Arrhythmia Variant Associations and Reclassifications in the eMERGE-III Sequencing Study.

BACKGROUND: Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability. METHODS: The eMERGE-III study (Electronic Medical Records and Genomics Phase III) is a multicenter prospective cohort that included 21 846 participants without previous indication for cardiac genetic testing. Participants were sequenced for 109 Mendelian disease genes, including 10 linked to arrhythmia syndromes. Variant carriers were assessed with electronic health record-derived phenotypes and follow-up clinical examination. Selected variants of uncertain significance (n=50) were characterized in vitro with automated electrophysiology experiments in HEK293 cells. RESULTS: As previously reported, 3.0% of participants had P/LP variants in the 109 genes. Herein, we report 120 participants (0.6%) with P/LP arrhythmia variants. Compared with noncarriers, arrhythmia P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records. Fifty-four participants had variant results returned. Nineteen of these 54 participants had inherited arrhythmia syndrome diagnoses (primarily long-QT syndrome), and 12 of these 19 diagnoses were made only after variant results were returned (0.05%). After in vitro functional evaluation of 50 variants of uncertain significance, we reclassified 11 variants: 3 to likely benign and 8 to P/LP. CONCLUSIONS: Genome sequencing in a large population without indication for arrhythmia genetic testing identified phenotype-positive carriers of variants in congenital arrhythmia syndrome disease genes. As the genomes of large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, electronic health record phenotypes, and in vitro functional studies. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier; NCT03394859.

High-Throughput Reclassification of SCN5A Variants.

Partial or complete loss-of-function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUSs). The designation of VUS is a barrier to the use of sequence data in clinical care. We selected 83 variants: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada syndrome-associated variants, selected on the basis of their frequency in the general population and in individuals with Brugada syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. The ten previously studied controls had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wild-type-like function. 22 suspected BrS variants had loss of channel function (<10% normalized peak current) and 22 variants had partial loss of function (10%-50% normalized peak current). The previously unstudied variants were initially classified as likely benign (n = 2), likely pathogenic (n = 10), or VUSs (n = 61). After the patch clamp studies, 16 variants were benign/likely benign, 45 were pathogenic/likely pathogenic, and only 12 were still VUSs. Structural modeling identified likely mechanisms for loss of function including altered thermostability and disruptions to alpha helices, disulfide bonds, or the permeation pore. High-throughput patch clamping enabled reclassification of the majority of tested VUSs in SCN5A.

Polygenic risk score for ACE-inhibitor-associated cough based on the discovery of new genetic loci.

AIMS: To search for sequence variants associated with ACEi discontinuation and to test their association with ACEi-associated adverse drug reactions (ADRs). METHODS AND RESULTS: A genome-wide association study (GWAS) on ACEi discontinuation was conducted, including 33 959 ACEi-discontinuers and 44 041 controls. Cases were defined as persons who switched from an ACEi treatment to an angiotensin receptor blocker. Controls were defined as persons who continued ACEi treatment for at least 1 year. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were computed for ACEi discontinuation risk by mixed model regression analysis. Summary statistics from the individual cohorts were meta-analyzed with a fixed-effects model. To test for association with specific ACEi-associated ADRs, any genome-wide significant (P < 5 × 10-8) ACEi discontinuation variants was tested for association with ACEi-associated cough and angioedema. A polygenetic risk score (PRS) based on ACEi discontinuation GWAS data was constructed and tested for association with ACEi-associated cough and angioedema in two population-based samples. In total, seven genetic genome-wide loci were identified, of which six were previously unreported. The strongest association with ACEi discontinuation was at 20q13.3 (NTSR1; OR: 1.21; 95% CI: 1.17-1.24; P = 2.1 × 10-34). Five of seven lead variants were associated with ACEi-associated cough, whereas none were associated with ACEi-associated angioedema. The ACEi discontinuation PRS was associated with ACEi-associated cough in a dose-response manner but not with ACEi-associated angioedema. ACEi discontinuation was genetically correlated with important causes for cough, including gastro-esophageal reflux disease, allergic rhinitis, hay fever, and asthma, which indicates partly shared genetic underpinning between these traits. CONCLUSION: This study showed the advantage of using prescription patterns to discover genetic links with ADRs. In total, seven genetic loci that associated with ACEi discontinuation were identified. There was evidence of a strong association between our ADR phenotype and ACEi-associated cough. Taken together, these findings increase insight into the pathophysiological processes that underlie ACEi-associated ADRs.

