Position of premature termination codons determines susceptibility of hERG mutations to nonsense-mediated mRNA decay in long QT syndrome

• Recognition of PTC-containing hERG transcripts by NMD machinery is studied.
• Positional requirements governing NMD susceptibility of LQT2 PTCs are identified.
• LQT2 PTC recognition and elimination by NMD require downstream intron.
• LQT2 PTCs > 54–60 nt upstream of the 3′-most exon–exon junction trigger NMD.
• The majority of reported LQT2 nonsense and frameshift mutations are targets of NMD.

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs*25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned > 54–60 nt upstream of the 3′-most exon–exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078*. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD.