Medeiros‐Domingo, Argelia

Precision medicine, which among other aspects includes an individual's genomic data in diagnosis and management, has become the standard‐of‐care for Mendelian cardiovascular disease (CVD). However, early identification and management of asymptomatic patients with potentially lethal and manageable Mendelian CVD through screening, which is the promise of precision health, remains an unsolved challenge. The reduced costs of genomic sequencing have enabled the creation of biobanks containing in‐depth genetic and health information, which have facilitated the understanding of genetic variation, penetrance, and expressivity, moving us closer to the genotype‐first screening of asymptomatic individuals for Mendelian CVD. This approach could transform health care by diagnostic refinement and facilitating prevention or therapeutic interventions. Yet, potential benefits must be weighed against the potential risks, which include evolving variant pathogenicity assertion or identification of variants with low disease penetrance; costly, stressful, and inappropriate diagnostic evaluations; negative psychological impact; disqualification for employment or of competitive sports; and denial of insurance. Furthermore, the natural history of Mendelian CVD is often unpredictable, making identification of those who will benefit from preventive measures a priority. Currently, there is insufficient evidence that population‐based genetic screening for Mendelian CVD can reduce adverse outcomes at a reasonable cost to an extent that outweighs the harms of true‐positive and false‐positive results. Besides technical, clinical, and financial burdens, ethical and legal aspects pose unprecedented challenges. This review highlights key developments in the field of genotype‐first approaches to Mendelian CVD and summarizes challenges with potential solutions that can pave the way for implementing this approach for clinical care.

Background: Potentially lethal syndromes such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) may account for a significant number of autopsy-negative sudden unexplained deaths (SUD). Here, the spectrum and prevalence of putative LQTS-/CPVT-causative mutations in autopsy- negative SUD was determined following a cardiac channel molecular autopsy. Methods: From September 1998 to October 2010, 173 cases of SUD (106 males, 18 ± 13 years, 89% white) were referred by Medical Examiners/Coroners for a cardiac channel molecular autopsy. Using PCR, DHPLC, and DNA sequencing, a comprehensive mutational analysis of the LQTS-susceptibility genes ( KCNQ1 , KCNH2 , SCN5A, KCNE1, and KCNE2 ) and a targeted analysis of the CPVT1-associated gene ( RYR2 ) was conducted. Results: Overall, 45 putative pathogenic mutations (20 RYR2 , 11 KCNQ1 , 6 KCNH2 , 6 SCN5A , and 2 KCNE2 , absent in 400 - 700 controls, were identified in 45 (26%) autopsy negative SUD cases. The yield was greatest for those with an exercise-induced death (16/46, 35%) or a positive personal/family history of a syncope (13/25, 52%), or sudden death (9/20, 45%). Females had a higher yield (26/67, 39%) than males (19/106, 18%, p < 0.005) especially if the SUD was during adolescence (12/25, 48% females vs. 9/50, 18% males). Females were more likely to have LQTS-associated mutations (65%) while males were more likely to have CPVT1-associated mutations (58%). Among SUD victims with exercise-induced death, the yield was much higher among the 1 - 10 year olds compared to the 11 -20 year olds (females, 71% vs 14%, p<0.05; males, 60% vs 15%, p = 0.07). However for those with death during a period of sleep, the 11 - 20 year olds had a much higher yield than the 1-10 year old group (females, 75% vs 0%, p<0.01; males 18% vs 6%). Conclusions: Over one-fourth of autopsy-negative SUD may stem from LQTS and CPVT. A cardiac channel molecular autopsy should be considered as a standard part of the evaluation of autopsy-negative SUD especially among children with exercise-induced SUD, adolescent females, and those with a positive personal/family history of cardiac events. A two-tiered approach starting with the LQTS-susceptibility genes in females and the CPVT-susceptibility genes in males may be prudent.

In most patients with mitral valve prolapse (MVP) without severe mitral regurgitation (MR), left ventricular (LV) function is preserved. There are, however, patients with MVP who have unexplained LV dilatation and/or decreased LV function. An association between MVP and sudden cardiac death has also been reported. LV size and function may be affected by the type of MVP, severity of regurgitation, and cause of MVP (myxomatous degeneration versus fibroelastic deficiency). There is increasing evidence suggesting an intrinsic cardiomyopathy associated with MVP. The cardiomyopathy associated with MVP can also affect the right ventricle (RV). Although the impact on ventricular dimensions and function are usually subtle, these abnormalities can affect clinical and echocardiographic estimation of the severity of MR and may thus have an impact on therapeutic decisions. Particularly in patients with the most extreme forms of MVP (Barlow disease), and in patients with Marfan syndrome or other connective tissue disorders, a cardiomyopathy affecting the LV and RV may thus occur occasionally. A better understanding of LV impairment associated with MVP is important for risk assessment and clinical decision-making.

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.

