Francesca Brun

Genetic Variation in Titin in Arrhythmogenic Right Ventricular Cardiomyopathy–Overlap Syndromes

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks. Methods and Results— All 312 titin exons known to be expressed in human cardiac titin and the complete 3′ untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families, including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in 1 large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain) located in the spring region of titin. Native gel electrophoresis, nuclear magnetic resonance, intrinsic fluorescence, and proteolysis assays of wild-type and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity for degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by a history of sudden death (5 of 7 families), progressive myocardial dysfunction causing death or heart transplantation (8 of 14 cases), frequent conduction disease (11 of 14), and incomplete penetrance (86%). Conclusions— Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks.

Utility of Cardiac Magnetic Resonance Imaging to Differentiate Cardiac Sarcoidosis from Arrhythmogenic Right Ventricular Cardiomyopathy

Some patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) are eventually found to have cardiac sarcoidosis (CS). Accurate differentiation between these 2 conditions has implications for immunosuppressive therapy and familial screening. We sought to determine whether cardiac magnetic resonance imaging (MRI) could be used to identify the characteristic findings to accurately differentiate between CS and ARVC. Consecutive patients with a diagnostic MRI scan indicating CS and/or ARVC constituted the cohort. All patients diagnosed with CS had histologic confirmation of sarcoidosis, and all patients with ARVC met the diagnostic task force criteria. The cardiac MRI data were retrospectively analyzed to identify possible differentiating characteristics. Of the patients, 40 had CS and 21 had ARVC. Those with CS were older and had more left ventricular scar. The presence of mediastinal lymphadenopathy or left ventricular septal involvement was seen exclusively in the patients with CS (p <0.001). A family history of sudden cardiac death was seen only in the ARVC group (p = 0.012). The right ventricular ejection fraction and ventricular volumes were also significantly different between the 2 groups. In conclusion, patients with CS have significantly different cardiac MRI characteristics than patients with ARVC. The cardiac volume, in addition to the degree and location of cardiac involvement, can be used to distinguish between these 2 disease entities. The presence of mediastinal lymphadenopathy and left ventricular septal scar favors a diagnosis of CS and not ARVC. Consideration of CS should be given if these MRI findings are observed during the evaluation for possible ARVC.

Multilevel analyses of SCN5A mutations in arrhythmogenic right ventricular dysplasia/cardiomyopathy suggest non-canonical mechanisms for disease pathogenesis.

Aims Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often associated with desmosomal mutations. Recent studies suggest an interaction between the desmosome and sodium channel protein Nav1.5. We aimed to determine the prevalence and biophysical properties of mutations in SCN5A (the gene encoding Nav1.5) in ARVD/C. Methods and results We performed whole-exome sequencing in six ARVD/C patients (33% male, 38.2 ± 12.1 years) without a desmosomal mutation. We found a rare missense variant (p.Arg1898His; R1898H) in SCN5A in one patient. We generated induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) from the patient’s peripheral blood mononuclear cells. The variant was then corrected (R1898R) using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology, allowing us to study the impact of the R1898H substitution in the same cellular background. Whole-cell patch clamping revealed a 36% reduction in peak sodium current (P = 0.002); super-resolution fluorescence microscopy showed reduced abundance of NaV1.5 (P = 0.005) and N-Cadherin (P = 0.026) clusters at the intercalated disc. Subsequently, we sequenced SCN5A in an additional 281 ARVD/C patients (60% male, 34.8 ± 13.7 years, 52% desmosomal mutation-carriers). Five (1.8%) subjects harboured a putatively pathogenic SCN5A variant (p.Tyr416Cys, p.Leu729del, p.Arg1623Ter, p.Ser1787Asn, and p.Val2016Met). SCN5A variants were associated with prolonged QRS duration (119 ± 15 vs. 94 ± 14 ms, P < 0.01) and all SCN5A variant carriers had major structural abnormalities on cardiac imaging. Conclusions Almost 2% of ARVD/C patients harbour rare SCN5A variants. For one of these variants, we demonstrated reduced sodium current, Nav1.5 and N-Cadherin clusters at junctional sites. This suggests that Nav1.5 is in a functional complex with adhesion molecules, and reveals potential non-canonical mechanisms by which Nav1.5 dysfunction causes cardiomyopathy.

Prognostic impact of familial screening in dilated cardiomyopathy.

