- Email: [email protected]
Allelic drop-out in long QT syndrome genetic testing: A possible mechanism underlying false negative results
Session 4 Quebec, Canada and Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan. Background: Severe bradycardia, including complete AV block, is known to precipitate QT prolongation and Torsades de Pointes (TdP) in man, a finding that has been attributed to the functional heart rate-QT interval relationship. However, it is also possible that slow heart rate affects ion-channel subunit transcription, a possibility that we assessed in the present study. Methods: We evaluated expression of mRNA and membrane protein of K-channel subunits underlying rapid ( IKr) and slow ( IKs) delayed rectifier K⫹ currents and transient outward current ( Ito) in right (RV) and left (LV) ventricular tissues from rabbits with 21-day RV pacing (Brady) at 60-90 bpm after creation of complete AV block, and compared these with shamoperated control rabbits with sinus rhythm (220⫾6 bpm). Results: Twenty three of 39 Brady rabbits died suddenly before day 21 and 16 survived for ⬎21 days, with 66⫾17 episodes of spontaneous TdP. ECGs showed prominent QT prolongation and QT dispersion (at day 0, 7 and 21, QT intervals at a pacing rate of 60 bpm: 256⫾3, 332⫾9 and 342⫾11 ms; QT dispersion: 41⫾3, 67⫾6 and 82⫾7 ms, respectively). All 5 Brady rabbits with telemetry died of TdP degenerating to Vf. KvLQT1, minK and ERG mRNA were downregulated in RV and LV from Brady rabbits (table). Protein levels of these subunits were reduced in parallel with those of mRNA. Neither Kv4.3 nor Kv1.4 was altered by bradypacing. Conclusions: Chronic bradycardia downregulates the expression of IKr and IKs, causing QT prolongation, TdP and sudden cardiac death in rabbits. Re-programming of ion-channel subunit gene expression may be an important contributor to the clinical observation of TdP and QT prolongation in patients with severe bradycardia, and may have important implications for understanding the effects of bradycardia-promoting drugs and interventions in man.
AB4-2 MISPROCESSING AS A MECHANISM FOR PHENOTYPIC VARIABILITY IN LQT3 Kai Liu, Tao Yang and Dan M. Roden, MD. Vanderbilt University, Nashville, TN. Variable penetrance is well-recognized in monogenic arrhythmia syndromes. We report here experiments using the cardiac Na channel SCN5A mutation L1825P (LP) to identify a novel underlying mechanism. Although LP displays persistent late INa typical of LQT3, the reported clinical phenotype is mild. As shown in the Figure, peak INa generated by LP (in CHO cells) is reduced, and far fewer cells display any current (3/22 vs 9/12 for wild-type [wt]). As one test of the idea that LP may not traffic normally to the cell surface, cells were grown in the presence of channel blockers (quinidine [Q] or lidocaine [L], 100 M); this intervention can increase cell surface expression of misprocessed channels. Confocal imaging of con-
S7 structs with an extracellular FLAG tag revealed markedly reduced surface expression with LP (12% of wt) which partially corrected with Q or L, to 61-84% of wt. Further, ⬎80% of LP-transfected cells grown in Q or L (and studied after drug washout) displayed current, and peak INa increased 3-4-fold, toward that seen with wt channels. However, with this correction, late INa was increased over wt: 40⫾3 pA (wt), 44⫾2 (LP), 146⫾9 (LP⫹Q), and 113⫾11 (LP⫹L). We conclude that the dissociation between the severe in vitro and mild clinical phenotype reflects misprocessing of the variant allele, resulting in decreased cell surface expression of channels generating the late current that underlies QT prolongation. AB4-3 ALLELIC DROP-OUT IN LONG QT SYNDROME GENETIC TESTING: A POSSIBLE MECHANISM UNDERLYING FALSE NEGATIVE RESULTS David J. Tester, BSc, Melissa L. Will, BSc, Benjamin A. Salisbury, PhD, Janet L. Carr, Vincent Schulz, PhD, Richard S. Judson, PhD and *Michael J. Ackerman, MD, PhD. Mayo Clinic College of Medicine, Rochester, MN and Genaissance Pharmaceuticals, New Haven, CT. Background: Congenital long QT syndrome (LQTS) genetic testing has been performed in research laboratories for the past decade. Approximately 75% of patients with high clinical probability for LQTS have a mutation in one of 5 LQTS-causing cardiac channel genes. Possible explanations for the remaining genotype negative cases include LQTS mimickers, novel LQTS-causing genes, unexplored regions of the known genes, and genetic testing detection failures. Here, we explore the possibility of allelic dropout as a possible mechanism underlying false negative test results. Methods: The published primers currently utilized by many research laboratories to conduct a comprehensive analysis of the 60 protein-encoding exons in the KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) genes were analyzed for the presence of common intronic single nucleotide polymorphisms. Repeat mutational analysis, following primer/amplicon redesign using polymerase chain reaction, denaturing high performance liquid chromatography, and DNA sequencing, was performed on a cohort of 388 consecutive, unrelated index cases referred for LQTS genetic testing. Results: Common intronic single nucleotide polymorphisms (SNP) residing within the primer sequence were found in the reverse primer used for exon 15 of KCNQ1 and the reverse primer used for exon 4 for KCNH2. The allelic frequency was 5% (1794⫹32 g⬎t, KCNQ1) and 39% (916⫹60 c⬎t, KCNH2). Following primer redesign to eliminate the possibility of allelic drop-out, 4 previously genotype negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 found in two unrelated cases. Repeat analysis of the amplicons in 400 reference alleles did not identify these or any additional amino acid variants. Conclusions: Allelic drop-out secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in 4 cases. Considering that many LQTS genetic testing research laboratories have utilized these primers, patients who are reportedly genotype negative may benefit from a re-examination of exon 15 in KCNQ1 and exon 4 in KCNH2. AB4-4 DOWNREGULATION OF ION CHANNEL SUBUNITS UNDERLYING ELECTRICAL REMODELING IN THE DOG WITH COMPLETE ATRIO-VENTRICULAR BLOCK: REGIONAL AND TEMPORAL CONSIDERATIONS Christian Ramakers, MS, Paul G. A. Volders, PhD, Dirk W. Donker, MD, Jet Beekman, BSc, Antoon F. M. Moorman, PhD and Marc A. Vos, PhD. University Medical Centre Utrecht, Utrecht, Netherlands, Cardiovascular Research Institute Maastricht, Maastricht, Netherlands and Experimental & Molecular Cardiology Group, Academic Medical Centre, Amsterdam, Netherlands.
