCAGAACACACATACCAAAGTCAGTACTGAGCACAACAAGGAATGTCTAATCAATATTTCCAAATACAAGTTTTCTTTGGTTATAAGCGGCCTCACTACTATTTTAAAGAATGTTAACTATATGAGAATATTTGGAGAAGCTGCTGAAAAAAATTTATATCTCTCTCAGTTGATTATATTGGATACACTGGAAAAATGTCTTGCTGGGCAACCAAAGGACACAATGAGATTAGATGAAACGATGCTGGTCAAACAGTTGCTGCCAGAAATCTGCCATTTTCTTCACACCTGTCGTGAAGGAAACCAGCATGCAGCTGAACTTCGGAATTCTGCCTCTGGGGTTTTATTTTCTCTCAGCTGCAACAACTTCAATGCAGTCTTTAGTCGCATTTCTACCAGGTTACAGGAATTAACTGTTTGTTCAGAAGACAATGTTGATGTTCATGATATAGAATTGTTACAGTATATCAATGTGGATTGTGCAAAATTAAAACGACTCCTGAAGGAAACAGCATTTAAATTTAAAGCCCTAAAGAAGGTTGCGCAGTTAGCAGTTATAAATAGCCTGGAAAAGGCATTTTGGAACTGGGTAGAAAATTATCCAGATGAATTTACAAAACTGTACCAGATCCCACAGACTGATATGGCTGAATGTGCAGAAAAGCTATTTGACTTGGTGGATGGTTTTGCTGAAAGCACCAAACGTAAAGCAGCAGTTTGGCCACTACAAATCATTCTCCTTATCTTGTGTCCAGAAATAATCCAGGATATATCCAAAGACGTGGTTGATGAAAACAACATGAATAAGAAGTTATTTCTGGACAGTCTACGAAAAGCTCTTGCTGGCCATGGAGGAAGTAGGCAGCTGACAGAAAGTGCTGCAATTGCCTGTGTCAAACTGTGTAAAGCAAGTACTTACATCAATTGGGAAGATAACTCTGTCATTTTCCTACTTGTTCAGTCCATGGTGGTTGATCTTAAGAACCTGCTTTTTAATCCAAGTAAGCCATTCTCAAGAGGCAGTCAGCCTGCAGATGTGGATCTAATGATTGACTGCCTTGTTTCTTGCTTTCGTATAAGCCCTCACAACAACCAACACTTTAAGATCTGCCTGGCTCAGAATTCACCTTCTACATTTCACTATGTGCTGGTAAATTCACTCCATCGAATCATCACCAATTCCGCATTGGATTGGTGGCCTAAGATTGATGCTGTGTATTGTCACTCGGTTGAACTTCGAAATATGTTTGGTGAAACACTTCATAAAGCAGTGCAAGGTTGTGGAGCACACCCAGCAATACGAATGGCACCGAGTCTTACATTTAAAGAAAAAGTAACAAGCCTTAAATTTAAAGAAAAACCTACAGACCTGGAGACAAGAAGCTATAAGTATCTTCTCTTGTCCATGGTGAAACTAATTCATGCAGCTCCAAAGCTCTTGCTTTGTAATCCAAGAAAACAGGGGCCCGAAACCCAAGGCAGTACAGCAGAATTAATTACAGGGCTCGTCCAACTGGTCCCTCAGTCACACATGCCAGAGATTGCTCAGGAAGCAATGGAGGCTCTGCTGGTTCTTCATCAGTTAGATAGCATTGATTTGTGGAATCCTGATGCTCCTGTAGAAACATTTTGGGAGATTAGCTCACAAATGCTTTTTTACATCTGCAAGAAATTAACTAGTCATCAAATGCTTAGTAGCACAGAAATTCTCAAGTGGTTGCGGGAAATATTGATCTGCAGGAATAAATTTCTTCTTAAAAATAAGCAGGCAGATAGAAGTTCCTGTCACTTTC

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?

Gene size

Number ofpatients

XXNumber ofcontrols

Dimension of the mutation detection study

frequent mutations

are known?

mutationscanning

SEQUENCINGSEQUENCING

screeningof recurrent mutations YESYES

NONO

mutationsare identified?

YESYES

NONO

General strategy for mutation detection

DMDDMDAA BB

BMDBMDCC DD

Log-PCR = 4 multiplex-PCR (2x20+2x18) with uniform spacing and gel position according to chromosomal

position

1 2 3 4 5 6

1: del ex 432: del ex 11, 17, 19, 213: del ex 17, 19, 214: del ex 50, 525: del ex 7, 11, 17, 196: del ex 61

1: no del 2: del ex 8, 12, 18, 20, 223: del ex 12, 18, 20, 224: del ex 46, 515: del ex 6, 8, 12, 186: del ex 62

MLPA ligation

Probes are ligated by a thermostable ligase

PCR amplification

A universal primer pair is used to amplify all ligated probesThe 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 Perfect match

Ligation of the two probe oligonucleotides Amplification product

Mismatch at the probe ligation site No ligation, no amplification product

MRC-Holland b.v.

Unmethylated Target

M

M

Methylated Target

Denaturation and Multiplex probe

hybridizationM

Only undigested (methylated) and ligated probes are exponentially amplified

Ligation and Digestion with

methylation sensitive

endonucleasesM

MS-MLPA

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

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 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

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

GoldenGate genotyping assay

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

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

DHPLCdenaturing HPLC from

Transgenomic

DHPLC analysis at different

temperatures of the column

DHPLC analysis of the CAPN3 gene (exon 11)

UV

UV

00

22

FL

UO

FL

UO

00

100100

1:2 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 wild-type 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 bpin 8 hours can be readAmbiguities 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 !!