Ackerman, Michael J.

Introduction: Long QT syndrome (LQTS) is a genetic disorder associated with abnormal ventricular repolarization and a predisposition for torsade de pointes and sudden cardiac death. Identification of markers to elucidate high risk patients is of major practical importance. Our aim was to use a novel, automated quantitative T wave analysis program to distinguish symptomatic from asymptomatic LQTS patients. Methods: We analyzed a genotyped cohort of 420 patients (22 ± 15.7years, 43% male) with either LQT1 (61%) or LQT2 (39%). ECG analysis was conducted using novel software that we created to automatically detect subtle changes in T wave morphology. Symptomatic patients were defined as those with a history of syncope or resuscitated cardiac arrest that was suspected of being LQTS triggered. Classification was performed using the top three T wave features in a linear discriminant classifier by 10x10 cross-validation. Results: Symptomatic LQT1 patients have a greater QTc duration in lead aVF (437 msec vs 416 msec, p <0.0001), a greater T wave center of gravity on the x axis (COGx) in lead V2 (0.304 vs 0.280, p <0.0001) and longer Tpeak-Tend/QT in lead II (0.208 vs 0.188, p < 0.0001) than asymptomatic patients with LQT1. Use of these 3 features enabled classification of symptomatic status in 72% of LQT1 cases, compared to 68% when QTc was used to classify alone. Symptomatic LQT2 patients had a longer QTc in Lead 1 (460 vs 418 msec, p < 0.0001), COGx in 4th segment of T wave was greater in lead V5 (0.399 vs 0.350, p<0.0001), and the Tpeak-Tend interval in V2 was longer (126 vs 99 msec, P =0.0004). Use of these 3 features enabled correct classification in 73% of LQT2 cases, compared to 67% when QTc was used alone. Conclusions: Application of an automated T wave analysis program was able to identify T wave features that distinguished the patients who presented as symptomatic LQTS, associated with syncope or resuscitated cardiac arrest. Use of these features could help assess arrhythmic risk and guide management strategies for patients with LQTS.

Background: Mutations in sarcomeric/myofilament proteins are commonly implicated in hypertrophic cardiomyopathy (HCM). However, the mechanisms underlying disease pathogenesis are not completely understood. Utilizing flash-frozen surgically resected left ventricular (LV) tissue from patients with obstructive HCM, we investigated the effect of sarcomeric HCM on tissue expression of various myofilament proteins. Methods: Frozen surgical specimens from 47 patients with HCM who underwent septal myectomy at the Mayo Clinic between 1999 and 2003 were examined. Using PCR, DHPLC, and direct DNA sequencing, all were genotyped for mutations involving the 8 myofilament-encoding genes that currently comprise the clinically available genetic test for sarcomeric HCM. Whole protein lysate was extracted from the frozen myectomy tissues and levels of myosin binding protein C, myosin heavy chain, alpha actin and alpha actinin were analyzed by Western Blot; GAPDH was used as a loading control. Frozen LV tissue derived from normal hearts of accidental death victims served as a reference group. Results: Overall, 25/47 (53%) patients had sarcomeric HCM including 13 patients with myosin binding protein C (MYBPC3) mutations and 10 with mutations in the beta myosin heavy chain (MYH7). Compared to normal heart tissue, the expression of myosin heavy chain was up-regulated significantly regardless of the underlying disease-causing genotype (genotype positive = genotype negative). However, there was gene-specific upregulation of myosin binding protein C with marked increases detected in patients with MYH7-HCM. In contrast, normal levels of myosin binding protein C were seen in patients with either MYBPC3-HCM or genotype-negative HCM. Conclusions: This is the first study to examine the expression pattern of sarcomeric proteins in a large collection of flash frozen human HCM myectomy tissue. Up-regulated, genotype-specific cardiac tissue expression of myosin binding protein C among patients with MYH7 (beta myosin heavy chain)-HCM suggests a novel compensatory pathway underlying the development of cardiac hypertrophy for one of most common genetic subtypes of HCM.

