Microsatellite Instability (MSI) and Mutation Detection in HNPCC

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Microsatellite Instability (MSI) and Mutation
Detection in HNPCC

• Jennie Bell
• West Midlands Regional Genetics Service

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Overview

• Introduction to HNPCC and MSI
• Current strategies
• Impact on mutation detection of MSI and IHC

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Contribution of familial cancer syndromes to
colorectal cancer
Lynch Cancer100, No1,2004
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Familial adenomatous polyposis (FAP)
Thousands of polyps in colon
Certain to become malignant by fourth decade
Prevention of cancer depends on regular
sigmoidoscopy
Molecular testing can be used to indicate those
requiring clinical screening
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Extracolonic manifestations
Congenital hypertrophy of the retinal pigment
epithelium (CHRPE)
Upper GI adenomas
Desmoid tumour
Epidermoid cysts
Osteomas
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The APC gene
Cabrera CM, López-Nevot MA. APC and chromosome
instability in colorectal cancer. Rev Esp Enferm
Dig 2005 97 738-743.
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Adenoma-Carcinoma Sequence
Renata dos Santos Coura Arq. Gastroenterol. vol.42
 no.2  São Paulo Apr./June 2005
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FAP vs HNPCC
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HNPCC

• Characterised on the basis of family pedigree -
  not on clinical grounds alone
• Termed the Amsterdam criteria
• One person affectedlt50
• At least two generations involved
• One person must be first degree relative of
  another
• FAP must be excluded

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HNPCC

• Early onset of colorectal cancer- fifth and sixth
  decade
• Associated with a range of other cancers
  including endometrial cancer, transitional cell
  carcinomas

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Mismatch Repair Genes

• Phenotype is caused by at least 5 different genes
• MSH2 (16 exons)
• MLH1 (19 exons)
• PMS 2 (and ?1)
• MSH6
• MSH3

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Mismatch repair
The EMBO Journal (1998) 17, 64276436
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Microsatellite instability (MSI)

• Comparison of normal with tumour DNA
• 7 microsatellite markers
• Additional peaks recorded as unstable
• gt30 markers unstable MSI-High
• lt30 markers unstable MSI-Low

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Example of MSI
Tumour DNA
Normal DNA
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White Paper (2004)

• Funding for 0.5 WTE for post in Department of
  Cellular Pathology at University of Birmingham
  Medical School for 2 years
• Impact has been outstanding
• MSI and IHC service working smoothly for last 12
  months

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Integration of MSI/IHC with Mutation Detection
Blocks returned
Dept. Cell. Pathol
Mutation screen
other path lab
Blocks sent
Blocks cut IHC
Request for blocks
Genetics Lab
Clinical Genetics
IHC/Amsterdam pos
IHC/Amsterdam neg
Sections for MSI
MSI positive
MSI negative
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Mutation detection

• Two genes analysed
• hMLH1 19 exons
• hMSH2 16 exons
• hMSH6 in development
• Point mutations (missense, nonsense, splice site)
• Small insertions and deletions
• Large deletions and duplications

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Automation Developments (2005)

• Sequence based analysis using robotics
• 2 x Beckman NX, 1 x Beckman FX
• 2 x ABI 3730 genetic analysers
• Mutation detection software (Mutation Surveyor)
• MLPA

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Mutation analysis
Sample received
report
Del/dup identified
MLPA
Del/dup not identified
Mutation identified
Mutation not identified
Sequence all exons
report
report
presymptomatic testing available
presymptomatic testing not available
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Mutation screening 2002 - 2005

• 270 patients screened by a combination of dHPLC,
  sequencing and MLPA
• 90 mutations reported
• Mutation detection rate approx 35

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Automated strategy Results since Jan 06

• 98 patients screened
• 39 variants identified
• 40 detection rate

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Contribution of Gene Mutations
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Positions of mutations identified
hMSH2
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hMLH1
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Summary of identified variants
Nature of variant No. identified (n/39)
Del/Dup 6 15
Nonsense/frameshift 9 23
Splice 11 28
Missense 11 28
Intronic variants 2 5
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Reporting times

• MSI
• 4 weeks (once sections cut)
• Mutation screen
• within 8 weeks

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Relationship between MSI/IHC and Mutation
Screening
624 screened
135 MSI ve
489 MSI neg
18 Mutation not identified
40 Mutation identified
77 Status unknown
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How does MSI/IHC help?

• Patient 1
• Missense p.Lys618Ala identified (exon 16 MLH1) by
  SSCP
• Unclear pathogenic significance
• Not able to offer presymptomatic testing to the
  family
• MSI and IHC undertaken to gain further
  information

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• Tumour tissue exhibited MSI and loss of MSH2
  antibody staining
• Repeat mutation screen by dHPLC
• Identified frameshift mutation in exon 1 of MSH2
• Clearly pathogenic
• Presymptomatic testing available

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How does MSI/IHC help?

• Increasing information on MSI IHC status
• Can give supporting evidence to interpretation of
  missense variants
• Identification of more mutations through
  screening more appropriate families

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Presymptomatic testing

• Aim of mutation screening is provide
  presymptomatic testing to at risk individuals
• Use of MSI and IHC along with pedigree
  information targets appropriate families
• Improved efficiency of mutation detection

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Presymptomatic testing
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The future of mutation detection

• Functional assays to define effect of missense
  variants
• RNA analysis to identify sequence variants within
  introns and exons that affect splicing machinery
• MSI and IHC will focus this analysis to
  appropriate families