Analysis of Rare APC Variants at the mRNA Level

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. Determining the pathogenicity of a germline mutation in an inherited disease is crucial for diagnostics and counseling and is particularly relevant for predictive testing in persons at risk. Generally, nonsense and frameshift mutations, large exonic deletions, and mutations in the conserved splice sequences in a gene are considered as pathogenic. In contrast, the pathological significance of rare variants predicted to result in amino acid substitutions (missense mutations), small in-frame deletions, or even same-sense mutations (silent variants) is often unclear. Similarly, the functional effect of DNA variants at less conserved positions in introns is difficult to predict. As a consequence, a growing number of single bp substitutions identified in routine diagnostics are denoted as unclassified variants (UVs) or variants of uncertain significance, respectively.Based on functional analysis, it has become evident in recent years that a substantial proportion of so-called UVs, particularly those located close to exon-intron boundaries, affect splicing because of the disruption of putative regulatory elements such as exonic splicing enhancer and silencer motifs, or composite exonic regulatory elements of splicing.1-8 Aberrant splicing as the underlying mechanism of presumed missense or silent variants has been detected in a number of genes involved in cancer predisposition such as MLH1, MSH2, BRCA1, BRCA2, RB1, NF1, and ATM. -17Familial adenomatous polyposis (FAP) (MIM no. 175100) is an autosomal-dominant precancerous condition characterized by the appearance of numerous colorectal adenomas, which, if not detected early and removed, invariably result in colorectal cancer. In classic FAP, patients develop hundreds to thousands of adenomatous poly...

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

“…Nevertheless, although it is becoming more clear that some alleles may defy the binary classification, depending on other co-factors to co-determine their penetrance, it remains a core feature of genetic services that when the bar of technological challenges is lowered and these VUS can be properly classified, satisfying answers may be provided to the families and care providers. Previous reports have clarified the contributions of APC exonic and splice-junction variants to disease (Aretz, et al, 2004; Goncalves, et al, 2009; Kaufmann, et al, 2009). …”

Novel strategies for comprehensive mutation screening of the APC gene

“…Nontruncating single-base substitutions in the coding APC sequence or unique variants in less conserved intronic regions close to the splice sites have rarely been reported in FAP. Most of these APC variants are pathogenic due to aberrant splicing [22]. Recent data suggest that gross alterations in the APC gene may have been underreported initially, with up to 20% of FAP families potentially carrying a gross alteration [23].…”

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

“…Skipping of exon 13 has been reported to be associated with a mutation in a highly conserved splice acceptor site (c.1627G>T, the first base of exon 13) in a patient with AFAP phenotype who underwent subtotal colectomy for carcinoma at the age of 60 [54]. In another report, skipping of exon 13 was found to be caused by a missense mutation in exon 13 (c.1742A>G) that was detected in a patient with attenuated FAP [59]. Splicing mutations resulting in the loss of exon 12 (p.Ala517_Gly542del) lead to an almost complete loss of ARM2.…”

“…Specifically, among the splicing mutations leading to an in-frame protein, 4/9 cause exon 12 or exon 13 skipping with loss of armadillo repeat 2 (ARM2) and armadillo repeat 3 (ARM3) in the APC N-terminal armadillo repeat domain. Of these, 3 were reported in patients with AFAP phenothype [45,54,59] (Table 1). The remaining molecularly characterized splicing mutations leading to an in-frame protein (5/9) result in skipping of exon 5, 7, 8, or (partially) 9 with loss of APC regions not encompassing known functional sites/domains [45,54,67] (Table S3).…”