Background: Apical hypertrophic cardiomyopathy (apical HCM) is a less common subtype of HCM characterized by a focal thickening in the left ventricular apex. “Classic” ECG features have been described, however, apical HCM can persist for many years without detection. We investigated the relationship between ECG findings and echocardiographic morphometry in a large referral series of patients with apical HCM. Methods: We enumerated all patients diagnosed with apical HCM prior to Sept. 30, 2006 using the Mayo Clinic HCM database. We compared echocardiographic measures separately for patients with positive status for two ECG indices of left ventricular hypertrophy (LVH); the Sokolow-Lyon index and the Romhilt-Estes (RE) point-score. We also compared echocardiographic measurements in patients with and without negative T-waves in the precordial leads. Results: Apical HCM was detected in 177 patients (111 men and 68 women). Only 51% had positive Sokolow criteria and 51% had positive RE criteria. The agreement between Sokolow and RE status was high (agreement = 75.0%; kappa = 0.50; 95% CI = 0.38 – 0.62). In particular, Sokolow positive patients had increased LV ejection fraction (P = 0.02), and decreased LV end-systolic diameter (P = 0.03) compared with Sokolow negative patients. The prevalence of right atrial enlargement (47 vs. 28%; P = 0.02) and intracavity obstruction (22 vs. 8%; P = 0.01) were more common in Sokolow positive patients. Positive RE criteria was associated with a greater thickness of the basal septal and basal posterior walls (P = 0.001 and 0.02, respectively), and with a higher frequency of intracavity obstruction (21 vs. 9%; P = 0.04). Most patients (89%) exhibited at least one negative T-wave in the precordial leads; however, only 10% of patients had a negative T-wave of greater than 1.0 mV. We found that patients with an inverted T-wave larger than 0.4 mV (median) had a significantly increased LV ejection fraction (P = 0.03) compared with patients who had smaller or no negative T-waves. Conclusions: Among patients with apical HCM, nearly half do not have ECG evidence of LVH based on classic criteria and most do not have marked T-wave inversions. However, the majority did have at least a mild expression of negative T-waves.

Introduction: Approximately 10 – 15% of sudden infant death syndrome (SIDS) may be caused by cardiac channelopathies including Brugada syndrome (BrS). Type 1 BrS (BrS1), due to mutations in the SCN5A-encoded sodium channel, accounts for approximately 20% of BrS. Recently, a novel mutation in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) disrupted trafficking of SCN5A in a multi-generational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of SIDS. Methods: Using DHPLC and direct DNA sequencing, we performed comprehensive open reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 228 anonymous cases of SIDS (86 females, 142 males, average age = 3 + 2 months, range 6 hours to 12 months). Results: Three putative, SIDS-associated GPD1-L missense mutations, E83K, I124V, and R273C, were discovered in a three-month-old white male, a five-week-old white female, and a one-month-old white male, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared to wild type GPD1-L, GPD1-L mutations co-expressed with SCN5A in heterologous HEK cells produced a 60% reduction in peak sodium current density (p<0.004). Adenovirus-mediated gene transfer of the E83K-GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current as well (p<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusion: This study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset (~1%) of SIDS via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal, BrS-like pro-arrhythmic substrate.

