- Email: [email protected]
AB1-3
Heart Rhythm (2006) 3, S1–S343
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ABSTRACTS ABSTRACT SESSION 1: BASIC/TRANSLATIONAL SCIENCE I: Genetics of Long QT Syndrome Thursday, May 18, 2006 8:00 a.m.–9:30 a.m. AB1-1 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF NOVEL CAV3-ENCODED CAVEOLIN-3 MUTATIONS IN CONGENITAL LONG QT SYNDROME Bin Ye, PhD, David J. Tester, BS, Matteo Vatta, PhD, Jonathan C. Makielski, MD and Michael J. Ackerman, MD, PhD. University of Wisconsin, Madison, WI, Mayo Clinic College of Medicine, Rochester, MN and Baylor College of Medicine, Houston, TX. Background: Approximately 75% of long QT syndrome (LQTS) is explained by pathogenic mutations scattered across three genes encoding key cardiac channel pore-forming subunits. Since the identification of LQTS-associated mutations in ANK2-encoded ankyrin B, genes encoding cardiac channel interacting proteins have become the target of candidate gene mutational analyses for genotype negative LQTS. Recently, we established CAV3 as a novel LQTS-associated gene with mutations in CAV3-encoded caveolin-3 producing a gain-of-function, LQT3-like molecular/cellular phenotype. Methods: Using PCR, DHPLC, and direct DNA sequencing, CAV3 mutational analysis was performed on a cohort of 541unrelated patients (358 females, average age at diagnosis, 24 years, and average QTc, 482 ms) who were referred to Mayo Clinic’s Sudden Death Genomics Laboratory for LQTS genetic testing. CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel. Results: In addition to the 2 patients from our original report that hosted T78M- and F97C-CAV3, we have identified 2 novel missense mutations, not seen in 400 reference alleles. K30R-CAV3 was detected in a white male diagnosed with LQTS at age 7 (QTc ⫽ 528 ms). No other LQTSassociated mutations were identified. L86P-CAV3 was discovered in a 12-year-old white male with a personal history of cardiac arrest while swimming and a negative family history for cardiac events. Comprehensive LQTS mutational analysis also identified a L273F-KCNQ1 mutation (LQT1). The functional effect of K30R- and L86P-CAV3 on the biophysical properties of stably expressed, wild type SCN5A was examined. Conclusions: Following our sentinel report of CAV3-encoded caveolin-3 as a novel pathogenetic mechanism for LQTS, we have now identified and functionally characterized 2 novel caveolin-3 mutations. Future studies should delineate whether or not the combination of LQT1 and CAV3LQTS confers synergistic heterozygosity and a distinct phenotype among family members hosting one or both variants. AB1-2 KCNJ2-S369X MUTATION CAUSES THE LOSS OF ER EXPORT MOTIF AND GENERATES MILD FORM OF ANDERSEN-TAWIL SYNDROME Takahiro Doi, MD, Hidetada Yoshida, MD, PhD, Atsushi Kobori, MD, PhD, Yoshisumi Haruna, MD, Takeru Makiyama, MD, Seiko Ono, MD, Keiko Tsuji, BSc, Yoshiaki