Pathogenic mutations in the RYR2 -encoded cardiac ryanodine receptor cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target three canonical domains encoded by < 40% of the translated exons. The extent of mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. Mutational analysis of all 105 RYR2 exons was performed using PCR, DHPLC, and DNA sequencing on 108 unrelated patients (57% females, 96% white, age at diagnosis 18 ± 13 years, mean QTc 424 ± 26 ms) with either an explicit referral diagnosis of CPVT (N = 60) or an initial diagnosis of exercise-induced long QT syndrome (LQTS) but with QTc < 480 ms and a subsequent negative LQTS genetic test (N = 48). Two hundred healthy individuals from the Human Genetic Cell Repository were examined to assess allelic frequency for all non-synonymous variants detected. Thirty-eight (15 novel) possible CPVT1-associated mutations absent in 400 reference alleles, were detected in 44 unrelated patients (41%). Three cases (7%) had >1 RYR2 mutation. Besides the 25 exons known previously to contain causative mutations, eight new mutation-containing exons were identified: 10, 12, 13, 21, 26, 40, 42, and 48. Nearly two-thirds of the CPVT1-positive patients had mutations that localized to one of only 7 exons: 8, 14, 47, 90, 93, 100, and 101. In addition, 5 (2 novel) common non-synonymous single nucleotide polymorphisms were identified. This study represents one of the largest cohorts of patients for which RYR2 was examined in its entirety. Possible CPVT1 mutations in RYR2 were identified in approximately 41% of both CPVT referrals and LQTS gene-negative patients with exercise-induced syncope and QTc <480 ms. Including the eight new exons hosting mutations in this study, 33 of the 105 translated exons are now known to host possible mutations. Considering that two-thirds of CPVT1-positive cases would be discovered by selective analysis of <10 exons, a tiered targeting strategy for mutation discovery may afford a more cost-effective approach to CPVT genetic testing.

Long QT syndrome (LQTS) is a heterogeneous group of channelopathies characterized by increased risk of potentially lethal ventricular arrhythmias. LQT1, LQT2, and LQT3 comprise 95% of genetically proven cases and exhibit a number of established genotype-phenotype correlations. The study aimed at examining the phenotypes of genotype-negative LQTS, accounting for ~25% of LQTS cases. An IRB-approved retrospective analysis was conducted on 56 patients (39 female, 25 ± 17 years) who, after genetic testing either in our sudden death genomics laboratory or with the commercially available Familion test, were negative for mutations in the 3 principal LQTS-susceptibility genes ( KCNQ1, KCNH2, and SCN5A), and the minor genes underlying LQT5 and LQT6. All had been diagnosed with LQTS, with a clinical diagnostic score of ≥ 3.5 or QTc ≥ 480 ms. The mean diagnostic score was 4.4 (95% CI 4.2 – 4.7); mean QTc was 525 ms (95% CI 508 – 543 ms). Two-thirds were symptomatic (syncope, cardiac arrest, and/or seizures) with exercise-triggered events in 10 (26%). Twenty-one (38%) had a family history of sudden cardiac arrest. ECG showed a T wave pattern suggestive of LQT1 in 32%, LQT2 in 43%, and LQT3 in 18%. In those with exercise-induced symptoms, the ECG was LQT2-like in 50% and LQT1-like in 30%. One patient had post-partum syncope with an LQT2-like ECG. None had an auditory trigger, but 3 patients, all with an LQT2-like ECG, had a family history of auditory-triggered events. One-third of the patients had received an ICD, 58% as secondary prevention. Over 2/3 were on beta-blockers. Among the 45 patients so far tested for mutations in minor LQTS-susceptibility genes, 2 had LQTS-causing mutations in ANKB (LQT4), 1 in SCN4B (LQT10), 1 in AKAP9 (LQT11) and 2 in SNTA1 (LQT12). Genotype-negative patients with a firm LQTS diagnosis show marked phenotypic heterogeneity, suggesting multiple underlying pathogenic pathways. Only a few patients have LQTS-causing mutations in minor genes after complete LQT1–12 genetic testing. Classifying genotype negative patients into LQT1-, LQT2-, or LQT3-like profiles may guide the discovery of novel genes encoding channel interacting proteins corresponding to those specific signaling pathways.

An estimated 10–15% of sudden infant death syndrome (SIDS) may be channelopathic with approximately half of the SIDS-associated channel mutations involving the sodium channel (NaV1.5) macromolecular complex. Recently, SNTA1, which encodes the sodium channel interacting protein (ChIP), syntrophin alpha, was discovered to be a novel long QT syndrome (LQTS) susceptibility gene (LQT12) with mutations causing increased late sodium current secondary to increased s-nitrosylation of the sodium channel’s alpha subunit. Therefore, SNTA1 is a plausible candidate gene for SIDS. In this IRB-approved study, mutational analysis of SNTA1’s eight translated exons was performed using PCR, DHPLC, and direct DNA sequencing of DNA derived from 286 SIDS cases (112 females, 197 white, average age 2.9 ± 1.9 months, range 3 days -12 months). The anonymized study design precluded a determination of mutation status as familial or sporadic. Overall, 8/286 cases of SIDS harbored missense mutations in SNTA1 that were absent in 800 reference alleles. Six distinct, rare SNTA1 missense mutations were detected in 7/112 (6.25%) female infants but only 1/174 (0.6%) male infants (p < 0.01). Comprehensive open reading frame analysis of all 800 reference alleles also revealed rare genetic variants in 1% of the control subjects. A common pair of non-synonymous single nucleotide polymorphisms (P74L/A257G), with a heterozygous frequency of 1%, was identified similarly in both cases and controls. This study provides the first molecular evidence implicating mutations in SNTA1 as a novel pathogenic substrate for channelopathic SIDS. However, given the ~1% background rate of rare variants in controls, mechanistic studies of each putative SIDS-associated SNTA1 mutation will be necessary to confirm that the mutant ChIP yields a functionally perturbed NaV1.5 macromolecular complex akin to the investigations that established SNTA1 as a novel LQTS-susceptibility gene.

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— 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 α1-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 (≈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 α1-syntrophin from similarly rare but innocuous ones.