Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

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.

Genome-wide association and pathway analysis of left ventricular function after anthracycline exposure in adults.

BACKGROUND: Anthracyclines are important chemotherapeutic agents, but their use is limited by cardiotoxicity. Candidate gene and genome-wide studies have identified putative risk loci for overt cardiotoxicity and heart failure, but there has been no comprehensive assessment of genomic variation influencing the intermediate phenotype of anthracycline-related changes in left ventricular (LV) function. The purpose of this study was to identify genetic factors influencing changes in LV function after anthracycline chemotherapy. METHODS: We conducted a genome-wide association study (GWAS) of change in LV function after anthracycline exposure in 385 patients identified from BioVU, a resource linking DNA samples to de-identified electronic medical record data. Variants with P values less than 1×10 were independently tested for replication in a cohort of 181 anthracycline-exposed patients from a prospective clinical trial. Pathway analysis was performed to assess combined effects of multiple genetic variants. RESULTS: Both cohorts were middle-aged adults of predominantly European descent. Among 11 candidate loci identified in discovery GWAS, one single nucleotide polymorphism near PR domain containing 2, with ZNF domain (PRDM2), rs7542939, had a combined P value of 6.5×10 in meta-analysis. Eighteen Kyoto Encyclopedia of Gene and Genomes pathways showed strong enrichment for variants associated with the primary outcome. Identified pathways related to DNA repair, cellular metabolism, and cardiac remodeling. CONCLUSION: Using genome-wide association we identified a novel candidate susceptibility locus near PRDM2. Variation in genes belonging to pathways related to DNA repair, metabolism, and cardiac remodeling may influence changes in LV function after anthracycline exposure.

A KCNJ8 mutation associated with early repolarization and atrial fibrillation.

AIM: The Kir 6.1 K(atp) channel is believed to play an important role in ventricular repolarization as determined from both functional and genetic studies of the potassium inwardly-rectifying channel, subfamily J, member 8 (KCNJ8)-S422L missense mutation in patients with J-wave syndromes. Although Kir6.1 is also present in atrial tissue, it is unknown whether this channel modulates atrial repolarization and hence whether the S422L mutation portends a greater risk of atrial arrhythmias. This study sought to examine whether there was an increased frequency of the KCNJ8-S422L mutation among patients with atrial fibrillation (AF) and early repolarization (ER) as a possible novel susceptibility gene for AF. METHODS AND RESULTS: A total of 325 lone AF probands were identified from the Vanderbilt AF Registry, a collection of clinical data and DNA from consented, consecutively enrolled participants. The coding regions of KCNJ8 were sequenced, and the patient's presenting electrocardiogram (ECG) was reviewed by two independent physicians for ER abnormalities. The KCNJ8-S422L mutation was identified in two AF probands while no other candidate gene variants were identified in these cases. Twenty-two (7%) patients were found to have ER on the ECG, including the two probands carrying the S422L variant. In one small AF kindred, the S422L variant co-segregated with AF and ER. CONCLUSIONS: The KCNJ8-S422L variant is associated with both increased AF susceptibility and ER indicating a role for Kir 6.1 K(atp) channel in both ventricular and atrial repolarization.

A new locus for autosomal dominant generalized epilepsy associated with mild mental retardation on chromosome 3p.

