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Aims: To determine the prevalence and in vitro electrophysiological (EP) phenotype of ultra-rare SCN5A variants of uncertain significance (VUS) identified in unexplained sudden cardiac arrest (SCA) survivors.Methods and Results: Retrospective review of 73 unexplained SCA survivors was used to identify all patients that underwent a form of genetic testing that included comprehensive SCN5A analysis. Ultra-rare SCN5A variants (minor allele frequency < 0.005) were adjudicated according to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines. Variants designated as VUS were expressed heterologously and characterized using the whole-cell patch clamp technique. Overall, 60/73 (82%; the average age at SCA 28 ± 12 years) unexplained SCA survivors had received SCN5A genetic testing. Of these, 5/60 (8.3%) had an ultra-rare SCN5A variant. All SCN5A variants were classified as VUS. Whereas the single SCN5A VUS (p.Asp872Asn-SCN5A) identified in an unexplained SCA survivor with PR interval prolongation and inferior early repolarization conferred a loss-of-function phenotype (46.2% reduction in peak current density; 16 ms slower recovery from inactivation), the four other SCN5A VUS (p.Glu30Gly-SCN5A, p.Gln245Lys-SCN5A, p.Pro648Leu-SCN5A, and p.Glu1240Gln-SCN5A) identified in unexplained SCA survivors without early repolarization/conduction delay were indistinguishable from wild-type Nav1.5 channels.Conclusion: In the absence of a phenotype(s) potentially attributable to sodium channel dysfunction, all SCN5A VUS identified in unexplained SCA survivors conferred a wild-type EP phenotype in vitro. As the background rate of SCN5A genetic variation is not trivial, great care must be taken to avoid prioritizing genotype over phenotype when attempting to ascertain the root cause of an individual's SCA. [ABSTRACT FROM AUTHOR]

Background: A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects.Methods and Results: Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in CaV1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current.Conclusions: Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype. [ABSTRACT FROM AUTHOR]

Background: SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity. Methods and Results: We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT. Conclusion: The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms. [ABSTRACT FROM AUTHOR]

Mutations in CACNA1C that increase current through the CaV1.2 L-type Ca2+ channel underlie rare forms of long QT syndrome (LQTS), and Timothy syndrome (TS). We identified a variant in CACNA1C in a male child of Filipino descent with arrhythmias and extracardiac features by candidate gene sequencing and performed functional expression studies to electrophysiologically characterize the effects of the variant on CaV1.2 channels. As a baby, the subject developed seizures and displayed developmental delays at 30 months of age. At age 5 years, he displayed a QTc of 520 ms and experienced recurrent VT. Physical exam at 17 years of age was notable for microcephaly, short stature, lower extremity weakness and atrophy with hyperreflexia, spastic diplegia, multiple dental caries and episodes of rhabdomyolysis. Candidate gene sequencing identified a G>C transversion at position 5731 of CACNA1C (rs374528680) predicting a glycine>arginine substitution at residue 1911 (p.G1911R) of CaV1.2. The allele frequency of this variant is 0.01 in Malays, but absent in 984 Caucasian alleles and in the 1000 genomes project. In electrophysiological analyses, the variant decreased voltage-dependent inactivation, thus causing a gain of function of CaV1.2. We also observed a negative shift of V1/2 of activation and positive shift of V1/2 of channel inactivation, resulting in an increase of the window current. Together, these suggest a gain-of-function effect on CaV1.2 and suggest increased susceptibility for arrhythmias in certain clinical settings. The p.G1911R variant was also identified in a case of sudden unexplained infant death (SUID), for which an increasing number of clinical observations have demonstrated can be associated with arrhythmogenic mutations in cardiac ion channels. In summary, the combined effects of the CACNA1C variant to diminish voltage-dependent inactivation of CaV1.2 and increase window current expand our appreciation of mechanisms by which a gain of function of CaV1.2 can contribute to QT prolongation. [ABSTRACT FROM AUTHOR]

R231H Causes a High Penetrance for Familial AF Background Atrial fibrillation (AF) is the most common cardiac arrhythmia, and its incidence is expected to grow. A genetic predisposition for AF has long been recognized, but its manifestation in these patients likely involves a combination of rare and common genetic variants. Identifying genetic variants that associate with a high penetrance for AF would represent a significant breakthrough for understanding the mechanisms that associate with disease. Method and Results Candidate gene sequencing in 5 unrelated families with familial AF identified the KCNQ1 missense mutation p.Arg231His (R231H). In addition to AF, several of the family members have abnormal QTc intervals, syncope or experienced sudden cardiac arrest or death. KCNQ1 encodes the voltage-gated K+ channel that conducts the slowly activating delayed rectifier K+ current in the heart. Functional and computational analyses suggested that R231H increases KCNQ1 current (IKCNQ1) to shorten the atrial action potential (AP) duration. R231H is predicted to minimally affect ventricular excitability, but it prevented the increase in IKCNQ1 following PKA activation. The unique properties of R231H appeared to be caused by a loss in voltage-dependent gating. Conclusions The R231H variant causes a high penetrance for interfamilial early-onset AF. Our study indicates R231H likely shortens atrial refractoriness to promote a substrate for reentry. Additionally, R231H might cause abnormal ventricular repolarization by disrupting PKA activation of IKCNQ1. We conclude genetic variants, which increase IKs during the atrial AP, decrease the atrial AP duration, and/or shorten atrial refractoriness, present a high risk for interfamilial AF. [ABSTRACT FROM AUTHOR]

