Promoter‐specific alterations of APC are a rare cause for mutation‐negative familial adenomatous polyposis

Abstract

n familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation-negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation-dependent probe amplification (MLPA, P043-B1). Promoter-specific events of APC were addressed by targeted resequencing, MLPA (P043-C1), methylation-specific MLPA, and Sanger sequencing of promoter regions. A novel 132-kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele-specific APC expression. No promoter-specific point mutations or hypermethylation were present in any family. In conclusion, promoter-specific alterations are a rare cause for mutation-negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele-specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations.

Pseudoexons provide a mechanism for allele-specific expression of APC in familial adenomatous polyposis

“…The families were associated with ASE imbalance of APC by SNuPE but no identifiable causative change in APC had been detected by PTT, Sanger sequencing of all exons and intron/exon borders, MLPA, and promoter mutation and methylation analyses (ref. [8] and this study). Only family 85 included several affected members.…”

“…This is likely attributable to some commonly observed disadvantages of PTT, such as decreased RNA stability and assay artifacts [24]. Instead of PTT, family 163 originally underwent an exon-by-exon screen in genomic DNA [8] that, obviously, was not able to capture deep intronic mutations.…”

“…In a typical PTT design, APC exons are examined in RNA, except for the last exon that is investigated in genomic DNA. Nevertheless, over 20% of classical FAP and up to 80% of AFAP patients remain APC mutation-negative, which may be attributable to methodological shortcomings in association with particular types of mutations [5–8], nontruncating alterations with uncertain pathogenic significance [2], and susceptibility associated with other genes than APC , such as MUTYH [3], POLE and POLD [9], and AXIN2 [10]. …”

Molecular approach to genetic and epigenetic pathogenesis of early-onset colorectal cancer

“…Although methylation caused the loosing of 1A isoform mRNA and a reduction in total APC transcript levels, APC gene expression was retained from promoter 1B [83] . Moreover, a recent study of Pavicic demonstrated that promoter 1B deletions of APC are not very common [84] . Thus, all these studies imply that > T were determined as associated with the FCCTX phenotype [61] .…”

“…Germline MMR gene mutations -Lindor et al [51] Klarskov et al [52] Sánchez-Tomé et al [53] Tumor supressor gene loci loss APC mutations 77% Francisco et al [56] KRAS mutations 46% Francisco et al [56] MGMT methylation 36% Francisco et al [56] Chromosome gains 20q, 19 and 17 Therkildsen et al [57] Chromosome loss 8p, 15, 18 Therkildsen et al [57] Signaling by G protein coupled receptor up-regulated Dominguez-Valentin et al [58] ( even though APC promoter methylation occurs in early during colon neoplasia progression, it does not result in complete gene inactivation or act as a "second hit" [84] and promoter-specific alterations of APC rarely leads to mutation-negative FAP [84] . In addition to APC, hypermethylation of other genes are usually observed in both FAP-related and sporadic duodenal carcinomas [85] .…”