Familial screening of patients with dilated cardiomyopathy (DCM) allows an early diagnosis of the disease in family members. It is unclear if familial forms (FDC) have a different long‐term outcome compared with sporadic DCM. Our aim was to compare the long‐term prognosis of FDC and sporadic forms in order to assess the role of familial screening.

Arrhythmogenic Phenotype in Dilated Cardiomyopathy: Natural History and Predictors of Life‐Threatening Arrhythmias

Background Patients with dilated cardiomyopathy (DCM) may present with ventricular arrhythmias early in the disease course, unrelated to the severity of left ventricular dysfunction. These patients may be classified as having an arrhythmogenic DCM (AR‐DCM). We investigated the phenotype and natural history of patients with AR‐DCM. Methods and Results Two hundred eighty‐five patients with a recent diagnosis of DCM (median duration of the disease 1 month, range 0 to 7 months) and who had Holter monitoring at baseline were comprehensively evaluated and followed for 107 months (range 29 to 170 months). AR‐DCM was defined by the presence of ≥1 of the following: unexplained syncope, rapid nonsustained ventricular tachycardia (≥5 beats, ≥150 bpm), ≥1000 premature ventricular contractions/24 hours, and ≥50 ventricular couplets/24 hours, in the absence of overt heart failure. The primary end points were sudden cardiac death (SCD), sustained ventricular tachycardia (SVT), or ventricular fibrillation (VF). The secondary end points were death from congestive heart failure or heart transplantation. Of the 285 patients, 109 (38.2%) met criteria for AR‐DCM phenotype. AR‐DCM subjects had a higher incidence of SCD/SVT/VF compared with non–AR‐DCM patients (30.3% vs 17.6%, P=0.022), with no difference in the secondary end points. A family history of SCD/SVT/VF and the AR‐DCM phenotype were statistically significant and cumulative predictors of SCD/SVT/VF. Conclusions One‐third of DCM patients may have an arrhythmogenic phenotype associated with increased risk of arrhythmias during follow‐up. A family history of ventricular arrhythmias in DCM predicts a poor prognosis and increased risk of SCD.

Role of Titin Missense Variants in Dilated Cardiomyopathy.

Background The titin gene (TTN) encodes the largest human protein, which plays a central role in sarcomere organization and passive myocyte stiffness. TTN truncating mutations cause dilated cardiomyopathy (DCM); however, the role of TTN missense variants in DCM has been difficult to elucidate because of the presence of background TTN variation. Methods and Results A cohort of 147 DCM index subjects underwent DNA sequencing for 313 TTN exons covering the N2B and N2BA cardiac isoforms of TTN. Of the 348 missense variants, we identified 44 “severe” rare variants by using a bioinformatic filtering process in 37 probands. Of these, 5 probands were double heterozygotes (additional variant in another DCM gene) and 7 were compound heterozygotes (2 TTN “severe” variants). Segregation analysis allowed the classification of the “severe” variants into 5 “likely” (cosegregating), 5 “unlikely” (noncosegregating), and 34 “possibly” (where family structure precluded segregation analysis) disease‐causing variants. Patients with DCM carrying “likely” or “possibly” pathogenic TTN “severe” variants did not show a different outcome compared with “unlikely” and noncarriers of a “severe” TTN variant. However, the “likely” and “possibly” disease‐causing variants were overrepresented in the C‐zone of the A‐band region of the sarcomere. Conclusions TTN missense variants are common and present a challenge for bioinformatic classification, especially when informative families are not available. Although DCM patients carrying bioinformatically “severe” TTN variants do not appear to have a worse clinical course than noncarriers, the nonrandom distribution of “likely” and “possibly” disease‐causing variants suggests a potential biological role for some TTN missense variants.

FLNC Gene Splice Mutations Cause Dilated Cardiomyopathy

Summary A genetic etiology has been identified in 30% to 40% of dilated cardiomyopathy (DCM) patients, yet only 50% of these cases are associated with a known causative gene variant. Thus, in order to understand the pathophysiology of DCM, it is necessary to identify and characterize additional genes. In this study, whole exome sequencing in combination with segregation analysis was used to identify mutations in a novel gene, filamin C (FLNC), resulting in a cardiac-restricted DCM pathology. Here we provide functional data via zebrafish studies and protein analysis to support a model implicating FLNC haploinsufficiency as a mechanism of DCM.

