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.

Keywords: APC; RNA-seq; allele-specific expression; familial adenomatous polyposis; pseudoexon.

Figure 1. Pedigrees of ASE families

Pedigrees of adenomatous polyposis families with ASE. Individuals with polyposis and/or colorectal cancer are indicated (see Table 1 for additional clinical details). Plus sign denotes carriers of deep intronic mutations associated with pseudoexons of APC. Index persons are marked with arrows.

Figure 2. RNA-seq (42, 85-2, and 163)

Sashimi plots to visualize splice junctions. IGV display of RNA-seq data is provided for an affected representative of each family and a healthy control individual for reference for each region. Sequence alignments are based on TopHat. The region between APC exons 6 and 7 (GRCh37/Hg19) is shown for FAP42 (Figure 2A) and that between exons 11 and 12 for FAP85 (individual 85-2) and AFAP163 (Figure 2B). The locations of pseudoexons are indicated by horizontal bars. A 54-bp in-frame insertion present in the normal reference sample, too, and not associated with any genomic change is denoted by a dashed bar (Figure 2B). The same insertion was discovered in an earlier investigation [12]. Numbers on the plots indicate APC exon coverages expressed as junction depth. Splice events corresponding to pseudoexons are boxed and those associated with the 54-bp insertion are underlined; the remaining ones represent canonical splicing.

Figure 3. RT-PCR (42, 85-1, 163, 103)

RT-PCR analysis of samples from ASE families. RT-PCR products separated by gel electrophoresis are shown. Arrows denote fragments with intronic insertions (pseudoexons). Fragment 2 (upper panel) encompasses a 615-bp cDNA segment from exon 4 to exon 9 [6] and shows a heterozygous 127-bp insertion in family 42. The wild-type size of the exon 11 - exon 13 fragment (lower panel) is 246 bp (Supplementary Table S1). An identical 83-bp insertion in families 85 (case 85-1) and 163 is evident. The RT-PCR products from the index persons and healthy controls were cloned and sequenced to verify their DNA sequences. In the exon 11 - exon 13 fragment, a 54-bp in-frame insertion (see legend for Figure 2) accompanied the pseudoexon and wild-type sequences in a proportion (up to one-third) of all clones and likely contributed to the multiplicity of fragments seen after gel electrophoresis.

Figure 4. WGS (42 and 85-2) + Sanger seq

Deep intronic mutations in APC. Upper panels provide IGV display of WGS data for intron 6 in FAP42 (Figure 4A) and intron 11 in FAP85, individual 85-2 (Figure 4B). Lower panels show Sanger sequence tracings of the mutations. In Figure 4B, the Sanger sequencing result of AFAP163 is also given (AFAP163 was not included in WGS analysis).

Figure 5. Schematic diagrams of pseudoexons

Schematic diagrams of APC pseudoexons identified. The canonical splice sites at the exon/intron borders, pseudoexons (underlined), and the responsible deep intronic mutations (in bold) are highlighted.