Citations

Background— An estimated 10% to 15% of sudden infant death syndrome (SIDS) cases may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1 -encoded gap junction channel protein connexin43 is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases, including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death. Methods and Results— GJA1 open reading frame mutational analysis was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole-cell patch-clamp studies were performed to determine the functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white boy and a 3-month-old white girl, respectively. Analysis of the E42K victim's parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in >1000 ethnically matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation, and S272P demonstrated wild-type junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss of function not rescued by wild type. Moreover, the E42K victim's cardiac tissue demonstrated a mosaic immunostaining pattern for connexin43 protein. Conclusions— This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-connexin43 demonstrated a trafficking-independent reduction in junctional coupling in vitro and a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of connexin43-associated sudden death.

OBJECTIVE: Long-QT syndrome (LQTS) is both underdiagnosed and overdiagnosed. Many patients are incorrectly diagnosed as having LQTS after presenting to an emergency department (ED) with presyncope/syncope and demonstrating “borderline” QT-interval prolongation (QTc ≥ 440 milliseconds) in a sentinel ED-obtained electrocardiogram (ECG). We sought to evaluate the distribution and clinical significance of QT intervals in the ED. METHODS: We retrospectively reviewed data for all patients 22 years of age or younger (N = 626; 369 females; age, mean ± SD: 17 ± 5 years) who had ECGs obtained in our ED between July 1, 2007, and June 30, 2008. A total of 223 patients were excluded because of known structural heart disease, arrhythmias, electrolyte abnormalities, or exposure to QT-interval–prolonging medications. RESULTS: The average QTc was 428 ± 28 milliseconds (range: 344–566 milliseconds), and approximately one-third of patients had QTc values of ≥440 milliseconds (females: 41%; males: 21%). Overall, 104 patients presented with presyncope/syncope, of whom 14 (13%) had follow-up ECGs. On follow-up, these patients demonstrated significant decreases in QTc values of 33 ± 43 milliseconds (P < .04). Only 8 (31%) of 26 patients with presyncope/syncope with borderline QT values had follow-up ECGs performed, in 5 of which the QTc values were decreased significantly. No patients ultimately received LQTS diagnoses. CONCLUSIONS: In the ED setting, approximately one-third of pediatric patients exhibited QTc values of ≥440 milliseconds and had significant normalization of QTc values in follow-up evaluations. First-time ECGs obtained after a syncopal episode must be interpreted with caution, particularly in the context of so-called borderline QT intervals.

Background— Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8 -encoded Kir6.1 (K ATP ) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8 -deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K ATP current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between −20 mV and 40 mV. Conclusions— Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

Background—Current clinical diagnosis of long-QT syndrome (LQTS) includes genetic testing of family members of mutation-positive patients. The present study was designed to assess the clinical course of individuals who are found negative for the LQTS-causing mutation in their families.Methods and Results—Multivariate Cox proportional hazards model was used to assess the risk for cardiac events (comprising syncope, aborted cardiac arrest [ACA], or sudden cardiac death [SCD]) from birth through age 40 years among 1828 subjects from the LQTS Registry who were found negative for their family LQTS-causing mutation. The median QTc of study subjects was 423 ms (interquartile range, 402–442 ms). The cumulative probability of a first syncope through age 40 years was 15%. However, only 2 patients (0.1%) had ACA, and none died suddenly during follow-up. Independent risk factors for syncope in genotype-negative subjects included female sex (hazard ratio [HR], 1.60;P=0.002), prolonged QTc (HR=1.63 per 100 ms increment,P=0.02), family history of ACA or SCD (HR=1.89,P=0.002), and LQT2 versus LQT1 family mutation (HR=1.41,P=0.03). Subgroup analysis showed that the presence of theK897Tpolymorphism in the LQT2 gene in an affected family was associated with an 11-fold (P=0.001) increase in the risk of recurrent syncope in genotype-negative subjects.Conclusions—Our findings suggest that cardiac events among genotype-negative family members of LQTS patients are dominated by nonfatal syncopal episodes without occurrence of sudden cardiac death. The risk for nonfatal events in this population may be mediated by the presence of common polymorphisms in LQTS genes.

