Truncating mutations in the 5’ half of APC (including exon 9)

Classical FAP is associated with APC mutations which produce truncating mutations (either frameshift or stop mutations) in the 5’ half of the APC gene (spanning exons 5 to the 5’ half of exon 15). The phenotype data from the published literature is summarised in Appendix 1.2, p 91, (Main FAP phenotype). The quality of the published literature is variable and depends partly on the focus of the publication. For example, reports from the early 1990s emphasise the classical nature of FAP phenotype (greater than 100 polyps, CHRPE, epidermoid cysts etc.) as a means to validate molecular or genetic analyses of the APC gene.

4.3.1.Ascertainment

The majority of reported cases have been ascertained by Polposis Registries, which have been referred proband cases and then identified other affected family members. Ascertainment is an important source of bias because the commonly quoted phenotype marker - age of onset of cancer/ symptoms - is heavily influenced by route of

ascertainment. For example, Bjork et al. (1999) clearly show that probands are diagnosed at later age (39 r: 11-71) when compared with screened cases (22 r:3-65). The incidence of cancer at diagnosis reflects this, screened cases have a similar age at

development of colorectal cancer to proband cases (38 vs 42, Bjork et al. 1999), but a much lower overall incidence (2% vs 48%) of cancer. This finding is also supported by FriedI et al. (2001) who found that 6% of screened cases had cancer at diagnosis. The majority of genotype-phenotype publications show colorectal cancer incidence at diagnosis intermediate in this range (see Appendix 1.2, p 91, and references therein).

4.3.2.Colon polyp number and general APC genotype

There is very limited published data that relates colonic adenoma/polyp number to APC genotype in a quantitative manner. Many publications simply state the adenoma number as a semi-quantitative value, e.g. ‘greater than 100’, ‘multiple’, ‘florid’, etc. For the main group of APC mutations there are two papers that give quantitative values of adenoma numbers for given APC mutations. Debinski et al. (1996) reported a median polyp number in a FAP colon of 842 (78-7500: n = 317) for all FAP cases without reference to mutation position. Ficari et al. (2000) reported 568 (r: 174-1750: n = 18) for mutations exclusive of exon 9 and codon 1309. Both of these papers base their figures on estimations of polyp number made from colectomy specimens and are therefore likely to be accurate.

4.3.3.Colon number and codon 1309 mutations

APC codon 1309 is frequently mutated in FAP and causes severe colonic features. Nugent et al. 1994 reported that (n = 27) patients with the 5 bp del 1309 have an average of 4000 (IQR: 3000 - 4875) polyps per colectomy specimen. This figure is confirmed directly by (Ficari et al. 2000) who reported 9 patients with this mutation who had an average of 2573 polyps per colectomy specimen. Because cancer risk in FAP is a function of polyp density (Debinski et al. 1996) other authors have indirectly

demonstrated that the 1309 mutation has the severest form of colonic FAP (Giardiello et al. 1994, FriedI et al. 2001). Consequently these patients undergo operation at an earlier age and tend to get colorectal cancer at an earlier age.

4.3.4.Colon polyp number and exon 9 mutations

Patients who have mutations within APC exon 9 (nt. 934 - 1322) may have attenuated FAP. Shortly after the discovery of APC as the gene responsible for FAP, a number of reports suggested that mutations at specific locations could result in low-density polyposis or attenuated FAP (i.e. fewer than 100 colonic polyps) - 1.2, p 89, (exon 9 section). Bunyan et al. (1995) suggested that mutations within exon 9 developed fewer than 100 polyps from a case series of 4 patients from the same pedigree (mutation at codon 366). van der Luijt et al. (1995) reported on 15 patients with the mutation n1069 INS Ibp (a) who had polyp counts in the range 10 to ‘> 1000’. The patients had an older age diagnosis 49 (r: 18-76) and an older age at diagnosis of cancer 61 (39-80) than would be expected for the typical FAP population.

