Whole exome sequencing in Brugada and long QT syndromes revealed novel rare and potential pathogenic mutations related to the dysfunction of the cardiac sodium channel.

BACKGROUND: Brugada syndrome (Brs) and long QT syndrome (LQTs) are the most observed "inherited primary arrhythmia syndromes" and "channelopathies", which lead to sudden cardiac death. METHODS: Detailed clinical information of Brs and LQTs patients was collected. Genomic DNA samples of peripheral blood were conducted for whole-exome sequencing on the Illumina HiSeq 2000 platform. Then, we performed bioinformatics analysis for 200 genes susceptible to arrhythmias and cardiomyopathies. Protein interaction and transcriptomic co-expression were analyzed using the online website and GTEx database. RESULTS: All sixteen cases of Brs and six cases of LQTs were enrolled in the current study. Four Brs carried known pathogenic or likely pathogenic of single-point mutations, including SCN5A p.R661W, SCN5A p.R965C, and KCNH2 p.R692Q. One Brs carried the heterozygous compound mutations of DSG2 p.F531C and SCN5A p.A1374S. Two Brs carried the novel heterozygous truncated mutations (MAF < 0.001) of NEBL (p.R882X) and NPPA (p.R107X), respectively. Except for the indirect interaction between NEBL and SCN5A, NPPA directly interacts with SCN5A. These gene expressions had a specific and significant positive correlation in myocardial tissue, with high degrees of co-expression and synergy. Two Brs carried MYH7 p.E1902Q and MYH6 p.R1820Q, which were predicted as "damaging/possibly damaging" and "damaging/damaging" by Polyphen and SIFT algorithm. Two LQTs elicited the pathogenic single splicing mutation of KCNQ1 (c.922-1G > C). Three LQTs carried a single pathogenic mutation of SCN5A p.R1880H, KCNH2 p.D161N, and KCNQ1 p.R243S, respectively. One patient of LQTs carried a frameshift mutation of KCNH2 p. A188Gfs*143. CONCLUSIONS: The truncated mutations of NEBL (p.R882X) and NPPA (p.R107X) may induce Brugada syndrome by abnormally affecting cardiac sodium channel. SCN5A (p.R661W, p.R965C and p.A1374S) and KCNH2 (p.R692Q) may cause Brugada syndrome, while SCN5A (p.R1880H), KCNQ1 (c.922-1G > C and p.R243S) and KCNH2 (p.D161N and p.A188Gfs*143) may lead to long QT syndrome.

Rare and potential pathogenic mutations of LMNA and LAMA4 associated with familial arrhythmogenic right ventricular cardiomyopathy/dysplasia with right ventricular heart failure, cerebral thromboembolism and hereditary electrocardiogram abnormality.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is associated with ventricular arrhythmia, heart failure (HF), and sudden death. Thromboembolism is also an important and serious complication of ARVC/D. However, the etiology of ARVC/D and thromboembolism and their association with genetic mutations are unclear. METHODS: Genomic DNA samples of peripheral blood were conducted for whole-exome sequencing (WES) and Sanger sequencing in the ARVC/D family. Then, we performed bioinformatics analysis for genes susceptible to cardiomyopathies and arrhythmias. Further, we analyzed how the potential pathogenic mutations were affecting the hydrophobicity and phosphorylation of amino acids and their joint pathogenicity by ProtScale, NetPhos and ORVAL algorisms. RESULTS: We discovered a Chinese Han family of ARVC/D with right ventricular HF (RVHF), cerebral thromboembolism, arrhythmias (atrial fibrillation, atrial standstill, multifocal ventricular premature, complete right bundle block and third-degree atrioventricular block) and sudden death. Based on the WES data, the variants of LMNA p.A242V, LAMA4 p.A225P and RYR2 p.T858M are highly conserved and predicated as "deleterious" by SIFT and MetaSVM algorithms. Their CADD predicting scores are 33, 27.4 and 25.8, respectively. These variants increase the hydrophobicity of their corresponding amino acid residues and their nearby sequences by 0.378, 0.266 and 0.289, respectively. The LAMA4 and RYR2 variants lead to changes in protein phosphorylation at or near their corresponding amino acid sites. There were high risks of joint pathogenicity for cardiomyopathy among these three variants. Cosegregation analysis indicated that LMNA p.A242V might be an important risk factor for ARVC/D, electrocardiogram abnormality and cerebral thromboembolism, while LAMA4 p.A225P may be a pathogenic etiology of ARVC/D and hereditary electrocardiogram abnormality. CONCLUSIONS: The LMNA p.A242V may participate in the pathogenesis of familial ARVC/D with RVHF and cerebral thromboembolism, while LAMA4 p.A225P may be associated with ARVC/D and hereditary electrocardiogram abnormality.