The article discusses research which examined the spectrum and prevalence of mutations in the three most common long QT syndrome (LQTS) susceptible genes. Intrauterine fetal death or stillbirth accounts for 50% of perinatal deaths. A total of 91 unexplained intrauterine fetal deaths are considered in the study. Denaturing high-performance liquid chromatography and direct DNA sequencing were used for mutational analyses of the genes. Findings revealed the association of missense mutations with LQTS susceptibility in three cases. In addition, genetic variants leading to dysfunctional LQTS-associated ion channels were found in 8 cases.

The article presents a study that determined the spectrum, prevalence, and function of KCNJ8 mutations in large sudden infant death syndrome (SIDS) cohort. The study used polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing to perform open reading frame/splice-site mutational analysis of KCNJ8 on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases. It proposes KCNJ8 mutations as a potential pathogenic substrate for SIDS.

Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1, KCNH2, and SCN5A, and the 2 minor genes, KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. [ABSTRACT FROM AUTHOR]

Background: Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in alpha1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I(Na)) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations.Methods and Results: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (approximately 3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I(Na) was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I(Na) and 2.3- to 2.7-fold increase in late I(Na) compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current.Conclusions: Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as alpha1-syntrophin from similarly rare but innocuous ones. [ABSTRACT FROM AUTHOR]

Background: KCNJ2 encodes Kir2.1, a pore-forming subunit of the cardiac inward rectifier current, I(K1). KCNJ2 mutations are associated with Andersen-Tawil syndrome and catecholaminergic polymorphic ventricular tachycardia. The aim of this study was to characterize the biophysical and cellular phenotype of a KCNJ2 missense mutation, V227F, found in a patient with catecholaminergic polymorphic ventricular tachycardia.Methods and Results: Kir2.1-wild-type (WT) and V227F channels were expressed individually and together in Cos-1 cells to measure I(K1) by voltage clamp. Unlike typical Andersen-Tawil syndrome-associated KCNJ2 mutations, which show dominant negative loss of function, Kir2.1WT+V227F coexpression yielded I(K1) indistinguishable from Kir2.1-WT under basal conditions. To simulate catecholamine activity, a protein kinase A (PKA)-stimulating cocktail composed of forskolin and 3-isobutyl-1-methylxanthine was used to increase PKA activity. This PKA-simulated catecholaminergic stimulation caused marked reduction of outward I(K1) compared with Kir2.1-WT. PKA-induced reduction in I(K1) was eliminated by mutating the phosphorylation site at serine 425 (S425N).Conclusions: Heteromeric Kir2.1-V227F and WT channels showed an unusual latent loss of function biophysical phenotype that depended on PKA-dependent Kir2.1 phosphorylation. This biophysical phenotype, distinct from typical Andersen-Tawil syndrome mutations, suggests a specific mechanism for PKA-dependent I(K1) dysfunction for this KCNJ2 mutation, which correlates with adrenergic conditions underlying the clinical arrhythmia. [ABSTRACT FROM AUTHOR]

The SCN5A-encoded Nav1.5 Na+ channel is expressed in interstitial cells of Cajal and smooth muscle in the circular layer of the human intestine. Patients with mutations in SCN5A are more likely to report gastrointestinal symptoms, especially abdominal pain. Twin and family studies of irritable bowel syndrome (IBS) suggest a genetic basis for IBS, but no genes have been identified to date. Therefore, our aims were to evaluate SCN5A as a candidate gene involved in the pathogenesis of lBS and to determine physiological consequences of identified mutations. Mutational analysis was performed on genomic DNA obtained from 49 subjects diagnosed with lBS who reported at least moderately severe abdominal pain. One patient hosted a loss-of-function missense mutation, G298S, that was not observed in >3,000 reference alleles derived from 1,500 healthy control subjects. Na+ currents were recorded from the four common human SCN5A transcripts in transfected HEK-293 cells. Comparing Nav1.5 with G298S-SCN5A versus wild type in HEK cells, Na+ current density was significantly less by 49-77%, and channel activation time was delayed in backgrounds that also contained the common H558R polymorphism. Single-channel measurements showed no change in Nav 1.5 conductance. Mechanosensitivity was reduced in the H558/Q1077de1 transcript but not in the other three backgrounds. In conclusion, the G298S-SCN5A missense mutation caused a marked reduction of whole cell Na+ current and loss of function of Nav1.5, suggesting SCN5A as a candidate gene in the pathophysiology of lBS. [ABSTRACT FROM AUTHOR]