Takahashi, MD, Toru Kita, MD, PhD and Minoru Horie, MD, PhD. Kyoto University Graduate School of Medicine, Kyoto, Japan, Takahashi Clinic for Pediatric Cardiology, Otsu, Japan and Shiga University of Medical Science, Otsu, Japan. Objective: Mutations in KCNJ2, the gene coding the human inward rectifier K⫹ channel Kir2.1, have been identified in Andersen-Tawil syndrome (ATS). ATS is an inherited disease characterized by (1) ventricular tachyarrhythmias associated with QT prolongation in ECG, (2) periodic paralysis, and (3) dysmorphic features. We identified a novel nonsense mutation producing the truncation of Kir2.1 in the middle of C-terminus and the elimination of the ER-to-Golgi export signals (FCYENE), in a 13-year-boy with periodic paralysis and long QT interval on ECG. Biophysical assay using the heterologous expression system revealed the mechanism underlying the generation of ATS in our patient. Methods and Results: Direct sequencing of KCNJ2 gene displayed a heterozygous point mutation, 1106 C ⬎ T (S369X), leading to a premature stop codon. The mutation causes a channel subunit lacking the ER export motif. To characterize the physiological consequences of the truncated Kir2.1 channel, CHO cells were transfected with wild-type (WT) or mutant S369X-KCNJ2 (0.5g each). Cells transfected with WT-KCNJ2 resulted in robust inward rectifying currents (⫺271.1 ⫾ 42.2 pA/pF at ⫺140 mV), but those with S369X-KCNJ2 expressed significantly smaller K⫹ currents (⫺28.4 ⫾ 4.5 pA/pF). Co-expression with WT and mutant at 1:1 ratio (1g each) displayed larger currents compared with WT alone (1g) (⫺723.6 ⫾ 97.9 pA/pF vs. ⫺571.1 ⫾ 86.7, p⫽0.30). Interestingly, increasing the mutant to 2 g (1:2 ratio) increased the resultant current densities to ⫺899.2 ⫾ 81.2 pA/pF (p⫽0.02 vs. WT alone). GFP-fused WT Kir2.1 exhibited robust plasma membrane expression on confocal microscopic images, but GFP-fused S369X remained in the cytoplasma. The impaired transport of S369X was partially rescued by co-expression with full-length Kir2.1, consistent with the electrophysiological data. Conclusion: S369X⫺KCNJ2 is a trafficking-refractory mutation, but the resultant truncated subunit can assemble with the WT protein and partially function. This may explain relatively mild phenotypes in our ATS patient, who has no ventricular arrhythmias and dysmorphic features. AB1-3 MINK AND MIRP1 FORM CARDIAC POTASSIUM CHANNEL COMPLEXES WITH KV2.1 Torsten K. Roepke, MD, Zoe A. McCrossan, PhD, Anthony Lewis, PhD, Gianina Panaghie, MS and Geoffrey W. Abbott, PhD. WMC Cornell University, New York, NY. Kv2.1 is a voltage-gated potassium (Kv) channel alpha subunit expressed in mammalian heart and brain. Kv2.1 reportedly contributes to both IK, slow and Iss in murine heart, and has been detected in human heart although a native current correlate there has not yet been established. MinK-related peptides (MiRPs), encoded by KCNE genes, are single transmembrane domain ancillary subunits that form complexes with Kv channel alpha subunits to modify their function. Mutations in MinK and MiRP1 are associated with inherited long QT syndrome (LQTS) in man.
1547-5271/$ -see front matter © 2006 Heart Rhythm Society. All rights reserved.
