Mutations In Titin Gene Research Articles

UNPREDICTABLE IDENTIFICATION OF A TITIN MUTATION IN A YOUNG PATIENT PRESENTING WITH INFLAMMATORY CARDIOMYOPATHY

Abstract Case Presentation A 23 years old patient, affected by type 1 diabetes, presented with fever, dyspnea and bilateral peripheral edemas. In the last week he received antibiotic treatment for a persistent dry cough and sore throat, and became symptomatic for exertional dyspnea from three days. His mother died at the age of 40 for sudden cardiac death. Blood pressure and oxygen saturation were normal: the ECG showed sinus tachycardia with symmetric negative T waves in all precordial leads. High sensitivity troponin T was 0.01 mcg/L (normal value < 0.014 mcg/L). All remaining blood tests were normal, except for CRP that was slightly elevated. His echocardiogram revealed left ventricular (LV) remodelling and dilation, severely reduced LV ejection fraction (LVEF 20%) with global hypokinesia, more pronounced at the level of the anterior wall, interventricular septum and apex and a dilated right ventricle (RV) with reduced systolic function. Cardiac magnetic resonance (CMR) confirmed severe biventricular disfunction (LVEF 19%; RVEF 23%) and showed a small area of subepicardial inferior junctional late gadolinium enhancement (LGE). The patient underwent an endomyocardial biopsy that showed a lymphocytic virus–negative myocarditis. Given his known type 1 diabetes, steroid treatment was not considered and anakinra was started. The patient was discharged after two weeks with a slight amelioration of the LV function (LVEF 27%), on optimal heart failure therapy and anakinra. At six months he was in NYHA class II and CMR showed slight reduction in LV dimensions and improvement of the LV and RV function (LVEF 39%; RVEF 45%), with no changes in the LGE pattern. A genetic test was performed and revealed two titin gene mutations, both categorized as likely pathogenic (TTN c.[11831T>A]; c.[72510_72517delGACAAATG]), responsible for a genetic form of dilated cardiomyopathy. Conclusion The present case suggests that in young patients presenting with clinical or histological diagnosis of myocarditis, genetic testing for cardiomyopathies should be considered especially in presence of a positive family history for sudden death or an incomplete recovery of the LV function at follow–up. Identification of a gene mutation in these patients allows a more tailored risk stratification of the index case and a proper management of the entire family.

Truncated titin is incorporated into the sarcomere in human cardiac samples with dilated cardiomyopathy

The most common genetic cause of dilated cardiomyopathy (DCM) is mutations in titin gene, TTN, with truncating variants (TTNtv). TTN encodes the giant protein titin, a large (3-4 MDa) and abundant protein that forms the third myofilament type of striated muscle. Titin spans half the sarcomere, from the Z-disk to the M-line. The underlying mechanisms by which titin mutations induce DCM are poorly understood.

Clinicopathological features of titinopathy from a Chinese neuromuscular center.

Titin, one of the largest proteins in humans, is a major component of muscle sarcomeres. Pathogenic variants in the titin gene (TTN) have been reported to cause a range of skeletal muscle diseases, collectively known as titinopathy. Titinopathy is a heterogeneous group of disabling diseases characterized by muscle weakness. In our study, we aimed to establish the clinicopathological-genetic spectrum of titinopathy from a single neuromuscular center. Three patients were diagnosed as having definite titinopathy, and additional three patients were diagnosed as having possible titinopathy according to the diagnostic criteria. All the patients showed initial symptoms from age one to 40 years. Physical examination revealed that five patients had muscle weakness, and that one patient experienced behavioral changes. Muscle biopsy specimens obtained from all six patients demonstrated multiple myopathological changes, including increased fiber size variation, muscle fiber hypertrophy or atrophy, formation of centralized cell nuclei, necklace cytoplasmic bodies, and formation of rimmed vacuoles and cores. Genetic testing revealed 11 different TTN alterations, including missense (6/11), nonsense (2/11), frameshift (2/11), and splicing (1/11) mutations. Our study provides further evidence that TTN mutations are more likely to be responsible for an increasing proportion of various myopathies, such as hereditary myopathy with early respiratory failure (HMERF), core myopathy, and distal myopathy with rimmed vacuoles, than currently recognized mutations. Our findings expand the clinical, pathohistological and genetic spectrum of titinopathy.

