Ethics, R&DGenetic testing

Testing function of genetic variant in Human cells

Functional testing in Ion Channel Arrhythmias

Fluorescent antibodies

Long QTdelayed repolarization of the heart and increases the risk of episodes of torsades de pointes

LQT-

Brugada syndrome-

The Ion channel genes that can cause LQT or Brugada Syndrome:

KCNQ1KCNH2SCN5AKCNE1KCNE2

70-80% of LQT mutations are in these genes &20% of Brugada mutations are in SCN5A

The flux of ions across membranes is responsible for the generation of the action potential

}} Β-subunits

KCNQ1 (676aa, 74.7kDa)

KCNE1 (129aa 14.7kDa)

SCN5A (2016 aa, 227kDa)

KCNH2 (1159aa, 126.7kDa)

Construct wildtype

Action potential

SCN5A + Nav1.5

KCNH2 - Kv11.1 (I kr)

KCNQ1 - Kv7.1 (Iks)

The model

To express channels of interest in human cells Flp-In 293using vector pcDNA5/FRT/V5-His TOPO

Except KCNE1 in mammalian blasticidin vector

Transfection into Flp-In cell system1. Allows integration and expression of your gene of

interest in mammalian cells at a specific genomic location.

2. Involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice (Flp-In 293).

3. The gene of interest is then integrated into the genome via Flp recombinase mediated DNA recombination at the FRT site (O'Gorman et al., 1991).

Site specific mutagenesis

MutagenesisKnown 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. 1. R1623Q results in a gain of function

related to LQT3 2. S910L results in a loss of function

related to Brugada syndrome

*KCNE1-wild type transfected as beta-subunit for KCNQ1

Confocal MicroscopyKCNQ1 RedV5 Green Nuclear Blue

Potassium fluxOR assay

Acknowledgments

Julian SampsonDhavendra Kumar

Chris GeorgeAlan WilliamsSam Mason

Peter O’CallaghanCath Owen