S2 Here, we show using native co-immunoprecipitations that both MinK and MiRP1 form native Kv channel complexes with Kv2.1 in rodent heart. In heterologous co-expression studies, human MiRP1 slowed Kv2.1 activation and deactivation, and increased inactivation. Despite complex formation, human MinK had no significant effects on Kv2.1 function, but rat MinK slowed Kv2.1 activation and deactivation, and increased inactivation. Inherited mutations in human MinK or MiRP1, previously associated with human LQTS, were also evaluated. In contrast to the lack of effects with wild-type human MinK, D76N-MinK and S74L-MinK reduced Kv2.1 current density, slowed deactivation, and D76N but not S74L slowed activation. Compared to wild-type human MiRP1-Kv2.1 complexes, channels formed with M54T or I57T MiRP1 showed slower activation, but only M54T increased inactivation and neither variant affected deactivation rate. The data broaden the known role of MinK and MiRP1 in cardiac physiology and lend support to the hypothesis that inherited mutations in either subunit may contribute to cardiac arrhythmia by multiple mechanisms. AB1-4 DRUGS THAT BLOCK KV11.1 (HERG) CURRENT WITH RAPID KINETICS INHIBIT THE PHARMACOLOGICAL RESCUE OF TRAFFICKING DEFICIENT KV11.1 MUTATIONS LINKED TO LONG QT SYNDROME Brian P. Delisle, PhD, Heather A. S. Underkofler, Corey L. Anderson, BS and Craig T. January, MD, PhD. University of Wisconsin, Madison, WI. KCNH2 or the human Ether-a-go-go Related Gene (hERG) encodes the Kv11.1 channel ␣subunits (Kv11.1) of the rapidly activating delayed rectifier K⫹ current in the heart. Hundreds of mutations in KCNH2 are linked to long QT syndrome (LQT2), and many reduce Kv11.1 current (IKv11.1) by preventing the trafficking of Kv11.1 to the cell surface. Western blot analyses of cells expressing WT-Kv11.1 show two protein bands, a core-glycosylated immature band (⬃135kDa) and a complex-glycosylated mature band (⬃155kDa). Trafficking deficient LQT2 channels are retained in the ER as immature protein. Western blot analyses of cells expressing F640V-Kv11.1 show only the 135kDa immature protein. Incubating cells expressing F640V-Kv11.1 in drugs block IKv11.1 with slow kinetics, E4031 (10M, 24h) or astemizole (10M, 24h), corrected the trafficking deficient phenotype (pharmacological rescue) and resulted in the appearance of the 155kDa mature protein. However, incubating cells expressing F640V-Kv11.1 in drugs that block IKv11.1 with rapid kinetics, verapamil (100M, 24hours) or chloroquine (100M, 24), did not cause pharmacological rescue. Furthermore, Western blot analyses showed that incubating cells in increasing concentrations of verapamil or chloroquine (1⫺100M, 24h) prevented pharmacological rescue of F640V-Kv11.1 by E4031 or astemizole (10M 24h). Similar results were observed for cells expressing trafficking deficient LQT2 G601S-Kv11.1. The peak IKv11.1 was also measured from cells (n⫽4⫺11) expressing F640V-Kv11.1 in control conditions (8 ⫾ 1pA/pF), after incubating in E4031 (81 ⫾ 13pA/pF, 10M, 24h), after incubating in verapamil (8 ⫾ 2pA/pF, 100M, 24h), or after incubating in E4031 and verapamil (15 ⫾ 1pA/pF). These data suggest that pharmacological rescue may depend on the kinetics of drug block of IKv11.1. Drugs that block IKv11.1 with relatively slow kinetics result in pharmacological rescue of trafficking deficient LQT2 channels, whereas drugs that block IKv11.1 with relatively rapid kinetics appear to inhibit pharmacological rescue of trafficking deficient LQT2 channels. AB1-5 GAIN OF FUNCTION KCNQ1 MUTATION ASSOCIATED WITH SUDDEN INFANT DEATH SYNDROME Troy E. Rhodes, MD, PhD, Lia Crotti, MD, Marianne Arnestad, MD, Roberto Insolia, BS, Matteo Pedrazzini, BS, Chiara Ferrandi, BS, Torleiv Rognum, MD, Peter J. Schwartz, MD and Alfred L. George, Jr., MD. Vanderbilt University Medical Center, Nashville, TN, IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy,
