Our analysis of a relatively large set of AFAP families has shown complexity in the phenotype and early genetic pathways of tumorigenesis. The two previous analyses of somatic APC
mutations in AFAP each focused on single families, one with a germline mutation in the 5′ region of the gene1
and the other with a mutation in exon 9.14
These two studies unequivocally provided the important and original finding that “three hits” (that is, two somatic mutations, including loss or mutation of the germline mutant allele) can occur in AFAP tumours. The restricted size of the two studies meant, however, that they were unable to provide further conclusions.
We have found that patients with germline APC
mutations in the 5′ and 3′ regions of the gene or the alternatively spliced region of exon 9 have a highly variable large bowel phenotype, in that the number of colorectal adenomas varies from almost none to the hundreds or thousands of lesions found in classical FAP.3
Although assessment methods necessarily differ among clinical centres, our analysis shows that patients with 5′ APC
mutations (codons 1–177) are likely to have a more severe phenotype than those with mutations in exon 9 or the 3′ end of the gene (>codon 1580). Phenotypic severity also tends to be similar within families, suggesting that restricting analyses to single kindreds may not provide accurate assessment of AFAP patients.
Our study has confirmed that “three hits” at APC
often occur in AFAP adenomas. In such polyps, the “third hit” appears to be required for initiation of tumorigenesis. Although “third hits” might occur at loci other than APC
, we have previously found no mutations at beta‐catenin in AFAP polyps (unpublished data). In polyps with “three hits” from exon 9 mutant and 3′ mutant patients, we have been able to identify specific combinations of APC
mutations which tend to occur. Exon 9 is alternatively spliced in all normal and neoplastic tissues which we have examined (not shown). The combinations of APC
mutations almost certainly produce a near optimal level of Wnt signalling, comparable with those found in classical FAP.9
Some of the combinations (such as R332X‐nt4661insA/LOH ) strongly suggest that the tumour has developed as a result of the functional effects of the germline mutant allele, but other combinations of mutations (such as truncating mutation leaving one 20AAR on the wild‐type with LOH of the germline mutant) might simply be indicative of a “sporadic” tumour occurring on the background of AFAP.
In our families, “third hits” were much rarer in 5′ and 3′ mutant patients than in the exon 9 mutants. These former families' somatic mutations usually, but not always, resembled those of classical FAP patients who have germline mutations before the first 20AAR of the APC
protein. In many ways, this is the result which would be predicted were the 5′ or 3′ mutations simply to cause absent or non‐functional protein. 5′ APC
mutations probably produce a small amount of partially functional APC through use of an internal ribosome entry site (IRES) at codon 18418
. 3′ Mutant proteins have been reported as being unstable19
although the reasons for this are unknown. It is entirely plausible that levels of functional APC protein vary among individuals with both 5′ and 3′ mutations, for example as a result of modifier alleles. Thus for an adenoma to form, some patients would tend to require “third hits” and others would not. The family of Spirio and colleagues,1
for example, may have been relatively efficient at use of the IRES. Formal testing of this hypothesis in vivo would require an exceptionally large unselected series of tumours and patients.
Our analysis of exon 9 mutant cases provides further evidence to show that not all AFAP patients are the same. “Third hits” were common in these patients' tumours. There was a markedly increased frequency of mutations which left three 20AARs on the germline mutant allele, particularly—but not exclusively—at nt4661, which appears to be a relatively hypermutable site. Our view differs somewhat from that of Su and colleagues14
who proposed that insAnt4661 mutations were overrepresented in AFAP polyps because both “strong” and “weak” mutations were sufficient to severely reduce the function of the exon 9 mutant allele. We suggest that mutations leaving three 20AARs on the germline mutant allele are common because the resulting allelotype R332X‐4661insA gives a near optimal genotype, taking into account loss of the germline wild‐type allele and alternative splicing of exon 9. Variation in splicing efficiency, again through modifier allele action, could explain phenotypic variability in exon 9 mutant AFAP but it appears that many of these patients produce sufficient functional protein by splicing out exon 9 that “third hits” are necessary in most polyps.
The reason why AFAP patients develop fewer polyps than classical FAP patients is evident, in that “three hits” are often needed to produce the near optimal genotype. We do not however claim that all polyps from patients with AFAP associated APC
mutations require “three hits”. Even allowing for the imperfections of mutation screening and LOH analysis in archival specimens, we were able to analyse the fresh frozen adenomas comprehensively and found many without “three hits”. Moreover, several polyps from our patients had somatic mutations which would have been predicted from a “two hit” model of optimal Wnt signalling. Currently, we cannot explain why in a single patient some polyps seem to require “three hits” and others do not, but it is possible that “third hits” at other loci can substitute for APC
mutation. Another possibility is that selective constraints on the diminished APC function needed for tumorigenesis are “just right”1,10
at some times, but weaker at others, for example during development or when tissue is undergoing repair.
Genetic analysis of colorectal tumours from patients with germline mutations in AFAP associated regions of APC
, in this study and others, has revealed a novel mechanism underlying the genotype‐phenotype association in this tumour syndrome—namely, a requirement for “three hits” in at least some AFAP adenomas. This finding must be viewed in the framework of the model of optimal combinations of APC
mutations, rather than simple loss of protein function. More than one different combination of APC
mutations can provide near optimal Wnt signalling in AFAP. However, not all AFAP patients are the same. Given that assembling a very large series of AFAP patients is extremely difficult, it is not easy to decide on what is the “typical” AFAP phenotype or somatic genotype. In the seven families with 5′ APC
mutations studied to date (Spirio and colleagues,1
Albuquerque and colleagues,10
and this study), approximately 15–20% of polyps seem to acquire “three hits”, but only Spirio and colleagues1
found a high frequency of nt4661insA. In the six 3′ mutant families studied (all from this study), the frequency of “third hits” seems similar to that of the 5′ mutants. Six exon 9 mutant families have been studied (Su and colleagues14
and this study) and almost all of these show evidence of a high frequency of “third hits”—we estimate a minimum of 50% in our study. In addition, there appear to be genetic factors apart from the germline APC
mutation that influence disease severity, as evidenced by the tendency for polyp numbers to be similar within families. The phenotypic and somatic molecular heterogeneity in AFAP means that clinical management of patients with AFAP associated mutations must be empirical. Accurate prediction of phenotype may only be possible when factors, such as modifier genes, that influence genetic pathways and disease severity are identified.