Large intron 14 rearrangement in APC results in splice defect and attenuated FAP

Abstract

Familial adenomatous polyposis [FAP (OMIM 175100)] is an autosomal dominant colorectal cancer predisposition syndrome characterized by hundreds to thousands of colonic polyps and, if untreated by a combination of screening and/or surgical intervention, an approximately 99% lifetime risk of colorectal cancer. A subset of FAP patients develop an attenuated form of the condition characterized by lower numbers of colonic polyps (highly variable, but generally less than 100) and a lower lifetime risk of colorectal cancer, on the order of 70%. We report the diagnosis of three attenuated FAP families due to a 1.4-kb deletion within intron 14 of APC, originally reported clinically as a variant of unknown significance (VUS). Sequence analysis suggests that this arose through an Alu-mediated recombination event with a locus on chromosome 6q22.1. This mutation is inherited by family members who presented with an attenuated FAP phenotype, with variable age of onset and severity. Sequence analysis of mRNA revealed an increase in the level of aberrant splicing of exon 14, resulting in the generation of an exon 13-exon 15 splice-form that is predicted to lead to a frameshift and protein truncation at codon 673. The relatively mild phenotypic presentation and the intra-familial variation are consistent with the leaky nature of exon 14 splicing in normal APC. The inferred founder of these three families may account for as yet undetected affected branches of this kindred. This and similar types of intronic mutations may account for a significant proportion of FAP cases where APC clinical analysis fails because of the current limitations of testing options.

Figure 1. Family pedigrees

Pedigrees of three kindreds, K495, K7652 and K485, presumed to be related by a common ancestor, showing AFAP clinical diagnoses. Haplotype markers are presented as 4X2 arrays, using allele numbers based on the number of repeats observed for each allele in the CEPH families for the four markers D5S2027, D5S2501, D5S346 and D5S421, which span the APC locus (http://genome.ucsc.edu). Where possible, parental haplotypes inferred from the available data are presented in parentheses. The intron 14-deletion-associated haplotype is presented in grey, and boxed, for ease of identification. Colonic phenotypes, including both single adenomas and multiple polyps detected on colonoscopy, reported colon cancers and surgeries and age at diagnosis are noted where known.

Figure 2. PCR and sequence amplification of genomic DNA

PCR analysis of genomic DNA from proband and related family members, as noted from each kindred, showing the normal 1936-bp allele, as well as the 534-bp allele detected in affected family members, carrying the shared haplotype. Samples are marked according to the position of the participant on the pedigree in Figure 1; unaffected; H₂O: no template control. B. Sequence of mutant allele. The upper panel shows the proximal portion of the rearranged allele (forward strand) and the lower panel, the distal portion (reverse strand). The upstream run of Ts depicted in Figure 3A is marked with a tan-colored box. The nucleotides that diverge from APC intron 14 sequence and share identity with the repetitive element on chromosome 6q22.1 are marked by red asterisks.

Figure 2. PCR and sequence amplification of genomic DNA

PCR analysis of genomic DNA from proband and related family members, as noted from each kindred, showing the normal 1936-bp allele, as well as the 534-bp allele detected in affected family members, carrying the shared haplotype. Samples are marked according to the position of the participant on the pedigree in Figure 1; unaffected; H₂O: no template control. B. Sequence of mutant allele. The upper panel shows the proximal portion of the rearranged allele (forward strand) and the lower panel, the distal portion (reverse strand). The upstream run of Ts depicted in Figure 3A is marked with a tan-colored box. The nucleotides that diverge from APC intron 14 sequence and share identity with the repetitive element on chromosome 6q22.1 are marked by red asterisks.

Figure 3. Schematic representation of proposed recombination event leading to APC intron 14 deletion

A. Schematic representation of recombination between Alu-containing repetitive element located on chromosome 6q22.1, which shares 100% identity with the insertion, and the APC locus on chromosome 5q22.1. The recombination appears to have been mediated on the proximal side by a run of T residues, and on the distal side by homology between the repetitive element on 6q22.1 and the third of three repetitive elements located in intron 14 of APC.

Figure 4. Discrete deletion of Exon 14 in mRNA from mutation carriers

(A) Results of RT-PCR analysis of mRNA from transformed lymphocytes; Amplification of transcripts encompassing exons 13–15. Unaffected individuals reveal a predominant fragment of 412 bp, and a trace of a fragment of 197 bp, while affected individuals reveal each fragment in approximately equal proportions. Samples are marked according to the position of the participant on the pedigree in Figure 1. (B) Sequence analysis, demonstrating absence of exon 14 in predominant splice-from, as indicated by vertical bar drawn through sequence trace. Forward and Reverse traces are shown for the K7652 proband and her mother. Printed sequence shows normal exon 13–15 sequence, with exon 14 colored in grey with experimental primers underlined.

Figure 4. Discrete deletion of Exon 14 in mRNA from mutation carriers

(A) Results of RT-PCR analysis of mRNA from transformed lymphocytes; Amplification of transcripts encompassing exons 13–15. Unaffected individuals reveal a predominant fragment of 412 bp, and a trace of a fragment of 197 bp, while affected individuals reveal each fragment in approximately equal proportions. Samples are marked according to the position of the participant on the pedigree in Figure 1. (B) Sequence analysis, demonstrating absence of exon 14 in predominant splice-from, as indicated by vertical bar drawn through sequence trace. Forward and Reverse traces are shown for the K7652 proband and her mother. Printed sequence shows normal exon 13–15 sequence, with exon 14 colored in grey with experimental primers underlined.