Sharon Graw

Truncations of Titin Causing Dilated Cardiomyopathy

BACKGROUND Dilated cardiomyopathy and hypertrophic cardiomyopathy arise from mutations in many genes. TTN, the gene encoding the sarcomere protein titin, has been insufficiently analyzed for cardiomyopathy mutations because of its enormous size. METHODS We analyzed TTN in 312 subjects with dilated cardiomyopathy, 231 subjects with hypertrophic cardiomyopathy, and 249 controls by using next-generation or dideoxy sequencing. We evaluated deleterious variants for cosegregation in families and assessed clinical characteristics. RESULTS We identified 72 unique mutations (25 nonsense, 23 frameshift, 23 splicing, and 1 large tandem insertion) that altered full-length titin. Among subjects studied by means of next-generation sequencing, the frequency of TTN mutations was significantly higher among subjects with dilated cardiomyopathy (54 of 203 [27%]) than among subjects with hypertrophic cardiomyopathy (3 of 231 [1%], P=3×10(-16)) or controls (7 of 249 [3%], P=9×10(-14)). TTN mutations cosegregated with dilated cardiomyopathy in families (combined lod score, 11.1) with high (>95%) observed penetrance after the age of 40 years. Mutations associated with dilated cardiomyopathy were overrepresented in the titin A-band but were absent from the Z-disk and M-band regions of titin (P≤0.01 for all comparisons). Overall, the rates of cardiac outcomes were similar in subjects with and those without TTN mutations, but adverse events occurred earlier in male mutation carriers than in female carriers (P=4×10(-5)). CONCLUSIONS TTN truncating mutations are a common cause of dilated cardiomyopathy, occurring in approximately 25% of familial cases of idiopathic dilated cardiomyopathy and in 18% of sporadic cases. Incorporation of sequencing approaches that detect TTN truncations into genetic testing for dilated cardiomyopathy should substantially increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy. Defining the functional effects of TTN truncating mutations should improve our understanding of the pathophysiology of dilated cardiomyopathy. (Funded by the Howard Hughes Medical Institute and others.).

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.

α-Myosin Heavy Chain A Sarcomeric Gene Associated With Dilated and Hypertrophic Phenotypes of Cardiomyopathy

Background—Mutations in the β-myosin heavy-chain (βMyHC) gene cause hypertrophic (HCM) and dilated (DCM) forms of cardiomyopathy. In failing human hearts, downregulation of αMyHC mRNA or protein has been correlated with systolic dysfunction. We hypothesized that mutations in αMyHC could also lead to pleiotropic cardiac phenotypes, including HCM and DCM. Methods and Results—A cohort of 434 subjects, 374 (134 affected, 214 unaffected, 26 unknown) belonging to 69 DCM families and 60 (29 affected, 30 unaffected, 1 unknown) in 21 HCM families, was screened for αMyHC gene (MYH6) mutations. Three heterozygous MYH6 missense mutations were identified in DCM probands (P830L, A1004S, and E1457K; 4.3% of probands). A Q1065H mutation was detected in 1 of 21 HCM probands and was absent in 2 unaffected offspring. All MYH6 mutations were distributed in highly conserved residues, were predicted to change the structure or chemical bonds of αMyHC, and were absent in at least 300 control chromosomes from an ethnically similar population. The DCM carrier phenotype was characterized by late onset, whereas the HCM phenotype was characterized by progression toward dilation, left ventricular dysfunction, and refractory heart failure. Conclusions—This study suggests that mutations in MYH6 may cause a spectrum of phenotypes ranging from DCM to HCM.

Prevalence of Desmin Mutations in Dilated Cardiomyopathy

Background— Desmin-related myofibrillar myopathy (DRM) is a cardiac and skeletal muscle disease caused by mutations in the desmin (DES) gene. Mutations in the central 2B domain of DES cause skeletal muscle disease that typically precedes cardiac involvement. However, the prevalence of DES mutations in dilated cardiomyopathy (DCM) without skeletal muscle disease is not known. Methods and Results— Denaturing high-performance liquid chromatography was used to screen DES for mutations in 116 DCM families from the Familial Dilated Cardiomyopathy Registry and in 309 subjects with DCM from the Beta-Blocker Evaluation of Survival Trial (BEST). DES mutations were transfected into SW13 and human smooth muscle cells and neonatal rat cardiac myocytes, and the effects on cytoskeletal desmin network architecture were analyzed with confocal microscopy. Five novel missense DES mutations, including the first localized to the highly conserved 1A domain, were detected in 6 subjects (1.4%). Transfection of DES mutations in the 2B domain severely disrupted the fine intracytoplasmic staining of desmin, causing clumping of the desmin protein. A tail domain mutation (Val459Ile) showed milder effects on desmin cytoplasmic network formation and appears to be a low-penetrant mutation restricted to black subjects. Conclusions— The prevalence of DES mutations in DCM is between 1% and 2%, and mutations in the 1A helical domain, as well as the 2B rod domain, are capable of causing a DCM phenotype. The lack of severe disruption of cytoskeletal desmin network formation seen with mutations in the 1A and tail domains suggests that dysfunction of seemingly intact desmin networks is sufficient to cause DCM.