A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the lKs potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of (i) regions on Yotiao critical to its binding to KCNQ1 and (ii) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the lKs channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Individual mutations in the SCN5A-encoding cardiac sodium channel α-subunit cause single cardiac arrhythmia disorders, but a few cause multiple distinct disorders. Here we report a family harboring an SCN5A mutation (L1821fs/10) causing a truncation of the C-terminus with a marked and complex biophysical phenotype and a corresponding variable and complex clinical phenotype with variable penetrance. Methods and results: A 12-year-old male with congenital sick sinus syndrome (SSS), cardiac conduction disorder (CCD), and recurrent monomorphic ventricular tachycardia (VT) had mutational analysis that identified a 4 base pair deletion (TCTG) at position 5464–5467 in exon 28 of SCN5A. The mutation was also present in six asymptomatic family members only two of which showed mild ECG phenotypes. The deletion caused a frame-shift mutation (L1821fs/10) with truncation of the C-terminus after 10 missense amino acid substitutions. When expressed in HEK-293 cells for patch-clamp study, the current density of L1821fs/10 was reduced by 90% compared with WT. In addition, gating kinetic analysis showed a 5-mV positive shift in activation, a 12-mV negative shift of inactivation and enhanced intermediate inactivation, all of which would tend to reduce peak and early sodium current. Late sodium current, however, was increased in the mutated channels. Conclusions: The L1821fs/10 mutation causes the most severe disruption of SCN5A structure for a naturally occurring mutation that still produces current. It has a marked loss-of-function and unique phenotype of SSS, CCD and VT with incomplete penetrance. [Copyright &y& Elsevier]

Background: Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality in the United States. Mutations in the RyR2-encoded cardiac ryanodine receptor cause the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT1) in the young.Objective: The purpose of this study was to determine the spectrum and prevalence of RyR2 mutations in a large cohort of SIDS cases.Methods: Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, a targeted mutational analysis of RyR2 was performed on genomic DNA isolated from frozen necropsy tissue on 134 unrelated cases of SIDS (57 females, 77 males; 83 white, 50 black, 1 Hispanic; average age = 2.7 months). RyR2 mutations were engineered by site-directed mutagenesis, heterologously expressed in HEK293 cells, and functionally characterized using single-channel recordings in planar lipid bilayers.Results: Overall, two distinct and novel RyR2 mutations were identified in two cases of SIDS. A 6-month-old black female hosted an R2267H missense mutation, and a 4-week-old white female infant harbored a S4565R mutation. Both nonconservative amino acid substitutions were absent in 400 reference alleles, involved conserved residues, and were localized to key functionally significant domains. Under conditions that simulate stress [Protein Kinase A (PKA) phosphorylation] during diastole (low activating [Ca2+]), SIDS-associated RyR2 mutant channels displayed a significant gain-of-function phenotype consistent with the functional effect of previously characterized CPVT-associated RyR2 mutations.Conclusions: Here we report a novel pathogenic mechanism for SIDS, whereby SIDS-linked RyR2 mutations alter the response of the channels to sympathetic nervous system stimulation such that during stress the channels become "leaky" and thus potentially trigger fatal cardiac arrhythmias. [ABSTRACT FROM AUTHOR]

Background: Loss-of-function mutations in the KCNJ2 cause approximately 50% of Andersen-Tawil Syndrome (ATS) characterized by a classic triad of periodic paralysis, ventricular arrhythmia, and dysmorphic features. Do KCNJ2 mutations occur in patients lacking this triad and lacking a family history of ATS?Objectives: The purpose of this study was to identify and characterize mutations in the KCNJ2-encoded inward rectifier potassium channel Kir2.1 from patients referred for genetic arrhythmia testing.Methods: Mutational analysis of KCNJ2 was performed for 541 unrelated patients. The mutations were made in wild type (WT) and expressed in COS-1 cells and voltage clamped for ion currents.Results: Three novel missense mutations (R67Q, R85W, and T305A) and one known mutation (T75M) were identified in 4/249 (1.6%) patients genotype-negative for other known arrhythmia genes with overall incidence 4/541 (0.74%). They had prominent U-waves, marked ventricular ectopy, and polymorphic ventricular tachycardia but no facial/skeletal abnormalities. Periodic paralysis was present in only one case. Outward current was decreased to less than 5% of WT for all mutants expressed alone. Co-expression with WT (simulating heterozygosity) caused a marked dominant negative effect for T75M and R82W, no dominant negative effect for R67Q, and a novel selective enhancement of inward rectification for T305A.Conclusions: KCNJ2 loss of function mutations were found in approximately 1% of patients referred for genetic arrhythmia testing that lacked criteria for ATS. Characterization of three new mutations identified a novel dominant negative effect selectively reducing outward current for T305A. These results extend the range of clinical phenotype and molecular phenotype associated with KCNJ2 mutations. [ABSTRACT FROM AUTHOR]