Digenic heterozygous mutations of KCNH2 and SCN5A induced young and early-onset long QT syndrome and sinoatrial node dysfunction.

INTRODUCTION: Long QT syndrome (LQTS) is a life-threatening inherited channelopathy, and prolonged QT intervals easily trigger malignant arrhythmias, especially torsades de pointes and ventricular fibrillation. MATERIALS AND METHODS: The proband with overlapped phenotypes of LQTS and sinoatrial node dysfunction underwent some necessary examinations, including echocardiography, electrocardiogram (ECG), and Holter monitoring. Next, whole-exome sequencing was performed, and candidate genes were validated by Sanger sequencing. RNA secondary structure and protein physical-chemical parameter analyses were used to predict the possible structural change of the proteins induced by the mutations. RESULTS: We identified the digenic heterozygous mutations of KCNH2 p.307_308del (NM_001204798, c.921_923del) and SCN5A p.R1865H (NM_001160160, c.G5594A) in the female and young proband (II: 1) of LQTS and ventricular fibrillation with repeat syncope at rest. Subsequently, she occurred with obvious sinus arrest with persistent ventricular pacing of implantable cardioverter-defibrillator. The heterozygous SCN5Ap.R1865H was carried by her father and sister but not carried by I:2. II:1 carried with KCNH2 p.307_308del as a de novo mutation, but not existed in other family members. RNA secondary structure of KCNH2 p.307_308del showed a false regional double helix, and its amino acids' hydrophobicity was significantly weakened. For the Nav 1.5 protein property, SCN5A p.R1865H slightly increased the molecular weight and aliphatic index but reduced the instability index. CONCLUSIONS: The digenic heterozygous KCNH2 and SCN5A mutations were associated with young early-onset long QT syndrome and sinoatrial node dysfunction.

Young and early-onset dilated cardiomyopathy with malignant ventricular arrhythmia and sudden cardiac death induced by the heterozygous LDB3, MYH6, and SYNE1 missense mutations.

BACKGROUND: The whole exome sequencing (WES) with targeted gene analysis is an effective diagnostic tool for cardiomyopathy. The early-onset sudden cardiac death (SCD) was commonly associated with dilated cardiomyopathy (DCM) induced by pathogenic genetic mutations. METHODS: In a Chinese Han family, the patient of 24 years old occurred with early-onset and DCM and died of SCD associated with ICD storms induced by repetitive ventricular tachycardia/fibrillation (VT/F). Genomic DNA samples of peripheral blood were conducted for WES and Sanger sequence. Then, we performed bioinformatics analysis for 200 genes susceptible to cardiomyopathies and arrhythmias. Further, we analyzed how the potential pathogenic mutations affecting the secondary structure, hydrophobicity, and phosphorylation of amino acids, protein properties, and their joint pathogenicity by ProtParam, SOPMA, and ORVAL algorisms. The protein-protein interaction was analyzed by STRING algorism. RESULTS: The mutations of LDB3 p.M456R, MYH6 p.S180Y, and SYNE1 p.S4607F were identified as "Damaging/Deleterious." The SYNE1 (p.S4607F) increased one of alpha helix and decreased one of beta sheet. The LDB3 (p.M456R) reduced one of beta sheet and increased one of beta turn. The MYH6 (p.S180Y) decreased two of beta sheets and four of beta turns, but significantly increased twelve coils. The hydrophobicity of amino acid residues and their adjacent sequences were decreased by LDB3 (p.M456R) and MYH6 (p.S180Y), and significantly increased by SYNE1 (p.S4607F). The mutations of LDB3 (p.M456R), SYNE1 (p.S4607F), and MYH6 (p.S180Y) resulted in the phosphorylation changes of the corresponding amino acid sites or the nearby amino acid sites. The pairwise combinations of LDB3, MYH6, and SYNE1 mutations have the high probability of causing disease, especially the highest probability for SYNE1 and LDB3 mutations. There was obviously indirect interaction of the proteins encoded by SYNE1, LDB3, and MYH6. CONCLUSIONS: The multiple heterozygous mutations of SYNE1, LDB3, and MYH6 may be associated with young and early-onset of DCM and SCD.

