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Background: Targeted postmortem genetic testing of the 4 major channelopathy-susceptibility genes (KCNQ1, KCNH2, SCN5A, and RYR2) have yielded putative pathogenic mutations in ≤30% of autopsy-negative sudden unexplained death in the young (SUDY) cases with highest yields derived from the subset of exertion-related SUDY. Here, we evaluate the role of whole-exome sequencing in exertion-related SUDY cases., Methods and Results: From 1998 to 2010, 32 cases of exertion-related SUDY were referred by Medical Examiners for a cardiac channel molecular autopsy. A mutational analysis of the major long-QT syndrome-susceptibility genes (KCNQ1, KCNH2, and SCN5A) and catecholaminergic polymorphic ventricular tachycardia-susceptibility gene (RYR2) identified a putative pathogenic mutation in 11 cases. Whole-exome sequencing was performed on the remaining 21 targeted gene-negative SUDY cases. After whole-exome sequencing, a gene-specific surveillance of all genes (N=100) implicated in sudden death was performed to identify putative pathogenic mutation(s). Three of these 21 decedents had a clinically actionable, pathogenic mutation (CALM2-F90L, CALM2-N98S, and PKP2-N634fs). Of the 18 remaining cases, 7 hosted at least 1 variant of unknown significance with a minor allele frequency <1:20 000. The overall yield of pathogenic mutations was higher among decedents aged 1 to 10 years (10/11, 91%) than those aged 11 to 19 years (4/21, 19%, P=0.0001)., Conclusions: Molecular screening in this clinical scenario is appropriate with a pathogenic mutation detection rate of 44% using direct DNA sequencing followed by whole-exome sequencing. Only 5 of the 100 interrogated sudden death genes hosted actionable pathogenic mutations for more than one third of these exertion-related, autopsy-negative SUDY cases., (© 2016 American Heart Association, Inc.)

Background: Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and a leading cause of identifiable sudden cardiac death (SCD) in the young. Herein, we sought to determine the genotype-phenotype correlations in a cohort of unrelated, genotyped patients diagnosed with HCM at a young age, as well as to characterize the differences between HCM diagnosed in adulthood and HCM diagnosed at a young age., Methods and Results: From 1999 to 2011, 1053 unrelated patients diagnosed with HCM were enrolled in research-based genetic testing. The electronic medical record was reviewed to identify those with HCM diagnosed at ≤21 years (N = 137, mean age at diagnosis 13.2 ± 6 years, 64% male). From this cohort of patients recruited from a tertiary care referral center, the genetic test was positive in 71 (52%), which was significantly higher than patients diagnosed >21 years (31%; P < .001). Genotype-positive patients had increased maximum left ventricular wall thickness (24.9 ± 8.0 vs. 21.6 ± 7.4 mm, P = .01) and higher incidence of reverse-curve ventricular septal morphology (71% vs. 40%, P < .001). Unrelated to genotype status, 26/137 patients (19%) experienced significant HCM-related morbidity/mortality including progressive heart failure symptoms in 12, transplantation in 4, and death in 10., Conclusions: Among patients diagnosed with HCM during the first two decades of life, the yield of genetic testing is significantly higher than when diagnosed at later age. While the phenotype of young HCM patients is worse than patients whose HCM is diagnosed at later age, the phenotypes of genotype-positive and genotype-negative young patients were similar. Independent of genotype, nearly 30% of the patients with follow-up in this study had symptom progression, transplant, or death., (© 2015 Wiley Periodicals, Inc.)

Background: Long-QT syndrome (LQTS) may result in syncope, seizures, or sudden cardiac arrest. Although 16 LQTS-susceptibility genes have been discovered, 20% to 25% of LQTS remains genetically elusive., Methods and Results: We performed whole-exome sequencing child-parent trio analysis followed by recessive and sporadic inheritance modeling and disease-network candidate analysis gene ranking to identify a novel underlying genetic mechanism for LQTS. Subsequent mutational analysis of the candidate gene was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing on a cohort of 33 additional unrelated patients with genetically elusive LQTS. After whole-exome sequencing and variant filtration, a homozygous p.D18fs*13 TRDN-encoded triadin frameshift mutation was discovered in a 10-year-old female patient with LQTS with a QTc of 500 milliseconds who experienced recurrent exertion-induced syncope/cardiac arrest beginning at 1 year of age. Subsequent mutational analysis of TRDN revealed either homozygous or compound heterozygous frameshift mutations in 4 of 33 unrelated cases of LQTS (12%). All 5 TRDN-null patients displayed extensive T-wave inversions in precordial leads V1 through V4, with either persistent or transient QT prolongation and severe disease expression of exercise-induced cardiac arrest in early childhood (≤3 years of age) and required aggressive therapy. The overall yield of TRDN mutations was significantly greater in patients ≤10 years of age (5 of 10, 50%) compared with older patients (0 of 24, 0%; P=0.0009)., Conclusions: We identified TRDN as a novel underlying genetic basis for recessively inherited LQTS. All TRDN-null patients had strikingly similar phenotypes. Given the recurrent nature of potential lethal arrhythmias, patients fitting this phenotypic profile should undergo cardiac TRDN genetic testing., (© 2015 American Heart Association, Inc.)

Objectives: To determine the prevalence and spectrum of mutations and genotype-phenotype relationships in the largest hypertrophic cardiomyopathy (HCM) cohort to date and to provide an easy, clinically applicable phenotype-derived score that provides a pretest probability for a positive HCM genetic test result., Patients and Methods: Between April 1, 1997, and February 1, 2007, 1053 unrelated patients with the clinical diagnosis of HCM (60% male; mean ± SD age at diagnosis, 44.4 ± 19 years) had HCM genetic testing for the 9 HCM-associated myofilament genes. Phenotyping was performed by review of electronic medical records., Results: Overall, 359 patients (34%) were genotype positive for a putative HCM-associated mutation in 1 or more HCM-associated genes. Univariate and multivariate analyses identified the echocardiographic reverse curve morphological subtype, an age at diagnosis younger than 45 years, a maximum left ventricular wall thickness of 20 mm or greater, a family history of HCM, and a family history of sudden cardiac death as positive predictors of positive genetic test results, whereas hypertension was a negative predictor. A score, based on the number of predictors of a positive genetic test result, predicted a positive genetic test result ranging from 6% when only hypertension was present to 80% when all 5 positive predictor markers were present., Conclusion: In this largest HCM cohort published to date, the overall yield of genetic testing was 34%. Although all the patients were diagnosed clinically as having HCM, the presence or absence of 6 simple clinical/echocardiographic markers predicted the likelihood of mutation-positive HCM. Phenotype-guided genetic testing using the Mayo HCM Genotype Predictor score provides an easy tool for an effective genetic counseling session., (Copyright © 2014 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Importance: Intrauterine fetal death or stillbirth occurs in approximately 1 out of every 160 pregnancies and accounts for 50% of all perinatal deaths. Postmortem evaluation fails to elucidate an underlying cause in many cases. Long QT syndrome (LQTS) may contribute to this problem., Objective: To determine the spectrum and prevalence of mutations in the 3 most common LQTS susceptible genes (KCNQ1, KCNH2, and SCN5A) for a cohort of unexplained cases., Design, Setting, and Patients: In this case series, retrospective postmortem genetic testing was conducted on a convenience sample of 91 unexplained intrauterine fetal deaths (mean [SD] estimated gestational age at fetal death, 26.3 [8.7] weeks) that were collected from 2006-2012 by the Mayo Clinic, Rochester, Minnesota, or the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. More than 1300 ostensibly healthy individuals served as controls. In addition, publicly available exome databases were assessed for the general population frequency of identified genetic variants., Main Outcomes and Measures: Comprehensive mutational analyses of KCNQ1 (KV7.1, LQTS type 1), KCNH2 (HERG/KV11.1, LQTS type 2), and SCN5A (NaV1.5, LQTS type 3) were performed using denaturing high-performance liquid chromatography and direct DNA sequencing on genomic DNA extracted from decedent tissue. Functional analyses of novel mutations were performed using heterologous expression and patch-clamp recording., Results: The 3 putative LQTS susceptibility missense mutations (KCNQ1, p.A283T; KCNQ1, p.R397W; and KCNH2 [1b], p.R25W), with a heterozygous frequency of less than 0.05% in more than 10 000 publicly available exomes and absent in more than 1000 ethnically similar control patients, were discovered in 3 intrauterine fetal deaths (3.3% [95% CI, 0.68%-9.3%]). Both KV7.1-A283T (16-week male) and KV7.1-R397W (16-week female) mutations were associated with marked KV7.1 loss-of-function consistent with in utero LQTS type 1, whereas the HERG1b-R25W mutation (33.2-week male) exhibited a loss of function consistent with in utero LQTS type 2. In addition, 5 intrauterine fetal deaths hosted SCN5A rare nonsynonymous genetic variants (p.T220I, p.R1193Q, involving 2 cases, and p.P2006A, involving 2 cases) that conferred in vitro electrophysiological characteristics consistent with potentially proarrhythmic phenotypes., Conclusions and Relevance: In this molecular genetic evaluation of 91 cases of intrauterine fetal death, missense mutations associated with LQTS susceptibility were discovered in 3 cases (3.3%) and overall, genetic variants leading to dysfunctional LQTS-associated ion channels in vitro were discovered in 8 cases (8.8%). These preliminary findings may provide insights into mechanisms of some cases of stillbirth.

Background: Approximately 75% of long QT syndrome (LQTS) has been explained genetically through research-based and, more recently, commercial genetic testing. While novel LQTS-susceptibility genes or mutations in unexplored regions of known genes underlie the genetic mechanism for some of the 25% "genotype-negative" remnant, it is likely that some cases represent false-negative test results owing to mutation detection failures., Objective: To determine the prevalence and etiology of false negatives that occurred with research-based mutational analysis involving denaturing high-performance liquid chromatography (DHPLC) followed by DNA sequencing (DHPLC-SEQ) in our previously published cohort of unrelated patients referred for LQTS genetic testing., Methods: Forty-four LQTS cases (29 men, average age 23 ± 15 years, average corrected QT interval 516 ± 56 ms) deemed genotype negative following DHPLC-SEQ were selected for repeat genetic testing using direct DNA sequencing., Results: LQTS-causing mutations were identified in 7 of 44 (16%) phenotype-positive/previously genotype-negative subjects, including 4 mutations in KCNQ1 (S225L, G568R, R591H, and R594Q), 2 in KCNH2 (H70R and G925R), and 1 in SCN5A (V411M). None of these variants were seen in more than 2600 reference alleles. Analysis of the misses revealed (1) normal DHPLC detection profile in 2, (2) allelic dropout in 2, (3) failure to correctly optimize DHPLC conditions in 1, and (4) failure to detect abnormal DHPLC signal in 2., Conclusions: Repeat genetic testing using direct DNA sequencing may be warranted for LQTS phenotype-positive individuals who were pronounced genotype negative during the decade of research-based mutational analysis that involved intermediate mutation detection methods such as DHPLC., (Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

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 © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: To perform long QT syndrome and catecholaminergic polymorphic ventricular tachycardia cardiac channel postmortem genetic testing (molecular autopsy) for a large cohort of cases of autopsy-negative sudden unexplained death (SUD)., Methods: From September 1, 1998, through October 31, 2010, 173 cases of SUD (106 males; mean ± SD age, 18.4 ± 12.9 years; age range, 1-69 years; 89% white) were referred by medical examiners or coroners for a cardiac channel molecular autopsy. Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, a comprehensive mutational analysis of the long QT syndrome susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) and a targeted analysis of the catecholaminergic polymorphic ventricular tachycardia type 1-associated gene (RYR2) were conducted., Results: Overall, 45 putative pathogenic mutations absent in 400 to 700 controls were identified in 45 autopsy-negative SUD cases (26.0%). Females had a higher yield (26/67 [38.8%]) than males (19/106 [17.9%]; P<.005). Among SUD cases with exercise-induced death, the yield trended higher among the 1- to 10-year-olds (8/12 [66.7%]) compared with the 11- to 20-year-olds (4/27 [14.8%]; P=.002). In contrast, for those who died during a period of sleep, the 11- to 20-year-olds had a higher yield (9/25 [36.0%]) than the 1- to 10-year-olds (1/24 [4.2%]; P=.01)., Conclusion: Cardiac channel molecular autopsy should be considered in the evaluation of autopsy-negative SUD. Several interesting genotype-phenotype observations may provide insight into the expected yields of postmortem genetic testing for SUD and assist in selecting cases with the greatest potential for mutation discovery and directing genetic testing efforts., (Copyright © 2012 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the prevalence and spectrum of mutations associated with long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) in a seemingly unexplained drowning cohort., Patients and Methods: From September 1, 1998, through October 31, 2010, 35 unexplained drowning victims (23 male and 12 female; mean ± SD age, 17±12 years [range, 4-69 years]) were referred for a cardiac channel molecular autopsy. Of these, 28 (20 male and 8 female) drowned while swimming, and 7 (3 male and 4 female) were bathtub submersions. Polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing were used for a comprehensive mutational analysis of the 3 major LQTS-susceptibility genes (KCNQ1, KCNH2, and SCN5A), and a targeted analysis of the CPVT1-associated, RYR2-encoded cardiac ryanodine receptor was conducted., Results: Of the 28 victims of swimming-related drowning, 8 (28.6%) were mutation positive, including 2 with KCNQ1 mutations (L273F, AAPdel71-73 plus V524G) and 6 with RYR2 mutations (R414C, I419F, R1013Q, V2321A, R2401H, and V2475F). None of the bathtub victims were mutation positive. Of the 28 victims who drowned while swimming, women were more likely to be mutation positive than men (5/8 [62.5%] vs 3/20 [15%]; P=.02). Although none of the mutation-positive, swimming-related drowning victims had a premortem diagnosis of LQTS or CPVT, a family history of cardiac arrest, family history of prior drowning, or QT prolongation was present in 50%., Conclusion: Nearly 30% of the victims of swimming-related drowning hosted a cardiac channel mutation. Genetic testing should be considered in the postmortem evaluation of an unexplained drowning, especially if a positive personal or family history is elicited.

Objectives: The aim of this study was to describe the clinical profile associated with triple sarcomere gene mutations in a large hypertrophic cardiomyopathy (HCM) cohort., Background: In patients with HCM, double or compound sarcomere gene mutation heterozygosity might be associated with earlier disease onset and more severe outcome. The occurrence of triple mutations has not been reported., Methods: A total of 488 unrelated index HCM patients underwent screening for myofilament gene mutations by direct deoxyribonucleic acid sequencing of 8 genes, including myosin binding protein C (MYBPC3), beta-myosin heavy chain (MYH7), regulatory and essential light chains (MYL2, MYL3), troponin-T (TNNT2), troponin-I (TNNI3), alpha-tropomyosin (TPM1), and actin (ACTC)., Results: Of the 488 index patients, 4 (0.8%) harbored triple mutations, as follows: MYH7-R869H, MYBPC3-E258K, and TNNI3-A86fs in a 32-year-old woman; MYH7-R723C, MYH7-E1455X, and MYBPC3-E165D in a 46-year old man; MYH7-R869H, MYBPC3-K1065fs, and MYBPC3-P371R in a 45-year old woman; and MYH7-R1079Q, MYBPC3-Q969X, and MYBPC3-R668H in a 50-year old woman. One had a history of resuscitated cardiac arrest, and 3 had significant risk factors for sudden cardiac death, prompting the insertion of an implantable cardioverter-defibrillator in all, with appropriate shocks in 2 patients. Moreover, 3 of 4 patients had a severe phenotype with progression to end-stage HCM by the fourth decade, requiring cardiac transplantation (n=1) or biventricular pacing (n=2). The fourth patient, however, had clinically mild disease., Conclusions: Hypertrophic cardiomyopathy caused by triple sarcomere gene mutations was rare but conferred a remarkably increased risk of end-stage progression and ventricular arrhythmias, supporting an association between multiple sarcomere defects and adverse outcome. Comprehensive genetic testing might provide important insights to risk stratification and potentially indicate the need for differential surveillance strategies based on genotype., (Copyright (c) 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Hypertrophic cardiomyopathy (HCM) can be classified into at least four major anatomic subsets based upon the septal contour, and the location and extent of hypertrophy: reverse curvature-, sigmoidal-, apical-, and neutral contour-HCM. Here, we sought to identify genetic determinants for sigmoidal-HCM and hypothesized that Z-disc-HCM may be associated preferentially with a sigmoidal phenotype. Utilizing PCR, DHPLC, and direct DNA sequencing, we performed mutational analysis of five genes encoding cardiomyopathy-associated Z-disc proteins. The study cohort consisted of 239 unrelated patients with HCM previously determined to be negative for mutations in the eight genes associated with myofilament-HCM. Blinded to the Z-disc genotype status, the septal contour was graded qualitatively using standard transthoracic echocardiography. Thirteen of the 239 patients (5.4%) had one of 13 distinct HCM-associated Z-disc mutations involving residues highly conserved across species and absent in 600 reference alleles: LDB3 (6), ACTN2 (3), TCAP (1), CSRP3 (1), and VCL (2). For this subset with Z-disc-associated HCM, the septal contour was sigmoidal in 11 (85%) and apical in 2 (15%). While Z-disc-HCM is uncommon, it is equal in prevalence to thin filament-HCM. In contrast to myofilament-HCM, Z-disc-HCM is associated preferentially with sigmoidal morphology.

Objectives: The purpose of this study was to examine the effect of clinical phenotype on the yield of genetic testing for congenital long QT syndrome (LQTS)., Background: Since the discovery of the first LQTS susceptibility genes in 1995, numerous genotype-phenotype relationships have emerged during the past decade of research genetic testing. In May 2004, LQTS genetic testing became a clinically available molecular diagnostic test., Methods: Blinded to genetic test results, analysis of the clinical phenotype was performed in 541 consecutive unrelated patients referred to Mayo Clinic's Sudden Death Genomics Laboratory for LQTS genetic testing from August 1997 to July 2004., Results: The yield of genetic testing correlated significantly with the corrected QT interval (QTc) and clinical diagnostic score ranging from 0% when QTc was <400 ms to 62% when QTc was >480 ms (p < 0.0001). Among those with the highest clinical probability, the yield was 72% (89 of 123). The yield fluctuated substantially depending on age at diagnosis in males. Among physicians who referred > or =5 patients, the yield ranged from 0% to 80% (p < 0.0001)., Conclusions: In this large cohort of unrelated patients referred for LQTS genetic testing, the clinical phenotype strongly correlated with the likelihood of elucidating a pathogenic mutation with the cardiac channel gene screen.

We tested the hypothesis that perturbations in metavinculin may provide a pathogenic substrate for hypertrophic cardiomyopathy (HCM). HCM and dilated cardiomyopathy (DCM) are partially allelic disorders whereby identical genes have been implicated in the pathogenesis of both diseases. Mutations in metavinculin, a muscle-specific isoform of vinculin, were identified previously in DCM and shown to alter in vitro organization of actin filaments. Using denaturing high performance liquid chromatography and direct DNA sequencing, mutational analysis of the metavinculin-specific exon of vinculin (VCL, exon 19) was performed in a cohort of 389 unrelated patients with clinical HCM, previously genotyped for the 8 most common HCM-associated myofilament-encoding genes. Overall, 3 non-synonymous single nucleotide polymorphisms (A934V, P943A, and R975W) were detected in 4 patients. One patient with severely obstructive, mid-ventricular and apical hypertrophy harbored the previously published DCM-associated mutation, R975W. R975 is a highly conserved residue and R975W was absent in over 1400 reference alleles. Immunohistochemical analysis of the proband's myectomy specimen revealed a paucity of vinculin/metavinculin in the intercalated discs. Metavinculin mutations are pathogenic substrates for both HCM and DCM, further highlighting the allelic nature of these cardiomyopathies. Mutations in functionally distinct regions of certain cardiomyopathy-associated genes may have a dominant effect in determining a remodeling pathway of either maladaptive hypertrophy or dilation. However, this study demonstrates that the same fundamental mutation in humans can yield either cardiomyopathic phenotype, underscoring a critical role for modifier genes and/or environmental stressors in cardiac remodeling.

Within the field of molecular cardiac electrophysiology, the previous decade of research elucidated the fundamental genetic substrate underlying many arrhythmogenic disorders such as long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), Andersen-Tawil syndrome, Brugada Syndrome, and Timothy syndrome. In addition, the genetic basis for cardiomyopathic processes vulnerable to sudden arrhythmic death-hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy-are understood now in greater detail. The majority of congenital LQTS is understood as a primary cardiac channelopathy that often but not always provides evidence of its presence via a prolonged QT interval on the 12-lead surface electrocardiogram. To date, more than 300 mutations have been identified in five genes encoding key ion channel sub units involved in the orchestration of the heart's action potential. LQTS genetic testing has been performed in research laboratories over the past decade, relying on the techniques of PCR, an intermediate mutation analysis platform such as single-stranded conformation polymorphism (SSCP) or denaturing high-performance liquid chromatography (dHPLC), and subsequent direct DNA sequencing to elucidate the genetic underpinnings of this disorder. Presently, LQTS genetic testing is a clinically available molecular diagnostic test that provides comprehensive open reading frame/splice site mutational analysis via high-throughput DNA sequencing. This chapter will focus on LQTS genetic testing employing the techniques of genomic DNA isolation from peripheral blood, exon-specific PCR amplification, dHPLC hetero-duplex analysis, and direct DNA sequencing.

Aims: The purpose of this study was to determine whether the deletion/insertion (D/I) polymorphism in the ACE-encoded angiotensin-converting enzyme or the pooled gene effect of five renin-angiotensin-aldosterone system (RAAS) polymorphisms were disease modifiers in a large cohort of unrelated patients with genotyped hypertrophic cardiomyopathy (HCM)., Methods and Results: Five different RAAS polymorphism genotypes were established by PCR amplification of the surrounding polymorphic regions of genomic DNA in a cohort of 389 unrelated patients comprehensively genotyped for HCM-causing mutations in eight sarcomeric/myofilament genes. Patient clinical data were archived in a database blinded both to the primary myofilament defect and the polymorphism genotype. Each patient was assessed with respect to ACE genotype as well as composite pro-left ventricular hypertrophy (LVH) RAAS polymorphism score (0-5). Overall, no clinical parameter correlated independently with ACE genotype. Subset analysis of the two most common genetic subtypes of HCM, MYBPC3 (myosin binding protein C) and MYH7 (beta myosin heavy chain), demonstrated a significant pro-LVH effect of DD-ACE only in patients with MYBPC3-HCM. In MYBPC3-HCM, left ventricular wall thickness was greater in patients with DD genotype (25.8+/-5 mm) compared with DI (21.8+/-4) or II genotype (20.8+/-5, P=0.01). Moreover, extreme hypertrophy (>30 mm) was only seen in MYBPC3-HCM patients who also hosted DD-ACE. An effect of RAAS pro-LVH score was evident only in the subgroup of patients with no previously identified myofilament mutation., Conclusion: This study demonstrates that RAAS genotypes may modify the clinical phenotype of HCM in a disease gene-specific fashion rather than indiscriminately.

Background: Mutations in the RyR2-encoded cardiac ryanodine receptor/calcium release channel cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1)., Objectives: Because CPVT and concealed long QT syndrome (LQTS) phenotypically mimic one other, we sought to determine the spectrum and prevalence of RyR2 mutations in a cohort of unrelated patients who were referred specifically for LQTS genetic testing., Methods: Using denaturing high-performance liquid chromatography and direct DNA sequencing, targeted mutational analysis of 23 RyR2 exons previously implicated in CPVT1 was performed on genomic DNA from 269 unrelated patients (180 females, average age at diagnosis 24 +/- 17 years) who were referred to Mayo Clinic's Sudden Death Genomics Laboratory for LQTS genetic testing. Previously, comprehensive mutational analysis of the five LQTS-associated cardiac channel genes proved negative for this entire subset of patients now designated as "genotype-negative" LQTS referrals., Results: Fifteen distinct RyR2 mutations (14 missense, 1 duplication/insertion, 12 novel) were found in 17 (6.3%) of 269 patients. None of these mutations were present in 400 reference alleles. Two mutations localized to the calstabin-2 (FKBP12.6) binding domain. Upon review of the clinical records, the referral diagnosis for all 17 patients was "atypical" or "borderline" LQTS., Conclusion: Putative pathogenic CPVT1-causing mutations in RyR2 were detected in 6% of unrelated, genotype-negative LQTS referrals. These findings suggest that CPVT may be underrecognized among physicians referring patients because of a suspected channelopathy. A diagnosis of "atypical LQTS" may warrant consideration of CPVT and analysis of RyR2 if the standard cardiac channel gene screen for LQTS is negative.

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.

Objectives: We sought to determine the frequency and phenotype of mutations in myosin binding protein C (MYBPC3) in a large outpatient cohort of patients with hypertrophic cardiomyopathy (HCM) seen at our tertiary referral center., Background: Mutations in MYBPC3 are one of the most frequent genetic causes of HCM and have been associated with variable onset of disease and prognosis. However, the frequency of mutations and associated clinical presentation have not been established in a large, unrelated cohort of patients., Methods: Using deoxyribonucleic acid from 389 unrelated patients with HCM, each protein coding exon of MYBPC3 was analyzed for mutations by polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. Clinical data were extracted from patient records blinded to patient genotype., Results: Of 389 patients with HCM, 71 (18%) had mutations in MYBPC3. In all, 46 mutations were identified, 33 of which were novel (72%). Patients with MYBPC3 mutations did not differ significantly from patients with thick filament-HCM, thin filament-HCM, or genotype-negative HCM with respect to age at diagnosis, degree of hypertrophy, incidence of myectomy, or family history of HCM or sudden death. Patients with multiple mutations (n = 10, 2.6%) had the most severe disease presentation., Conclusions: This study defines the frequency and associated phenotype for MYBPC3 and/or multiple mutations in HCM in the largest cohort to date. In this cohort, unrelated patients with MYBPC3-HCM virtually mimicked the phenotype of those with mutations in the beta-myosin heavy chain. Patients with multiple mutations had the most severe phenotype.

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.

