Helen Stuart, Cardiff

1. HNPCC and FAP are both associated with familial colon cancer but differ in many aspects. Describe the similarities and differences between these conditions. How does MYH polyposis differ from both? Helen Stuart, Cardiff

2. Essay plan

3. Colorectal cancer • Third most common cause of cancer-related death in men and women in the western hemisphere. • Among colon cancer patients, hereditary risk contributes ~20%. Ref: S Narayan and D Roy Molecular Cancer 2003, 2:41  2 major forms of hereditary colorectal cancer are: • Hereditary non-polyposis colorectal cancer (HNPCC) [~2-7% of total colorectal cancer] Ref: A Muller and R Fishel Cancer Investigation 2002; 20(1):102-109 • Familial adenomatous polyposis (FAP)

4. Diagnosis of hereditary colorectal cancer HNPCC • Lacks clear phenotypic characteristics • Diagnosis based on: • Family history (Amsterdam or Bethesda criteria) inc earlier age of onset than gen pop (45yrs vs 63yrs) • Demonstration of defective MMR (MSI) • Confirmed by identification of mutation FAP • Presence of >100 adenomatous polyps and microadenomas in the large intestine. • Diagnosis based on: • Polyps • Family history • Confirmed by identification of mutation

5. Cancer predisposition HNPCC • 50-80% lifetime risk of colon cancer • Proximal colon cancer • Extra-colonic manifestations: • Endometrium, ovarian, stomach tumours. • Also: CNS tumours (Turcot), skin tumours (Muir-Torre) FAP • Virtually 100% lifetime risk of colon cancer • Distal colon cancer • Extra-colonic manifestations: • Desmoids, gastric adenomas, osteomas. • Also: CNS tumours (Turcot), osseous, dental and cutaneous anomalies (Gardner) Ref: S Baglioni and M Genuardi 2004 Am J Med Gen 129C:35-43

6. Inheritance HNPCC Autosomal dominant inherited predisposition FAP Autosomal dominant inherited predisposition • Both involve inherited germline mutation + extra somatic mutation. • Tumours usually arise when a second somatic hit affects the wild-type allele. This is termed ‘loss of heterozygosity’. Knudson Two-Hit Hypothesis of Tumorigenesis: • First Hit: The first hit is classically thought of as a point mutation that inactivates one copy of a gene (e,g, tumour suppressor gene - TSG). In hereditary cancer syndromes, individuals are born with the first hit. The individual does not develop cancer at this point because the remaining TSG on the other allele is still functioning normally. • Second Hit: The second hit results in loss of functioning TSG allele. This leaves only a non-functioning copy of the TSG, and the individual goes on to develop cancer.

7. Gene/s involved HNPCC Mismatch repair genes MLH1, MSH2, MSH6, PMS2, (PMS1) FAP Tumour suppressor gene APC (5q) Note: A mutation in the PMS1 gene was originally reported in a single HNPCC family; however, this alteration was not found to segregate with the cancers in the family. Further analysis identified a mutation in the MSH2 gene. Therefore, the role of PMS1 in HNPCC is currently being questioned. Ref: GeneReviews

8. Mismatch repair genes (MMR)e.g. MLH1, MSH2, MSH6, PMS2 • MMR is critical for the maintenance of genomic stability. • MMR deals with correcting mismatches of bases (i.e. not Watson-Crick base pairing A•T, C•G). • MMR increases fidelity of DNA replication by identifying and excising single-base mismatches and insertion-deletion loops that may arise during DNA replication. • There are at least 6 proteins required for the complete MMR system: MSH2, MLH1, PMS1, PMS2, MSH3 and MSH6. • Recognition of a mismatch requires several different proteins (including MSH2), which binds to the mismatch site. • Subsequently, other MMR proteins (including MLH1) are recruited to the site and perform the excision, re-synthesis and ligation of the DNA. • As a result of MMR deficiency, replication misincorporation errors accumulate, resulting in a mutator phenotype. • Mutation of ‘gatekeeper’ genes, such as those involved in apoptosis and cell cycle control (e.g. APC or K-ras) leads to tumour development. • Hypermutability associated with MMR deficiency mainly affects small repeated seqs (microsatellites), causing microsatellite instability. • Genes with coding microsatellites (e.g. TCF4) accumulate frame-shift mutations and lose function, so speeding up cancer progression. Note: MMR may play a role in the development of 15–25% of sporadic tumours that occur in a number of tissues. Ref: P Peltomaki 2003 J Clinical Oncol 21(6): 1174-1179