Background: Sudden infant death syndrome (SIDS) is one of the leading causes of death during the first year of life. Long QT syndrome (LQTS)-associated mutations may be responsible for 5% to 10% of SIDS cases. We recently established CAV3-encoded caveolin-3 as a novel LQTS-associated gene with mutations producing a gain-of-function, LQT3-like molecular/cellular phenotype.Objective: The purpose of this study was to determine the prevalence and functional properties of CAV3 mutations in SIDS.Methods: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, postmortem genetic testing of CAV3 was performed on genomic DNA isolated from frozen necropsy tissue on a population-based cohort of unrelated cases of SIDS (N = 134, 57 females, average age = 2.7 months). CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel.Results: Overall, three distinct CAV3 mutations (V14L, T78M, and L79R) were identified in three of 50 black infants (6-month-old male, 2-month-old female, and 8 month-old female), whereas no mutations were detected in 83 white infants (P <.05). CAV3 mutations were more likely in decedents 6 months or older (2/12) than in infants who died before 6 months (1/124, P = .02). Voltage clamp studies showed that all three CAV3 mutations caused a significant fivefold increase in late sodium current compared with controls.Conclusion: This study provides the first molecular and functional evidence implicating CAV3 as a pathogenic basis of SIDS. The LQT3-like phenotype of increased late sodium current supports an arrhythmogenic mechanism for some cases of SIDS. [ABSTRACT FROM AUTHOR]

Background: Genetic testing for congenital long QT syndrome (LQTS) 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 five 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. Objectives: The purpose of this study was to explore the possibility of allelic dropout as a possible mechanism underlying false-negative test results. Methods: The published primers currently used by many research laboratories to conduct a comprehensive analysis of the 60 translated 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 (SNPs). 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 541 consecutive, unrelated patients referred for LQTS genetic testing. Results: Common (>1% minor allele frequency) intronic SNPs were discovered within the primer sequences of five of 60 translated exons. Following primer redesign to eliminate the possibility of allelic dropout, four previously genotype-negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 in two unrelated cases. Repeat examination of these two amplicons in 400 reference alleles did not identify these or any additional amino acid variants. Conclusion: Allelic dropout secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in four cases. Considering that many LQTS genetic testing research laboratories have used these primers, patients who reportedly are genotype negative may benefit from re-examination of those regions susceptible to allelic dropout due to primer-disrupting SNPs, particularly exon 15 in KCNQ1 and exon 4 in KCNH2. [ABSTRACT FROM AUTHOR]