Objectives: We sought to determine the prevalence and phenotype of beta-myosin heavy chain gene MYH7 mutations in a large cohort of unrelated patients with hypertrophic cardiomyopathy (HCM)., Background: Hypertrophic cardiomyopathy is a heterogeneous cardiac disease. MYH7 mutations are one of the most common genetic causes of HCM and have been associated with severe hypertrophy, young age of diagnosis, and high risk of sudden cardiac death. However, these clinical findings from large, family studies have not been confirmed in a large unrelated cohort., Methods: Deoxyribonucleic (DNA) samples obtained from 389 HCM outpatients seen at this tertiary referral center were analyzed for mutations, using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing for all 38 protein-coding exons of MYH7. Clinical data were extracted from patient records blinded to patient genotype., Results: Fifty-eight patients (15%) harbored 40 different mutations in MYH7. Compared with HCM patients without MYH7 mutations, HCM patients with MYH7 were younger at diagnosis (32.9 vs. 42.7 years, p = 0.0002), had more hypertrophy (left ventricular wall thickness of 24.2 vs. 21.1 mm, p = 0.0009), and more frequently underwent myectomy (60% vs. 38%, p = 0.002). The HCM patients with MYH7 mutations more often had a family history of HCM (43% vs. 29%, p = 0.006), but there was no difference in family history of sudden death (16% vs. 14%, p = NS)., Conclusions: In this setting, HCM patients with MYH7 were diagnosed at a younger age and had more hypertrophy, but they had no greater frequency of sudden death among first-degree relatives. Although these associations may prove useful for targeted gene screening, caution should be exercised in terms of using pathogenic status in risk stratification.

Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas drug-induced LQTS is caused primarily by HERG channel block. Many common polymorphisms are functionally silent and have been traditionally regarded as benign and without physiological consequence. However, the identification of common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid coding variants) with functional phenotypes in the SCN5A Na(+) channel and MiRP1 K(+) channel beta-subunit have challenged this viewpoint. In this report, we test the hypothesis that common missense HERG polymorphisms alter channel physiology. Comprehensive mutational analysis of HERG was performed on genomic DNA derived from a population-based cohort of sudden infant death syndrome and two reference allele cohorts derived from 100 African American and 100 Caucasian individuals. Amino acid-encoding variants were considered common polymorphisms if they were present in at least two of the three study cohorts with an allelic frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R. Wild-type (WT) and polymorphic channels were heterologously expressed in human embryonic kidney cells, and biochemical and voltage-clamp techniques were used to characterize their functional properties. All channel types were processed similarly, but several electrophysiological differences were identified: 1) K897T current density was lower than the other polymorphic channels; 2) K897T channels activated at more negative potentials than WT and R1047L; 3) K897T and Q1068R channels inactivated and recovered from inactivation faster than WT, P967L, and R1047L channels; and 4) K897T channels showed subtle differences compared with WT channels when stimulated with an action potential waveform. In contrast to K897T and Q1068R channels, P967L and R1047L channels were electrophysiologically indistinguishable from WT channels. All HERG channels had similar sensitivity to block by cisapride. Therefore, some HERG polymorphic channels are electrophysiologically different from WT channels.

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.

Objective: To determine the spectrum, frequency, and ethnic-specificity of channel variants in the potassium channel genes implicated in congenital long QT syndrome (LQTS) among healthy subjects., Subjects and Methods: Genomic DNA from 744 apparently healthy individuals-305 black, 187 white, 134 Asian, and 118 Hispanic--was subject to a comprehensive mutational analysis of the 4 LQTS-causing potassium channel genes: KCNQ1 (LQT1), KCNH2 (LQT2), KCNE1 (LQT5), and KCNE2 (LQT6)., Results: Overall, 49 distinct amino acid-altering variants (36 novel) were identified: KCNQ1 (n = 16), KCNH2 (n = 25),KCNE1 (n = 5), and KCNE2 (n = 3). More than half of these variants (26/49) were found exclusively in black subjects. The known K897T-HERG and the G38S-min K common polymorphisms were identified in all 4 ethnic groups. Excluding these 2 common polymorphisms, 25% of black subjects had at least 1 nonsynonymous potassium channel variant compared with 14% of white subjects (P < .01)., Conclusions: To our knowledge, this study represents the first comprehensive determination of the frequency and spectrum of cardiac channel variants found among healthy subjects from 4 major ethnic groups. Defining the population burden of genetic variants in these critical cardiac ion channels is crucial for proper interpretation of genetic test results of individuals at risk for LQTS. This compendium provides a resource for epidemiological and functional investigation of variant effects on the repolarization properties of cardiac tissues, including susceptibility to lethal cardiac arrhythmias.