PURPOSE: Generalized epilepsies are clinically and genetically heterogeneous syndromes. Idiopathic generalized epilepsy (IGE), which has a strong genetic background, is not associated with any additional clinical features, such as mental retardation (MR). Herein we report results of linkage analysis in a large family with autosomal dominant (AD) generalized epilepsy associated with MR. METHODS: We identified a four-generation kindred with several affected members with generalized epilepsy without any evidence for secondary causes. Electroencephalography (EEG) studies and magnetic resonance imaging (MRI) results were reviewed when available. We performed a genome-wide linkage analysis. KEY FINDINGS: Fourteen individuals were classified as affected and an additional three were considered as nonpenetrant obligatory carriers. Thirteen affected individual had a history of generalized tonic-clonic seizures, and absence seizures were reported in nine affected individuals. There was no history of preceding febrile seizures. MR was present in nine affected individuals with epilepsy but the other affected individuals had normal intelligence. Neuroimaging did not reveal any structural abnormalities and EEG studies were consistent with IGE rather than symptomatic generalized epilepsy. Genetic analysis detected a group of markers with logarithmic (base 10) of odds (LOD) score >3 on chromosome 3p spanning a 5.5 Mbp region. Sequencing of several candidate genes, including dynein light chain-A, golgin subfamily a4, leucine rich repeat (in FLII) interacting gene, serine/threonine-protein kinase DCAMKL3 (doublecortin- like and CAM kinase-like 3), laforin (EPM2A) interacting protein 1 (EPM2AIP1, programmed cell death 6 interacting protein, and CLIP-associating protein 2 (cytoplasmic linker-associated protein 2) (hOrbit2) genes did not identify the disease-causing mutations. SIGNIFICANCE: We report the identification of a genetic locus for generalized epilepsy associated with MR on chromosome 3p. Affected individuals have a form of genetic epilepsy with generalized seizures variably associated with MR. Despite the presence of MR in several affected patients, epilepsy phenotype was not fully consistent with symptomatic epilepsy and suggests a biologic continuum between symptomatic epilepsies and IGE.

Reappraisal of the role of the DRD3 gene in essential tremor.

OBJECTIVES: Analyze the distribution of polymorphism in the dopamine receptor D3 (DRD3) gene, which was previously reported as a susceptibility risk for essential tremor (ET), in a large cohort of ET. METHODS: The role of 312G>A DRD3 polymorphism was analyzed using linkage analysis, association study and transmission disequilibrium test in a group of 433 ET patients, and two unrelated control groups with 121 and 151 individuals. RESULTS: Allelic frequencies of glycine and serine forms of the DRD3 gene did not differ between patients and both control groups, and were in Hardy-Weinberg equilibrium. Linkage analysis identified obligatory recombinants in every large pedigree, even in those with relatively high frequency of glycine allele, thus excluding the linkage to this locus. Both alleles were transmitted with an equal likelihood to affected offspring. We also failed to replicate the relationship between glycine homozygosity and an earlier age of onset or more severe tremor course. CONCLUSIONS: Our comprehensive genetic analysis in a large ET cohort strongly argues against the role of the DRD3 gene in ET pathogenesis.

Infantile onset of hereditary spastic paraplegia poorly predicts the genotype.

Age of symptom onset of hereditary spastic paraplegia varies from infancy to the eighth decade. Infantile onset of hereditary spastic paraplegia without a positive family history may cause difficulties in reaching the correct diagnosis and misdiagnosis as a diplegic form of cerebral palsy is particularly common. Infantile onset of hereditary spastic paraplegia caused by mutations in the spastin gene (SPAST) is very rare and previously was mostly associated with codominant mutations in this gene. We present a kindred with infantile onset of spastic paraplegia in three successive generations caused by confirmed de novo novel mutation 1537G>A (G471D) in SPAST. Several family members were previously diagnosed as having cerebral palsy. Infantile onset of hereditary spastic paraplegia may be caused by mutations in multiple genes, and this phenotype does not reliably predict the genotype. Pediatric neurologists need to be aware of relatively frequent de novo mutations in hereditary spastic paraplegia genes and a possibility that this condition presents in infancy without a positive family history.

Mutations in GABRA1, GABRA5, GABRG2 and GABRD receptor genes are not a major factor in the pathogenesis of familial focal epilepsy preceded by febrile seizures.

GABA(A) receptors mutations have been reported in few epilepsy families with febrile seizures (FS) followed by generalized epilepsy. It is not known if such mutations may underlie FS followed by partial epilepsy, which is a more common type of epilepsy. We searched for disease-causing mutations in the genes of the alpha1, alpha5, gamma2 and delta subunits of the GABA-A receptor that were previously shown to contain epilepsy-causing mutations or epilepsy susceptibility polymorphisms. All coding and untranslated exons of these four GABA(A) subunit genes were screened in 74 unrelated patients with familial partial epilepsy preceded by FS. Most patients had temporal lobe epilepsy (TLE). We did not detect any disease-causing mutations that would be consistent with missense, nonsense or splice site mutations in any of the four analyzed genes. We conclude that these genes are not a major genetic factor in familial TLE preceded by FS.