Genetic Variants on SCN5A, KCNQ1, and KCNH2 in Patients with Ventricular Arrhythmias during Acute Myocardial Infarction in a Chinese Population.

OBJECTIVE: Acute myocardial infarction (AMI) remains a leading cause of morbidity and mortality worldwide. About half of sudden deaths from AMI are mainly because of malignant ventricular arrhythmias (VA) after AMI. The sodium channel gene SCN5A and potassium channel genes KCNQ1 and KCNH2 have been widely reported to be genetic risk factors for arrhythmia including Brugada syndrome and long QT syndrome (LQTS). A few studies reported the association of SCN5A variant with ventricular tachycardia (VT)/ventricular fibrillation (VF) complicating AMI. However, little is known about the role of KCNQ1 and KCNH2 in AMI with VA (AMI_VA). This study focuses on investigating the potential variants on SCN5A, KCNQ1, and KCNH2 contributing to AMI with VA in a Chinese population. MATERIALS AND METHODS: In total, 139 patients with AMI_VA, and 337 patients with AMI only, were included. Thirty exonic sites were selected to be screened. Sanger sequencing was used to detect variants. A subsequent association study was also performed between AMI_VA and AMI. RESULTS: Twelve variants [5 on KCNH2(NM_000238.3), 3 on KCNQ1(NM_000218.2), and 4 on SCN5A(NM_198056.2)] were identified in AMI_VA patients. Only 5 (KCNH2: c.2690A>C; KCNQ1: c.1927G>A, c.1343delC; SCN5A: c.1673A>G, c.3578G>A) of them are missense variants. Two (KCNQ1: c.1343delC and SCN5A: c.3578G>A) of the missense variants were predicted to be clinically pathogenic. All these variants were further genotyped in an AMI without VA group. The association study identified a statistically significant difference in genotype frequency of KCNH2: c.1539C>T and KCNH2: c.1467C>T between the AMI and AMI_VA groups. Moreover, 2 rare variants (KCNQ1: c.1944C>T and SCN5A: c.3621C>T) showed an elevated allelic frequency (more than 1.5-fold) in the AMI_VA group when compared to the AMI group. CONCLUSION: Twelve variants (predicting from benign/VUS to pathogenic) were identified on KCNH2, KCNQ1, and SCN5A in patients with AMI_VA. Genotype frequency comparison between AMI_VA and AMI identified 2 significant common variants on KCNH2. Meanwhile, the allelic frequency of 2 rare variants on KCNQ1 and SCN5A, respectively, were identified to be enriched in AMI_VA, although there was no statistical significance. The present study suggests that the ion-channel genes KCNH2, KCNQ1, and SCN5A may contribute to the pathogenesis of VA during AMI.

Compound and heterozygous mutations of DSG2 identified by Whole Exome Sequencing in arrhythmogenic right ventricular cardiomyopathy/dysplasia with ventricular tachycardia.

BACKGROUNDS: This study was designed to identify the pathogenic mutations in two Chinese families of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) using the Whole Exome Sequencing (WES). METHODS AND RESULTS: The proband 1 (Family 1, II:1) and proband 2 (Family 2, II:1) underwent the WES of DNA from peripheral blood. The genes susceptible to arrhythmias and cardiomyopathies were analyzed and both the probands carried the same exonic mutation of DSG2 p.F531C (NM_001943, exon 11: c.T1592G). The proband 1 also carried the splicing mutation of DSG2 (NM_001943: exon 4:c.217-1G>T), and proband 2 carried the intronic mutation of DSG2 (NM_001943: exon 6: c.524-3C>G) that potentially influenced the splicing function predicted by Human Splicing Finder. The compound heterozygous mutations of the two probands inherited from their paternal and maternal side, respectively. The carriers with DSG2 p.F531C showed early abnormal electrocardiograms, characterized as the subclinical phenotype of ARVC/D. CONCLUSIONS: The DSG2 p.F531C was the main reason for ARVC/D. More severe phenotypes of ARVC/D occurred when coexisting with 217-1G>T or 524-3C>G mutation that potentially affecting the splicing function, as a compound heterozygous recessive inheritance.

Case report of familial sudden cardiac death caused by a DSG2 p.F531C mutation as genetic background when carrying with heterozygous KCNE5 p.D92E/E93X mutation.