Identification of Novel TTN Mutations in Three Chinese Familial Dilated Cardiomyopathy Pedigrees by Whole Exome Sequencing

Familial dilated cardiomyopathy (DCM) is associated with numerous genes, especially those of the sarcomere family. The titin gene (TTN) consists of 365 exons and encodes the largest sarcomere protein (titin) in our bodies. Titin is associated with many diseases, such as hypertrophic cardiomyopathy and DCM. Here we screened three Chinese families affected by DCM, and found that each harbors a stop-gain or splice site mutation in TTN (c.G20137T,c. G52522T,c.44610-2A>C). Assessment of the probands by electrocardiogram, B-mode echocardiography, and cardiac magnetic resonance imaging revealed impaired cardiac function, arrhythmia, or abnormal cardiac structure. In conclusion, using whole exome sequencing, we found three unreported TTN mutations associated with DCM. This has expanded the TTN mutation spectrum of Chinese DCM patients, especially in Henan, the most populous province. These data provide new genetic targets for the diagnosis and treatment of DCM, and will increase our understanding of the relationship between TTN mutation and DCM clinical symptoms.

Delineating the phenotypes of early onset myopathy due to novel titin gene mutations

Delineating the phenotypes of early onset myopathy due to novel titin gene mutations

A mechanical model of the half-sarcomere which includes the contribution of titin.

The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A. F. Huxley and his collaborators in 1981, which provides that the half-sarcomere (hs) compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length (SL) the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of hs stiffness at increasing SL, which is important either in relation to the mechanism of stabilisation of SL against the consequence of sarcomere inhomogeneity in active force generation, or for investigations on the role of titin as mechano-sensor in thick filament regulation. Moreover the model opens the possibility for understanding the functional differences related to the titin isoform of various muscle types and the mechanism by which mutations in titin gene lead to myopathies.

Effect of Truncated Mutations in the Titin Gene on Cardiac Function

Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and impaired systolic function, where up to 40% of cases are associated with mutations. Truncating mutations in the TTN gene are the most common cause of familial DCM. While it is known that titin is the major contributor to myocardial stiffness and is an essential component of the Frank-Starling mechanism of the heart, its function is not fully established. Titin mechanical properties are particularly important in assisting with myofibril viscoelasticity and consequently contribute to the work output characteristic of myocardium. In contrast, changes in myofilament Ca2+-sensitivity and loss of its modulation by phosphorylation are involved in the proposed mechanism for disease-causing mutations in contractile proteins but not necessary in the TTN gene. We studied mechanical properties and isometric contractility in single cardiac myofibrils of DCM patients with and without TTN mutations. We found that all studied DCM mutations in contractile protein genes (TNNC1, TNNI3, and MYH7) including truncations in the titin gene (TTN), result in lower passive stiffness compared to donor samples. Furthermore we showed that the myofilament Ca2+-sensitivity was higher than donor and without any difference in myofibril kinetics. In fact, heart function is highly coordinated and strongly dependent on spatiotemporal control during the cardiac cycle so data obtained in isometric conditions may be inadequate to understand the effect of mutations. To further characterize mutations and the impact of myofibril length changes on cardiac output, we now use cyclic length and Ca2+ concentration changes to imitate the changes in single myofibril similar to those that have place during the cardiac work-producing cycle.

Recovery in Patients With Dilated Cardiomyopathy With Loss-of-Function Mutations in the Titin Gene

This study compares the prevalence of titin gene mutations in patients with dilated cardiomyopathy receiving standard pharmacological therapy with and without recovery of systolic function.

Titin Gene and Protein Functions in Passive and Active Muscle.

The thin and thick filaments of muscle sarcomeres are interconnected by the giant protein titin, which is a scaffolding filament, signaling platform, and provider of passive tension and elasticity in myocytes. This review summarizes recent insight into the mechanisms behind how titin gene mutations cause hereditary cardiomyopathy and how titin protein is mechanically active in skeletal and cardiac myocytes. A main theme is the evolving role of titin as a modulator of contraction. Topics include strain-sensing via titin in the sarcomeric A-band as the basis for length-dependent activation, titin elastic recoil and refolding of titin domains as an energy source, and Ca2+-dependent stiffening of titin stretched during eccentric muscle contractions. Findings suggest that titin stiffness is a principal regulator of the contractile behavior of striated muscle. Physiological or pathological changes to titin stiffness therefore affect contractility. Taken together, titin emerges as a linker element between passive and active myocyte properties.