Dominant negative effects of SCN5A missense variants.

PURPOSE: Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. METHODS: We identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. RESULTS: In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). CONCLUSION: Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.

Singlet glycine riboswitches bind ligand as well as tandem riboswitches.

The glycine riboswitch often occurs in a tandem architecture, with two ligand-binding domains (aptamers) followed by a single expression platform. Based on previous observations, we hypothesized that "singlet" versions of the glycine riboswitch, which contain only one aptamer domain, are able to bind glycine if appropriate structural contacts are maintained. An initial alignment of 17 putative singlet riboswitches indicated that the single consensus aptamer domain is flanked by a conserved peripheral stem-loop structure. These singlets were sorted into two subtypes based on whether the active aptamer domain precedes or follows the peripheral stem-loop, and an example of each subtype of singlet riboswitch was characterized biochemically. The singlets possess glycine-binding affinities comparable to those of previously published tandem examples, and the conserved peripheral domains form A-minor interactions with the single aptamer domain that are necessary for ligand-binding activity. Analysis of sequenced genomes identified a significant number of singlet glycine riboswitches. Based on these observations, we propose an expanded model for glycine riboswitch gene control that includes singlet and tandem architectures.

Multifocal Ectopic Purkinje Premature Contractions due to neutralization of an SCN5A negative charge: structural insights into the gating pore hypothesis.

Background: We identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein NaV1.5 and generate a gating pore current. Methods and Results: E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of NaV1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. Conclusion: Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.

Safety and efficacy of desmopressin (DDAVP) in preventing hematoma expansion in intracranial hemorrhage associated with antiplatelet drugs use: A systematic review and metaanalysis.

INTRODUCTION: One of the most serious complications associated with antiplatelet agents is antiplatelet-associated intracranial hemorrhage (AA-ICH). Desmopressin is a synthetic antidiuretic hormone (ADH) analog. It has been linked to improving patient outcomes in antiplatelet-induced intracranial hemorrhage. The secondary outcomes included the incidence of thrombotic complications and neurological outcomes. METHODS: A systematic search was conducted on three databases (PubMed, Cochrane, and ClinicalTrials.gov) to find eligible literature that compares desmopressin (DDAVP) versus controls in patients with AA-ICH. The Mantel-Haenszel statistic was used to determine an overall effect estimate for each outcome by calculating the risk ratios and 95% confidence intervals (CI). Heterogeneity was measured using the I2 test. The risk of bias in studies was calculated using the New Castle Ottowa Scale. RESULTS: Five studies were included in the analysis with a total of 598 patients. DDAVP was associated with a nonsignificant decrease in the risk of hematoma expansion (RR = .8, 95% CI,.51-1.24; p = .31, I2 = 44%). It was also associated with a non-significant decrease in the risk of thrombotic events (RR,.83; 95% CI,.25-2.76; p = .76, I2 = 30%). However, patients in the DDAVP group demonstrated a significant increase in the risk of poor neurological outcomes (RR, 1.31; 95% CI, 1.07-1.61; p = .01, I2 = 0%). The risk of bias assessment showed a moderate to low level of risk. CONCLUSION: DDAVP was associated with a nonsignificant decrease in hematoma expansion and thrombotic events. However, it was also associated with a significantly poor neurological outcome in the patients. Thus, until more robust clinical trials are conducted, the use of DDAVP should be considered on a case-to-case basis.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

BACKGROUND: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. METHODS: In this study, we leveraged the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein-coding KCNE1 variants. RESULTS: We comprehensively assayed KCNE1 variant cell surface expression (2554/2709 possible single-amino-acid variants) and function (2534 variants). Our study identified 470 loss- or partial loss-of-surface expression and 574 loss- or partial loss-of-function variants. Of the 574 loss- or partial loss-of-function variants, 152 (26.5%) had reduced cell surface expression, indicating that most functionally deleterious variants affect channel gating. Nonsense variants at residues 56-104 generally had WT-like trafficking scores but decreased functional scores, indicating that the latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation (with > 70% loss-of-function variants) were in predicted close contact with binding partners KCNQ1 or calmodulin. Our functional assay data were consistent with gold standard electrophysiological data (ρ = - 0.64), population and patient cohorts (32/38 presumed benign or pathogenic variants with consistent scores), and computational predictors (ρ = - 0.62). Our data provide moderate-strength evidence for the American College of Medical Genetics/Association of Molecular Pathology functional criteria for benign and pathogenic variants. CONCLUSIONS: Comprehensive variant effect maps of KCNE1 can both provide insight into I Ks channel biology and help reclassify variants of uncertain significance.