Background: Mutations in the RyR2-encoded cardiac ryanodine receptor/calcium release channel and in CASQ2-encoded calsequestrin cause catecholaminergic polymorphic ventricular tachycardia (CPVT1 and CPVT2, respectively).Objectives: The purpose of this study was to evaluate the extent of genotypic and phenotypic heterogeneity among referrals for CPVT genetic testing.Methods: Using denaturing high-performance liquid chromatography and DNA sequencing, mutational analysis of 23 RyR2 exons previously implicated in CPVT1, comprehensive analysis of all translated exons in CASQ2 (CPVT2), KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), KCNE2 (LQT6), and KCNJ2 (Andersen-Tawil syndrome [ATS1], also annotated LQT7), and analysis of 10 ANK2 exons implicated in LQT4 were performed on genomic DNA from 11 unrelated patients (8 females) referred to Mayo Clinic's Sudden Death Genomics Laboratory explicitly for CPVT genetic testing.Results: Overall, putative disease causing mutations were identified in 8 patients (72%). Only 4 patients (3 males) hosted CPVT1-associated RyR2 mutations: P164S, V186M, S3938R, and T4196A. Interestingly, 4 females instead possessed either ATS1- or LQT5-associated mutations. Mutations were absent in >400 reference alleles.Conclusion: Putative CPVT1-causing mutations in RyR2 were seen in <40% of unrelated patients referred with a diagnosis of CPVT and preferentially in males. Phenotypic mimicry is evident with the identification of ATS1- and LQT5-associated mutations in females displaying a normal QT interval and exercise-induced bidirectional VT, suggesting that observed exercise-induced polymorphic VT in patients may reflect disorders other than CPVT. Clinical consideration for either Andersen-Tawil syndrome or long QT syndrome and appropriate genetic testing may be warranted for individuals with RyR2 mutation-negative CPVT, particularly females. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To perform a molecular autopsy Involving the RyR2-encoded cardiac ryanodine receptor/calcium release channel to determine whether mutations responsible for catecholaminergic polymorphic ventricular tachycardla (CPVT) represent a novel pathogenic basis for unexplained drownings. METHODS: A cardiac channel molecular autopsy was performed on 2 individuals who died of unexplained drowning and whose cases were referred to the Sudden Death Genomics Laboratory at the Mayo Clink in Rochester Minn. Comprehensive mutational analysis of all 60 protein-encoded exons of the 5 long QT syndrome- causing cardiac channel genes and a targeted analysis of 18 RyR2 exons known to host RyR2-medilated CPVT-causing mutations (CPVT1) was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing. RESULTS: Both Individuals harbored novel mutations In RyR2. Postmortem mutational analysis revealed a familial missense mutation in non 14, R414C, in a 16-year-old girl. A 9-year-old boy possessed a sporadic missense mutation in exon 49, V2475F. Both amino acid positions involve highly conserved residues that localize to critical functional domains in the calcium release channel. Neither substitution was present in 1000 reference shies. CONCLUSIONS: This molecular autopsy study provides proof of principle that RyR2 mutations can underlie some unexplained drownings. A population-based genetic epidemiology study that Involves molecular autopsies of individuals who die of unexplained drowning Is needed to determine the prevalence and spectrum of KCNQ1 and now RyR2 mutations as potential pathogenic mechanisms for drowning. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to determine the spectrum and prevalence of cardiac channel mutations among a large cohort of consecutive, unrelated patients referred for long QT syndrome (LQTS) genetic testing.Background: Congenital LQTS is a primary cardiac channelopathy. More than 300 mutations have been identified in five genes encoding key ion channel subunits. Until the recent release of the commercial clinical genetic test, LQTS genetic testing had been performed in research laboratories during the past decade.Methods: A cardiac channel gene screen for LQTS-causing mutations in KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) was performed for 541 consecutive, unrelated patients (358 females, average age at diagnosis 24 +/- 16 years, average QTc 482 +/- 57 ms) referred to Mayo Clinic's Sudden Death Genomics Laboratory for LQTS genetic testing between August 1997 and July 2004. A comprehensive open reading frame and splice site analysis of the 60 protein-encoding exons was conducted using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing.Results: Overall, 211 putative pathogenic mutations in KCNQ1 (88), KCNH2 (89), SCN5A (32), KCNE1 (1), and KCNE2 (1) were found in 272 unrelated patients (50%). Among the genotype positive patients (N = 272), 243 had single pathogenic mutations (LQT1: n = 120 patients; LQT2: n = 93; LQT3: n = 26; LQT5: n = 3; LQT6: n = 1), and 29 patients (10% of genotype-positive patients and 5% overall) had two LQTS-causing mutations. The majority of mutations were missense mutations (154/210 [73%]), singletons (identified in only a single unrelated patient: 165/210 [79%]), and novel (125/211 [59%]). None of the mutations identified were seen in more than 1,500 reference alleles. Those patients harboring multiple mutations were younger at diagnosis (15 +/- 11 years vs 24 +/- 16 years, P = .003).Conclusions: In this comprehensive cardiac channel gene screen of the largest cohort of consecutive, unrelated patients referred for LQTS genetic testing, half of the patients had an identifiable mutation. The majority of mutations continue to represent novel singletons that expand the published compendium of LQTS-causing mutations by 35%. These observations should facilitate diagnostic interpretation of the clinical genetic test for LQTS. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To perform a molecular autopsy of the RyR2-encoded cardiac ryanodine receptor/calcium release channel In medical examiner/coroner's cases of sudden unexplained death (SUD). METHODS: From September 1998 to March 2004, 49 cases of SUD were referred by medical examiners/coroners to the Sudden Death Genomics Laboratory at the Mayo Clinic In Rochester, Minn, for a cardiac channel molecular autopsy. Mutational analysis of LB exons of RyR2 implicated previously in the pathogenesis of catechoiaminergic polymorphic ventricular tachycardia (CPVT) was performed on genomic DNA using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing. RESULTS: This cohort of 49 cases of SW) included 30 males, 13 with a family history of syncope, cardiac arrest, or sudden cardiac death (mean ± SD age at death, 14.2&plusm;10.9 years). Six distinct RyR2 missense mutations (3 novel) were discovered In 7 cases (14%, 6 males, mean ± SD age at death, 13.6±11.2 years) of SUD. The activities at the time of SUD were exertion (3), emotion (1), and unknown (3). The mutations, R420W, S2246L, N4097S, E4146K, T4158P, and R4491C, involved nonconservative amino acid substitutions in highly conserved residues across species and were not seen In 400 reference alleles. CONCLUSIONS: This study represents the first molecular autopsy of RyR2 In medical examiner-referred cases of SUD. A targeted analysis of only 18 of the 105 protein-encoding exons of the cardiac ryanodine receptor/calcium release channel revealed potential CPVTI-causing RyR2 mutations in 1 of every 7 cases of SUD. These findings suggest that postmortem genetic testing of RyR2 should be considered as a part of the comprehensive medicolegal autopsy investigation of a SUD case and that this potentially heritable and often elusive arrhythmia syndrome be scrutinized carefully in family members of those who experience SUD. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to determine the prevalence and spectrum of nonsynonymous polymorphisms (amino acid variants) in the cardiac sodium channel among healthy subjects.Background: Pathogenic mutations in the cardiac sodium channel gene, SCN5A, cause approximately 15 to 20% of Brugada syndrome (BrS1), 5 to 10% of long QT syndrome (LQT3), and 2 to 5% of sudden infant death syndrome.Methods: Using single-stranded conformation polymorphism, denaturing high-performance liquid chromatography, and/or direct DNA sequencing, mutational analysis of the protein-encoding exons of SCN5A was performed on 829 unrelated, anonymous healthy subjects: 319 black, 295 white, 112 Asian, and 103 Hispanic.Results: In addition to the four known common polymorphisms (R34C, H558R, S1103Y, and R1193Q), four relatively ethnic-specific polymorphisms were identified: R481W, S524Y, P1090L, and V1951L. Overall, 39 distinct missense variants (28 novel) were elucidated. Nineteen variants (49%) were found only in the black cohort. Only seven variants (18%) localized to transmembrane-spanning domains. Four variants (F1293S, R1512W, and V1951L cited previously as BrS1-causing mutations and S1787N previously published as a possible LQT3-causing mutation) were identified in this healthy cohort.Conclusions: This study provides the first comprehensive determination of the prevalence and spectrum of cardiac sodium channel variants in healthy subjects from four distinct ethnic groups. This compendium of SCN5A variants is critical for proper interpretation of SCN5A genetic testing and provides an essential hit list of targets for future functional studies to determine whether or not any of these variants mediate genetic susceptibility for arrhythmias in the setting of either drugs or disease. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to elucidate the genetic basis for long QT syndrome (LQTS) in patients with a personal or family history of postpartum cardiac events.Background: The postpartum period is a time of increased arrhythmogenic susceptibility in women with LQTS.Methods: Between August 1997 and May 2003, 388 unrelated patients (260 females, average age at diagnosis, 23 years, and average QTc, 482 ms) were referred to Mayo Clinic's Sudden Death Genomics Laboratory for LQTS genetic testing. Comprehensive mutational analysis of the 5 LQTS-causing channel genes was performed. The postpartum period was defined as the 20 weeks after delivery. Cardiac events included sudden cardiac death, aborted cardiac arrest, and syncope. The presence of a personal and/or family history of cardiac events during postpartum period was determined by review of the medical records and/or phone interviews and was blinded to the status of genetic testing.Results: Fourteen patients (3.6% of cohort) had personal (n = 4) and/or family history (n = 11) of cardiac events during the defined postpartum period. Thirteen of 14 patients (93%) possessed an LQT2 mutation and 1 had an LQT1 mutation. Postpartum cardiac events were found more commonly in patients with LQT2 (13 of 80, 16%) than in patients with LQT1 (1 of 103, <1%, P = .0001).Conclusions: There is a relatively gene-specific molecular basis underlying cardiac events during the postpartum period in LQTS. Along with previous gene-specific associations involving swimming and LQT1 as well as auditory triggers and LQT2, this association between postpartum cardiac events and LQT2 can facilitate strategic genotyping. [ABSTRACT FROM AUTHOR]

