Introduction Genetic mutations in KCNQ2 which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause Long QT Syndrome, an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed Long QT Syndrome are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest. Methods and Results Here, we demonstrate through electrophysiology techniques and immunochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of two previously reported inadvertent cDNA variants on hERG channel expression and half-life. Conclusion Our results underscore the importance of careful characterization of the impact of epitope fusion tags and conformational sequencing prior to genotype-phenotype studies for ion channel proteins such as hERG.
Synonymous nucleotide variation is increasingly recognized as a factor than can affect protein expression, but the underlying mechanisms are incompletely understood. Here, we investigated whether synonymous changes could affect expression of the potassium voltage-gated channel subfamily H member 2 () gene, encoding the human ether-a-go-go-related gene (hERG) ion channel, which is linked to hereditary cardiac arrhythmia. We examined a previously described synthetic version (hERG-codon modified (CM)) with synonymous substitutions designed to reduce GC content, rare codons, and mRNA secondary structure relative to the native construct (hERG-NT). hERG-CM exhibited lower protein expression than hERG-NT in HEK293T cells. We found that the steady-state abundance of hERG-NT mRNA was greater than hERG-CM because of an enhanced transcription rate and increased mRNA stability for hERG-NT. Translation of hERG-CM was independently reduced, contributing to the overall greater synthesis of hERG-NT channel protein. This was partially offset, however, by a higher aggregation of a newly synthesized hERG-NT channel, resulting in nonfunctional protein. Regional mRNA analyses of chimeras of hERG-NT and hERG-CM revealed that synonymous changes in the 5' segments of the coding region had the greatest influence on hERG synthesis at both the mRNA and protein levels. Taken together, these results indicate that synonymous nucleotide variations within the coding region, particularly in the 5' region of the hERG mRNA, can affect both transcription and translation. These findings support the notion that greater attention should be given to the effects of synonymous genetic variation when analyzing hERG DNA sequences in the study of hereditary cardiac disease.
Background: Most late-stage breast cancer occurs in those who have never been screened. Uninsured women have lower screening rates than insured women. Student-run free clinics (SRFCs) caring for uninsured women can test interventions that attempt to increase screening rates.Methods: The United States Preventive Task Force guidelines were used to determine patient eligibility for screening mammography in a SRFC. Medical students began receiving education on screening mammography on May 31, 2013 while a simultaneous intervention to streamline workflow related to mammography referrals was implemented. We assessed the change in counseling and referral rates pre- and post-intervention as well as the impact of race/ethnicity, chronic disease status, and preferred language on the likelihood of getting counseled and referred for screening mammography. We also investigated the impact of the intervention on screening mammography attendance rates.Results: We collected data from the medical records of 106 women (171 patient visits) pre-intervention and 113 women (193 patient visits) post-intervention. The intervention significantly improved the rate of mammography counseling (from 54.4% to 81.9%) and mammography screening referrals (from 37.7% to 70.6%). Preferred language, race/ethnicity, and chronic disease status were not associated with counseling and referral rates. The intervention did not improve mammography attendance.Conclusions: An education and workflow intervention in a SRFC that improved counseling and referral rates did not improve mammography attendance. Further studies are needed to investigate patient-level barriers that may be affecting mammography appointment attendance.
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