Mutations in the GABRA1 and EFHC1 genes are rare in familial juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME), accounting for approximately 25% of idiopathic generalized epilepsies, is genetically heterogeneous. Mutations in the alpha-1 subunit of the GABAA receptor (GABRA1) and EFHC1 genes have been reported in a few families with autosomal dominant (AD) JME. We have investigated the contribution of these two genes to familial JME in our cohort of 54 JME Caucasian families. Syndromic classification of JME was based on previously published criteria. We considered kindreds with at least one affected first-degree relative and the evidence of a vertical transmission as definite AD JME, and families with at least one affected second-degree relative as probable AD JME. We included 33 families meeting criteria for definitive AD JME and 21 that were classified as probable AD JME. None of these families were considered informative enough to analyze candidate loci for JME using linkage analysis. We have systematically screened coding exons of these two genes using temperature gradient capillary electrophoresis. Every heteroduplex with an abnormal mobility was sequenced. No disease-causing mutations in the GABRA1 gene were identified. Analysis of EFHC1 gene found one putative disease-causing mutation R221H that was previously reported as a tandem mutation. Several synonymous and non-synonymous coding polymorphisms were identified but the allelic frequency did not differ between controls and affected individuals. Our data suggests that the majority of familial AD JME is not caused by mutations in the GABRA1 and EFHC1 genes.

Genetic and clinical risk prediction model for postoperative atrial fibrillation.

BACKGROUND: Postoperative atrial fibrillation (PoAF) is common after coronary artery bypass grafting. We previously showed that atrial fibrillation susceptibility single nucleotide polymorphisms (SNPs) at the chromosome 4q25 locus are associated with PoAF. Here, we tested the hypothesis that a combined clinical and genetic model incorporating atrial fibrillation risk SNPs would be superior to a clinical-only model. METHODS AND RESULTS: We developed and externally validated clinical and clinical/genetic risk models for PoAF. The discovery and validation cohorts included 556 and 1164 patients, respectively. Clinical variables previously associated with PoAF and 13 SNPs at loci associated with atrial fibrillation in genome-wide association studies were considered. PoAF occurred in 30% and 29% of patients in the discovery and validation cohorts, respectively. In the discovery cohort, a logistic regression model with clinical factors had good discrimination, with an area under the receiver operator characteristic curve of 0.76. The addition of 10 SNPs to the clinical model did not improve discrimination (area under receiver operator characteristic curve, 0.78; P=0.14 for difference between the 2 models). In the validation cohort, the clinical model had good discrimination (area under the receiver operator characteristic curve, 0.69) and addition of genetic variables resulted in a marginal improvement in discrimination (area under receiver operator characteristic curve, 0.72; P<0.0001). CONCLUSIONS: We developed and validated a model for the prediction of PoAF containing common clinical variables. Addition of atrial fibrillation susceptibility SNPs did not improve model performance. Tools to accurately predict PoAF are needed to risk stratify patients undergoing coronary artery bypass grafting and identify candidates for prophylactic therapies.

A genome-wide association study to identify genomic modulators of rate control therapy in patients with atrial fibrillation.

