Pseudoexons may underlie unbalanced expression of APC alleles in familial adenomatous polyposis

NIEMINEN T.; JÄRVINEN HEIKKI J.; PAVICIC W.; LEPISTÖ ANNA; PORKKA NOORA; PELTOMÄKI P.

Congreso; AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA; 2016

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC gene. Clinically, the disease can be subdivided into classical FAP with hundreds or thousands of polyps throughout the colon and rectum and Attenuated Familial Adenomatous Polyposis (AFAP) with a milder polyposis. Conventional screening protocols fail to detect any APC mutation in a considerable fraction of adenomatous polyposis families, especially those representing AFAP. We investigated 56 FAP and AFAP families from Finland that had remained APC mutation-negative after protein truncation test, exon-specific Sanger sequencing, and multiplex ligation-dependent probe amplification. Of particular interest were four families with constitutionally unbalanced mRNA expression of APC alleles by Single Nucleotide Primer Extension. These families were interrogated by next generation sequencing of the whole genomes (WGS) and RNA (RNA-seq); additionally, APC promoter methylation was addressed by methylation-specific multiplex ligation-dependent probe amplification. RNA-seq raised the suspicion of a pseudoexon (erroneous inclusion of intronic sequence in the mature mRNA) in three families. A pseudoexon between exons 5 and 6 of the APC gene was present in one family and a pseudoexon between exons 10 and 11 in two families. By WGS, the exon 5-6 pseudoexon consisted of c.646-1806 T>G, which generated a cryptic splice site leading to a 127-bp intronic insertion in the APC mRNA. This pseudoexon was associated with classical FAP. Two alternative genetic changes underlay the exon 10-11 pseudoexon: c.1408+729 A>G was present in one family (AFAP) and c.1408+731 C>T in another family (FAP); both created a new splice donor site leading to an identical 83-bp insertion in APC mRNA. The exon 10-11 pseudoexon resulting from c.1408+729 A>G has previously been described in 2 unrelated German patients with FAP; the remaining two pseudoexon-associated mutations are novel. All three pseudoexons were predicted to cause frameshifts and premature stop codons leading to APC protein truncation. Sanger sequencing was used to confirm the mutations and to test their presence in the entire series of 56 families; no additional mutation-positive cases were detected. Taken together, deep intronic mutations of the APC gene may explain a proportion of FAP and AFAP families that screen mutation-negative by traditional methods. Specifically, such mutations accounted for three out of four families with the unbalanced expression phenotype of APC in our study. We further conclude that RNA-seq is an effective method to reveal deep intronic mutations and is worth considering in apparent mutation-negative families.