Background: Long QT Syndrome (LQTS) genetic testing has been available clinically since August 2004. In cases of bona fide LQTS, the yield of genetic testing is approximately 75%. Recently (HRS 2007), Priori and colleagues have suggested that referral for LQTS genetic testing should be based on the screening QTc citing a yield of < 2% for cases referred to their research program with a QTc < 440 ms. Here, we analyze the effect of QTc on the yield in two of the largest assembled cohorts of unrelated patients (pts) referred for LQTS genetic testing. Methods: From August 1997 to July 2006, 1125 unrelated pts (717 females, average age at diagnosis 23.5 yrs, average QTc 473 ms) were referred to either the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory (N = 541) for research-based LQTS genetic testing or to PGxHealth (N = 574) for the FAMILION ® LQTS genetic test. Comprehensive open reading frame and splice site analysis (60 exons) for LQTS-causing mutations involving KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) was conducted by either dHPLC/DNA sequencing (Mayo), or by direct DNA sequencing (FAMILION). Results: The probability of a positive genetic test increased dramatically with increasing QTc, ranging from 5% for the 38 pts referred for genetic testing despite a QTc < 400 ms to 60% for the 168 pts with a QTc > 500 ms (p < 0.00001). Akin to the Priori cohort, the yield of the genetic test was similar among the subset of pts with a QTc < 470 ms (64% Priori, 61% Mayo cohort, and 53% FAMILION, p = NS). However, the yield for those pts with a QTc between 440 and 470 ms was much greater in the Mayo (49%) and FAMILION (36%) cohorts compared to the Priori cohort (14%, p < 0.0005). In contrast to the 2% yield observed in Italy, 22% of pts with QTc < 440 ms referred to Mayo Clinic and 14.5% of the FAMILION referrals had a positive genetic test (p < 0.001). Conclusions: Whereas only 2% of patients with a QTc < 440 ms had a positive genetic test in Italy, the yield was ten times greater among those referred to Mayo Clinic despite the same QTc cut-off. The cumulative index of suspicion for LQTS rather than a pre-specified QTc cut-off should guide clinical decision making as to the appropriate utilization of the genetic test.

Background: Genes encoding sodium channel interacting proteins (ChIPs) have been identified as novel long QT syndrome (LQTS)-susceptibility genes. Approximately 25% of patients with LQTS remain genotype negative. ChIPs comprising the sodium channel macromolecular complex represent attractive candidates for disease susceptibility. Previously, in human jejunal circular smooth muscle, we demonstrated that syntrophin γ2 regulates sodium channel gating by a PDZ-domain interaction involving the C-terminus. In the heart, syntrophin α is more highly expressed than γ2, therefore, syntrophin α was evaluated as a possible LQTS-susceptibility ChIP. Methods: Comprehensive open reading frame/splice mutational analysis of syntrophin α was performed using PCR, DHPLC, and direct DNA sequencing on a cohort of 50 unrelated patients with LQT1–10 negative/phenotype positive LQTS (34 females; average age at diagnosis 26 ± 16 years; average QTc 531 ± 60.4 ms; QTc range 480 –759 ms). Mutations were engineered by site-directed mutagenesis and transiently expressed in HEK293 cells containing the stably-expressed SCN5A- encoded sodium channel α subunit (hNaV1.5). Results: Analysis of syntrophin α, encoded by SNTA1 , revealed 3 missense mutations in 2 patients (4%). A female diagnosed with LQTS at 13 years (QTc, 480 ms) with family history of autopsy-negative unexplained sudden death, was compound heterozygous for P74L/A257G while a male, diagnosed with LQTS at 18 years following syncopal episodes (QTc, 529 ms), hosted the A390V mutation. These mutations were absent in 600 reference alleles and A257G and A390V involved highly conserved residues. Functional studies revealed a marked leftward shift in sodium current activation for A257G and a significant rightward shift in channel inactivation for A390V-SNTA1. Both kinetic perturbations precipitated a ``gain-of-function” increase in the sodium channel window current. Conclusion: We provide the sentinel report of SNTA1 as a novel LQTS-susceptibility gene with a putative pathogenic mechanism involving a secondary ``gain-of-function” in the NaV1.5 sodium channel. Thus, CAV3-LQTS (LQT9), SCN4B-LQTS (LQT10), and now SNTA1-LQTS represent functional homologues with LQT3-like perturbations.

