Download Cardiovascular – Dr Sharon Coleman

Functional testing in Ion Channel Arrhythmias
Ethics, R&D
Genetic testing
Fluorescent antibodies
Testing function
of genetic variant
in
Human cells
Long QT
LQT- delayed repolarization of the heart and increases the
risk of episodes of torsades de pointes
Brugada syndrome-
The Ion channel genes that can cause LQT or Brugada Syndrome:
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KCNQ1
KCNH2
SCN5A
KCNE1
KCNE2
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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
Construct wildtype
KCNQ1 (676aa, 74.7kDa)
KCNE1
(129aa 14.7kDa)
SCN5A (2016 aa, 227kDa)
KCNH2 (1159aa, 126.7kDa)
Action potential
SCN5A +
Nav1.5
KCNQ1
-
Kv7.1 (Iks)
KCNH2
-
Kv11.1 (I kr)
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
Transfection into Flp-In cell system
1. 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
Mutagenesis
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.
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 betasubunit for KCNQ1
Confocal Microscopy
KCNQ1 Red
V5 Green
Nuclear Blue
Potassium fluxOR assay
Acknowledgments
Julian Sampson
Dhavendra Kumar
Chris George
Alan Williams
Sam Mason
Peter O’Callaghan
Cath Owen