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  • Mismatch repair – corrects bases which are not paired according to the Watson-Crick base pairing A-T and C-G.
  • Mismatch repair – corrects bases which are not paired according to the Watson-Crick base pairing A-T and C-G.
  • Mismatch repair – corrects bases which are not paired according to the Watson-Crick base pairing A-T and C-G.

Transcript

  • 1. Factors influencing decisions genetic syndromes ESO-ESSO Masterclass Cascais, Portugal 2011 Professor Sir John Burn MD FRCP FRCPCH FRCOG FMedSci Institute of Human Genetics, Newcastle University, UK With thanks to Professor Marco Novelli UCL London for several teaching slides used in this presentation Institute of Human Genetics Newcastle University Centre for Life, Newcastle UK
  • 2.
        • Lynch Syndrome (HNPCC)
        • Familial Adenomatous Polyposis
        • MYH Polyposis
        • Hyperplastic polyposis
        • Mixed Polyposis
        • Peutz Jegher’s
        • Juvenile polyposis
    Finding the 5%: early onset, strong family history, multiple polyps Exracolonic features, tumour histopathology
  • 3.
    • Run PCR
    • Hand to technician who puts on machine.
    2010 Automated sequencing
  • 4. Roche Genome Sequencer FLX First run: 1 week 124 Mb of E coli sequence analysed Using Sanger sequencing manually In 1990 Would have taken a lifetime
  • 5. New Technology: Sequence Capture Elute Fragment and hybridize to NimbleGen capture array 454 Sequencing Analyze Exon or loci sequences www.nimblegen.com gDNA Exon 1 Exon 2 Exon 3 Exon 4 Exon 5
  • 6. Examples – complex mutations BRCA1 exon 16 (2) c.4964_4982del19 p.Ser1655TyrfsX15 BRCA2 exon 10 (1) c.891_899del9ins10 Newgene Ltd BRCA 1&2 600 euros 4 weeks
  • 7.
    • Most common form of familial CRC
    • Autosomal dominant inheritance.
    • Mutation in mismatch repair gene (MMR)
        • - Family of DNA repair genes.
        • - MLH1, MSH2 , MSH6 + PMS2.
    • Usually inherited, but may be new mutation.
    Lynch syndrome
  • 8. Lynch Syndrome (HNPCC)
    • Various older names:
      • Lynch type I + II syndromes.
      • HNPCC.
      • Muir Torre syndrome.
      • ?Turcot’s syndrome
    • Multiple adenocarcinomas in colon (synchronous/metachronous tumours).
    • Tumours at other sites:
      • Endometrial, stomach, uroepithelial, small bowel, bladder, pancreatobiliary, brain, skin .
    • Early age of tumour development (40+).
  • 9. The two hit hypothesis Predisposed tumour All cells second hit
  • 10. Lynch syndrome Endometrial Carcinoma Bertha Ted 3 CRC 9 skin cancers Family from which DNA used to Demonstrate hMSH2 mutation by Kolodner’s group 1993: 1 st of the MISMATCH REPAIR GENE DEFECTS 14 years ago
  • 11. MMR genes are caretaker genes. Think of computer spell checker
  • 12.
      • Microsatellites are lengths of short repeated sequences usually
      • mononucleotides eg BAT25
      • tgttttgtttttttga ttttttttttttttttttttttttt gagaacagagca
      • or dinucleotides eg D5S346
      • aattt acacacacacacacacacacacacacac agagta
    Microsatellites (old name based on old separation technique
  • 13. Important genes containing repeat sequences
    • TGFBRII
    • BAX
    • IGFIIR
    • E2F4
    • TCF4
    • hMSH6
    ggatactcc atatatatatatatatatatatatatata ggcttcgta
  • 14. MSS MSS MSS MSI-H MSI-H BAT26 NR21 BAT25 NR24 MONO27 Normal pattern MSI testing with quasimonomorphic 5 marker panel: no need to test normal tissue Normal pattern
  • 15. Deletion in upstream EPCAM gene removes MSH2 promoter leaving gene methylated Deletions Removing the Last Exon of TACSTD1 Constitute a Distinct Class of Mutations Predisposing to Lynch Syndrome Marietta E. Kovacs,1 Janos Papp,1 Zoltan Szentirmay,2 Szabolcs Otto,3 and Edith Olah Human Mutation 2009;30:197 TACSTD1 MSH2 5KB
  • 16. Loss of MLH1/PMS2 expression MSH2 MSH6 PMS2 MLH1
  • 17. Implications of loss of expression of Mismatch Repair proteins.
