APC Splicing Mutations Leading to In-Frame Exon 12 or Exon 13 Skipping Are Rare Events in FAP Pathogenesis and Define the Clinical Outcome

Abstract

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.

Keywords: APC; FAP pathogenesis; exon skipping; familial adenomatous polyposis; splicing.

Figure 1

Pedigree of the Italian family involved in this study. Squares indicate men, circles represent women. The arrow indicates the index case. Unfilled symbols indicate unaffected individuals. Slashed symbols denote dead individuals. Black-filled symbols denote individuals with colorectal cancer and polyposis, while gray-filled symbols correspond to patients with colorectal polyposis. The following information is given below each filled symbol: clinical manifestations (CRC = colorectal cancer; CPs = colon polyps, with the number of polyps indicated in square brackets; Pca = prostate cancer), age at diagnosis (y = years).

Figure 2

Characterization of APC splicing mutation c.1626_1627+7del. (A) Sequencing electropherograms of genomic DNA from a healthy control individual and the proband, showing the splicing mutation c.1626_1627+7del. (B) Left: Agarose gel electrophoresis showing the RT-PCR analysis of mRNA extracted from peripheral blood of the patient (P) carrying the APC c.1621_1626+7del mutation and a healthy control (C). Amplified products obtained with primers spanning APC exon 10–11 and 13–14 boundaries were separated on 3% agarose gel and independently sequenced. Center: Schematic diagrams showing the wild type amplification product (356 bp) and the altered-splicing amplification product lacking APC exon 12 (277 bp). Right: Sequencing electropherograms of cDNA splicing isoforms generated from the wild type and mutant RT-PCR products. Bottom: Diagram showing the localization of the primers (indicated as arrows) used for RT-PCR experiments.

Figure 3

Splicing minigene reporter assay based on the pSPL3 exon-trapping vector. (A) Schematic representation of the pSPL3 minigene reporter used for the molecular characterization of APC splicing mutation c.1621_1626+7del. The pSPL3 vector contains two exons (SD and SA) and a functional intron, with transcription beginning after the SV40 promoter and ending at the late poly(A) signal (LPAS). EcoRI and BamHI indicate the cloning sites used to subclone the genomic APC fragments obtained from the wild type and mutant alleles (c.1621_1626+7del). (B) RT-PCR analysis of transcripts derived from the indicated pSPL3 reporter minigenes transfected in HEK-293 cells. Left: Agarose gel electrophoresis showing the RT-PCR products obtained with the SD6 and SA2 primers from HEK-293 cells transfected with the pSPL3 empty vector (263 bp), the pSPL3 vector with the genomic APC fragment from the wild type allele (341 bp), or the pSPL3 vector with the genomic APC fragment from the mutant allele (263 bp), and untransfected HEK-293 cells (negative control). Center: Schematic diagrams showing the RT-PCR products obtained. Right: Sequencing electropherograms of the RT-PCR products obtained.

Figure 4

APC coding region. (A) Schematic diagram of wild type APC protein, depicting conserved regions and domains that interact with other proteins. (B) Schematic diagram of APC truncated proteins that result from total removal of ARM2 and/or ARM3 along with disruption of all downstream APC domains and are associated with the classic FAP phenotype. (C) Schematic diagram of APC variants that result from exon 12 or exon 13 splicing mutations leading to an in-frame protein and are associated with the AFAP phenotype.