9. Tumour suppressor genes e.g. APC • Classical ‘gatekeeper’ tumour suppressor gene involved in the control of cell proliferation of intestinal epithelial cells through the b-catenin/WNT signalling pathway. • APC binds to beta-catenin, axin and glycogen synthase kinase 3beta to form a large protein complex, in which beta-catenin is phosphorylated and broken down, resulting in negative regulation of the WNT signalling pathway. • The WNT signalling pathway regulates around 50 genes, including genes involved in cell proliferation, and cell-cell interactions. • Many of the APC mutations in colorectal tumours result in a lack of the beta-catenin-binding regions, so fail to inhibit WNT signalling, leading to constitutive activation of the pathway and over-proliferation of tumour cells. • Also involved in the maintenance of chromosome stability. Note: Inappropriate activation of the WNT pathway are observed in several human cancers (Spink et al., 2000). Ref: S Baglioni and M Genuardi 2004 Am J Med Gen 129C:35-43 Ref: Senda T et al 2005 Anat Sci Int. 80(3):121-31

10. Microsatellite instability (MSI) vs chromosomal instability (CIN) • 2 main types of genetic instability • MSI leads to a 1,000-fold increase in the rate of subtle DNA changes. • CIN enhances the rate at which gross chromosomal changes occur during cell division such as chromosome breaks, duplication, rearrangements, and deletions. • CIN may provide additional growth advantage to the cancer cell by accelerating the rate of LOH at tumour suppressor loci and/or by amplifying chromosomal regions encompassing oncogenes.

11. Types of mutation HNPCC • Point mutations and single and multiple exon dels/dups • 40% MLH1 • 40% MSH2 Ref: A Muller and R Fishel Cancer Investigation 2002; 20(1):102-109 • No common mutations • 29% MLH1 and 16% MSH2 are missense FAP • Point mutations and single and multiple exon dels/dups • No common mutations • But: mostly 5’ end of gene • And: 80% cause premature protein truncation in classic FAP. Note: In many sporadic colon cancers, hypermethylation of the MLH1 gene promoter resulting in its transcriptional silencing has been observed more than mutations.Ref: S Narayan and D Roy Molecular Cancer 2003, 2:41  This epigenetic change is not heritable.

12. Testing methods HNPCC • MSI • Found in ~15% of CRCs • ~7% due to HNPCC • Immunohistochemistry • Shows abnormal MMR protein expression • ~15% of sporadic colon cancers show loss of staining of MLH1 • Sequencing MMR genes (MLH1 & MSH2) • MLPA (MLH1 & MSH2) FAP • Protein truncation test • Sequencing APC gene • MLPA (APC)

13. Surveillance HNPCC • Colonoscopy (annual from 25yrs) and prophylactic surgery FAP • Colonoscopy (annual from 10-12yrs) and prophylactic surgery

14. MYH polyposis (differences between this and HNPCC & FAP) • Diagnosis: • MYH gene implicated in FAP cases with lower number of polyps and more advanced age of CRC diagnosis. • FAP-associated extra-intestinal manifestations have been reported in MYH-related cases; including duodenal polyps and very early-onset gastric cancer. • A relatively high prevalence of breast cancer in MAP patients was found in the Nielson et al study 2005; the role of MYH in breast cancer needs to be investigated further. • Cancer predisposition: The 2005 study by Nielson et al stated that ~50% of the MAP patients studied developed colorectal cancer. • Inheritance: Autosomal recessive • Gene involved: mutY human homologue (MYH) on 1p • Biallelic MYH mutations - carriers are homozygous or compound heterozygous for germline mutations in MYH gene, • MYH encodes for a DNA glycosylase involved in BER pathway. • MYH dysfunction increases the somatic mutation rate within APC, which drives neoplastic transformation. • Types of mutation: 2 common mutations (Y165C and G382D) account for 75% of MYH mutations in Caucasians. • Surveillance: Colonoscopy (every 2yrs from 25-30yrs) and prophylactic surgery. • Genetic counselling should be focussed on the probands’ siblings, who are at 25% risk of carrying biallelic MYH mutations. (compared to all relatives of APC/HNPCC)

15. MYH polyposis: Base Excision Repair (BER) The BER steps: • Removal of the damaged base by a DNA glycosylase. • There are at least 8 genes encoding different DNA glycosylases ,each enzyme responsible for identifying and removing a specific kind of base damage. • Removal of its deoxyribose phosphate in the backbone, producing a gap. • There are 2 genes encoding enzymes with this function. • Replacement with the correct nucleotide. • This relies on DNA polymerase beta. • Ligation of the break in the strand. • 2 enzymes are known that can do this; both require ATP.

16. References • S Narayan and D Roy Molecular Cancer 2003, 2:41  • A Muller and R Fishel Cancer Investigation 2002; 20(1):102-109 • P Peltomaki (2003) J Clinical Oncol 21(6): 1174-1179 • S Baglioni and M Genuardi 2004 Am J Med Gen 129C:35-43 • Senda T, Shimomura A, Iizuka-Kogo A 2005 Anat Sci Int. 80(3):121-31