Heart Rhythm, Vol 3, No 5, May Supplement 2006 Institute of Forensic Medicine, Rikshospitalet University Hospital, Oslo, Norway and IRCCS Policlinico S. Matteo, Pavia, Italy. Sudden infant death syndrome (SIDS) is a leading cause of death during the first year of life. Mutations in genes responsible for the congenital long QT syndrome (LQTS) have been reported in some cases. Rarely, putative KCNQ1 mutations have been identified in SIDS. A large, retrospective cohort of Norwegian SIDS cases (N ⫽ 201) was examined for genetic variants in the major LQTS genes. We report the functional characterization of a novel missense KCNQ1 mutation identified by this effort. The mutation, I274V, alters a highly conserved amino acid residue in the fifth transmembrane domain of the KCNQ1 potassium channel. Wild-type (WT) and mutant KCNQ1 channels were heterologously expressed in cultured mammalian cells (CHO-K1) with and without KCNE1, and whole-cell patch clamp recording was performed to functionally characterize the variant. In the absence of KCNE1, I274V-KCNQ1 generated the typical voltage-dependent, rapidly activating, and slowly deactivating outward potassium current with current density and biophysical properties similar to WT-KCNQ1. When expressed with KCNE1, I274V generated currents resembling the slow component of the cardiac delayed rectifier current (IKs) but with significantly increased amplitude compared to cells expressing WT-KCNQ1 with KCNE1 (pA/pF at 60 mV: WT-KCNQ1 ⫹ KCNE1, 395.4 ⫾ 47.2, n⫽9; I274V ⫹ KCNE1, 696.0 ⫾ 94.8, n⫽6; p⬍0.05). The time course of deactivation was significantly slower for I274V-IKs compared to WT-IKs (time constant at ⫺80mV: WT-KCNQ1 ⫹ KCNE1, 640.1 ⫾ 47.0 ms, n⫽8; I274V ⫹ KCNE1, 989.3 ⫾ 94.8 ms, n⫽6; p⫽0.004). In the presence of KCNE1, I274V causes a gain-of-function in IKs characterized by increased current density and slower deactivation. Our study demonstrates for the first time a gain-of-function KCNQ1 mutation associated with SIDS. This mutation is predicted to enhance cardiac repolarization resulting in a shortened QT interval and an increased risk of atrial and ventricular tachyarrhythmias, features typical of the short QT syndrome. AB1-6 SCREENING FOR KNOWN MUTATIONS IN LQTS: A NOVEL WAY TO FAST AND LOW COST GENOTYPING? Carlo Napolitano, MD, PhD, Silvia G. Priori, MD, PhD, Peter J. Schwartz, MD, PhD, Raffaella Bloise, MD, PhD, Elena Ronchetti, PhD, Marina Cerrone, MD, Giuseppe Celano, MD and Andrea Capoferri, MD. IRCCS Fondazione S. Maugeri, Pavia, Italy and IRCCS San Matteo, Universitu of Pavia, Pavia, Italy. Nearly 15 years after the initial identification of the long QT syndrome (LQTS) genes, the clinical relevance of genetic testing for risk stratification, diagnosis and therapy is undisputed. The weaknesses of such process are slow turnaround times, costs and poor availability of genetic analysis. Here we report the results of a systematic screening of one of the largest group of consecutively genotyped patients in order to: 1) define the yield of genetic testing; 2) define the type and the prevalence of mutations; 3) propose a novel strategy for genotyping. We evaluated 480 LQTS probands (PB) consecutively screened on the entire coding regions of KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 by DHPLC and DNA sequencing. A separate cohort of 75 consecutive PB was used as a prospective validation cohort to assess our genotyping strategy. We identified 325 mutations in 310 (72%) PB; fourteen of them (4.5%) were compound heterozygous of 2 (n⫽13) or 3 (n⫽1) mutations. Among 1115 family members tested, we identified 521 genetically affected family members (GAFM). GAFM presented significantly shorted QTc (PB QTc 496⫾46ms vs. GAFM QTc 461 ⫾ 40ms; p⬍0.001) and incomplete penetrance (60%). Overall there were 235 different mutations (138 novel): 49% KCNQ1, 39% KCNH2; 10% SCN5A; 1.7% KCNE1; 0.7% KCNE2. Among such non-redundant genetic defects we identified a set of 64 codons in KCNQ1, KCNH2 and SCN5A genes that represented the LQTS-causing mutation in 58% percent of PB. A similar occurrence of mutations at these codons (52%) was confirmed in the prospective cohort and it was further validated against the
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ABSTRACTS ABSTRACT SESSION 1: BASIC/TRANSLATIONAL SCIENCE I: Genetics of Long QT Syndrome Thursday, May 18, 2006 8:00 a.m.–9:30 a.m. AB1-1 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF NOVEL CAV3-ENCODED CAVEOLIN-3 MUTATIONS IN CONGENITAL LONG QT SYNDROME Bin Ye, PhD, David J. Tester, BS, Matteo Vatta, PhD, Jonathan C. Makielski, MD and Michael J. Ackerman, MD, PhD. University of Wisconsin, Madison, WI, Mayo Clinic College of Medicine, Rochester, MN and Baylor College of Medicine, Houston, TX. Background: Approximately 75% of long QT syndrome (LQTS) is explained by pathogenic mutations scattered across three genes encoding key cardiac channel pore-forming subunits. Since the identification of LQTS-associated mutations in ANK2-encoded ankyrin B, genes encoding cardiac channel interacting proteins have become the target of candidate gene mutational analyses for genotype negative LQTS. Recently, we established CAV3 as a novel LQTS-associated gene with mutations in CAV3-encoded caveolin-3 producing a gain-of-function, LQT3-like molecular/cellular phenotype. Methods: Using PCR, DHPLC, and direct DNA sequencing, CAV3 mutational analysis was performed on a cohort of 541unrelated patients (358 females, average age at diagnosis, 24 years, and average QTc, 482 ms) who were referred to Mayo Clinic’s Sudden Death Genomics Laboratory for LQTS genetic testing. CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel. Results: In addition to the 2 patients from our original report that hosted T78M- and F97C-CAV3, we have identified 2 novel missense mutations, not seen in 400 reference alleles. K30R-CAV3 was detected in a white male diagnosed with LQTS at age 7 (QTc ⫽ 528 ms). No other LQTSassociated mutations were identified. L86P-CAV3 was discovered in a 12-year-old white male with a personal history of cardiac arrest while swimming and a negative family history for cardiac events. Comprehensive LQTS mutational analysis also identified a L273F-KCNQ1 mutation (LQT1). The functional effect of K30R- and L86P-CAV3 on the biophysical properties of stably expressed, wild type SCN5A was examined. Conclusions: Following our sentinel report of CAV3-encoded caveolin-3 as a novel pathogenetic mechanism for LQTS, we have now identified and functionally characterized 2 novel caveolin-3 mutations. Future studies should delineate whether or not the combination of LQT1 and CAV3LQTS confers synergistic heterozygosity and a distinct phenotype among family members hosting one or both variants. AB1-2 KCNJ2-S369X MUTATION CAUSES THE LOSS OF ER EXPORT MOTIF AND GENERATES MILD FORM OF ANDERSEN-TAWIL SYNDROME Takahiro Doi, MD, Hidetada Yoshida, MD, PhD, Atsushi Kobori, MD, PhD, Yoshisumi Haruna, MD, Takeru Makiyama, MD, Seiko Ono, MD, Keiko Tsuji, BSc, Yoshiaki
