All studies were performed with Institutional Review Board approval at Massachusetts General Hospital. Prior to any study procedures, written informed consent was obtained from the study patient. Each patient underwent a physical examination, standardized interview, 12-lead electrocardiogram, echocardiogram, and laboratory studies including a TSH. Patients in sinus rhythm had a signal-averaged electrocardiogram of the P wave (PHiRes, GE, Inc.). Three individuals with a noise level exceeding 1.0 μV on the signal averaged electrocardiogram were excluded from further analysis (Subjects III-5, III-7 and III-8).
We defined affected individuals as those with electrocardiographically documented AF. We defined as unaffected only those individuals greater than 40 years of age, with no personal history of AF, and no offspring with a history of AF. All other family members were classified as unknown for the purpose of the genetic analyses.
A blood sample (16 ml.) was obtained in acid citrate dextrose from every patient and DNA was isolated using the Puregene DNA Purification Kit (Gentra Systems, Inc., Minneapolis, Minnesota). Genetic analyses were performed on all available individuals, regardless of affection status. Polymorphic genomic short tandem repeat markers were used for mapping and amplified by PCR using standard conditions. Genotypes were ascertained without knowledge of clinical status. Oligonucleotide primers for the coding region of KCNQ1 were designed using the known cDNA and genomic sequence. PCR was performed using standard conditions, amplicons were purified, and DNA was sequenced using the ABI PRISM dye terminator method (Model 3730XL, Applied Biosystems, Foster City, California).
Two point and multipoint logarithm of the odds (LOD) scores were calculated assuming a disease penetrance of 0.95. Allele frequencies were estimated from the population. Two point LOD scores were calculated with the MLINK program. Continuous variables were tested for normality of distribution and two-sided t-tests were used for comparisons of means. P values of 0.05 or less were considered significant. All statistical analyses were performed using STATA version 10.0 (StataCorp LP, College Station, Texas).
Mutagenesis, Cloning, and Cell culture
Mammalian expression vectors pCI-KCNQ1 and pCI-KCNQ1-EGFP were obtained courtesy of Y.H. Chen11
and M. Horie15
, respectively (KCNQ1 reference sequence AF000571). Mutations in KCNQ1 were introduced into the wildtype KCNQ1 constructs using site directed mutagenesis (QuikChange kit, Stratagene, La Jolla, California) and the following primers: S209P Forward: CATCGTGGTCGTGGCCCCCATGGTGGTCCTCTG, S209P Reverse: CAGAGGACCACCATGGGGGCCACGACCACGATG, S209F Forward: CGTGGTCGTGGCCTTCATGGTGGTCCTCTGC, S209F Reverse: GCAGAGGACCACCATGAAGGCCACGACCACG. After introduction of the mutation, the coding region was sequenced to exclude the introduction of additional variants.
The coding region of KCNE1 (NM 000219) was amplified from human right ventricle cDNA (BD Clontech, Mountain View, California) by PCR reaction with primers: 5' hKCNE1: TGAGCCGAGGATCCATTGGAGGAAGG, 3' hKCNE1: GATCGCGGCCGCGGATGTGTCCAGTTTTAGCCAGTGGTGG. The 5' BamHI and 3' Not I fragment was ligated into pXOOM and pcDNA3 mammalian expression vectors16
for expression under a CMV promoter.
COS-7 cells were transfected with 4 μg of KCNQ1-WT, S209P or S209F and 1 μG of pXOOM-KCNE1 using SuperFect transfection reagent (Qiagen Inc. Valencia, California). GFP positive cells were identified for electrophysiologic recording 2–3 days after transfections. Signals were amplified using an Axon 200B amplifier (Molecular Devices, Inc., Sunnyvale, CA), digitized with a Digidata 1322A A/D converter (Molecular Devices, Inc., Sunnyvale, CA) and analyzed with Clampex 9.2, Clampfit 9.2 (Molecular Devices, Inc., Sunnyvale, CA) or SigmaPlot 9.0 (Systat Software Inc., Point Richmond, CA). Specific recording protocols are provided as an inset in each figure. All experiments were performed at room temperature. Solutions used were as follows: extracellular solution 145 mM sodium gluconate, 4 mM potassium gluconate, 7 mM calcium gluconate, 4 mM magnesium gluconate, 5 mM HEPES, 5 mM glucose, 20 mM mannitol pH 7.4; intracellular solution was 145 mM potassium gluconate, 5 mM HEPES, 2 mM EGTA, 2 mM magnesium gluconate, pH 7.2.
The proportion of current due to constitutive, voltage independent current was calculated by taking the ratio of the instantaneously appearing current after a depolarization from a holding potential of −80mV to +80mV to the maximal current achieved at the end of the depolarization.
COS-7 cells grown on 18 mm coverslips were transfected with 2 μg of KCNQ1−GFP or S209P−GFP with or without pcDNA3−KCNE1. Thirty six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde and counterstained with DAPI. Coverslips were mounted onto glass slides and visualized using confocal microscopy using the Zeiss LSM system (Carl Zeiss MicroImaging, Inc. Thornwood, New York). Images were acquired digitally at the same gain and magnification for each condition. Cells were scored for membrane staining versus intracellular staining in 3 separate views for each transfected construct in a blinded fashion.