A-Kinase Anchoring Protein 9 (AKAP9), or Yotiao, binds to the carboxy-terminus (C-T) of the IKs channel alpha subunit KCNQ1 and recruits cAMP-dependent protein kinase A (PKA) and protein phosphatase 1 (PP1) to the channel to control its phosphorylation state. Previously, an inherited LQT1-causing mutation in the KCNQ1 C-T/Yotiao binding domain was shown to disrupt the complex and render the channel functionally insensitive to cAMP-dependent regulation. Despite the critical role of Yotiao in mediating this fundamental physiological response, the large size of the Yotiao protein (over 200 kD) has hindered patient screens and, to date, no disease-linked mutations have been found in this adaptor protein. The objectives of the present study were first to locate reciprocal binding site(s) for KCNQ1 on Yotiao and to test whether mutation(s) in the binding module(s) might cause LQTS. Using GST fusion protein pull down and immunoprecipitation (IP) techniques, we identified two regions in Yotiao necessary for binding to KCNQ1: one within the Yotiao N-terminus (residues 29 – 46), and a second within its C-terminus (1574 –1643). Targeted sequence analysis was then carried out in fifty clinically diagnosed but genotype negative LQTS patients in the coding regions for these two KCNQ1 binding domains in AKAP9-encoded Yotiao. This analysis identified a novel missense mutation, S1570L-Yotiao, present in two siblings diagnosed with LQTS but absent in the normal population. We found that the S1570L mutation, located close to the Yotiao C-T binding motif for KCNQ1, disrupted the interaction between the two proteins and significantly reduced both basal and exogenous cAMP-induced KCNQ1 phosphorylation. Perforated patch experiments in CHO cells revealed that expression of S1570L-Yotiao significantly reduced basal IKS channel activity and completely ablated the response of the IKs channel to cAMP plus the phosphatase inhibitor okadaic acid. 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 for the first time a link between an inherited AKAP mutation and human disease, LQTS.

Background Recently, genetic testing for sarcomeric hypertrophic cardiomyopathy (HCM) became a commercially available diagnostic test involving analysis of 8 HCM-associated myofilament genes, four of which encode thin filament proteins - TNNT2- encoded cardiac troponin T, TPM1 -encoded alpha tropomyosin, TNNI3- encoded cardiac troponin I, and ACTC- encoded cardiac actin. Compared to the two most common subtypes of sarcomeric HCM, much less is known about the prevalence and spectrum of thin filament HCM. Methods Comprehensive open reading frame/splice site mutational analysis of these 4 thin filament-encoding genes was performed, using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, on the largest assembled cohort of unrelated patients (N = 1025, 61% male, 49 ± 18 years at diagnosis, maximal left ventricular wall thickness, LVWT, 22.3 ± 7 mm) with clinically diagnosed HCM that were seen at the Mayo Clinic. Results Nineteen distinct missense mutations, involving conserved residues and absent in 600 reference alleles, were identified in 36 of 1025 patients (3.5%) including TNNT2 (16 patients), TNNI3 (12), TPM1 (7) and ACTC (1). Among these 36 patients with thin filament-HCM, their average age at diagnosis was 33.2 ± 16 years and the average LVWT was 21 ± 8 mm. Eighteen patients (50%) had evidence for familial HCM and 13% had a positive history for sudden cardiac death. Only 13 patients (36%) had a reverse septal curvature by echocardiography. Instead, 12 patients (34%) had either sigmoidal or neutral septal contours and 9 patients (25%) had apical variant-HCM. Conclusion Here, we present the largest cohort analysis of thin filament HCM to date and detail the prevalence and spectrum of HCM-associated mutations in these 4 genes. Compared to the strong association between reverse septal curvature and thick filament HCM, the septal morphology associated with thin filament HCM was quite varied. Contrary to published estimates of the frequency of troponin T-HCM (5 – 10%) and the other subtypes of thin filament HCM, the prevalence of thin filament HCM was very low in this cohort. If confirmed in other cohorts, a revised cascade for HCM genetic testing should be considered.