Deletion of a 5-cM Region at Chromosome 8p23 Is Associated With a Spectrum of Congenital Heart Defects

BACKGROUND Cytogenetic evidence suggests that the haploinsufficiency of > or =1 gene located in 8p23 behaves as a dominant mutation, impairing heart differentiation and leading to a wide spectrum of congenital heart defects (CHDs), including conotruncal lesions, atrial septal defects, atrioventricular canal defects, and pulmonary valve stenosis. An 8p heart-defect-critical region was delineated, and the zinc finger transcription factor GATA4 was considered a likely candidate for these defects. We narrowed this region and excluded a major role of GATA4 in these CHDs. METHODS AND RESULTS We studied 12 patients (7 had CHD and 5 did not) with distal 8p deletions from 9 families by defining their chromosome rearrangements at the molecular level by fluorescent in situ hybridization and short-tandem repeat analysis. Subjects with 8p deletions distal to D8S1706, at approximately 10 cM from the 8p telomere, did not have CHD, whereas subjects with a deletion that included the more proximal region suffered from the spectrum of heart defects reported in patients with 8p distal deletions. The 5-cM critical region is flanked distally by D8S1706 and WI-8327, both at approximately 10 cM, and proximally by D8S1825, at 15 cM. Neither GATA4 nor angiopoietin-2 (ANGPT2; a gene in 8p23 involved in blood vessel formation) were found to be deleted in some of the critical patients. We also found that CHDs are not related to the parental origin of deletion. CONCLUSIONS Haploinsufficiency for a gene between WI-8327 and D8S1825 is critical for heart development. A causal relationship does not seem to exist between GATA4 and ANGPT2 haploinsufficiency and CHDs.

Danon disease presenting with dilated cardiomyopathy and a complex phenotype

AbstractX-linked dilated cardiomyopathy (XLCM) was first described in 1987 and associated with dystrophin gene (DMD) mutations a decade later in one of the original two families. Here we report long-term follow-up of the second family (XLCM-2), for which a DMD mutation was never found. Analysis of the lysosome-associated membrane protein-2 (LAMP-2) gene detected a novel mutation, confirming a diagnosis of Danon disease. The broad phenotype in this family included dilated and hypertrophic cardiomyopathy, cardiac pre-excitation, skeletal myopathy with high serum creatine kinase, cognitive impairment (in males), and a pigmentary retinopathy in affected females. Cardiac biopsy specimens showed extensive vacuolar changes in an affected adult male. Remarkably, the skeletal muscle biopsy in a 13-month-old mutation-carrying male showed no vacuolization by standard histology. We conclude that XLCM may be the presenting sign of Danon disease and, in the presence of a familial history of HCM, pre-excitation, skeletal muscle involvement and retinal pigmentary dystrophy should prompt LAMP-2 clinical testing. Furthermore, the absence of vacuolar myopathy in biopsies from young patients may not exclude Danon disease.

The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is a candidate gene for schizophrenia and an important drug target for cognitive deficits in the disorder. Activation of the α7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca(2+), that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of α7*nAChR, as measured by binding of the ligand [(125)I]-α-bungarotoxin (I-BTX). The structure of the gene, CHRNA7, is complex. During evolution, CHRNA7 was partially duplicated as a chimeric gene (CHRFAM7A), which is expressed in the human brain and elsewhere in the body. The association between a 2bp deletion in CHRFAM7A and schizophrenia suggested that this duplicate gene might contribute to cognitive impairment. To examine the putative contribution of CHRFAM7A on receptor function, co-expression of α7 and the duplicate genes was carried out in cell lines and Xenopus oocytes. Expression of the duplicate alone yielded protein expression but no functional receptor and co-expression with α7 caused a significant reduction of the amplitude of the ACh-evoked currents. Reduced current amplitude was not correlated with a reduction of I-BTX binding, suggesting the presence of non-functional (ACh-silent) receptors. This hypothesis is supported by a larger increase of the ACh-evoked current by the allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596) in cells expressing the duplicate than in the control. These results suggest that CHRFAM7A acts as a dominant negative modulator of CHRNA7 function and is critical for receptor regulation in humans.