BACKGROUND: Sudden cardiac death (SCD) induced by malignant ventricular tachycardia (MVT) among young adults with right ventricular cardiomyopathy/dysplasia (ARVC/D) is a devastating event. Parts of ARVC/D patients have a mutation in genes encoding components of cardiac desmosomes, such as desmoglein-2 (DSG2), plakophilin-2 and desmoplakin. CASE PRESENTATION: Here we report a potentially pathogenic mutation in the DSG2 gene, which was identified in a family with ARVC/D using Whole Exome Sequencing (WES) and Sanger Sequencing. In all, Patient III:1 with ARVC/D carried the compound heterozygous mutations of DSG2 p.F531C and KCNE5 p.D92E/E93X, which were both inherited from her mother (II:2), who died of SCD. Carriers of DSG2p.F531C showed various phenotypes, such as ARVC/D, SCD, MVT and dilated cardiomyopathy. For III:1, there were significant low-voltage regions in the inferior-apical, inferior-lateral wall of the right ventricular epicardium and outflow tracts of the right ventricle. Under the guidance of a three-dimensional mapping system, MVT was successfully ablated with an epicardial-endocardial approach targeting for late, double or fragmental potentials after implantable cardioverter-defibrillator (ICD) electrical storms. No VT recurrence was observed during the one year of follow-up. CONCLUSIONS: When coexisting with heterozygous KCNE5 p.D92E/E93X, heterozygous DSG2 p.F531C as a genetic background was found to predispose to ARVC/D, SCD and MVT, which were successfully ablated using an epicardial-endocardial approach.

Whole exome sequencing identified a pathogenic mutation in RYR2 in a Chinese family with unexplained sudden death.

OBJECTIVE: This study aimed to identify the pathogenic mutation in a Chinese family with unexplained sudden death (USD) or occasional syncope. MATERIALS AND METHODS: Whole exome sequencing and target capture sequencing were respectively conducted for two related patients. The genetic data was screened using the 1000 genomes project and SNP database (PubMed), and the identified mutations were assessed for predicted pathogenicity using the SIFT and Polyphen-2 algorithms. RESULTS: We identified a heterozygous mutation in the RYR2 gene at c.490C>T (p.P164S), highly conserved across all species, in three family members of USD, syncope and malignant ventricular tachycardias induced by treadmill exercise test, while another heterozygous de novo mutation in SCN5A at c.5576G>A p.R1859H was detected in one family member. Both variants were verified by Sanger sequencing. Importantly, RYR2 p.P164S is associated with the risk of sudden cardiac death, such as in catecholaminergic polymorphic ventricular tachycardia. CONCLUSIONS: A pathogenic mutation in RYR2 (p.P164S) is the likely cause of USD in a Chinese family associated with malignant ventricular arrhythmias. Whole exome and target capture sequencing can be useful for discovering the genetic causes of USD.

Whole Genome Sequence Identified a Rare Homozygous Pathogenic Mutation of the DSG2 Gene in a Familial Arrhythmogenic Cardiomyopathy Involving Both Ventricles.

BACKGROUND: This study was designed to identify the pathogenic mutation in a Chinese family with arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) using whole genome sequencing (WGS). METHODS AND RESULTS: Probands II:1 and II:2 underwent routine examinations for diagnosis. Genomic DNA was extracted from the peripheral blood of family members and analyzed using WGS. A total of 60,285 single-nucleotide polymorphisms (SNP) and 13,918 insertions/deletions (InDel) occurring in the exonic regions of genes and predisposing to cardiomyopathies and arrhythmias were identified. When filtered using the 1000 Genomes Project (2014 version), NHLBI ESP6500, and ExAC databases, 12 missense SNP and 2 InDel in exonic regions remained, the allele frequencies of which were <0.01 or unknown. The potentially pathogenic mutations that occurred in the genes DSG2, PKP4, PRKAG2, FOXD4, CTTN, and DMD, which were identified by SIFT or PolyPhen-2 software as "damaging," were validated using Sanger sequencing. Probands II:1 and II:2 shared an extremely rare homozygous mutation in the DSG2 (p.F531C) gene, which was also demonstrated using intersection analysis of WGS data from probands II:1 and II:2. Electron microscopy and histological staining of myocardial biopsies showed widened and destroyed intercalated discs, and interrupted, atrophic, and disarranged myocardial fibers, and hyperplastic interstitial fibers, collagen fibers, and adipocytes were infiltrated and invaded. CONCLUSIONS: A homozygous mutation of DSG2 p.F531C was identified as the pathogenic mutation in patients with ARVC/D involving both ventricles, as a result of widened and impaired intercalated discs, interrupted myocardial fibers, and abnormally hyperplastic interstitial fibers, collagen fibers, and adipocytes.