Takahashi, MD, Toru Kita, MD, PhD and Minoru Horie, MD, PhD. Kyoto University Graduate School of Medicine, Kyoto, Japan, Takahashi Clinic for Pediatric Cardiology, Otsu, Japan and Shiga University of Medical Science, Otsu, Japan. Objective: Mutations in KCNJ2, the gene coding the human inward rectifier K⫹ channel Kir2.1, have been identified in Andersen-Tawil syndrome (ATS). ATS is an inherited disease characterized by (1) ventricular tachyarrhythmias associated with QT prolongation in ECG, (2) periodic paralysis, and (3) dysmorphic features. We identified a novel nonsense mutation producing the truncation of Kir2.1 in the middle of C-terminus and the elimination of the ER-to-Golgi export signals (FCYENE), in a 13-year-boy with periodic paralysis and long QT interval on ECG. Biophysical assay using the heterologous expression system revealed the mechanism underlying the generation of ATS in our patient. Methods and Results: Direct sequencing of KCNJ2 gene displayed a heterozygous point mutation, 1106 C ⬎ T (S369X), leading to a premature stop codon. The mutation causes a channel subunit lacking the ER export motif. To characterize the physiological consequences of the truncated Kir2.1 channel, CHO cells were transfected with wild-type (WT) or mutant S369X-KCNJ2 (0.5g each). Cells transfected with WT-KCNJ2 resulted in robust inward rectifying currents (⫺271.1 ⫾ 42.2 pA/pF at ⫺140 mV), but those with S369X-KCNJ2 expressed significantly smaller K⫹ currents (⫺28.4 ⫾ 4.5 pA/pF). Co-expression with WT and mutant at 1:1 ratio (1g each) displayed larger currents compared with WT alone (1g) (⫺723.6 ⫾ 97.9 pA/pF vs. ⫺571.1 ⫾ 86.7, p⫽0.30). Interestingly, increasing the mutant to 2 g (1:2 ratio) increased the resultant current densities to ⫺899.2 ⫾ 81.2 pA/pF (p⫽0.02 vs. WT alone). GFP-fused WT Kir2.1 exhibited robust plasma membrane expression on confocal microscopic images, but GFP-fused S369X remained in the cytoplasma. The impaired transport of S369X was partially rescued by co-expression with full-length Kir2.1, consistent with the electrophysiological data. Conclusion: S369X⫺KCNJ2 is a trafficking-refractory mutation, but the resultant truncated subunit can assemble with the WT protein and partially function. This may explain relatively mild phenotypes in our ATS patient, who has no ventricular arrhythmias and dysmorphic features. AB1-3 MINK AND MIRP1 FORM CARDIAC POTASSIUM CHANNEL COMPLEXES WITH KV2.1 Torsten K. Roepke, MD, Zoe A. McCrossan, PhD, Anthony Lewis, PhD, Gianina Panaghie, MS and Geoffrey W. Abbott, PhD. WMC Cornell University, New York, NY. Kv2.1 is a voltage-gated potassium (Kv) channel alpha subunit expressed in mammalian heart and brain. Kv2.1 reportedly contributes to both IK, slow and Iss in murine heart, and has been detected in human heart although a native current correlate there has not yet been established. MinK-related peptides (MiRPs), encoded by KCNE genes, are single transmembrane domain ancillary subunits that form complexes with Kv channel alpha subunits to modify their function. Mutations in MinK and MiRP1 are associated with inherited long QT syndrome (LQTS) in man.
1547-5271/$ -see front matter © 2006 Heart Rhythm Society. All rights reserved.