Introduction : Type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1) is pursuant to mutations in the RyR2- encoded cardiac ryanodine receptor. Recently, we discovered nearly as many CPVT1-causing mutations as LQT3-causing mutations in a large cohort of unrelated LQTS referral patients. Here, we examine potential predictors of RyR2 genotype (CPVT1) among LQTS referral patients. Methods : Previously, comprehensive mutational analysis of the LQT1–10 susceptibility genes was completed for 541 unrelated patients (358 females, average age at diagnosis = 24 years, average QTc = 482 ms) referred for LQTS genetic testing. Using PCR, DHPLC, and DNA sequencing, we performed targeted analysis of RyR2 involving 44 exons (8–15, 37–49, 83–105) for the 260 unrelated referrals (172 females, average age at diagnosis = 25 years, average QTc = 470 ms) that remained “genotype-negative” following LQTS genetic testing. Results : Overall, 22 distinct RyR2 mutations (19 novel) were seen in 23 (8.8%, 12 females, average age at diagnosis = 22 ±14 years) unrelated “genotype-negative” LQTS individuals. Compared to patients with either genetically proven LQTS or the remaining genotype negative cohort, the RyR2 -positive cohort had significantly lower average QTc (419 ms vs 494 ms, p =2.4 ×10 −6 ; vs 472 ms, p = 0.005) and was more likely to experience either syncope (88% vs 66%, p = 0.06 vs 53%, p = 0.008) or cardiac arrest (56% vs 23%, p = 0.006 vs 17%, p = 0.001). Overall, 123 of the 541 LQTS referrals (23%) exhibited exertion-induced cardiac events (68 LQTS gene positive; 42 gene negative, and 13 RyR2-positive). Every RyR2 -positive case with an exertionally-triggered event had a QTc ≤ 460 ms, compared to only 17% of LQTS gene positive cases (p = 0.1 × 10 −8 ) and 24% of LQT1 cases. None of the exertion-induced LQTS gene positive cases had a QTc ≤ 420 ms. Conclusions: Failure to distinguish CPVT from LQTS can be a fatal mistake, as individuals with CPVT1 have a more severe phenotype and are much less protected by beta blocker therapy than those with LQT1. CPVT1, rather than concealed LQT1, should lead the differential diagnosis in the setting of syncope/cardiac arrest during exercise and a normal resting QTc.

Background: Bicuspid aortic valve disease (BAV), the most common congenital cardiovascular malformation, has an incidence of 0.5–1.0% of live births. While most cases of BAV appear to be sporadic, familial inheritance patterns have been observed consistent with autosomal dominant inheritance with variable penetrance. However, little is known about specific genetic loci responsible for familial BAV. Here, we performed linkage analysis on a large multi-generational pedigree affected with BAV. Methods: We identified a large, five-generation pedigree (136 family members) with 10 individuals having BAV. Two-dimensional echocardiography was used to assign aortic valve phenotype. Genome-wide linkage analysis using 430 microsatellite markers (Marshfield Clinic) and fine mapping using 100 single nucleotide polymorphisms (Affymetrix) on chromosome 9 was performed on genomic DNA from all available family members. Logarithm of odds (LOD) scores of >2.0 were considered suggestive of linkage. Comprehensive splice site/open reading frame mutational analysis of candidate genes residing in the putative locus is underway using PCR, DHPLC, and DNA sequencing. A candidate gene, KLF9, Krüppel-like factor 9 was analyzed for mutations because of its role in cardiogenesis. Results: Multi-point genome-wide linkage analysis demonstrated a 7 cM region on chromosome 9q21 that was suggestive of linkage for familial BAV with a maximum multipoint LOD score of 2.8 flanked by the microsatellite markers GATA7D12 and D9S1834. This region contains several candidate genes with biological plausibility for BAV phenotype. KLF9- encoded Krüppel-like factor 9, localized to chromosome 9q21, was targeted as a prime candidate gene for familial BAV. However, no mutations involving the translated exons of KLF9 were detected. Further fine mapping studies and candidate gene analysis are currently underway. Conclusions: We report a novel susceptibility locus on chromosome 9q21 for BAV in a large multi-generational family. Although coding region mutations in KLF9 are not responsible for BAV in this pedigree, several candidate genes with biological plausibility for the development of congenital BAV lie within this region and warrant further scrutiny.