Synonymous Nucleotide Modification alters hERG synthesis
Osterbur Badhey, Marika L
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Synonymous variation, a change at the gene level that does not affect the protein sequence, has long been considered functionally silent. However, recent evidence demonstrates that synonymous variations are phenotypically important. These variations can impact transcription and translation through mechanisms other than amino acid change Synonymous variation can cause changes in codon frequency, mRNA structure, mRNA stability, and translation rate, leading to changes in protein quantity, folding and function. Recognition that synonymous variation may impact in human genetic disease is relatively new and may be important in the era of precision medicine. KCNH2 is an interesting candidate gene in which to investigate the role of synonymous variation. Pathogenic variants in KCNH2 are the cause of 25-30% of Long QT Syndrome, a disease known for causing sudden cardiac death due to changes in cardiac electrophysiology. KCNH2 encodes the alpha subunit of the potassium voltage-gated channel subfamily H, member 2, known as hERG, which tetramerizes and forms a potassium ion channel responsible for the IKr current. This current is essential for cardiac repolarization after the heartbeat. Synonymous variation in this gene as cause for LQT2 is of potential interest for two reasons: (1) Although there are numerous genetic loci in hereditary LQTS, approximately 20% of clinically diagnosed patients, have no identifiable genetic mutations within these loci. (2) KCNH2 is an unusual gene in that the coding region exhibits 66% GC content and has a high frequency of rare codons (<10% usage). Evolutionary selection of these novel features may facilitate co-translational protein folding and associations, and synonymous variation disrupting these features could potentially affect protein folding, trafficking and function. To investigate the functional impact of the unique features of the KCNH2 gene, Sroubek, et. al created a codon-modified hERG construct (hERG-CM) with reduced GC content (51%), no use of rare codons and a decrease in complementary regions in the mRNA while maintaining the native hERG protein sequence. The goal of these changes was to "optimize" the expression of the hERG protein, but actually resulted in decreased protein expression. The current study investigates impact of synonymous changes of the KCNH2 nucleotide sequence on hERG protein synthesis. The role of synonymous modification in hERG-CM on the hERG protein was investigated by assessing multiple aspects of hERG synthesis. hERG-CM was determined to have a reduced mRNA expression level due to both a decreased rate of transcription and a reduced mRNA half-life. Next, translation was assessed. There was a decreased rate of translation for hERG-CM, as noted by pulse-chase studies and ribosomal profiling. Finally, protein folding and function were assessed, and hERG-CM had significantly less protein aggregation, a longer protein half-life, and distinct electrophysiology patterns. Dissection of the regions of the construct responsible for the transcription and translational features of the hERG-CM construct revealed that the first 51 nucleotides were sufficient to determine the mRNA and protein expression differences. With the demonstration that synthetic synonymous modification resulted in changes in hERG protein folding, function and expression, we examined human variants identified in Long QT Syndrome patients. First, we characterized 7 novel synonymous variants in patients with no previously described genetic cause of their LQTS. Then, we investigated the impact of slowing the translation rate on non-synonymous variants known to cause LQT2 through a trafficking-deficient mechanism, as we have demonstrated that slowing down translation increases protein trafficking to the surface of the cell. Although synonymous variants responsible for LQTS were not identified and slowing down translation did not improve current density for trafficking-deficient variants, these investigations highlight the need for further characterization of synonymous variants and hERG trafficking mechanisms. The impact of synonymous nucleotide variation on functional expression of the clinically important hERG protein was demonstrated at every level of synthesis. Using the hERG-CM construct, transcription, translation, protein folding, and channel function were shown to be impacted by synonymous variation. The first 51 nucleotides of bERGCM and hERG-NT were sufficient to facilitate either -CM or -NT phenotype mRNA and protein expression, highlighting the importance of the 5' region of the coding sequence of hERG. This study led to the conclusion that synonymous variants have the potential to play a role in the disease mechanism of Long QT Syndrome. This work also highlights the biology of the hERG protein that is still unknown and discusses the importance understanding the synthesis and trafficking patterns of hERG, so that proper characterization of variation, both synonymous and non-synonymous, can occur.