Accurate pre-mRNA splicing is essential for the conversion of nascent transcripts to mRNA templates for protein synthesis. Pre-mRNA splicing depends upon the recognition of the 5' and 3' splice sites as well as the branch point. These sequences appear to be vulnerable to disease-causing mutations. It has been estimated that up to 15% of all point mutations causing human genetic diseases result in an mRNA splicing defect [15
]. The 2398+1G>C mutation in hERG has been identified in three unrelated kindreds with long QT syndrome [1
]. This mutation involves the +1 position of the 5' donor splice site of intron 9. When the 2398+1G>C mutation was first identified, it was predicted to result in protein truncation due to potential abnormal splicing [1
]. However, our present analysis at the mRNA level demonstrates that the 2398+1G>C mutation leads to the activation of a cryptic site 54 bp downstream from the normal site of intron 9. Thus, rather than protein truncation this mutation results in the production of a full-length channel with the insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. This is the first report that a splice site mutation in hERG results in the utilization of a cryptic splice site and leads to a large in-frame insertion in the hERG channel. Our findings underscore the importance of studying LQT2 splice site mutations at the mRNA level.
Using RNA isolated from the lymphocytes of the patients carrying the 2398+1G>C mutation, we were able to show that the endogenous mutant RNA undergoes cryptic splicing. The use of RNA from lymphocytes for studying splice site mutations has been reported in several human heart diseases including KCNQ1 in LQT1 and SCN5A in Brugada syndrome [23
]. One limitation of using lymphocytes is that the aberrant splicing observed in lymphocytes may not be present in the heart. In the present study we have used a minigene system to demonstrate that the same cryptic site as that observed in lymphocytes was used in neonatal rat ventricular myocytes. This result strongly suggests that the cryptic splicing remains in the mammalian ventricular myocyte and is likely to occur in the human heart of subjects carrying the mutation. Our present findings also show that lymphocytes may be a convenient source of endogenous hERG mRNA for studying other LQT2 splice site mutations when the affected heart tissues are not available.
In general, mutation of a splice site can result in one or a combination of splicing defects. These defects include the failure to recognize the affected exon resulting in exon skipping, activation of a cryptic site, and inability to recognize the affected intron resulting in whole intron retention. For the 2398+1G>C mutation, the activation of a cryptic site in intron 9 might be due to the intrinsic strength of the cryptic site. The sequence of the cryptic 5' splice site in intron 9 is GAG/gtgcga. We calculated the consensus value (CV) scores of the normal, mutant and cryptic splice sites using the method described by Shapiro and Senapathy [28
]. The consensus value reflects the similarity of a splice site to the consensus sequence, and most splice donor sites have a score of 70 or higher. The CV scores of WT, 2398+1G>C and the cryptic splice site are 76.3, 58.0 and 79.6, respectively. Thus, the cryptic splice site has an intrinsic strength comparable to that of the normal splice site of intron 9. However, in normal conditions the cryptic site is completely inactive even though its CV score is slightly higher than that of the normal site. The cryptic site is activated when the normal site is disrupted by the mutation with a dramatic decrease in the CV score.
The functional studies indicate that the insertion of 18 amino acids in the cyclic nucleotide binding domain results in defective trafficking of the mutant hERG channel. Defective trafficking of mutant hERG channels has been shown to be the most common mechanism in LQT2 missense mutations [22
]. These trafficking deficient hERG mutants are retained in the endoplasmic reticulum due to prolonged association with chaperone proteins calnexin, Hsp70 or Hsp90, and are rapidly degraded by the ubiquitin proteasome pathway [29
]. It is noted that all LQT2 mutations in the cyclic nucleotide binding domain lead to defective trafficking of mutant channels [22
]. The large insertion caused by the cryptic splicing involves a highly ordered structure in β5 region of the cyclic nucleotide binding domain, which has been shown to play an important role in hERG channel trafficking [32
]. The present results also show that coexpression of mutant and WT channels leads to a dominant negative suppression of WT channel function by intracellular retention of heteromeric channels. The presence of the dominant negative effect is consistent with the observed clinical presentation of the patients carrying the 2398+1G>C mutation. Four of seven mutation carriers had syncope episodes. Previous phenotype-genotype correlation studies have shown that patients with mutations in the pore region of the hERG gene are at markedly increased risk for arrhythmia-related cardiac events compared with patients with nonpore mutations [33
]. One of the families (family 2) in the present study was also included in that report. Although this family was assigned to the nonpore mutation group, the mutation carriers from this family appear to have a higher risk for arrhythmia-related cardiac events than other patients with nonpore mutations. Three of five mutation carriers in this family had syncope episodes and one of them developed cardiac arrest. Two patients in this family required ICD therapy. Our present study suggests that the severe clinical phenotype of this family might be explained by the presence of the dominant negative effect observed in the 2398+1G>C mutation.
In conclusion, the 2398+1G>C mutation disrupts the normal splicing and leads to the activation of a downstream cryptic site. The cryptic splicing results in a full-length hERG channel with an insertion of 18 amino acids, leading to a trafficking defect of the mutant channel. These findings indicate that functional analyses at both mRNA and protein levels are important for obtaining a full understanding of putative splice site mutations in hERG. The identification of the pathogenic mechanisms of hERG mutations will facilitate the studies of genotype-phenotype relationship in LQT2.