Mutations at the Intron 9 Donor Splice Site in hERG Lead to Cryptic Splicing in LQT2

Abstract

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of LQT2 mutations are nonsense, frameshift, or splice site mutations that may affect mRNA stability and splicing. To date, relatively few studies have focused on the pathogenesis of hERG splice site mutations. We characterized three LQT2 mutations in the 5′ donor splice site of intron 9: 2398G>T, 2398+3A>T, and 2398+5G>T. G2398 is the last nucleotide of exon 9 and 2398G>T has been previously classified as a missense mutation (G800W). The functional consequences of these mutations were studied by RT-PCR analysis of RNA collected from HEK293 cells transfected with minigenes containing the wild-type or mutant genomic sequence spanning exon 8 to exon 11 of hERG. All three splice site mutants disrupt normal splicing and produce an aberrantly spliced transcript. Sequence analysis showed that this transcript results from the use of a cryptic 5′ donor splice site in intron 9 located 54 nt downstream of the normal site. Translation of this transcript would result in an in-frame insertion of 18 amino acids in the cyclic nucleotide binding domain. A full length hERG cDNA construct including the 2398G>T mutation and the additional 54 nt from intron 9 was expressed in HEK293 cells. Patch clamp studies revealed that the splice mutant channels did not produce hERG current. Western blot analysis showed that the mutant expressed the immature form of the hERG protein indicating defective channel trafficking. These studies underscore the importance of RNA analysis in describing the pathogenesis of LQT2. The intron 9 donor splice site appears to be a localized hot-spot for LQT2 mutations.