Informed written consent was obtained in accordance with study protocols approved by the Mayo Foundation Institutional Review Board. Between August 1997 and July 2004, 541 consecutive, unrelated patients with a suspected clinical diagnosis of congenital long QT syndrome (LQTS) were referred to Mayo Clinic’s Sudden Death Genomics Laboratory for molecular genetic testing9
. See for characteristics of the total cohort. Comprehensive mutational analysis of all 60 translated exons of the 5 cardiac channel LQTS-associated genes (LQT1-3, 5–6) and targeted analysis of ANKB-
associated LQT4 and RyR2-
associated catecholaminergic polymorphic ventricular tachycardia (CPVT) was performed previously9,10,11
. The investigation conforms with the principles obtained in the Declaration of Helsinki 12
Phenotypic comparison of LQTS gene-positive, LQTS gene-negative, RyR2-positive, and KCNJ2-positive cohorts
KCNJ2 Mutational Analysis
Comprehensive mutational analysis of KCNJ2 was performed using polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and DNA sequencing. DNA amplification of the entire single exon coding region of the KCNJ2 gene was conducted using five overlapping fragments with PCR primers designed using Oligo Primer Analysis Software version 6.63 (Molecular Biology Insights, Inc., Cascade, Colorado) (primers, PCR and DHPLC conditions are available upon request). Control genomic DNA, comprised of 100 healthy white and 100 healthy black subjects, was obtained from the Human Genetic Cell Repository sponsored by the National Institute of General Medical Sciences and the Coriell Institute for Medical Research (Camden, New Jersey).
The investigators constructing KCNJ2 mutations, performing and analyzing cell trafficking and electrophysiological experiments, were blinded to the clinical data until after the experiments were completed and analyzed.
KCNJ2 construction and mutagenesis
Wild-type (WT) human Kir2.1 was isolated from human cardiac cDNA using PCR, forward primer atgggcagtgtgcgaaccaac and reverse primer tcatatctccgactctcgccgtaagg. Sequence integrity was verified with sequence analysis. KCNJ2 mutations were constructed using the Stratagene ExSite site directed mutagenesis kit using the following primers: for R67Q forward agtacctcgcagacatcttca and reverse gttgccccttctcacccac, T75M forward ccatgtgtgtggacattcgct and reverse tgaagatgtctgcgaggtacc, R82W forward gtggtggatgctggttatcttct and reverse cagcgaatgtccacacacgt, T305A forward gctgccatgacgacacagtg and reverse ggcttccaccatttgccttc. WT and mutant DNA was sub-cloned into mammalian expression vector pcDNA3.1 (Invitrogen). The WT and mutant DNA were also sub-cloned into pcDNA3.1-NT-GFP-TOPO vector for sub-cellular localization. All constructs were verified by sequence analysis.
Transfection and cell culture
COS-1 cells were cultured in DMEM (Invitrogen) with 10% FBS. For electrophysiological experiments, cells were transiently transfected using SuperFect method (Qiagen) using 2.5μg of WT and/or mutant KCNJ2 both with green fluorescent protein cDNA in a pRK5 vector (GFP-pRK5, Clonetech, Palo Alto, CA). For fluorescence studies, WT or mutant KCNJ2 DNA in pcDNA3.1-NT-GFP-TOPO were transfected using the SuperFect into COS-1 cells using 2.5μg DNA. After 48 hours, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min.
Patch clamp experiments were carried out 24 hours after transfection using the ruptured patch whole cell technique at room temperature and recorded with an Axopatch 200B amplifier. Borcillica glass pipettes were pulled to resistances of 2–4 MΩ. Cells were identified by GFP fluorescence under fluorescent microscopy (Olympus Optical Corp). Bath solution contained (mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 0.5, HEPES 5, NaH2PO4 0.33, D-glucose 5.5 and pH adjusted to 7.4. Pipette solution contained (mM): KCl 30, K Aspartate 85, MgCl2 5, KH2PO4 10, K2EGTA 2, K2ATP 2, and HEPES 5 and pH adjusted to 7.2. Control experiments in WT were done contemporaneously with the mutations. From a holding potential of −70mV, voltages were applied from −140 to 40mV in 20mV increments for 100ms. Data were filtered at 10kHz and digitized using a Digidata 1200 (Axon Instruments). Analysis of data was done using pClamp 8 and Origin 6.1.
Cells obtained 24 hours after transfection were fixed to coverslips with 4% paraformaldehyde and washed with PBS. The fixed cells were washed again with PBS and the coverslips containing the cells were mounted on slides using a 50% glycerol/50% PBS solution. A Bio-Rad MRC 1024 laser scanning system with 15 mW mixed gas (krypton/argon) laser was utilized to view GFP labeled cells. The Bio-Rad MRC 1024 system was mounted on a Nikon Diaphot 200 inverted microscope. Images of the fluorescent-labeled cells were scanned under a x40 objective with normal speed, x2 zoom. The confocal system was set to 3.6 for iris, laser power at 100%, and camera sensitivity gain to 900. A Kalman collection filter with three frames per image was applied to record the image.