Context: Fatal arrhythmias from occult long QT syndrome may be responsible for some cases of sudden infant death syndrome (SIDS). Because patients who have long QT syndrome with sodium channel gene (SCN5A) defects have an increased frequency of cardiac events during sleep, and a recent case is reported of a sporadic SCN5A mutation in an infant with near SIDS, SCN5A has emerged as the leading candidate ion channel gene for SIDS.Objective: To determine the prevalence and functional properties of SCN5A mutations in SIDS.Design, Setting, and Subjects: Postmortem molecular analysis of 93 cases of SIDS or undetermined infant death identified by the Medical Examiner's Office of the Arkansas State Crime Laboratory between September 1997 and August 1999. Genomic DNA was extracted from frozen myocardium and subjected to SCN5A mutational analyses. Missense mutations were incorporated into the human heart sodium channel alpha subunit by mutagenesis, transiently transfected into human embryonic kidney cells, and characterized electrophysiologically.Main Outcome Measures: Molecular and functional characterization of SCN5A defects.Results: Two of the 93 cases of SIDS possessed SCN5A mutations: a 6-week-old white male with an A997S missense mutation in exon 17 and a 1-month old white male with an R1826H mutation in exon 28. These 2 distinct mutations occurred in highly conserved regions of the sodium channel and were absent in 400 control patients (800 alleles). Functionally, the A997S and R1826H mutant channels expressed a sodium current characterized by slower decay and a 2- to 3-fold increase in late sodium current.Conclusion: Approximately 2% of this prospective, population-based cohort of SIDS cases had an identifiable SCN5A channel defect, suggesting that mutations in cardiac ion channels may provide a lethal arrhythmogenic substrate in some infants at risk for SIDS. [ABSTRACT FROM AUTHOR]

The article discusses a study on the currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells in relation to long-QT syndrome. It was found that hERG 1a currents are smaller than heteromeric hERG 1a/1b currents. This finding is said to correspond with a 2-fold increase in the rates of activation and recovery from inactivation. Also, It was observed that mutations disrupting hERG 1b function can reduce cardiac IKr and increase drug sensitivity.

Objectives: The aim of this study was to provide the spectrum and prevalence of mutations in the 12 Brugada syndrome (BrS)–susceptibility genes discovered to date in a single large cohort of unrelated BrS patients. Background: BrS is a potentially lethal heritable arrhythmia syndrome diagnosed electrocardiographically by coved-type ST-segment elevation in the right precordial leads (V1 to V3; type 1 Brugada electrocardiographic [ECG] pattern) and the presence of a personal/family history of cardiac events. Methods: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive mutational analysis of BrS1- through BrS12-susceptibility genes was performed in 129 unrelated patients with possible/probable BrS (46 with clinically diagnosed BrS [ECG pattern plus personal/family history of a cardiac event] and 83 with a type 1 BrS ECG pattern only). Results: Overall, 27 patients (21%) had a putative pathogenic mutation, absent in 1,400 Caucasian reference alleles, including 21 patients with an SCN5A mutation, 2 with a CACNB2B mutation, and 1 each with a KCNJ8 mutation, a KCND3 mutation, an SCN1Bb mutation, and an HCN4 mutation. The overall mutation yield was 23% in the type 1 BrS ECG pattern-only patients versus 17% in the clinically diagnosed BrS patients and was significantly greater among young men <20 years of age with clinically diagnosed BrS and among patients who had a prolonged PQ interval. Conclusions: We identified putative pathogenic mutations in ∼20% of our BrS cohort, with BrS genes 2 through 12 accounting for <5%. Importantly, the yield was similar between patients with only a type 1 BrS ECG pattern and those with clinically established BrS. The yield approaches 40% for SCN5A-mediated BrS (BrS1) when the PQ interval exceeds 200 ms. Calcium channel–mediated BrS is extremely unlikely in the absence of a short QT interval. [Copyright &y& Elsevier]