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

BACKGROUND: Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. METHODS: An in vitro SCN5A-Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. RESULTS: Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. CONCLUSIONS: This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Variant-based heritability assessment of dexmedetomidine and fentanyl clearance in pediatric patients.

Despite complex pathways of drug disposition, clinical pharmacogenetic predictors currently rely on only a few high effect variants. Quantification of the polygenic contribution to variability in drug disposition is necessary to prioritize target drugs for pharmacogenomic approaches and guide analytic methods. Dexmedetomidine and fentanyl, often used in postoperative care of pediatric patients, have high rates of inter-individual variability in dosing requirements. Analyzing previously generated population pharmacokinetic parameters, we used Bayesian hierarchical mixed modeling to measure narrow-sense (additive) heritability ( h SNP 2 ) of dexmedetomidine and fentanyl clearance in children and identify relative contributions of small, moderate, and large effect-size variants to h SNP 2 . We used genome-wide association studies (GWAS) to identify variants contributing to variation in dexmedetomidine and fentanyl clearance, followed by functional analyses to identify associated pathways. For dexmedetomidine, median clearance was 33.0 L/h (interquartile range [IQR] 23.8-47.9 L/h) and h SNP 2 was estimated to be 0.35 (90% credible interval 0.00-0.90), with 45% of h SNP 2 attributed to large-, 32% to moderate-, and 23% to small-effect variants. The fentanyl cohort had median clearance of 8.2 L/h (IQR 4.7-16.7 L/h), with estimated h SNP 2 of 0.30 (90% credible interval 0.00-0.84). Large-effect variants accounted for 30% of h SNP 2 , whereas moderate- and small-effect variants accounted for 37% and 33%, respectively. As expected, given small sample sizes, no individual variants or pathways were significantly associated with dexmedetomidine or fentanyl clearance by GWAS. We conclude that clearance of both drugs is highly polygenic, motivating the future use of polygenic risk scores to guide appropriate dosing of dexmedetomidine and fentanyl.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

Background: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. Results: Here, we demonstrate the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein coding KCNE1 variants. We comprehensively assayed KCNE1 variant cell surface expression (2,554/2,709 possible single amino acid variants) and function (2,539 variants). We identified 470 loss-of-surface expression and 588 loss-of-function variants. Out of the 588 loss-of-function variants, only 155 had low cell surface expression. The latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were highly concordant with gold standard electrophysiological data (ρ = -0.65), population and patient cohorts (32/38 concordant variants), and computational metrics (ρ = -0.55). Our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. Conclusions: Comprehensive variant effect maps of KCNE1 can both provide insight into IKs channel biology and help reclassify variants of uncertain significance.

Multi-site validation of a functional assay to adjudicate SCN5A Brugada Syndrome-associated variants.

Brugada Syndrome (BrS) is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in the cardiac sodium channel gene, SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging and ~79% of SCN5A missense variants in ClinVar are currently classified as Variants of Uncertain Significance (VUS). An in vitro SCN5A-BrS automated patch clamp assay was generated for high-throughput functional studies of NaV1.5. The assay was independently studied at two separate research sites - Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute - revealing strong correlations, including peak INa density (R2=0.86). The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. The assay accurately distinguished benign controls (24/25) from pathogenic controls (23/24). Odds of Pathogenicity values derived from the experimental results yielded 0.042 for normal function (BS3 criterion) and 24.0 for abnormal function (PS3 criterion), resulting in up to strong evidence for both ACMG criteria. The calibrated assay was then used to study SCN5A VUS observed in four families with BrS and other arrhythmia phenotypes associated with SCN5A loss-of-function. The assay revealed loss-of-function for three of four variants, enabling reclassification to likely pathogenic. This validated APC assay provides clinical-grade functional evidence for the reclassification of current VUS and will aid future SCN5A-BrS variant classification.