S2 Here, we show using native co-immunoprecipitations that both MinK and MiRP1 form native Kv channel complexes with Kv2.1 in rodent heart. In heterologous co-expression studies, human MiRP1 slowed Kv2.1 activation and deactivation, and increased inactivation. Despite complex formation, human MinK had no significant effects on Kv2.1 function, but rat MinK slowed Kv2.1 activation and deactivation, and increased inactivation. Inherited mutations in human MinK or MiRP1, previously associated with human LQTS, were also evaluated. In contrast to the lack of effects with wild-type human MinK, D76N-MinK and S74L-MinK reduced Kv2.1 current density, slowed deactivation, and D76N but not S74L slowed activation. Compared to wild-type human MiRP1-Kv2.1 complexes, channels formed with M54T or I57T MiRP1 showed slower activation, but only M54T increased inactivation and neither variant affected deactivation rate. The data broaden the known role of MinK and MiRP1 in cardiac physiology and lend support to the hypothesis that inherited mutations in either subunit may contribute to cardiac arrhythmia by multiple mechanisms. AB1-4 DRUGS THAT BLOCK KV11.1 (HERG) CURRENT WITH RAPID KINETICS INHIBIT THE PHARMACOLOGICAL RESCUE OF TRAFFICKING DEFICIENT KV11.1 MUTATIONS LINKED TO LONG QT SYNDROME Brian P. Delisle, PhD, Heather A. S. Underkofler, Corey L. Anderson, BS and Craig T. January, MD, PhD. University of Wisconsin, Madison, WI. KCNH2 or the human Ether-a-go-go Related Gene (hERG) encodes the Kv11.1 channel ␣subunits (Kv11.1) of the rapidly activating delayed rectifier K⫹ current in the heart. Hundreds of mutations in KCNH2 are linked to long QT syndrome (LQT2), and many reduce Kv11.1 current (IKv11.1) by preventing the trafficking of Kv11.1 to the cell surface. Western blot analyses of cells expressing WT-Kv11.1 show two protein bands, a core-glycosylated immature band (⬃135kDa) and a complex-glycosylated mature band (⬃155kDa). Trafficking deficient LQT2 channels are retained in the ER as immature protein. Western blot analyses of cells expressing F640V-Kv11.1 show only the 135kDa immature protein. Incubating cells expressing F640V-Kv11.1 in drugs block IKv11.1 with slow kinetics, E4031 (10M, 24h) or astemizole (10M, 24h), corrected the trafficking deficient phenotype (pharmacological rescue) and resulted in the appearance of the 155kDa mature protein. However, incubating cells expressing F640V-Kv11.1 in drugs that block IKv11.1 with rapid kinetics, verapamil (100M, 24hours) or chloroquine (100M, 24), did not cause pharmacological rescue. Furthermore, Western blot analyses showed that incubating cells in increasing concentrations of verapamil or chloroquine (1⫺100M, 24h) prevented pharmacological rescue of F640V-Kv11.1 by E4031 or astemizole (10M 24h). Similar results were observed for cells expressing trafficking deficient LQT2 G601S-Kv11.1. The peak IKv11.1 was also measured from cells (n⫽4⫺11) expressing F640V-Kv11.1 in control conditions (8 ⫾ 1pA/pF), after incubating in E4031 (81 ⫾ 13pA/pF, 10M, 24h), after incubating in verapamil (8 ⫾ 2pA/pF, 100M, 24h), or after incubating in E4031 and verapamil (15 ⫾ 1pA/pF). These data suggest that pharmacological rescue may depend on the kinetics of drug block of IKv11.1. Drugs that block IKv11.1 with relatively slow kinetics result in pharmacological rescue of trafficking deficient LQT2 channels, whereas drugs that block IKv11.1 with relatively rapid kinetics appear to inhibit pharmacological rescue of trafficking deficient LQT2 channels. AB1-5 GAIN OF FUNCTION KCNQ1 MUTATION ASSOCIATED WITH SUDDEN INFANT DEATH SYNDROME Troy E. Rhodes, MD, PhD, Lia Crotti, MD, Marianne Arnestad, MD, Roberto Insolia, BS, Matteo Pedrazzini, BS, Chiara Ferrandi, BS, Torleiv Rognum, MD, Peter J. Schwartz, MD and Alfred L. George, Jr., MD. Vanderbilt University Medical Center, Nashville, TN, IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy,
