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1.
J Med Genet ; 61(4): 369-377, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-37935568

RESUMO

BACKGROUND: Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS: Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS: Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION: Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.


Assuntos
Miopatias Distais , Humanos , Conectina/genética , Miopatias Distais/genética , Variações do Número de Cópias de DNA/genética , Músculo Esquelético/patologia , Mutação/genética , Fenótipo
2.
Ann Neurol ; 83(6): 1105-1124, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29691892

RESUMO

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.


Assuntos
Cardiomiopatia Dilatada/congênito , Conectina/genética , Proteínas Musculares/genética , Músculo Esquelético/patologia , Feminino , Humanos , Masculino , Mutação/genética , Fenótipo , Isoformas de Proteínas/genética
3.
Hum Mutat ; 35(12): 1418-26, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25205138

RESUMO

A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.


Assuntos
Proteínas Musculares/genética , Doenças Musculares/genética , Mutação , Processamento Alternativo , Animais , Cromossomos Humanos Par 2 , Bases de Dados Genéticas , Éxons , Genótipo , Humanos , Modelos Animais , Doenças Musculares/classificação , Fenótipo
4.
Hum Mutat ; 35(7): 779-90, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24692096

RESUMO

Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.


Assuntos
Estudos de Associação Genética , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação , Tropomiosina/genética , Actinas/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Bases de Dados Genéticas , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/diagnóstico , Fenótipo , Fosforilação , Ligação Proteica , Alinhamento de Sequência , Tropomiosina/química , Tropomiosina/metabolismo , Adulto Jovem
5.
Brain ; 136(Pt 2): 494-507, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23378224

RESUMO

Mutations in the TPM2 gene, which encodes ß-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of ß-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-ß-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-ß-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.


Assuntos
Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mutação/fisiologia , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Tropomiosina/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Galinhas , Feminino , Estudos de Associação Genética/métodos , Triagem de Portadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Ratos , Prevenção Secundária , Suínos
6.
Biochem J ; 442(1): 231-9, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22084935

RESUMO

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding ß-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.


Assuntos
Actinas/metabolismo , Miopatias da Nemalina/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Animais , Humanos , Miopatias da Nemalina/genética , Miopatias da Nemalina/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Proteínas Recombinantes , Spodoptera
7.
Front Genet ; 12: 763159, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34777479

RESUMO

We present a method for communicating personalized genetic risk information to citizens and their physicians using a secure web portal. We apply the method for 3,177 Finnish individuals in the P5 Study where estimates of genetic and absolute risk, based on genetic and clinical risk factors, of future disease are reported to study participants, allowing individuals to participate in managing their own health. Our method facilitates using polygenic risk score as a personalized tool to estimate a person's future disease risk while offering a way for health care professionals to utilize the polygenic risk scores as a preventive tool in patient care.

8.
Artigo em Inglês | MEDLINE | ID: mdl-31127036

RESUMO

Next-generation sequencing has led to transformative advances in our ability to diagnose rare diseases by simultaneously sequencing dozens, hundreds, or even entire genomes worth of genes to efficiently identify pathogenic mutations. These studies amount to multiple hypothesis testing on a massive scale and not infrequently lead to discovery of multiple genetic variants whose relative contributions to a patient's disease are unclear. Panel testing, in particular, can be problematic because each of the many genes being sequenced might represent a plausible explanation for a given case. We performed targeted gene panel analysis of 43 established neuromuscular disease genes in a patient with congenital fiber-type disproportion (CFTD) and fatal infantile cardiomyopathy. Initial review of variants identified changes in four genes that could be considered relevant candidates to cause this child's disease. Further analysis revealed that two of these are likely benign, but a homozygous frameshift variant in the myosin light chain 2 gene, MYL2, and a heterozygous nonsense mutation in the nebulin gene, NEB, met criteria to be classified as likely pathogenic or pathogenic. Recessive MYL2 mutations are a rare cause of CFTD associated with both skeletal and cardiomyopathy, whereas recessive NEB mutations cause nemaline myopathy. Although the proband's phenotype is likely largely explained by the MYL2 variant, the heterozygous pathogenic NEB variant cannot be ruled out as a contributing factor. This case illustrates the complexity when analyzing large numbers of variants from targeted gene panels in which each of the genes might plausibly contribute to the patient's clinical presentation.