Genome-Wide Approach to Measure Variant-Based Heritability of Drug Outcome Phenotypes.

Pharmacogenomic studies have successfully identified variants-typically with large effect sizes in drug target and metabolism enzymes-that predict drug outcome phenotypes. However, these variants may account for a limited proportion of phenotype variability attributable to the genome. Using genome-wide common variation, we measured the narrow-sense heritability ( h SNP 2 ) of seven pharmacodynamic and five pharmacokinetic phenotypes across three cardiovascular drugs, two antibiotics, and three immunosuppressants. We used a Bayesian hierarchical mixed model, BayesR, to model the distribution of genome-wide variant effect sizes for each drug phenotype as a mixture of four normal distributions of fixed variance (0, 0.01%, 0.1%, and 1% of the total additive genetic variance). This model allowed us to parse h SNP 2 into bins representing contributions of no-effect, small-effect, moderate-effect, and large-effect variants, respectively. For the 12 phenotypes, a median of 969 (range 235-6,304) unique individuals of European ancestry and a median of 1,201,626 (range 777,427-1,514,275) variants were included in our analyses. The number of variants contributing to h SNP 2 ranged from 2,791 to 5,356 (median 3,347). Estimates for h SNP 2 ranged from 0.05 (angiotensin-converting enzyme inhibitor-induced cough) to 0.59 (gentamicin concentration). Small-effect and moderate-effect variants contributed a majority to h SNP 2 for every phenotype (range 61-95%). We conclude that drug outcome phenotypes are highly polygenic. Thus, larger genome-wide association studies of drug phenotypes are needed both to discover novel variants and to determine how genome-wide approaches may improve clinical prediction of drug outcomes.

Mitochondrial Haplogroups Modify the Effect of Diabetes Duration and HbA1c on Proliferative Diabetic Retinopathy Risk in Patients With Type 2 Diabetes.

Purpose: We previously demonstrated an association between European mitochondrial haplogroups and proliferative diabetic retinopathy (PDR). The purpose of this study was to determine how the relationship between these haplogroups and both diabetes duration and hyperglycemia, two major risk factors for diabetic retinopathy (DR), affect PDR prevalence. Methods: Our population consisted of patients with type 2 diabetes with (n = 377) and without (n = 480) DR. A Kruskal-Wallis test was used to compare diabetes duration and hemoglobin A1c (HbA1c) among mitochondrial haplogroups. Logistic regressions were performed to investigate diabetes duration and HbA1c as risk factors for PDR in the context of European mitochondrial haplogroups. Results: Neither diabetes duration nor HbA1c differed among mitochondrial haplogroups. Among DR patients from haplogroup H, longer diabetes duration and increasing HbA1c were significant risk factors for PDR (P = 0.0001 and P = 0.011, respectively). Neither diabetes duration nor HbA1c was a significant risk factor for PDR in DR patients from haplogroup UK. Conclusions: European mitochondrial haplogroups modify the effects of diabetes duration and HbA1c on PDR risk in patients with type 2 diabetes. In our patient population, longer diabetes duration and higher HbA1c increased PDR risk in patients from haplogroup H, but did not affect PDR risk in patients from haplogroup UK. This relationship has not been previously demonstrated and may explain, in part, why some patients with nonproliferative DR develop PDR and others do not, despite similar diabetes duration and glycemic control.

Delayed Presentation of Forgotten Thyroid Goiter - 25 Years After Thyroidectomy.

Forgotten goiter is a rare occurrence (2 - 16% of retrosternal thyroid cases) that depicts recurrence of retrosternal thyroid mass due to growth of remnant thyroid tissue overlooked during an initial thyroidectomy. The patient is a 59-year female who presented with dyspnea and stridor 25 years after total thyroidectomy. She was diagnosed as having a mediastinal mass on radiographic imaging. There were only localized pressure symptoms due to mass effect. The mass was excised employing an extra-cervical approach and using a partial sternotomy. The profuse extension of thyroid tissue, adherent to vital structures in the thorax and the extra-cervical thyroidectomy was a novel experience. The mass was completely excised and the patient's symptoms alleviated after the surgery. The patient recovered without any complications and there was no recurrence at 1 year of follow-up.