Background: Genotype-phenotype correlative studies have implicated 8 particular mutations that cause hypertrophic cardiomyopathy (HCM) as "benign defects," associated with near-normal survival: N232S, G256E, F513C, V606M, R719Q, and L908V of beta-myosin heavy chain (MYH7); S179F of troponin T (TNNT2); and D175N of alpha-tropomyosin (TPM1). Routine genetic screening of HCM patients for specific mutations is anticipated to provide important diagnostic and prognostic information. The frequency and associated phenotype of these mutations in a large, unselected cohort of HCM is unknown., Methods and Results: A total of 293 unrelated HCM patients were genotyped for the presence of a benign mutation. DNA was obtained after informed consent; specific MHY7, TNNT2, and TPM1 fragments were amplified by polymerase chain reaction; and the mutations were detected by denaturing high-performance liquid chromatography and automated DNA sequencing. Only 5 (1.7%) of the 293 patients possessed a benign mutation. Moreover, all 5 subjects with an ascribed benign mutation had already manifested clinically severe expression of HCM, with all 5 requiring surgical myectomy, 3 of the 5 having a family history of sudden cardiac death, and 1 adolescent requiring an orthotopic heart transplant., Conclusions: These findings demonstrate the rarity of specific mutations in HCM and challenge the notion of mutation-specific clinical outcomes. Fewer than 2% of the subjects harbored a benign mutation, and those patients with a benign mutation experienced a very serious clinical course.

We sought to define the pathogenic mutation in a family with hypertrophic cardiomyopathy (HC) and a markedly arrhythmogenic phenotype. The proband was an 8-year-old female with a sentinel event of sudden death. Screening echocardiograms revealed HC in 2 of her 3 siblings and her father. Her youngest male sibling was diagnosed with HC at age 2 years and died suddenly at age 6 years from ventricular fibrillation despite an implanted cardioverter defibrillator. Using DNA extracted from peripheral lymphocytes, linkage exclusion was performed by haplotype analysis of polymorphic markers for the HC genes. Genes not excluded by linkage were analyzed for mutations using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. Using this strategy, a 610 T>G nucleotide substitution in the alpha-tropomyosin gene (TPM1) was identified resulting in a novel L185R (Leucine [L] to Arginine [R]) missense mutation. This mutation was a spontaneous germ-line mutation originating in the proband's father. L185R-TPM1 cosegregated with family members having clinical evidence of HC, including the proband as confirmed by molecular autopsy. The mutation was not present in 400 reference alleles. Thus, a novel missense mutation in TPM1 was discovered in a family with HC and sudden death in childhood. Unlike previously defined mutations that may disrupt the interactions between alpha-tropomyosin monomers, the L185R mutation may affect troponin-T binding. Defining the pathogenic mutation enabled definitive molecular diagnosis of 2 surviving children.

Objectives: The goal of this study was to determine the prevalence of "malignant" mutations in hypertrophic cardiomyopathy (HCM)., Background: Previous genotype-phenotype studies have implicated four mutations (R403Q, R453C, G716R and R719W) as highly malignant defects in the beta-myosin heavy chain (MYH7). In the cardiac troponin T gene (TNNT2), a specific mutation (R92W) has been associated with high risk of sudden death. Routine clinical screening for these malignant mutations has been suggested to identify high-risk individuals., Methods: We screened 293 unrelated individuals with HCM seen at the Mayo Clinic in Rochester, Minnesota, between April 1997 and October 2000. Deoxyribonucleic acid (DNA) was obtained after informed consent; amplification of MYH7 exons 13 (R403Q), 14 (R453C) and 19 (G716R and R719W), and TNNT2 exon 9 (R92W) was performed by polymerase chain reaction. The mutations were detected using denaturing high-performance liquid chromatography and automated DNA sequencing., Results: The mean age at diagnosis was 42 years with 53 patients diagnosed before age 25. The mean maximal left ventricular wall thickness was 21 mm. Nearly one-third of cases were familial and one-fourth had a family history of sudden cardiac death. Only 3 of the 293 patients possessed one of the five "malignant" mutations, and all 3 patients were <25 years of age at presentation (p < 0.006)., Conclusions: This finding underscores the profound genetic heterogeneity in HCM. Only 1% of unrelated individuals seen at a tertiary referral center for HCM possessed one of the five "malignant" mutations that were examined. Routine clinical testing for these specific mutations is of low yield.

A host-cell protein impurity found in preparations of recombinant human acidic fibroblast growth factor (aFGF) was identified. Samples of aFGF examined by western blot analysis employing antiserum raised against an Escherichia coli cell lysate contained an immunoreactive protein with a molecular weight of approximately 26,000. The impurity was chromatographically isolated and the N-terminal sequence was determined. Comparing the sequence to a protein database provisionally identified the isolated impurity as the S3 ribosomal protein of E. coli. Monoclonal antibodies recognizing three separate epitopes of S3 confirmed the identity of the impurity in western blots of aFGF samples. The monoclonal antibodies were also used to estimate S3 levels in various preparations of aFGF.