Objectives: This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). Background: Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. Methods: Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 ± 15 years, mean QTc 428 ± 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). Results: Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. Conclusions: Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that ≈65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered. [Copyright &y& Elsevier]

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na+ channels in excitable cells results in influx of Na+ and cellular depolarization. The function of Nav1.5, an Na+ channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming α-subunit of this channel is encoded by SCNSA. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCNSA are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na+ channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Nav1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Nav1.5 kinetics and may play a role in intestinal pseudo-obstruction. [ABSTRACT FROM AUTHOR]

Background: A 4-year-old boy born at 37 weeks' gestation with intrauterine growth retardation presented with developmental delay with pronounced language and gross motor delay, axial hypotonia, and dynamic hypertonia of the extremities. Investigations including the Minnesota Newborn Screen, thyroid stimulating hormone/thyroxin, and inborn errors of metabolism screening were negative. Cerebral magnetic resonance imaging and spectroscopy were normal. Genetic testing was negative for coagulopathy, Smith-Lemli-Opitz, fragile X, and Prader-Willi/Angelman syndromes. Whole genome array analysis was unremarkable.Methods: Whole exome sequencing was performed through a commercial testing laboratory to elucidate the underlying etiology for the child's presentation. A de novo mutation was hypothesized. In attempt to establish pathogenicity of our candidate variant, cellular electrophysiologic functional analysis of the putative de novo mutation was performed using patch-clamp technology.Results: Whole exome sequencing revealed a p.P1353L variant in the CACNA1A gene, which encodes for the α1-subunit of the brain-specific P/Q-type calcium channel (CaV2.1). This presynaptic high-voltage-gated channel couples neuronal excitation to the vesicular release of neurotransmitter and is implicated in several neurologic disorders. DNA Sanger sequencing confirmed that the de novo mutation was absent in both parents and present in the child only. Electrophysiologic analysis of P1353L-CACNA1A demonstrated near complete loss of function, with a 95% reduction in peak current density.Conclusions: Whole exome sequencing coupled with cellular electrophysiologic functional analysis of a de novoCACNA1A missense mutation has elucidated the probable underlying pathophysiologic mechanism responsible for the child's phenotype. Genetic testing of CACNA1A in patients with congenital hypotonia and developmental delay may be warranted. [ABSTRACT FROM AUTHOR]

Background—Type 2 long QT syndrome involves mutations in the human ether a-go-go—related gene (hERG or KCNH2). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes. Methods and Results—Isolated adult rat ventricular myocytes were infected with wild-type (WT)-Kv 11.1- and T421M-Kv11.1-expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current (IKv11.1) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant IKv11.1 resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak IKv11.1 density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to β-adrenergic stimulation by increasing IKv11.1. Conclusions—The T421M-Kv11.1 mutation caused a loss of IKv11.1 through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT+T421 M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to β-adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K+ channel (Kv7.1) that underlies the slowly activating delayed rectifier K+ current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K+-K+ repulsive forces required for rapid K+ permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K+ channel selectivity filter. [ABSTRACT FROM AUTHOR]

Objectives: The aims of this study were to determine the spectrum and prevalence of “background genetic noise” in the arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC) genetic test and to determine genetic associations that can guide the interpretation of a positive test result. Background: ARVC is a potentially lethal genetic cardiovascular disorder characterized by myocyte loss and fibrofatty tissue replacement of the right ventricle. Genetic variation among the ARVC susceptibility genes has not been systematically examined, and little is known about the background noise associated with the ARVC genetic test. Methods: Using direct deoxyribonucleic acid sequencing, the coding exons/splice junctions of PKP2, DSP, DSG2, DSC2, and TMEM43 were genotyped for 93 probands diagnosed with ARVC from the Netherlands and 427 ostensibly healthy controls of various ethnicities. Eighty-two additional ARVC cases were obtained from published reports, and additional mutations were included from the ARVD/C Genetic Variants Database. Results: The overall yield of mutations among ARVC cases was 58% versus 16% in controls. Radical mutations were hosted by 0.5% of control individuals versus 43% of ARVC cases, while 16% of controls hosted missense mutations versus a similar 21% of ARVC cases. Relative to controls, mutations in cases occurred more frequently in non-Caucasians, localized to the N-terminal regions of DSP and DSG2, and localized to highly conserved residues within PKP2 and DSG2. Conclusions: This study is the first to comprehensively evaluate genetic variation in healthy controls for the ARVC susceptibility genes. Radical mutations are high-probability ARVC-associated mutations, whereas rare missense mutations should be interpreted in the context of race and ethnicity, mutation location, and sequence conservation. [Copyright &y& Elsevier]