Targeted Next-Generation Sequencing Reveals Novel TTN Mutations Causing Recessive Distal Titinopathy.

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

P.79 - Titin gene mutations presenting as centronuclear myophathies

P.79 - Titin gene mutations presenting as centronuclear myophathies

Heart Failure: From Protein to Phenotype37MicroRNA-494 reduces ATF3 expression and promotes heart failure in cardiac hypertrophic remodeling in vivo38A novel recessive plakophilin-2 gene mutation causes severe arrhythmogenic dilated cardiomyopathy and sudden cardiac death at young age39Investigation of titin expression in explanted hearts with familial dilated cardiomyopathy and TTN truncating variants

Heart Failure: From Protein to Phenotype37MicroRNA-494 reduces ATF3 expression and promotes heart failure in cardiac hypertrophic remodeling in vivo38A novel recessive plakophilin-2 gene mutation causes severe arrhythmogenic dilated cardiomyopathy and sudden cardiac death at young age39Investigation of titin expression in explanted hearts with familial dilated cardiomyopathy and TTN truncating variants

G.P.323 - Targeted next-generation sequencing reveals novel TTN mutations causing recessive distal titinopathy

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

Titin gene mutations are common in families with both peripartum cardiomyopathy and dilated cardiomyopathy

Peripartum cardiomyopathy (PPCM) can be an initial manifestation of familial dilated cardiomyopathy (DCM). We aimed to identify mutations in families that could underlie their PPCM and DCM. We collected 18 families with PPCM and DCM cases from various countries. We studied the clinical characteristics of the PPCM patients and affected relatives, and applied a targeted next-generation sequencing (NGS) approach to detect mutations in 48 genes known to be involved in inherited cardiomyopathies. We identified 4 pathogenic mutations in 4 of 18 families (22%): 3 in TTN and 1 in BAG3. In addition, we identified 6 variants of unknown clinical significance that may be pathogenic in 6 other families (33%): 4 in TTN, 1 in TNNC1, and 1 in MYH7. Measurements of passive force in single cardiomyocytes and titin isoform composition potentially support an upgrade of one of the variants of unknown clinical significance in TTN to a pathogenic mutation. Only 2 of 20 PPCM cases in these families showed the recovery of left ventricular function. Targeted NGS shows that potentially causal mutations in cardiomyopathy-related genes are common in families with both PPCM and DCM. This supports the earlier finding that PPCM can be part of familial DCM. Our cohort is particularly characterized by a high proportion of TTN mutations and a low recovery rate in PPCM cases.

Atypical phenotypes in titinopathies explained by second titin mutations

Several patients with previously reported titin gene (TTN) mutations causing tibial muscular dystrophy (TMD) have more complex, severe, or unusual phenotypes. This study aimed to clarify the molecular cause of the variant phenotypes in 8 patients of 7 European families. Clinical, histopathological, and muscle imaging data of patients and family members were reanalyzed. The titin protein was analyzed by Western blotting and TTN gene by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Western blotting showed more pronounced C-terminal titin abnormality than expected for heterozygous probands, suggesting the existence of additional TTN mutations. RT-PCR indicated unequal mRNA expression of the TTN alleles in biopsies of 6 patients, 3 with an limb-girdle muscular dystrophy type 2J (LGMD2J) phenotype. Novel frameshift mutations were identified in 5 patients. A novel A-band titin mutation, c.92167C>T (p.P30723S), was found in 1 patient, and 1 Portuguese patient with a severe TMD phenotype proved to be homozygous for the previously reported Iberian TMD mutation. The unequal expression levels of TTN transcripts in 5 probands suggested severely reduced expression of the frameshift mutated allele, probably through nonsense-mediated decay, explaining the more severe phenotypes. The Iberian TMD mutation may cause a more severe TMD rather than LGMD2J when homozygous. The Finnish patient compound heterozygous for the FINmaj TMD mutation and the novel A-band titin missense mutation showed a phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by TTN mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism explaining phenotype variability.

