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Familial adenomatous polyposis is a Mendelian syndrome in which germline loss-of-function mutations of APC are associated with multiple adenomatous polyps of the large bowel, a multiplicity of extracolonic features, and a high lifetime risk of colorectal cancer. Different APC germline mutations have been identified, including sequence changes, genomic rearrangements, and expression defects. Recently, very rare families have been associated with constitutive large deletions encompassing the APC-5' regulatory region, while leaving the remaining gene sequence intact; the regulatory region contains a proximal and a distal promoter, called 1A and 1B, respectively. We identified a novel deletion encompassing promoter 1B in a large Italian family that manifested polyposis in three of the six branches descending from a founding couple married in 1797. By combining different molecular approaches on both DNA and RNA, we precisely mapped this deletion (6858 bp in length) that proved to be associated with APC allele silencing. The finding of the same deletion in two additional polyposis families pointed to a founder mutation in Italy. Deletion carriers from the three families all showed a "classical" polyposis phenotype. To explore the molecular mechanisms underlying promoter deletions, we performed an in silico analysis of the breakpoints of 1A and 1B rearrangements so far reported in the literature; moreover, to decipher genotype-phenotype correlations, we critically reviewed current knowledge on deletions versus point mutations in the APC-5' regulatory region., (© 2017 Wiley Periodicals, Inc.)

Chain-terminating germline APC mutations are responsible for adenomatous polyposis coli (APC). In the present work, we tested the hypothesis that germline APC mutations may be present in some patients with a milder phenotype, i.e., multiple synchronous colorectal adenomas. Eighteen patients with 3 or more colorectal adenomas at endoscopy (within a 6-month period) were ascertained from a series of subjects undergoing endoscopic examination. Their blood DNAs were analysed for the presence of germline mutations in the APC coding region by single-strand polymorphism analysis. Ten unrelated polyp-free subjects and 101 unrelated APC patients were used as controls in the molecular analyses. Five of the eighteen patients carried novel germline APC variants or rare polymorphisms. These were various in site (from the splice acceptor site of intron 7 to the end of exon 15) and type (splice-site, missense, and chain-terminating mutations). Only one of ten polyp-free individuals carried a silent APC variant and none of these variants was found in the 101 APC controls. A first- or second-degree family history of colorectal cancer was reported by 4 of the 5 patients carrying a germline APC variant. In conclusion, novel APC germline variants were detected in patients with multiple synchronous adenomas. This suggests that the development of sporadic adenomas, in some instances, is associated with the presence of minor germline variants of the APC gene and that the spectrum of germline APC functional mutations may be larger than previously thought.

APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5' of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5' of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level.