Changes of Virtual Planar QRS and T Vectors Derived from Holter in the Populations with and without Diabetes Mellitus.

AIMS: Research related to type 2 diabetes mellitus (DM) and parameters of electrocardiography (ECG) was limited. Patients with and without DM (NDM) were randomly enrolled in a study to exploit the influence of DM on planar QRS and T vectors derived from the Virtual Holter process. METHODS: A total of 216 (NDM) and 127 DM patients were consecutively and randomly recruited. We selected a 1-minute length of ECG, which was scheduled for analysis at 4 AM. After a series of calculating algorisms, we received the virtual planar vector parameters. RESULTS: Patients with DM were elderly (65.61 ± 12.08 vs 59.41 ± 16.86 years, P < 0.001); higher morbidity of hypertension (76.38% vs 58.14%, P < 0.001) and coronary artery disease (44.09% vs 32.41%, P = 0.03); thicker interventricular septum (10.92 ± 1.77 vs 10.08 ± 1.96 mm, P < 0.001) and left ventricular posterior wall (9.84 ± 1.38 vs 9.39 ± 1.66 mm, P = 0.03); higher lipid levels and average heart rate (66.67 ± 12.04 vs 61.87 ± 13.36 bpm, P < 0.01); higher angle of horizontal QRS vector (HQRSA, -2.87 ± 48.48 vs -19.00 ± 40.18 degrees, P < 0.01); lower maximal magnitude of horizontal T vector (HTV, 2.33 ± 1.47 vs 2.88 ± 1.89 mm, P = 0.01) and maximal magnitude of right side T vector (2.77 ± 1.55 vs 3.27 ± 1.92 mm, P = 0.03), and no difference in angle of frontal QRS-T vector (FQRSTA, 32.77 ± 54.20 vs 28.39 ± 52.87 degrees, P = 0.74) compared with patients having NDM. After adjusting for confounding factors, DM was significantly effective on FQRSTA (regression coefficient -40.0, 95%CI -66.4 to -13.6, P < 0.01), HQRSA (regression coefficient 22.6, 95%CI 2.5 to 42.8, P = 0.03), and HTV (regression coefficient 0.9, 95%CI 0.2 to 1.7, P = 0.01). Confounding factors included: sex, 2-hour postprandial blood glucose, smoking, triglyceride, apolipoprotein A, creatinine, left ventricular ejection fraction, and average heart rate. CONCLUSIONS: The risk factors of DM and lipid metabolism abnormality particularly apolipoprotein A significantly modified parameters of virtual planar QRS and T vector, including frontal QRS-T angle.

[Chronic outcome of patients with paroxysmal atrial fibrillation post catheter ablation].

OBJECTIVE: High short-term successful rate was reported for catheter ablation in patients with paroxysmal atrial fibrillation (AF), we analyzed the long-term outcome (success rate, anticoagulation therapy and embolism event, anti-arrhythmic therapy and death post procedure) of catheter ablation for paroxysmal AF in this study. METHODS: From January 2000 to December 2004, 106 consecutive patients with drug-refractory paroxysmal AF underwent catheter ablation and were followed-up for (60.7 + or - 11.8) months. Segmental pulmonary vein isolation (SPVI) was routinely performed by radiofrequency energy under the guidance of circular mapping catheter. The patients were followed up with 24 h-holter, ECG, telephone or letter. Data on recurrence of AF, the anticoagulation medication and the incidence of embolism, anti-arrhythmic therapy were obtained. RESULTS: There were 9 patients lost to follow up. In the remaining 97 patients [65 males, (54.8 + or - 11.2) years old], 3 cases died from cancer, sinus rhythm was maintained in 68 patients (Group S, 72.3%) and AF recurrence evidenced in 26 patients (Group R, 27.7%). In Group S, 56 patients (82.4%) discontinued anticoagulation medication, and 12 patients continued to take aspirin. There was no embolism event in Group S during follow-up. In Group R, 1 patient continued to take warfarin; 11 patients continued to take aspirin and 2 patients suffered from cerebral embolism. Anticoagulation medication was discontinued in 14 patients (53.8%) and 1 patient suffered form cerebral embolism. The incidence of embolism event in Group R is significantly higher than in Group S (P < 0.01). More patients discontinued anti-arrhythmic medication in Group S than in Group R (80.9% vs. 56.0%, P < 0.05). CONCLUSION: Catheter ablation is associated with satisfactory long-term success rate, reduced anti-arrhythmia medication, improved quality of life in patients with paroxysmal AF.