For many patients with atrial fibrillation, ventricular rate control with atrioventricular (AV) nodal blockers is considered first-line therapy, although response to treatment is highly variable. Using an extreme phenotype of failure of rate control necessitating AV nodal ablation and pacemaker implantation, we conducted a genome-wide association study (GWAS) to identify genomic modulators of rate control therapy. Cases included 95 patients who failed rate control therapy. Controls (n = 190) achieved adequate rate control therapy with ≤2 AV nodal blockers using a conventional clinical definition. Genotyping was performed on the Illumina 610-Quad platform, and results were imputed to the 1000 Genomes reference haplotypes. A total of 554,041 single-nucleotide polymorphisms (SNPs) met criteria for minor allele frequency (>0.01), call rate (>95%), and quality control, and 6,055,224 SNPs were available after imputation. No SNP reached the canonical threshold for significance for GWAS of p <5 × 10(-8). Sixty-three SNPs with p <10(-5) at 6 genomic loci were genotyped in a validation cohort of 130 cases and 157 controls. These included 6q24.3 (near SAMD5/SASH1, p = 9.36 × 10(-8)), 4q12 (IGFBP7, p = 1.75 × 10(-7)), 6q22.33 (C6orf174, p = 4.86 × 10(-7)), 3p21.31 (CDCP1, p = 1.18 × 10(-6)), 12p12.1 (SOX5, p = 1.62 × 10(-6)), and 7p11 (LANCL2, p = 6.51 × 10(-6)). However, none of these were significant in the replication cohort or in a meta-analysis of both cohorts. In conclusion, we identified several potentially important genomic modulators of rate control therapy in atrial fibrillation, particularly SOX5, which was previously associated with heart rate at rest and PR interval. However, these failed to reach genome-wide significance.

A common variant on chromosome 4q25 is associated with prolonged PR interval in subjects with and without atrial fibrillation.

Single nucleotide polymorphisms (SNPs) at chromosome 4q25 (near PITX2) are strongly associated with atrial fibrillation (AF). We assessed whether a 4q25-tagging SNP (rs2200733) is associated with PR interval duration in patients with lone and typical AF and controls. Patients with lone (n = 169) and typical (n = 269) AF enrolled in the Vanderbilt AF registry and controls (n = 1,403) derived from the Vanderbilt DNA Biobank were studied. Carriage of the rs2200733T allele (CT or TT genotype) was more common in patients with lone (39%) than typical (25%) AF or controls (21%, p <0.01 for both comparisons). In both AF cohorts, we observed an association between genotype and PR interval duration (median PR interval for CC, CT, and TT: 162, 178, and 176 ms, respectively, for lone, p = 0.038 and 166, 180, and 196 ms, respectively, for typical, p = 0.001). After adjustment for covariates, the association between T allele and PR prolongation persisted, with mean effect size of 10.9, 12.8, and 4.4 ms for patients with lone and typical AF and controls, respectively (p <0.05 for each comparison). We found that a common 4q25 AF susceptibility allele (rs2200733) is associated with PR interval prolongation in patients with lone and typical AF and controls with no AF. Given that prolonged PR interval is an established risk factor for AF, this observation, in the context of previously described functional effects of PITX2 deficiency, provides further knowledge about the pathophysiological link of 4q25 variants with AF.

Common genetic polymorphism at 4q25 locus predicts atrial fibrillation recurrence after successful cardioversion.

BACKGROUND: Genome-wide association studies have identified numerous common polymorphisms associated with atrial fibrillation (AF). The 3 loci most strongly associated with AF occur at chromosome 4q25 (near PITX2), 16q22 (in ZFHX3), and 1q21 (in KCNN3). OBJECTIVE: To evaluate whether timing of AF recurrence after direct current cardioversion (DCCV) is modulated by common AF susceptibility alleles. METHODS: A total of 208 patients (age 65 ± 11 years; 77% men) with persistent AF underwent successful DCCV and were prospectively evaluated at 3, 6, and 12 months for AF recurrence. Four single nucleotide polymorphisms--rs2200733 and rs10033464 at 4q25, rs7193343 in ZFHX3, and rs13376333 in KCNN3--were genotyped. RESULTS: The final study cohort consisted of 184 patients. In 162 (88%) patients, sinus rhythm was restored with DCCV, of which 108 (67%) had AF recurrence at a median of 60 (interquartile range 29-176) days. In multivariable analysis, the presence of any common single nucleotide polymorphism (rs2200733, rs10033464) at the 4q25 locus was an independent predictor of AF recurrence (hazard ratio 2.1; 95% confidence interval 1.21-3.30; P = .008). Furthermore, rs2200733 exhibited a graded allelic dose response for early AF recurrence (homozygous variants: 7 [interquartile range 4-56] days; heterozygous variants: 54 [28-135] days; and wild type: 64 [29-180] days; P = .03). CONCLUSIONS: To our knowledge, this is the first study to evaluate whether genomic markers can predict timing of AF recurrence in patients undergoing elective DCCV. Our findings show that a common polymorphism on chromosome 4q25 (rs2200733) is an independent predictor of AF recurrence after DCCV and point to a potential role of stratification by genotype.