Heart Rhythm, Vol 3, No 5, May Supplement 2006 Institute of Forensic Medicine, Rikshospitalet University Hospital, Oslo, Norway and IRCCS Policlinico S. Matteo, Pavia, Italy. Sudden infant death syndrome (SIDS) is a leading cause of death during the first year of life. Mutations in genes responsible for the congenital long QT syndrome (LQTS) have been reported in some cases. Rarely, putative KCNQ1 mutations have been identified in SIDS. A large, retrospective cohort of Norwegian SIDS cases (N ⫽ 201) was examined for genetic variants in the major LQTS genes. We report the functional characterization of a novel missense KCNQ1 mutation identified by this effort. The mutation, I274V, alters a highly conserved amino acid residue in the fifth transmembrane domain of the KCNQ1 potassium channel. Wild-type (WT) and mutant KCNQ1 channels were heterologously expressed in cultured mammalian cells (CHO-K1) with and without KCNE1, and whole-cell patch clamp recording was performed to functionally characterize the variant. In the absence of KCNE1, I274V-KCNQ1 generated the typical voltage-dependent, rapidly activating, and slowly deactivating outward potassium current with current density and biophysical properties similar to WT-KCNQ1. When expressed with KCNE1, I274V generated currents resembling the slow component of the cardiac delayed rectifier current (IKs) but with significantly increased amplitude compared to cells expressing WT-KCNQ1 with KCNE1 (pA/pF at 60 mV: WT-KCNQ1 ⫹ KCNE1, 395.4 ⫾ 47.2, n⫽9; I274V ⫹ KCNE1, 696.0 ⫾ 94.8, n⫽6; p⬍0.05). The time course of deactivation was significantly slower for I274V-IKs compared to WT-IKs (time constant at ⫺80mV: WT-KCNQ1 ⫹ KCNE1, 640.1 ⫾ 47.0 ms, n⫽8; I274V ⫹ KCNE1, 989.3 ⫾ 94.8 ms, n⫽6; p⫽0.004). In the presence of KCNE1, I274V causes a gain-of-function in IKs characterized by increased current density and slower deactivation. Our study demonstrates for the first time a gain-of-function KCNQ1 mutation associated with SIDS. This mutation is predicted to enhance cardiac repolarization resulting in a shortened QT interval and an increased risk of atrial and ventricular tachyarrhythmias, features typical of the short QT syndrome. AB1-6 SCREENING FOR KNOWN MUTATIONS IN LQTS: A NOVEL WAY TO FAST AND LOW COST GENOTYPING? Carlo Napolitano, MD, PhD, Silvia G. Priori, MD, PhD, Peter J. Schwartz, MD, PhD, Raffaella Bloise, MD, PhD, Elena Ronchetti, PhD, Marina Cerrone, MD, Giuseppe Celano, MD and Andrea Capoferri, MD. IRCCS Fondazione S. Maugeri, Pavia, Italy and IRCCS San Matteo, Universitu of Pavia, Pavia, Italy. Nearly 15 years after the initial identification of the long QT syndrome (LQTS) genes, the clinical relevance of genetic testing for risk stratification, diagnosis and therapy is undisputed. The weaknesses of such process are slow turnaround times, costs and poor availability of genetic analysis. Here we report the results of a systematic screening of one of the largest group of consecutively genotyped patients in order to: 1) define the yield of genetic testing; 2) define the type and the prevalence of mutations; 3) propose a novel strategy for genotyping. We evaluated 480 LQTS probands (PB) consecutively screened on the entire coding regions of KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 by DHPLC and DNA sequencing. A separate cohort of 75 consecutive PB was used as a prospective validation cohort to assess our genotyping strategy. We identified 325 mutations in 310 (72%) PB; fourteen of them (4.5%) were compound heterozygous of 2 (n⫽13) or 3 (n⫽1) mutations. Among 1115 family members tested, we identified 521 genetically affected family members (GAFM). GAFM presented significantly shorted QTc (PB QTc 496⫾46ms vs. GAFM QTc 461 ⫾ 40ms; p⬍0.001) and incomplete penetrance (60%). Overall there were 235 different mutations (138 novel): 49% KCNQ1, 39% KCNH2; 10% SCN5A; 1.7% KCNE1; 0.7% KCNE2. Among such non-redundant genetic defects we identified a set of 64 codons in KCNQ1, KCNH2 and SCN5A genes that represented the LQTS-causing mutation in 58% percent of PB. A similar occurrence of mutations at these codons (52%) was confirmed in the prospective cohort and it was further validated against the