Assuntos
Miosinas Cardíacas/genética , Miopatias Congênitas Estruturais/diagnóstico , Miopatias Congênitas Estruturais/genética , Cadeias Leves de Miosina/genética , Cardiomiopatias/genética , Cardiomiopatias/mortalidade , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Mutação/genética , Miopatias da Nemalina/genética , Doenças Neuromusculares/genética , Fenótipo
9.
Neuromuscul Disord ; 17(6): 433-42, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17434307

RESUMO

"Cap myopathy" or "cap disease" is a congenital myopathy characterised by cap-like structures at the periphery of muscle fibres, consisting of disarranged thin filaments with enlarged Z discs. Here we report a deletion in the beta-tropomyosin (TPM2) gene causing cap disease in a 36-year-old male patient with congenital muscle weakness, myopathic facies and respiratory insufficiency. The mutation identified in this patient is an in-frame deletion (c.415_417delGAG) of one codon in exon 4 of TPM2 removing a single glutamate residue (p.Glu139del) from the beta-tropomyosin protein. This is expected to disrupt the seven-amino acid repeat essential for making a coiled coil, and thus to impair tropomyosin-actin interaction. Missense mutations in TPM2 have previously been found to cause rare cases of nemaline myopathy and distal arthrogryposis. This mutation is one not previously described and the first genetic cause identified for cap disease.


Assuntos
Deleção de Genes , Heterozigoto , Mutação de Sentido Incorreto/genética , Miopatias Congênitas Estruturais/genética , Tropomiosina/genética , Actinas/genética , Actinas/metabolismo , Adulto , Sequência de Aminoácidos , Sequência de Bases , Fácies , Regulação da Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Debilidade Muscular/patologia , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Insuficiência Respiratória/patologia , Tropomiosina/metabolismo
10.
Skelet Muscle ; 4: 15, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25110572

RESUMO

BACKGROUND: Nemaline myopathy (NM) is a rare genetic muscle disorder, but one of the most common among the congenital myopathies. NM is caused by mutations in at least nine genes: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), ß-tropomyosin (TPM2), troponin T (TNNT1), cofilin-2 (CFL2), Kelch repeat and BTB (POZ) domain-containing 13 (KBTBD13), and Kelch-like family members 40 and 41 (KLHL40 and KLHL41). Nebulin is a giant (600 to 900 kDa) filamentous protein constituting part of the skeletal muscle thin filament. Around 90% of the primary structure of nebulin is composed of approximately 35-residue α-helical domains, which form super repeats that bind actin with high affinity. Each super repeat has been proposed to harbor one tropomyosin-binding site. METHODS: We produced four wild-type (WT) nebulin super repeats (S9, S14, S18, and S22), 283 to 347 amino acids long, and five corresponding repeats with a patient mutation included: three missense mutations (p.Glu2431Lys, p.Ser6366Ile, and p.Thr7382Pro) and two in-frame deletions (p.Arg2478_Asp2512del and p.Val3924_Asn3929del). We performed F-actin and tropomyosin-binding experiments for the nebulin super repeats, using co-sedimentation and GST (glutathione-S-transferase) pull-down assays. We also used the GST pull-down assay to test the affinity of WT nebulin super repeats for WT α- and ß-tropomyosin, and for ß-tropomyosin with six patient mutations: p.Lys7del, p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro. RESULTS: WT nebulin was shown to interact with actin and tropomyosin. Both the nebulin super repeats containing the p.Glu2431Lys mutation and nebulin super repeats lacking exon 55 (p.Arg2478_Asp2512del) showed weak affinity for F-actin compared with WT fragments. Super repeats containing the p.Ser6366Ile mutation showed strong affinity for actin. When tested for tropomyosin affinity, super repeats containing the p.Glu2431Lys mutation showed stronger binding than WT proteins to tropomyosin, and the super repeat containing the p.Thr7382Pro mutation showed weaker binding than WT proteins to tropomyosin. Super repeats containing the deletion p.Val3924_Asn3929del showed similar affinity for actin and tropomyosin as that seen with WT super repeats. Of the tropomyosin mutations, only p.Glu41Lys showed weaker affinity for nebulin (super repeat 18). CONCLUSIONS: We demonstrate for the first time the existence of direct tropomyosin-nebulin interactions in vitro, and show that nebulin interactions with actin and tropomyosin are altered by disease-causing mutations in nebulin and tropomyosin.

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