P.15.7 A founder mutation in the titin gene is a common cause of myofibrillar myopathy with early respiratory failure

Titin gene (TTN) mutations have been described in 8 families with hereditary myopathy with early respiratory failure (HMERF) a rare condition with features resembling myofibrillar myopathy (MFM). In order to verify if TTN mutations could be a cause of MFM, especially in presence of early respiratory involvement, we carried out a clinical and genetic analysis of 127 undiagnosed patients with a clinical presentation compatible with MFM. Sanger sequencing for the TTN mutations previously described in patients with HMERF (p.C30071R and p.R32450W) was performed in all patients. We identified 5 families with the p.C30071R mutation (3.9% of study population) likely due to a British founder effect. Affected subjects showed phenotypic features similar to previously reported cases, and muscle pathology demonstrated diagnostic features of MFM. Two further families had novel variants in the 119th FN3 domain (p.P30091L and p.N30145K). Although still with uncertain pathogenicity, these new variants suggest that other TTN mutations in this domain may cause MFM. Our results indicate that mutations in TTN gene cause MFM and titinopathy is more common than previously thought. We suggest that HMERF due to mutations in the TTN gene be nosologically classified as MFM-titinopathy.

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins

BackgroundHereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins.MethodsWhole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy.ResultsA novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.ConclusionsMissense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

C.O.6 Complete genetic analysis by whole exome sequencing of a cohort with centronuclear myopathy identifies titin gene mutations

Centronuclear myopathies (CNM) are a group of rare heterogeneous congenital myopathies characterized by muscle weakness and low tone of varying onset, often requiring respiratory, feeding and/or ambulatory assistance. Frequent central nuclei are the primary pathological finding in affected skeletal muscles. CNM may be caused by mutations in the MTM1, DNM2, RYR1 and BIN1 genes. MTM1 mutations, which cause X-linked myotubular myopathy, are thought to account for about 50% of CNM cases, while DNM2 and RYR1 account for ∼15% each, and BIN1 for <5%, leaving about ∼15–20% of cases without a known disease gene. To identify known and novel gene mutations responsible for CNM, we performed whole exome sequencing (WES) in 28 probands with sporadic CNM from a population already depleted by clinical testing for MTM1 and DNM2 mutations. RYR1 and BIN1 had largely not been sequenced because of the large gene size (RYR1) and rarity (BIN1) of these mutations. WES was performed by Axeq Technologies using the Illumina HiSeq 2000 platform, which generated 10× coverage of 84–88% across the target regions in these samples. Concomitantly, one of these 28 patients was identified to have MTM1 duplication on further clinical testing. Of the remaining 27, two had DNM2 and nine had RYR1 mutations that were considered probably pathogenic. Remarkably, we identified five cases with compound heterozygote mutations of the titin gene (TTN), the majority being frameshift insertions, deletions, or stopgain mutations. Analysis of the remaining cases has identified at least one additional new candidate gene, and further testing is underway. In summary, exome sequencing has identified the molecular basis for roughly 60% of these patients with CNM. Work is underway to identify novel genes in the remaining 11 exomes.

Hereditary myopathy with early respiratory failure associated with a mutation in A-band titin

Hereditary myopathy with early respiratory failure and extensive myofibrillar lesions has been described in sporadic and familial cases and linked to various chromosomal regions. The mutated gene is unknown in most cases. We studied eight individuals, from three apparently unrelated families, with clinical and pathological features of hereditary myopathy with early respiratory failure. The investigations included clinical examination, muscle histopathology and genetic analysis by whole exome sequencing and single nucleotide polymorphism arrays. All patients had adult onset muscle weakness in the pelvic girdle, neck flexors, respiratory and trunk muscles, and the majority had prominent calf hypertrophy. Examination of pulmonary function showed decreased vital capacity. No signs of cardiac muscle involvement were found. Muscle histopathological features included marked muscle fibre size variation, fibre splitting, numerous internal nuclei and fatty infiltration. Frequent groups of fibres showed eosinophilic inclusions and deposits. At the ultrastructural level, there were extensive myofibrillar lesions with marked Z-disc alterations. Whole exome sequencing in four individuals from one family revealed a missense mutation, g.274375T>C; p.Cys30071Arg, in the titin gene (TTN). The mutation, which changes a highly conserved residue in the myosin binding A-band titin, was demonstrated to segregate with the disease in all three families. High density single nucleotide polymorphism arrays covering the entire genome demonstrated sharing of a 6.99 Mb haplotype, located in chromosome region 2q31 including TTN, indicating common ancestry. Our results demonstrate a novel and the first disease-causing mutation in A-band titin associated with hereditary myopathy with early respiratory failure. The typical histopathological features with prominent myofibrillar lesions and inclusions in muscle and respiratory failure early in the clinical course should be incentives for analysis of TTN mutations.