Mutation in KIF5A can also cause adult-onset hereditary spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD HSP) linked to chromosome 12q (SPG10) is caused by mutations in the neuronal kinesin heavy-chain KIF5A gene. This is a rare cause of AD HSP, and only two disease-causing mutations have been reported thus far. In both instances, affected individuals harboring mutations in the KIF5A gene displayed symptom onset at a very early age. Here we present the results of clinical and genetic analyses of a large kindred with uncomplicated AD HSP. We were able to establish a definitive linkage to the SPG10 locus, and sequencing of the KIF5A gene revealed a heterozygous missense mutation 1,035 A>G in exon 10, resulting in tyrosine-to-cysteine substitution. This mutation is located in a highly conserved kinesin motor domain of the neuronal kinesin heavy-chain protein, but in contrast to two previously reported missense mutations, the age of symptom onset in our family was much later, with an average age of 36.1+/-4 years. Our results demonstrate that mutations in the KIF5A gene can also be associated with an adult age of onset of AD HSP.

Identification of a novel locus for febrile seizures and epilepsy on chromosome 21q22.

PURPOSE: To report results of linkage analysis in a large family with autosomal dominant (AD) febrile seizures (FS) and epilepsy. BACKGROUND: AD FS and epilepsy is clinically and genetically a heterogeneous group of epilepsies, frequently inherited. The most notable, generalized epilepsy with febrile seizures plus (GEFS+), is characterized by heterogeneous phenotypes including FS persisting beyond the usual age of remission or coexisting with afebrile seizures. Mutations in three subunits of sodium channel genes and one GABA(A)-receptor subunit gene have been identified in some GEFS+ pedigrees. Six genetic loci for FS have been reported so far, but the molecular basis of FS remains unknown. METHODS: We identified a five-generation family with 13 individuals affected by FS. Evidence was found for coexisting afebrile seizures in some affected individuals. Evaluation included a detailed history and neurologic examination, as well as collection of DNA. After excluding previously identified loci associated with FS and epilepsy, a genome-wide search was performed. RESULTS: Two affected individuals reported only a single FS, whereas the other affected individuals had a history of repeated FS. Coexisting afebrile seizures developed in three individuals. The mode of inheritance was consistent with AD inheritance with an incomplete penetrance. Tight linkage to a group of markers on chromosome 21q22 was identified with flanking markers D21S1909 and D21S1444, and maximum 2-point lod score 3.35 for markers D21S1910 and D21S1894. We excluded four ion-channel genes within this 6.5-cM locus as a cause of FS and epilepsy in this family. CONCLUSIONS: We report a novel locus on chromosome 21q22 for AD FS. Identification of the gene causing epilepsy on chromosome 21q22 will advance our understanding of inherited epilepsy and FS, and possibly other types of epilepsies.

Familial essential tremor with apparent autosomal dominant inheritance: should we also consider other inheritance modes?

A positive family history is present in many patients with essential tremor (ET), but twin studies and segregation analysis have suggested that ET is not entirely a genetic disorder. Two genetic loci have been identified in autosomal dominant (AD) ET and polymorphisms in the DRD3 and HS1-BP3 genes have been proposed as the possible susceptibility factors for ET. There is also evidence for further genetic heterogeneity. We evaluated 4 unrelated large kindreds with ET with an apparent AD mode of transmission. Each kindred spanned at least 3 generations and contained at least 13 living affected subjects who met criteria for definitive ET. None of the pedigrees had evidence for inheritance of ET from both parents. Known genetic ET loci were excluded in these families. We detected a preferential transmission of ET in every kindred and the proportion of affected offspring varied from 75% to 90% (P < 0.05) in the generations with complete ascertainment. Our data indicate that non-Mendelian preferential transmission of an affected allele is a feature in many ET kindreds with multiple affected members and an apparent AD mode of inheritance. ET may have a complex etiology. Additional genetic models need to be considered, including an interaction of susceptibility genes and environmental risk factors.