    • Not diagnostic of Lynch syndrome.
    • Up to 10% of sporadic tumours lose MLH1 / PMS2 expression (due to MLH1 promoter methylation).
    • Strong +ve correlation of loss of staining MSH2/6 with germline mutation.
    • Suggests further genetic analysis may be required.
  • 18. Newcastle 2009 285 samples 214 both normal 2 MSI failed IHC normal 58 samples 5 IHC failed 6 wrong tissue 13 IHC normal 26 IHC abnormal 19 other IHC combinations 12 MSI-L 1 MSI-H,+BRAF 1 MLH1 1 7 PMS2 6* 9 MLH1/PMS2 7 3 MSH6 2** 6 MSH2/6 6 Gene MSI-H, -BRAF 14 MSI-S 1 MSI-L 1 MSI-H, +BRAF 1 MSI-H, -BRAF (MSH2/6 nata) *1 MSI failed ** 1 MSI-S
  • 19.
      • Two pathways to evolution of CRC?
        • Classic adenoma-carcinoma sequence
        • serrated pathway of neoplasia associated with MSI, MLH1 promoter methylation and activating mutations in BRAF
        • Young et al. (2005) Clinical Gastroenterology and hepatology 3 :254-263
    MSI and BRAF
  • 20.
      • BRAF is a kinase encoding gene from the RAS/RAF/MAPK pathway
      • The BRAF-V599E hotspot mutation is found in 40% of sporadic MSI-H tumours and 0% of HNPCC tumours
      • Presence of V599E mutations excludes diagnosis of HNPCC
    MSI and BRAF
  • 21. Bayes probability sporadic MSI H Lynch syndrome PRIOR 0.16 0.03 BRAF-ive 0.6 1 JOINT 0.096 0.03 “ posterior” NEW ODDS 0.096: 0.03 3.2 : 1 only a quarter have LS
  • 22.
    • 9 year old male
    • NF1 style café au lait patches
    • Multiple colorectal carcinomas/adenomas.
    • Glioblastoma multiforme
    CHILHOOD CANCER SYNDROME
  • 23. Childhood cancer syndrome (?“Turcot syndome”)
    • “ Autosomal recessive” MMR deficiency.
    • Biallelic mutations in PMS2, MSH2, MSH6 or MLH1 (compound or homozygous).
    • Phenotype:
      • Café-au-lait spots (NF1 type features).
      • Colorectal cancers and adenomas.
      • Brain tumours (Gliomas, PNETs, meduloblastomas).
      • Haematological malignancies (leukaemias and diffuse large B cell lymphoma).
  • 24. FAP Simon Burn et al J Med Genet 1990 Exon 11 Exon 15 1 2 2 1 22 11 1 2 2 1 1 2 2 1
  • 25. Familial Adenomatous Polyposis
    • > 100 polyps predominantly in large intestine (typically 1000s).
    • Colorectal adenocarcinomas < 40 years old (10-15 years after appearance of polyps)
    • Commonest cause death now duodenal carcinoma and desmoids.
