Objectives The aim of this study was to define the genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC). Background Arrhythmogenic right ventricular cardiomyopathy, characterized ...by right ventricular fibrofatty replacement and arrhythmias, causes sudden death. Autosomal dominant inheritance, reduced penetrance, and 7 desmosome-encoding causative genes are known. The basis of low penetrance is poorly understood. Methods Arrhythmogenic right ventricular cardiomyopathy probands and family members were enrolled, blood was obtained, lymphoblastoid cell lines were immortalized, deoxyribonucleic acid was extracted, polymerase chain reaction (PCR) amplification of desmosome-encoding genes was performed, PCR products were sequenced, and diseased tissue samples were studied for intercellular junction protein distribution with confocal immunofluorescence microscopy and antibodies against key proteins. Results We identified 21 variants in plakophilin-2 ( PKP2 ) in 38 of 198 probands (19%), including missense, nonsense, splice site, and deletion/insertion mutations. Pedigrees showed wide intra-familial variability (severe early-onset disease to asymptomatic individuals). In 9 of 38 probands, PKP2 variants were identified that were encoded in trans (compound heterozygosity). The 38 probands hosting PKP2 variants were screened for other desmosomal genes mutations; second variants (digenic heterozygosity) were identified in 16 of 38 subjects with PKP2 variants (42%), including desmoplakin ( DSP ) (n = 6), desmoglein-2 ( DSG2 ) (n = 5), plakophilin-4 ( PKP4 ) (n = 1), and desmocollin-2 ( DSC2 ) (n = 1). Heterozygous mutations in non -PKP 2 desmosomal genes occurred in 14 of 198 subjects (7%), including DSP (n = 4), DSG2 (n = 5), DSC2 (n = 3), and junctional plakoglobin ( JUP ) (n = 2). All variants occurred in conserved regions; none was identified in 700 ethnic-matched control subjects. Immunohistochemical analysis demonstrated abnormalities of protein architecture. Conclusions These data suggest that the genetic basis of ARVC includes reduced penetrance with compound and digenic heterozygosity. Disturbed junctional cytoarchitecture in subjects with desmosomal mutations confirms that ARVC is a disease of the desmosome and cell junction.
...currently the interpretation of genetic tests is subjected to the complexity of the molecular basis of LQTS, and, even more than before, a comprehensive clinical approach along with pre- and ...post-genetic counseling is necessary to assess each variant effect and weight in the patient phenotype, as well as which variant(s) to be tested for in the proband's first-degree relatives to identify at-risk family members.
Objectives We evaluated ankyrin repeat domain 1 ( ANKRD1) , the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation ...analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. Background CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. Methods In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription–polymerase chain reaction. Results Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. Conclusions ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.
Sudden infant death syndrome (SIDS) is one of the leading causes of death during the first year of life. Long QT syndrome (LQTS)-associated mutations may be responsible for 5% to 10% of SIDS cases. ...We recently established CAV3-encoded caveolin-3 as a novel LQTS-associated gene with mutations producing a gain-of-function, LQT3-like molecular/cellular phenotype.
The purpose of this study was to determine the prevalence and functional properties of CAV3 mutations in SIDS.
Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, postmortem genetic testing of CAV3 was performed on genomic DNA isolated from frozen necropsy tissue on a population-based cohort of unrelated cases of SIDS (N = 134, 57 females, average age = 2.7 months). CAV3 mutations were engineered using site-directed mutagenesis and heterologously expressed in HEK293 cell lines stably expressing the SCN5A-encoded cardiac sodium channel.
Overall, three distinct CAV3 mutations (V14L, T78M, and L79R) were identified in three of 50 black infants (6-month-old male, 2-month-old female, and 8 month-old female), whereas no mutations were detected in 83 white infants (P <.05). CAV3 mutations were more likely in decedents 6 months or older (2/12) than in infants who died before 6 months (1/124, P = .02). Voltage clamp studies showed that all three CAV3 mutations caused a significant fivefold increase in late sodium current compared with controls.
This study provides the first molecular and functional evidence implicating CAV3 as a pathogenic basis of SIDS. The LQT3-like phenotype of increased late sodium current supports an arrhythmogenic mechanism for some cases of SIDS.
Objectives Four variants (K60N, Q128R, G202R, and A592E) in the nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if ...these mutations are cardiomyopathy causing. Background Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. Methods We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain. Results We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. Conclusions Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.
Abstract Sudden cardiac death (SCD) is a leading cause of mortality worldwide. Although coronary artery disease remains the most common substrate for SCD, primary cardiac genetic diseases, presenting ...with or without structural heart abnormalities, play a significant role. In the last 30 years, the study of large family pedigrees allowed the discovery of causative genes unveiling the genetic basis of diseases such as primary cardiomyopathies and arrhythmia syndromes, which are known to increase the risk of SCD. However, recent technological advancement with the ability to perform massive parallel sequencing and analyze the entire genome has uncovered a higher level of complexity in the genetic predisposition for cardiac diseases, which are usually characterized by Mendelian inheritance patterns. Clinical genetic testing, historically shaped around a monogenic Mendelian disorder paradigm, is now facing the challenge to adopt and adapt to a more complex model in which a significant portion of subjects may present with multi-allelic inheritance involving additional genes that could modulate the severity and type of disease-related phenotypes. Here, we will try to provide a viewpoint that will hopefully foster further debate in the field.
Apamin-sensitive small-conductance calcium-activated potassium (SK) channels are gated by intracellular Ca(2+) through a constitutive interaction with calmodulin.
We hypothesize that arrhythmogenic ...human calmodulin mutations impede activation of SK channels.
We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G, and F90L). Plasmids encoding either wild-type or mutant calmodulin were transiently transfected into human embryonic kidney 293 cells that stably express subtype 2 of SK protein channels (SK2 cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (n=7) or without (n=3) pretreatment with sea anemone toxin.
SK2 cells transfected with wild-type calmodulin exhibited an apamin-sensitive current density of 33.6 pA/pF (31.4-36.5 pA/pF) (median and confidence interval 25th-75th percentile), which was significantly higher than that observed for cells transfected with N54I (17.0 pA/pF 14.0-27.7 pA/pF; P = .016), F90L (22.6 pA/pF 20.3-24.3 pA/pF; P = .011), D96V (13.0 pA/pF 10.9-15.8 pA/pF; P = .003), N98S (13.7 pA/pF 8.8-20.4 pA/pF; P = .005), and D130G (17.6 pA/pF 13.8-24.6 pA/pF; P = .003). The decrease in SK2 current densities was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular action potential duration at 80% repolarization (from 79.6 ms 63.4-93.3 ms to 121.8 ms 97.9-127.2 ms; P = .010) in hearts pretreated with anemone toxin but not in control hearts.
Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in human embryonic kidney 293 cells.
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