Danon Disease Clinical Features, Evaluation, and Management

Danon disease is an X-linked dominant skeletal and cardiac muscle disorder with multisystem clinical manifestations. It was first described in boys presenting with cardiomyopathy, skeletal myopathy, and varying degrees of intellectual disability.1 As histological findings of glycogen buildup in muscle tissue similar to those seen in Pompe disease were noted, the condition was originally considered to be a lysosomal storage disease and was termed glycogen storage disease type IIb. In 2000, Nishino et al2 identified the genetic defects in the lysosome-associated membrane protein 2 ( LAMP2 ) gene, encoding the LAMP2 protein. Most Danon disease mutations lead to an absence of LAMP2 protein expression,2 a situation more problematic in men who are hemizygous for LAMP2 . For reasons not yet fully understood, reduction in LAMP2 disrupts intracytoplasmic trafficking and leads to accumulation of autophagic material and often glycogen in skeletal muscle and cardiac muscle cells (Figure 1).2 Major clinical features include skeletal and cardiac myopathy, cardiac conduction abnormalities, mild intellectual difficulties, and retinal disease. Men are typically affected earlier and more severely than women. The disease is unfamiliar to many practitioners, and the majority of published data stem from case reports with a brief clinical review published in 2002.4 Our aim was to perform a systematic review of Danon disease, provide a comprehensive clinical and molecular update, and propose diagnostic and management guidelines for clinicians and researchers working with patients with Danon disease. Figure 1. Histological images from skeletal muscle biopsy and endomyocardial biopsy.3 Electron microscopy shows intracytoplasmic vacuoles (arrows) containing autophagic material and glycogen in both ( A ) skeletal muscle (bar 1 μm) and ( B ) endomyocardial tissue biopsy (bar 1 μm). Reprinted from Taylor et al3 with permission of the publisher. Copyright

Reduced Chrna7 expression in mice is associated with decreases in hippocampal markers of inhibitory function: implications for neuropsychiatric diseases

The α7* nicotinic acetylcholine receptor encoded by CHRNA7 (human)/Chrna7 (mice) regulates the release of both the inhibitory neurotransmitter GABA and the excitatory neurotransmitter glutamate in the hippocampal formation. A heterozygous (Het) deletion at 15q13.3 containing CHRNA7 is associated with increased risk for schizophrenia, autism, and epilepsy. Each of these diseases are characterized by abnormalities in excitatory and inhibitory hippocampal circuit function. Reduced Chrna7 expression results in decreased hippocampal α7* receptor density, abnormal hippocampal auditory sensory processing, and increased hippocampal CA3 pyramidal neuron activity in C3H mice Het for a null mutation in Chrna7. These abnormalities demonstrate that decreased Chrna7 expression alters hippocampal inhibitory circuit function. The current study examined the specific impact of reduced Chrna7 expression on hippocampal inhibitory circuits by measuring the levels of GABA, GABA(A) receptors, the GABA synthetic enzyme l-glutamic acid decarboxylase-65 (GAD-65), and the vesicular GABA transporter 1 (GAT-1) in wild-type (Chrna7 +/+) and Het (Chrna7 +/-) C3H α7 mice of both genders. GAD-65 levels were significantly decreased in male and female Het C3H α7 mice, whereas GABA(A) receptors were significantly reduced only in male Het C3H α7 mice. No changes in GABA and GAT-1 levels were detected. These data suggest that reduced CHRNA7 expression may contribute to the abnormalities in hippocampal inhibitory circuits observed in schizophrenia, autism, and/or epilepsy.

A Review of the Giant Protein Titin in Clinical Molecular Diagnostics of Cardiomyopathies

Titin (TTN) is known as the largest sarcomeric protein that resides within the heart muscle. Due to alternative splicing of TTN, the heart expresses two major isoforms (N2B and N2BA) that incorporate four distinct regions termed the Z-line, I-band, A-band, and M-line. Next-generation sequencing allows a large number of genes to be sequenced simultaneously and provides the opportunity to easily analyze giant genes such as TTN. Mutations in the TTN gene can cause cardiomyopathies, in particular dilated cardiomyopathy (DCM). DCM is the most common form of cardiomyopathy, and it is characterized by systolic dysfunction and dilation of the left ventricle. TTN truncating variants have been described as the most common cause of DCM, while the real impact of TTN missense variants in the pathogenesis of DCM is still unclear. In a recent population screening study, rare missense variants potentially pathogenic based on bioinformatic filtering represented only 12.6% of the several hundred rare TTN missense variants found, suggesting that missense variants are very common in TTN and are frequently benign. The aim of this review is to understand the clinical role of TTN mutations in DCM and in other cardiomyopathies. Whereas TTN truncations are common in DCM, there is evidence that TTN truncations are rare in the hypertrophic cardiomyopathy (HCM) phenotype. Furthermore, TTN mutations can also cause arrhythmogenic right ventricular cardiomyopathy (ARVC) with distinct clinical features and outcomes. Finally, the identification of a rare TTN missense variant cosegregating with the restrictive cardiomyopathy (RCM) phenotype suggests that TTN is a novel disease-causing gene in this disease. Clinical diagnostic testing is currently able to analyze over 100 cardiomyopathy genes, including TTN; however, the size and presence of extensive genetic variation in TTN presents clinical challenges in determining significant disease-causing mutations. This review discusses the current knowledge of TTN genetic variations in cardiomyopathies and the impact of the diagnosis of TTN pathogenic mutations in the clinical setting.