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

Abstract

Allele-specific expression (ASE) of the Adenomatous Polyposis Coli (APC) gene occurs in up to one-third of families with adenomatous polyposis (FAP) that have screened mutation-negative by conventional techniques. To advance our understanding of the genomic basis of this phenomenon, 54 APC mutation-negative families (21 with classical FAP and 33 with attenuated FAP, AFAP) were investigated. We focused on four families with validated ASE and scrutinized these families by sequencing of the blood transcriptomes (RNA-seq) and genomes (WGS). Three families, two with classical FAP and one with AFAP, revealed deep intronic mutations associated with pseudoexons. In all three families, intronic mutations (c.646-1806T>G in intron 6, c.1408+729A>G in intron 11, and c.1408+731C>T in intron 11) created new splice donor sites resulting in the insertion of intronic sequences (of 127 bp, 83 bp, and 83 bp, respectively) in the APC transcript. The respective intronic mutations were absent in the remaining polyposis families and the general population. Premature stop of translation as the predicted consequence as well as co-segregation with polyposis supported the pathogenicity of the pseudoexons. We conclude that next generation sequencing on RNA and genomic DNA is an effective strategy to reveal and validate pseudoexons that are regularly missed by traditional screening methods and is worth considering in apparent mutation-negative polyposis families.

Biallelic germline nonsense variant of MLH3 underlies polyposis predisposition

“…1a. In the first phase, blood DNAs from 40 unrelated cases/families with adenomatous polyposis from Finland, without any detectable pathogenic sequence changes in known polyposis-associated genes 2 and in most cases sporadic at presentation, were submitted to exome sequencing (ES). Based on polyp count (with 100 polyps as a divider), 14 represented FAP and 26 AFAP.…”

“…1 ). Significant proportions of polyposis cases (10–80% depending on polyp count) remain molecularly unexplained, 2 encouraging searches for novel susceptibility genes.…”

“…To this end, we undertook an exome sequencing study of 40 index cases from a cohort with adenomatous polyposis from Finland in which pathogenic variants in known polyposis-associated genes had been excluded. 2 Results from this cohort, supplemented with panel sequencing data from some 1000 cases with various colorectal phenotypes from Sweden, unravel a novel polyposis syndrome associated with biallelic germline nonsense variant of MLH3 (MutL Homolog 3).…”

Candidate Gene Discovery in Hereditary Colorectal Cancer and Polyposis Syndromes–Considerations for Future Studies

“…Moreover, WGS of patient cohorts might facilitate discovery of missed non-coding variants in known hCRC and polyposis genes. In the past, deep-intronic and promoter variants were described in tumor suppressor genes APC and PTEN , which makes sequencing of these non-coding regions of particular interest for unresolved hCRC and polyposis patients [ 98 , 99 , 100 , 101 , 102 , 103 ]. Long-read sequencing and optical mapping techniques might be valuable as well, as these techniques are specifically directed to the detection of complex and structural variants, and allow alignment and variant mapping in regions that used to be uncovered in the past due to their nucleotide composition (e.g., extreme GC-rich, and multiple short repeats) [ 104 , 105 ].…”

Current status of the genetic susceptibility in attenuated adenomatous polyposis

“…Thanks to the advance of sequencing technologies, new genes have been recently associated with primary predisposition to the development of adenomas by genome/exome sequencing studies in unexplained AP cohorts[13-16]. In the same way, other genetic alterations not detected by conventional coding germline DNA sequencing screening strategies have also been described in the APC gene, such as mutations in the promoter[17] or introns[18], large inversions[19] or mosaicism phenomes[20]. In addition, the use of wide gene panels for the genetic diagnosis of AP has incidentally revealed an overlap between different polyposis syndromes[21].…”