Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Abstract

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.

Assessing the Disease-Liability of Mutations in CFTR

“…Measurements of CFTR function in vivo in two R1162X homozygotes were similar to those recorded in 74 F508del homozygotes (Stanke et al 2008). Another exception is the mutation E831X ( p.Glu831X) that occurs at the 5 0 splice site of exon 14a (Hinzpeter et al 2010). The mutation alters splicing of CFTR such that three nucleotides encoding the mutant nonsense codon are omitted and a protein is synthesized missing the glutamic acid residue at codon 831.…”

“…The mutation alters splicing of CFTR such that three nucleotides encoding the mutant nonsense codon are omitted and a protein is synthesized missing the glutamic acid residue at codon 831. Although the resulting protein is stable, it has reduced function and is associated with CF, albeit a milder pancreatic sufficient form (Hinzpeter et al 2010). Thus, PTC mutations either cause a loss of the RNA transcript and absence of protein or, in a few rare cases, cause stable RNA transcript but a modified protein that is either degraded or malfunctional.…”

Genetics of cystic fibrosis: CFTR mutation classifications toward genotype-based CF therapies

“…Skipping of the PTC harboring exon should also be taken into account as it will also reduce the amount of target transcripts (Hinzpeter et al, 2013). Indeed, taking into account these two parameters, it appeared that 8% of the CFTR transcripts would be targeted by readthrough therapy in a patient homozygous for the p.Glu831X mutation, which would be predictive of poor treatment efficiency (Hinzpeter et al, 2010).…”

The Cystic Fibrosis Gene: A Molecular Genetic Perspective

“…For some mutations, however, the resulting mRNA species with exon skipping can persist and produce a dysfunctional protein (Hull et al 1994). In the case of the c.2491G.T ( p.Glu831X, also known as 2623G.T, E831X) variant, the presence of an alternative spliced mutant transcript may explain a mild disease presentation and pancreatic sufficiency in patients carrying this nonsense mutation (Hinzpeter et al 2010).…”

“…Based on studies with minigene constructs, Hinzpeter et al (2010) have proposed that the c.2491G.T mutation not only introduces a premature stop codon, but also creates a binding site (CAGTAG) for the splicing factor that recognizes the consensus sequence NAGNAG. The study shows that the mutation can lead to the production of three different mRNAs: CFTR_831-873del, CFTR_E831X, and CFTR_ E831del, and, unexpectedly, the latter transcript could bypass the premature termination codon and produce a functional p.E831del protein.…”