AB4-2 MISPROCESSING AS A MECHANISM FOR PHENOTYPIC VARIABILITY IN LQT3 Kai Liu, Tao Yang and Dan M. Roden, MD. Vanderbilt University, Nashville, TN. Variable penetrance is well-recognized in monogenic arrhythmia syndromes. We report here experiments using the cardiac Na channel SCN5A mutation L1825P (LP) to identify a novel underlying mechanism. Although LP displays persistent late INa typical of LQT3, the reported clinical phenotype is mild. As shown in the Figure, peak INa generated by LP (in CHO cells) is reduced, and far fewer cells display any current (3/22 vs 9/12 for wild-type [wt]). As one test of the idea that LP may not traffic normally to the cell surface, cells were grown in the presence of channel blockers (quinidine [Q] or lidocaine [L], 100 M); this intervention can increase cell surface expression of misprocessed channels. Confocal imaging of con-
S7 structs with an extracellular FLAG tag revealed markedly reduced surface expression with LP (12% of wt) which partially corrected with Q or L, to 61-84% of wt. Further, ⬎80% of LP-transfected cells grown in Q or L (and studied after drug washout) displayed current, and peak INa increased 3-4-fold, toward that seen with wt channels. However, with this correction, late INa was increased over wt: 40⫾3 pA (wt), 44⫾2 (LP), 146⫾9 (LP⫹Q), and 113⫾11 (LP⫹L). We conclude that the dissociation between the severe in vitro and mild clinical phenotype reflects misprocessing of the variant allele, resulting in decreased cell surface expression of channels generating the late current that underlies QT prolongation. AB4-3 ALLELIC DROP-OUT IN LONG QT SYNDROME GENETIC TESTING: A POSSIBLE MECHANISM UNDERLYING FALSE NEGATIVE RESULTS David J. Tester, BSc, Melissa L. Will, BSc, Benjamin A. Salisbury, PhD, Janet L. Carr, Vincent Schulz, PhD, Richard S. Judson, PhD and *Michael J. Ackerman, MD, PhD. Mayo Clinic College of Medicine, Rochester, MN and Genaissance Pharmaceuticals, New Haven, CT. Background: Congenital long QT syndrome (LQTS) genetic testing has been performed in research laboratories for the past decade. Approximately 75% of patients with high clinical probability for LQTS have a mutation in one of 5 LQTS-causing cardiac channel genes. Possible explanations for the remaining genotype negative cases include LQTS mimickers, novel LQTS-causing genes, unexplored regions of the known genes, and genetic testing detection failures. Here, we explore the possibility of allelic dropout as a possible mechanism underlying false negative test results. Methods: The published primers currently utilized by many research laboratories to conduct a comprehensive analysis of the 60 protein-encoding exons in the KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) genes were analyzed for the presence of common intronic single nucleotide polymorphisms. Repeat mutational analysis, following primer/amplicon redesign using polymerase chain reaction, denaturing high performance liquid chromatography, and DNA sequencing, was performed on a cohort of 388 consecutive, unrelated index cases referred for LQTS genetic testing. Results: Common intronic single nucleotide polymorphisms (SNP) residing within the primer sequence were found in the reverse primer used for exon 15 of KCNQ1 and the reverse primer used for exon 4 for KCNH2. The allelic frequency was 5% (1794⫹32 g⬎t, KCNQ1) and 39% (916⫹60 c⬎t, KCNH2). Following primer redesign to eliminate the possibility of allelic drop-out, 4 previously genotype negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 found in two unrelated cases. Repeat analysis of the amplicons in 400 reference alleles did not identify these or any additional amino acid variants. Conclusions: Allelic drop-out secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in 4 cases. Considering that many LQTS genetic testing research laboratories have utilized these primers, patients who are reportedly genotype negative may benefit from a re-examination of exon 15 in KCNQ1 and exon 4 in KCNH2. AB4-4 DOWNREGULATION OF ION CHANNEL SUBUNITS UNDERLYING ELECTRICAL REMODELING IN THE DOG WITH COMPLETE ATRIO-VENTRICULAR BLOCK: REGIONAL AND TEMPORAL CONSIDERATIONS Christian Ramakers, MS, Paul G. A. Volders, PhD, Dirk W. Donker, MD, Jet Beekman, BSc, Antoon F. M. Moorman, PhD and Marc A. Vos, PhD. University Medical Centre Utrecht, Utrecht, Netherlands, Cardiovascular Research Institute Maastricht, Maastricht, Netherlands and Experimental & Molecular Cardiology Group, Academic Medical Centre, Amsterdam, Netherlands.