M. Marttila

Cap disease caused by heterozygous deletion of the β-tropomyosin gene TPM2

‘‘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 b-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 b-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. � 2007 Elsevier B.V. All rights reserved.

Mutation update and genotype-phenotype correlations of novel and previously described mutations in TPM2 and TPM3 causing congenital myopathies.

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 Ca2+ 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.

Mutation Update: The Spectra of Nebulin Variants and Associated Myopathies

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.

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy.

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 β-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.

K7del is a common TPM2 gene mutation associated with nemaline myopathy and raised myofibre calcium sensitivity

Mutations in the TPM2 gene, which encodes β-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 β-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-β-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-β-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.

C.P.15 K7del is a recurrent TPM2 nemaline myopathy mutation associated with joint contractures and increased calcium sensitivity

Abstract The TPM2 gene encodes beta-tropomyosin (βTm). A range of TPM2 mutations have been associated with several congenital myopathies and distal arthrogryposis. We report a novel dominant TPM2 mutation, K7del, in five unrelated families (de novo occurrence was likely in four families and could be confirmed in two). This mutation removes a highly conserved lysine residue within the N-terminal region that mediates βTm head-to-tail polymerisation. Patients had postnatal large joint contractures and little skeletal muscle weakness until adulthood. Muscle histology showed nemaline bodies and core-like areas, and there was marked fatty infiltration of lower limb muscles by mid-adulthood on muscle MRI. We performed a wide range of studies to investigate the causes of muscle dysfunction. 2D-gel electrophoresis on patient muscle and primary patient cultured myotubes suggested that mutant protein incorporates poorly into sarcomeres and most resides in nemaline rods. Recombinant K7del protein showed a reduced ability to polymerise into long filaments and reduced actin affinity but bound to thin filaments when mixed with wild-type β Tm and caused an in myofibrillar calcium sensitivity. Single dissected patient muscle fibres also showed increased calcium sensitivity of force generation. In summary, K7del is a recurrent TPM2 mutation that causes mild nemaline myopathy with core-like features and postnatal contractures. Mutant K7del protein incorporates poorly into sarcomeres likely due to abnormal head-to-tail polymerisation and reduced actin affinity. Joint contractures may arise from muscle hypercontraction due to increased 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.

C.P.16 Mutations in the β-tropomyosin (TPM2) and γ-tropomyosin (TPM3) genes causing a spectrum of neuromuscular disorders

Abstract Published case reports of mutations in the tropomyosin genes describe a variety of neuromuscular disorders. The tropomyosins (Tms) exist as coiled-coil homo- or heterodimers forming head-to-tail polymers, running along the length of the actin molecule. They are encoded by four different genes; αTm (TPM1), βTm (TPM2), γTm (TPM3), and δTm (TPM4), generating more than 40 different tropomyosin isoforms. The tropomyosin genes TPM1, TPM2 and TPM3 are expressed in skeletal muscle encoding isoforms αTmfast, βTm, and δTmslow respectively. We extend the mutational and clinical spectrum of disorders caused by mutations of the β-tropomyosin (TPM2) and γ-tropomyosin (TPM3) genes and provide an overview of new and published cases. The clinical and histological findings of patients with cap myopathy, nemaline myopathy, CFTD, DA1A, DA2B and Escobar syndrome are correlated to the patients’ genotypes. We have also characterized the phosphorylation pattern of β-tropomyosin, both wt and β-tropomyosins containing patient mutations, and identified four novel phosphorylation sites.