    • 50-85% sporadic CRCs have somatic APC mutations
  • 26. APC protein domains and FAP phenotype association with (truncating) germline mutation position Heptad repeats (dimerisation) EB1 and HDLG binding sites Armadillo repeats 15-amino acid repeats (  -catenin binding) 20-amino acid repeats (  -catenin binding, GSK3  phosphorylation Basic domain (Microtubule binding, tubulin polymerisation) 6 57 453 767 1020 1169 1262 2033 2200 2400 2560 2843 1395 2000 Desmoids SAMP1 NESR3 1580 AAPC 457 1444 CHRPE 1250 1464 severe FAP 1309 Classical FAP 168 1580 400 AAPC 78
  • 27. Truncation of APC Courtesy Inke Nathke Why is FAP so highly penetrant? Accumulation Aneuploidy Transformation Cell migration Chromosome segregation Deregulation of  -catenin
  • 28. Attenuated FAP
    • Typically less than 100 adenomas in colon.
    • Mean 25 adenomas (0 - 470).
    • Concentrated in proximal colon.
    • Some extra-colonic manifestations.
    • Colorectal carcinoma in 70% by 80 years of age.
  • 29. Testing for FAP
    • APC is a large gene (15 exons, 2843 amino acids).
    • Classical FAP – most mutations around hot spot
    • Attenuated FAP – mutations more likely at 3’ or 5’ ends of gene
      • -> Initial mutation screening (SSCP, PTT) followed by direct sequencing
  • 30. Oxidative damage of DNA
    • Oxidative damage - 10 4 lesions/cell/day.
    • 8-oxo-G most deleterious.
    • 8-oxo-G mis-pairs with adenine residues
    • Normal G:C Damaged G o :A
    • G:C  T:A mutation
    • Role of MYH is to excise mispaired adenines (Base excision repair BER).
  • 31. MYH (MUTYH) gene
    • base excision repair (BER)
    • 1p35, 16 exons
    • protects against mutagenesis caused by oxidative DNA damage (8-oxoG)
    • MYH: excision of adenine mismatched with 8-oxoG
    • MYH biallelic mutations increase somatic G:C>T:A transversions in other genes like APC
    • MYH-associated Polyposis (MAP)
    • AUTOSOMAL RECESSIVE
    Cheadle et al., HMG, 2002 Stefan Aretz Bonn 2006 MYH
  • 32. MYH-associated Polyposis
    • Autosomal recessive inheritance.
    • Penetrance approaching 100%
    • Clinically very similar to attenuated FAP.
      • Typically <100 polyps (but may show classical FAP phenotype).
      • Extra colonic manifestations
    • May account for up to 10% of FAP cases with no documented Apc mutation.
  • 33. MYH-associated Polyposis
    • Due to biallelic mutations in MYH gene.
    • Thought that there is a gradual accumulation oxidative damage causing tumorigenesis (found in Apc and kRas genes).
    • Mean age cancer diagnosis 50 years old.
    • Up to 30% of European patients with > 10 adenomas carry biallelic MYH mutations.
  • 34.
    • Autosomal dominant inheritance.
    • Gene LKB1 (p53-mediated apoptosis)
    • 39% lifetime risk CRC.
    • 93% lifetime risk of developing malignancy.
      • Carcinomas of colorectum, stomach, small intestine (from polyps).
      • Melanoma, sex cord tumours, uterine, breast, lung,pancreatobiliary cancers
    Peutz-Jeghers syndrome
  • 35.
    • Mucocutaneous pigmentation
    • Hamartomatous polyps – GI tract
    Peutz-Jeghers syndrome
  • 36. Peutz-Jegher’s polyp
  • 37. Juvenile Polyposis Syndrome (JPS)
    • Autosomal dominant
    • Hamartomatous polyps of the GI tract
    • 10-38% lifetime risk CRC
    • Mean age diagnosis CRC 34 years
    • SMAD4 and BMPR1A/ALK3 genes
      • involved in TGF-B signalling
      • account for 40 – 50% cases
  • 38. Juvenile Polyp
  • 39. Familial Colorectal Cancer Syndromes
        • Familial Adenomatous Polyposis (AD)
        • Lynch syndrome (HNPCC) (AD)
        • Familial CRC “type X”
        • MYH Polyposis (AR)
        • Peutz Jegher’s (AD)
        • Juvenile polyposis (AD)
        • Others (e.g. Hyperplastic polyposis)