Background—Women with congenital long-QT syndrome experience an increased risk for cardiac events after the onset of adolescence that is more pronounced among carriers of the LQT2 genotype. We hypothesized that the hormonal changes associated with menopause may affect clinical risk in this population.Methods and Results—We used a repeated-events analysis to evaluate the risk for recurrent syncope during the menopause transition and postmenopausal periods (5 years before and after the age at onset of menopause, respectively) among 282 LQT1 (n=151) and LQT2 (n=131) women enrolled in the Long-QT Syndrome Registry. Multivariate analysis showed that the risk for recurrent syncope (n=150) among LQT2 women was significantly increased during both menopause transition (hazard ratio, 3.38;P=0.005) and the postmenopausal period (hazard ratio, 8.10;P<0.001) compared with the reproductive period. The risk increase was evident among women who did or did not receive estrogen therapy. In contrast, among LQT1 women, the onset of menopause was associated with a reduction in the risk for recurrent syncope (hazard ratio, 0.19;P=0.05;P=0.02 for genotype-by-menopause interaction). Only 22 women (8%) experienced aborted cardiac arrest or sudden cardiac death during follow-up. The frequency of aborted cardiac arrest/sudden cardiac death showed a similar genotype-specific association with the onset of menopause.Conclusions—The onset of menopause is associated with a significant increase in the risk of cardiac events (dominated by recurrent episodes of syncope) in LQT2 women, suggesting that careful follow-up and continued long-term therapy are warranted in this population.

The common polymorphism SCN5A-S1103Y (∼13% allelic frequency in African Americans) is a risk factor for arrhythmia, sudden unexplained death (SUD), and sudden infant death syndrome. Prompted by a case of autopsy-negative SUD in a 23-year-old African American man who collapsed while playing football, we hypothesized that S1103Y interacted with other SCN5A variants to pathologically modify sodium current ( I Na ). Mutational analysis of arrhythmia-associated genes in the victim revealed the variants SCN5A-R680H and SCN5A-S1103Y. These variants were made both separately and in the same cDNA construct of the alternative splice variant backgrounds (SCN5A-Q1077del and Q1077) and expressed in HEK293 cells. In the most abundant SCN5A-Q1077del, late I Na for S1103Y alone was not significantly different from wild type (WT). However, late I Na for R680H, R680H+S1103Y (coexpressed), and R680H/S1103Y (on the same cDNA) was increased 2.1-, 3.4-, and 3.6-fold, respectively, compared with WT. Intracellular acidosis (pH 6.7) increased late I Na for S1103Y, R680H, R680H+S1103Y, and R680H/S1103Y by 2.2-, 2.4-, 5.0-, and 5.5-fold, respectively, compared with WT at pH 6.7. Expression in the less abundant SCN5A-Q1077 showed no increased late I Na . This is the initial report of a functional interaction for the common polymorphism S1103Y with another mutation in the major transcript Q1077del of SCN5A. The “double hit” and environmental factor of acidosis may have converged to cause arrhythmic sudden death in this case.

Background— Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca 2+ channels and Ca 2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca 2+ -induced Ca 2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results— To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma–sarcoplasmic reticulum distance. Conclusions— Loss of JPH2 had profound effects on Ca 2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca 2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA–mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca 2+ release in the heart.

Background— Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca 2+ ) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca 2+ channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca 2+ handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca 2+ handling in a prohypertrophic manner. Methods and Results— JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca 2+ -handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca 2+ transient amplitudes that were insensitive to L-type Ca 2+ channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca 2+ levels were observed with potentially increased total Ca 2+ stores. Spontaneous Ca 2+ oscillations were elicited at a higher extracellular [Ca 2+ ] and with decreased frequency in JPH2 knockdown cells. Conclusions— Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca 2+ homeostasis, which may be associated with prohypertrophic remodeling.