Scalability of split-gate charge trap memories down to 20nm for low-power embedded memories

In this work, split-gate charge trap memories with electrical gate length down to 20nm are presented for the 1^st time. Silicon nanocristals (Si-ncs), or silicon nitride (Si<inf>3</inf>N<inf>4</inf>) and hybrid Sinc/SiN based split-gate memories, with SiO<inf>2</inf> or Al<inf>2</inf>O<inf>3</inf> control dielectrics, are compared in terms of program erase and retention. Then, the scalability of split-gate charge trap memories is studied, investigating the impact of gate length reduction on the memory window, retention and consumption. The results are analyzed by means of TCAD simulations.

Titin and desmosomal genes in the natural history of arrhythmogenic right ventricular cardiomyopathy

Background Genotype–phenotype correlations are poorly characterised in arrhythmogenic right ventricular cardiomyopathy (ARVC). We investigated whether carriers of rare variants in desmosomal genes (DC) and titin gene (TTN) display different phenotypes and clinical outcomes compared with non-carriers (NT-ND). Methods and results Thirty-nine ARVC families (173 subjects, 67 affected) with extensive follow-up (mean 9 years), prospectively enrolled in the International Familial Cardiomyopathy Registry since 1991, were screened for rare variants in TTN and desmosomal genes (DSP, PKP2, DSG2, DSC2). Multiple clinical and outcome variables were compared between three genetic groups (TTN, DC, NT-ND) to define genotype–phenotype associations. Of the 39 ARVC families, 13% (5/39) carried TTN rare variants (11 affected subjects), 13% (5/39) DC (8 affected), while 74% (29/39) were NT-ND (48 affected). When compared with NT-ND, DC had a higher prevalence of inverted T waves in V2-3 (75% vs 31%, p=0.004), while TTN had more supraventricular arrhythmias (46% vs 13%, p=0.013) and conduction disease (64% vs 6% p<0.001). When compared with the NT-ND group, the DC group experienced a worse prognosis (67% vs 11%, p=0.03) and exhibited a lower survival free from death or heart transplant (59% vs 95% at 30 years, and 31% vs 89% at 50 years, HR 9.66, p=0.006), while the TTN group showed an intermediate survival curve (HR 4.26, p=0.037). Conclusions TTN carriers display distinct phenotypic characteristics including a greater risk for supraventricular arrhythmias and conduction disease. Conversely, DC are characterised by negative T waves in anterior leads, severe prognosis, high mortality and morbidity.

Clinical Spectrum of PRKAG2 Syndrome

PRKAG2 syndrome (PS) is a rare, early-onset autosomal dominant inherited disease, characterized by ventricular pre-excitation, supraventricular arrhythmias, and cardiac hypertrophy. It is frequently accompanied by chronotropic incompetence and advanced heart blocks, leading to premature pacemaker implantation. The association of these clinical features had previously been recognized by several studies since the second half of the 20th century.1–3 In 1991, PRKAG2 syndrome was mapped to the locus 7q 36,4 and in 2001, Gollob et al5 identified the responsible gene. The syndrome is caused by mutations in the gene encoding for the 5′ AMP-activated protein kinase (AMPK), specifically for its γ2 regulatory subunit (PRKAG2). AMPK is an enzyme deeply involved in cellular ATP metabolic regulation.6 PRKAG2 genetic mutations are rare and have been recognized mainly in the context of patients with nonsarcomeric familial hypertrophic cardiomyopathy associated with Wolff–Parkinson–White syndrome.7 PS can show different expressivity both of ventricular hypertrophy and arrhythmic features, ranging from an asymptomatic condition to sudden cardiac death (SCD). PS can occasionally lead to heart failure (HF) or demonstrate systemic involvement.7–9 This review aims to describe the various features and clinical implications of PS, providing clinicians and researchers with a summary of the published literature to improve the diagnosis and to better manage the clinical course of the disease. A search of the English literature was performed using PubMed up to September 2014 on the clinical features, genetics, and pathophysiology of PS syndrome. The term PRKAG2 combined with either cardiomyopathy, Wolff–Parkinson–White syndrome, atrial fibrillation, familial, left ventricular hypertrophy, atrioventricular (AV) block, pacemaker, SCD, HF, clinical characteristics, genotype, phenotype, or mutations was used. Observational studies, case reports, and reviews were included in our search. References were carefully evaluated for …