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Ethics, R&D

Functional testing in Ion Channel Arrhythmias. Testing function of genetic variant in Human cells. Ethics, R&D. Genetic testing. Fluorescent antibodies. Long QT. LQT-. delayed repolarization of the heart and increases the risk of episodes of torsades de pointes.

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Ethics, R&D

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  1. Functional testing in Ion Channel Arrhythmias Testing function of genetic variant in Human cells Ethics, R&D Genetic testing Fluorescent antibodies

  2. Long QT LQT- delayed repolarization of the heart and increases the risk of episodes of torsades de pointes

  3. Brugada syndrome-

  4. The Ion channel genes that can cause LQT or Brugada Syndrome: } • KCNQ1 • KCNH2 • SCN5A • KCNE1 • KCNE2 70-80% of LQT mutations are in these genes & 20% of Brugada mutations are in SCN5A } Β-subunits The flux of ions across membranes is responsible for the generation of the action potential

  5. Construct wildtype KCNQ1 (676aa, 74.7kDa) KCNE1 (129aa 14.7kDa) SCN5A (2016 aa, 227kDa) KCNH2 (1159aa, 126.7kDa)

  6. Action potential SCN5A + Nav1.5 KCNQ1 - Kv7.1 (Iks) • KCNH2 - Kv11.1 (I kr)

  7. The model To express channels of interest in human cells Flp-In 293 using vector pcDNA5/FRT/V5-His TOPO Except KCNE1 in mammalian blasticidin vector

  8. Transfection into Flp-In cell system • Allows integration and expression of your gene of interest in mammalian cells at a specific genomic location. • Involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice (Flp-In 293). • The gene of interest is then integrated into the genome via Flprecombinase mediated DNA recombination at the FRT site (O'Gorman et al., 1991).

  9. Site specific mutagenesis

  10. Known gene variants identified can be introduced and compared to wildtype for RISK STRATIFICATION KCNQ1, - 11 variants constructed and 6 transfected with beta-subunit* KCNH2- only wildtype. SCN5A – 2 variants constructed and transfected. R1623Q results in a gain of function related to LQT3 S910L results in a loss of function related to Brugada syndrome *KCNE1-wild type transfected as beta-subunit for KCNQ1 Mutagenesis

  11. Confocal Microscopy KCNQ1 Red V5 Green Nuclear Blue

  12. Potassium fluxOR assay

  13. Acknowledgments Julian Sampson Dhavendra Kumar Chris George Alan Williams Sam Mason Peter O’Callaghan Cath Owen

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