Published series of exon 9 mutations after this (Young et al. 1998, Soravia et al. 1998, Rozen et al. 1999, Ficari et al. 2000, Su et al. 2000, Scott et al. 2001) have supported the original observation. The typical exon 9 patient (mutation nucleotide range nt 996 - 1311) reported in the literature has a very variable colonic polyp number (range 0-310: possibly >1000). The majority of affected individuals seem to have less than 100 colonic adenomas, for example, Rozen et al. (1999) reported 5 of 11 patients had no adenomas visible at colonoscopy although most were young adults. Ficari et al. (2000) reported a colectomy specimen from a 38 year old patient with just 22 polyps

macroscopically visible (codon 367). Young et al. (1998) found that age at diagnosis tended to be older than typical for FAP (45 years: 34-68: c398 del 2 bp). Although caution may be appropriate for some of these cases because of the accuracy of non- dye-spray colonoscopy it is difficult to dismiss, entirely, the suggestion that exon 9 APC mutations produce at the least ‘hypervariable’ attenuated FAP.

4.3.5. The colonic phenotype of extreme 5’ APC mutations.

Extreme 5’ FAP causing APC mutations have been claimed to produce attenuated FAP (Spirio et al. 1992, Spirio et al. 1993, Spirio et al. 1999, Leppert et al. 1990,

c158:Giardiello et al. 1997, c168:Soravia et al. 1998) although only a minority of causative FAP mutations localize to this region (5%). The ‘typical’ atypical patient is said to have fewer adenomas (less than 100), of right-sided distribution and a tendency to colorectal cancer at an older age. The papers that describe these mutations are summarised in Appendix 1.3 (5’ APC mutation FAP phenotype, p 93). Although many published reports state that 5’ APC mutations can generate fewer than 100 colonic polyps, many have also demonstrated that extreme 5’ APC mutations can produce moderately severe polyposis (Walon et al. 1997, Nasioulas et al. 2001).

The precise boundary of extreme 5’ mutations is not clearly defined in the literature. The usually cited boundary is between codons 159-168 (nt 477-504) (Spirio et al. 1993, Walon et al. 1997) this region approximately coincides with the 3’ end of exon 4 (nt 423-531). However, the literature is inconsistent: for example, Nasioulas et al. (2001) reported a family that consistently produced moderate degrees of colonic polyposis (500-1500 adenomas) but was caused by germiine APC mutation at nt 348 del ttcat (i.e. codons 116-118 inclusive, exon 3) - well inside the proposed limits. Extreme 5’ mutations are also interesting because of the proposed mechanisms that may generate the colonic adenomas. This topic is discussed in more detail in the section on the APC gene.

4.3.6.. The colonic phenotype of distal 3’ APC mutations

FAP is associated with a distribution of germiine APC mutations that is confined to the 5’-half of the gene (approx. nt 150 - 4500). Mutations which are distal to the 3’

boundary are often associated with milder colonic polyp burdens. There are two (not necessarily mutually exclusive) competing theories for this observation. Firstly, the carboxy-terminus function(s) may be less critical for the formation of polyps than the amino-terminus’s functions. Some researchers (van der Luijt et al. 1996) have suggested that extreme 3’ mutations of the APC gene render either the mRNA or translated protein product unstable, effectively resulting in a null-APC gene, cf above.

The majority of published reports (Appendix 1.4, p94: FAP phenotype caused by 3’ APC mutations) are fairly consistent, stating that 3’ mutations generate either low density or attenuated FAP. The boundary of where the transition in phenotype occurs is less clear. It is probably about codon 1550 (nt 4650), which is located about half way through the section of the gene which contains the 20 amino acid repeats. It is notable that this is very close to the location of the first SAMP repeat (cl 581 - 1584: nt 4741 -

4752), which is absolutely required for (3-catenin degradation. An alternative

explanation may rest with the nearby third 20 aa repeat (c l491 -1510: nt 4471- 4530), suggesting a dosing effect for the number of these repeats.

The genotype-phenotype association of 3' mutations has implications for the

pathological mechanism of mutant APC. APC is conventionally considered a tumour suppressor gene. This is given a fuller discussion above.