Objectives: The purpose of this study was to investigate the effect of location, coding type, and topology of KCNH2(hERG) mutations on clinical phenotype in type 2 long QT syndrome (LQTS). Background: Previous studies were limited by population size in their ability to examine phenotypic effect of location, type, and topology. Methods: Study subjects included 858 type 2 LQTS patients with 162 different KCNH2 mutations in 213 proband-identified families. The Cox proportional-hazards survivorship model was used to evaluate independent contributions of clinical and genetic factors to the first cardiac events. Results: For patients with missense mutations, the transmembrane pore (S5-loop-S6) and N-terminus regions were a significantly greater risk than the C-terminus region (hazard ratio [HR]: 2.87 and 1.86, respectively), but the transmembrane nonpore (S1–S4) region was not (HR: 1.19). Additionally, the transmembrane pore region was significantly riskier than the N-terminus or transmembrane nonpore regions (HR: 1.54 and 2.42, respectively). However, for nonmissense mutations, these other regions were no longer riskier than the C-terminus (HR: 1.13, 0.77, and 0.46, respectively). Likewise, subjects with nonmissense mutations were at significantly higher risk than were subjects with missense mutations in the C-terminus region (HR: 2.00), but that was not the case in other regions. This mutation location–type interaction was significant (p = 0.008). A significantly higher risk was found in subjects with mutations located in α-helical domains than in subjects with mutations in β-sheet domains or other locations (HR: 1.74 and 1.33, respectively). Time-dependent β-blocker use was associated with a significant 63% reduction in the risk of first cardiac events (p < 0.001). Conclusions: The KCNH2 missense mutations located in the transmembrane S5-loop-S6 region are associated with the greatest risk. [Copyright &y& Elsevier]

Abstract: Hypertrophic Cardiomyopathy (HCM) is a common primary cardiac disorder defined by a hypertrophied left ventricle, is one of the main causes of sudden death in young athletes, and has been associated with mutations in most sarcomeric proteins (tropomyosin, troponin T and I, and actin, etc.). Many of these mutations appear to affect the functional properties of cardiac troponin C (cTnC), i.e., by increasing the Ca2+-sensitivity of contraction, a hallmark of HCM, yet surprisingly, prior to this report, cTnC had not been classified as a HCM-susceptibility gene. In this study, we show that mutations occurring in the human cTnC (HcTnC) gene (TNNC1) have the same prevalence (~0.4%) as well established HCM-susceptibility genes that encode other sarcomeric proteins. Comprehensive open reading frame/splice site mutation analysis of TNNC1 performed on 1025 unrelated HCM patients enrolled over the last 10 years revealed novel missense mutations in TNNC1: A8V, C84Y, E134D, and D145E. Functional studies with these recombinant HcTnC HCM mutations showed increased Ca2+ sensitivity of force development (A8V, C84Y and D145E) and force recovery (A8V and D145E). These results are consistent with the HCM functional phenotypes seen with other sarcomeric-HCM mutations (E134D showed no changes in these parameters). This is the largest cohort analysis of TNNC1 in HCM that details the discovery of at least three novel HCM-associated mutations and more strongly links TNNC1 to HCM along with functional evidence that supports a central role for its involvement in the disease. This study may help to further define TNNC1 as an HCM-susceptibility gene, a classification that has already been established for the other members of the troponin complex. [Copyright &y& Elsevier]

Abstract: Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype–phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids, which leads to a trafficking defect of the mutant channel. [Copyright &y& Elsevier]

Congenital long QT syndrome (LQTS) is a genetic disease that predisposes affected individuals to arrhythmias, syncope, and sudden death. Mutations in several ion channel genes have been discovered in different families with LQTS: KCNQ1 (KVLQT1, LQT1), KCNH2 (HERG, LQT2), SCN5A (LQT3), KCNE1 (minK, LQT5), and KCNE2 (MiRP1, LQT6). Previously, the P448R-KVLQT1 missense mutation has been reported as an LQT1-causing mutation. In this report, we demonstrate the presence of the P448R polymorphism in two, unrelated Chinese LQTS families. Although absent from 500 reference alleles derived from 150 white and 100 African-American subjects, P448R was present in 14% of healthy Chinese volunteers. Given the inconsistencies between the genotype (LQT1) and clinical phenotype (LQT2) in our two LQTS families, together with the finding that the P448R appears to be a common, ethnic-specific polymorphism, mutational analysis was extended to the other LQTS-causing genes resulting in the identification of distinct HERG missense mutations in each of these two families. Heterologous expression of P448R-KVLQT1 yielded normal, wild-type (WT) currents. In contrast, the two unique HERG mutations resulted in dominant-negative suppression of the WT HERG channel. Our study has profound implications for those engaged in genetic research. Importantly, one child of the original proband was initially diagnosed with LQT1 based upon the presence of P448R-KVLQT1 and was treated with beta-blockers. However, he did not possess the subsequently determined LQT2-causing mutation. On the other hand, his untreated P448R-negative brother harbored the true, disease-causing HERG mutation. These findings underscore the importance of distinguishing channel polymorphisms from mutations pathogenic for LQTS and emphasize the importance of using appropriate ethnically matched controls in the genotypic analysis of LQTS. [Copyright &y& Elsevier]