Analysis of rare APC variants at the mRNA level: six pathogenic mutations and literature review

Abstract

In monogenic disorders, the functional evaluation of rare, unclassified variants helps to assess their pathogenic relevance and can improve differential diagnosis and predictive testing. We characterized six rare APC variants in patients with familial adenomatous polyposis at the mRNA level. APC variants c.531 + 5G>C and c.532-8G>A in intron 4, c.1409-2_1409delAGG in intron 10, c.1548G>A in exon 11, and a large duplication of exons 10 and 11 result in a premature stop codon attributable to aberrant transcripts whereas the variant c.1742A>G leads to the in-frame deletion of exon 13 and results in the removal of a functional motif. Mutation c.1548G>A was detected in the index patient but not in his affected father, suggesting mutational mosaicism. A literature review shows that most of the rare APC variants detected by routine diagnostics and further analyzed at the transcript level were evaluated as pathogenic. The majority of rare APC variants, particularly those located close to exon-intron boundaries, could be classified as pathogenic because of aberrant splicing. Our study shows that the characterization of rare variants at the mRNA level is crucial for the evaluation of pathogenicity and underlying mutational mechanisms, and could lead to better treatment modalities.

Figure 1

Characterization of variant c.531 + 5G>C in intron 4 of the APC gene (patient 1159). A: Sequencing pattern of genomic DNA reveals the heterozygous substitution G>C. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 3F and in exon 5R in patient 1159, in patient 1007 with the mutation c.531 + 1G>A, and a control (C). C: Diagram representing the mutation in genomic DNA (top) and the resulting aberrant splicing and premature stop codon analyzed by RT-PCR (bottom; primers indicated as arrows; boxes with numbers denote individual exons; the mutation and surrounding sequences are indicated). D: Sequencing pattern of the entire RT-PCR product showing the heterozygous deletion of exon 4.

Figure 2

Characterization of the variant c.532-8G>A in intron 4 of the APC gene (patient 1398). A: The sequencing pattern of genomic DNA reveals the heterozygous substitution G>A. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 2F and in exon 5R in the patient (P) and in two control samples (C). C: Diagram representing the mutation in genomic DNA and the aberrant splicing analyzed by RT-PCR (primers indicated as arrows) resulting in a premature stop codon (TAG). D: Sequencing pattern of the RT-PCR product showing the heterozygous insertion of six intronic nucleotides in the patient's sample. The sequences of the normal and mutant forward primer are marked by arrows.

Figure 3

Characterization of variant c.1409-2_1409delAGG (patient 1476). A: Sequencing pattern of genomic DNA reveals the mutation in the splice acceptor site of exon 11. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 9F and in exon 13R in patient 1476 (P) and in a control (C), indicated by a white arrow. C: Diagram representing the mutation on genomic DNA and the two aberrant transcripts detected by RT-PCR leading to premature stop codons. Arrows indicate primer positions.

Figure 4

Characterization of variant c.1548G>A in exon 11 of the APC gene (patient 5). A: Sequencing pattern of genomic DNA reveals the heterozygous substitution G>A, arrows, in the index patient but not in his affected father. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 9F and in exon 13R (P) and a control (C). C: Sequencing pattern of the 290-bp and 430-bp fragments excised from the gel showing the deletion of exon 11 in the short fragment and the complete lack of the mutant allele in the full-length fragment. Arrow indicates the position of the mutation. D: Haplotype analysis in family 5 shows that the mutation in the index patient occurred in the paternal haplotype. T, C, the two alleles of the SNP at nucleotide position 1458 (codon 486); Mut, mutation (G>A, arrow) at nucleotide position 1548; and n, normal sequence (G). E: Schematic diagram representing the mutation in genomic DNA and the aberrant splicing variant detected by RT-PCR leading to a premature stop codon. Arrows indicate primer positions, arrowhead indicates location of the G>A mutation.

Figure 5

Characterization of variant c.1742A>G in exon 13 of the APC gene (patient 1172). A: Sequencing pattern of genomic DNA reveals the heterozygous substitution A>G. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 11F and in exon 15A-R in the patient (P) and a control (C). C: Diagram representing the normal and aberrant transcript. Arrows indicate primer positions. D: Sequencing pattern of the 501-bp and 618-bp fragments excised from the gel showing the deletion of exon 13 in the short fragment (above) and the (almost) complete lack of the mutant allele (arrow) in the full-length fragment.

Figure 6

Characterization of the duplication of exons 10 to 11 of the APC gene (patient 1199) detected by MLPA. A: Normalized peak areas showing the duplication of exons 10, 10a, and 11. B: Agarose gel showing the RT-PCR product obtained with primers localized in exon 9F and 13R in the patient (P) and a control (C). The faint bands above the main bands represent the products containing the alternatively spliced exon 10a (small arrows). C: Partial sequence showing the junction of exon 11 and exon 10 in the duplicated fragment (666 bp) excised from the agarose gel (without exon 10a). D: Diagram representing the order of the exons and the resulting premature stop codon in the duplicated exon 10. Arrows indicate primer positions.