To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients.
We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy.
MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes.
Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.
Key Clinical Message
Autosomal recessive spastic ataxia of Charlevoix‐Saguenay is a rare disorder outside Quebec causing childhood‐onset cerebellar ataxia, peripheral neuropathy, and pyramidal tract signs. A Finnish family with milder form of ARSACS was found to harbor three mutations, p.E1100K, p.N1489S, and p.M1359T, in SACS gene. The mutations segregated with the disease.
Autosomal recessive; SACS gene; spastic ataxia
Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD; OMIM #226670) is an autosomal recessive disorder characterized by neonatal blistering and later-onset muscle weakness.
To identify the genetic etiology and characterize the clinicopathologic features of a novel distal myopathy.
We performed whole-exome sequencing on a family with an autosomal dominant distal myopathy and targeted exome sequencing in 1 patient with sporadic distal myopathy, both with rimmed vacuolar pathology. We also evaluated the pathogenicity of identified mutations using immunohistochemistry, Western blot analysis, and expression studies.
Sequencing identified a likely pathogenic c.1165+1 G>A splice donor variant in SQSTM1 in the affected members of 1 family and in an unrelated patient with sporadic distal myopathy. Affected patients had late-onset distal lower extremity weakness, myopathic features on EMG, and muscle pathology demonstrating rimmed vacuoles with both TAR DNA-binding protein 43 and SQSTM1 inclusions. The c.1165+1 G>A SQSTM1 variant results in the expression of 2 alternatively spliced SQSTM1 proteins: 1 lacking the C-terminal PEST2 domain and another lacking the C-terminal ubiquitin-associated (UBA) domain, both of which have distinct patterns of cellular and skeletal muscle localization.
SQSTM1 is an autophagic adaptor that shuttles aggregated and ubiquitinated proteins to the autophagosome for degradation via its C-terminal UBA domain. Similar to mutations in VCP, dominantly inherited mutations in SQSTM1 are now associated with rimmed vacuolar myopathy, Paget disease of bone, amyotrophic lateral sclerosis, and frontotemporal dementia. Our data further suggest a pathogenic connection between the disparate phenotypes.
To identify the genetic defects present in 3 families with muscular dystrophy, contractures, and calpain 3 deficiency.
We performed targeted exome sequencing on one patient presenting a deficiency in calpain 3 on Western blot but for which mutations in the gene had been excluded. The identification of a homozygous truncating mutation in the M-line part of titin prompted us to sequence this region in 2 additional patients presenting similar clinical and biochemical characteristics.
The 3 patients shared similar features: coexistence of limb-girdle weakness and early-onset diffuse joint contractures without cardiomyopathy. The biopsies showed rimmed vacuoles, a dystrophic pattern, and secondary reduction in calpain 3. We identified a novel homozygous mutation in the exon Mex3 of the TTN gene in the first patient. At protein level, this mutation introduces a stop codon at the level of Mex3. Interestingly, we identified truncating mutations in both alleles in the same region of the TTN gene in patients from 2 additional families. Molecular protein analyses confirm loss of the C-ter part of titin.
Our study broadens the phenotype of titinopathies with the report of a new clinical entity with prominent contractures and no cardiac abnormality and where the recessive mutations lead to truncation of the M-line titin and secondary calpain 3 deficiency.
Glycogen storage disease V (GSDV, McArdle disease) and GSDVII (Tarui disease) are the most common of the rare disorders of glycogen metabolism. Both are associated with low lactate levels on exercise. Our aim was to find out whether lactate response associated with exercise testing could distinguish between these disorders.
Two siblings with Tarui disease, two patients with McArdle disease and eight healthy controls were tested on spiroergometric exercise tests with follow-up of venous lactate and ammonia.
A late increase of lactate about three times the basal level was seen 10–30 min after exercise in patients with Tarui disease being higher than in McArdle disease and lower than in the controls. Ammonia was increased in Tarui disease.
Our results suggest that follow-up of lactate associated with exercise testing can be utilized in diagnostics to distinguish between different GSD diseases.
ammonia; McArdle disease; lactate; muscle phosphofructokinase; pentose phosphate pathway; spiroergometry; Tarui disease; muscle metabolism
The objective of this study was to characterize and compare muscle histopathological findings in 3 different genetic motor neuron disorders. We retrospectively re-assessed muscle biopsy findings in 23 patients with autosomal dominant lower motor neuron disease caused by p.G66V mutation in CHCHD10 (SMAJ), 10 X-linked spinal and bulbar muscular atrophy (SBMA) and 11 autosomal dominant c9orf72-mutated amyotrophic lateral sclerosis (c9ALS) patients. Distinct large fiber type grouping consisting of non-atrophic type IIA muscle fibers were 100% specific for the late-onset spinal muscular atrophies (SMAJ and SBMA) and were never observed in c9ALS. Common, but less specific findings included small groups of highly atrophic rounded type IIA fibers in SMAJ/SBMA, whereas in c9ALS, small group atrophies consisting of small-caliber angular fibers involving both fiber types were more characteristic. We also show that in the 2 slowly progressive motor neuron disorders (SMAJ and SBMA) the initial neurogenic features are often confused with considerable secondary “myopathic” changes at later disease stages, such as rimmed vacuoles, myofibrillar aggregates and numerous fibers reactive for fetal myosin heavy chain (dMyHC) antibodies. Based on our findings, muscle biopsy may be valuable in the diagnostic work-up of suspected motor neuron disorders in order to avoid a false ALS diagnosis in patients without clear findings of upper motor neuron lesions.
Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy.
After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy.
Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, αB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31.
Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 – a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis.
LGMD1D; DNAJB6; Myopathy; Autophagy; CASA
Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/β-cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalised skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families the grandfather of the proband was found to be a mosaic for the MYH7 mutation. In eight cases de novo mutation appeared to have occurred, which was proven in three. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalised floppiness and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in 9 of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases.
MYH7; Laing distal myopathy; MPD1
Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.
To elaborate the diagnostic methods used as “gold standard” in one of the most common glycogen storage diseases (GSDs), Tarui disease (GSDVII).
Two siblings with disease suggestive of GSD underwent thorough clinical analysis, including muscle biopsy, muscle MRI, exercise tests, laboratory examinations, and whole-exome sequencing (WES).
Both siblings had juvenile-onset exercise intolerance with cramping and infrequent myoglobinuria. Muscle biopsy showed extralysosomal glycogen accumulation, but because of normal phosphofructokinase histochemistry, GSDVII was thought to be excluded. However, WES revealed a causative homozygous PFKM gene defect, R39Q, in both siblings, establishing the diagnosis of GSDVII, which was confirmed by very low residual phosphofructo-1-kinase (PFK) enzyme activity in biochemical studies.
We suggest that in patients with suspicion of GSD and extralysosomal glycogen accumulation, biochemical activity assay of PFK followed by molecular genetics should be performed even when enzyme histochemistry is normal.
Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild-to-moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe for the first time the clinical and morphological characteristics of recessive myosin IIa myopathy associated with MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild-to-moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated small or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that mild muscle weakness and ophthalmoplegia in combination with muscle biopsy demonstrating small or absent type 2A muscle fibers are the hallmark of recessive myopathy associated with MYH2 mutations.
Myosin heavy chain; MYH2; ophthalmoplegia; myosin myopathy
The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.
Myotonic dystrophy; DM1; DM2; aberrant isoform expression; missplicing
Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery–Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.
Aberrant transcription and mRNA processing of multiple genes due to RNA-mediated toxic gain-of-function has been suggested to cause the complex phenotype in myotonic dystrophies type 1 and 2 (DM1 and DM2). However, the molecular basis of muscle weakness and wasting and the different pattern of muscle involvement in DM1 and DM2 are not well understood. We have analyzed the mRNA expression of genes encoding muscle-specific proteins and transcription factors by microarray profiling and studied selected genes for abnormal splicing. A subset of the abnormally regulated genes was further analyzed at the protein level. TNNT3 and LDB3 showed abnormal splicing with significant differences in proportions between DM2 and DM1. The differential abnormal splicing patterns for TNNT3 and LDB3 appeared more pronounced in DM2 relative to DM1 and are among the first molecular differences reported between the two diseases. In addition to these specific differences, the majority of the analyzed genes showed an overall increased expression at the mRNA level. In particular, there was a more global abnormality of all different myosin isoforms in both DM1 and DM2 with increased transcript levels and a differential pattern of protein expression. Atrophic fibers in DM2 patients expressed only the fast myosin isoform, while in DM1 patients they co-expressed fast and slow isoforms. However, there was no increase of total myosin protein levels, suggesting that aberrant protein translation and/or turnover may also be involved.
Myotonic dystrophy type 1 (DM1); Myotonic dystrophy type 2 (DM2); Skeletal muscle; Aberrant splicing; Microarray expression profiling
Because of their central role in muscle development and maintenance, MEF2 family members represent excellent candidate effectors of the muscle pathology in myotonic dystrophy (DM). We investigated the expression and alternative splicing of all four MEF2 genes in muscle from neuromuscular disorder (NMD) patients, including DM1 and DM2. We observed MEF2A and MEF2C overexpression in all NMD muscle, including 12 MEF2-interacting genes. Exon 4 and 5 usage in MEF2A and MEF2C was different between DM and normal muscle, with DM showing the embryonic isoform. Similar splicing differences were observed in other NMD muscle. For MEF2C, missplicing was more pronounced in DM than in other dystrophies. Our data confirm dysregulation of MEF2A and MEF2C expression and splicing in several NMD, including DM. Our findings demonstrate that aberrant splicing in NMD is independent from expression of mutant repeats, and suggests that some aberrant splicing, even in DM, may be compensatory rather than primary.
dysregulation; MADS-domain transcription enhancer factor 2; MEF2; myotonic dystrophy; splicing
Based on previous reports the frequency of co-segregating recessive chloride channel (CLCN1) mutations in families with myotonic dystrophy type 2 (DM2) was suspected to be increased. We have studied the frequency of CLCN1 mutations in two separate patient and control cohorts from Germany and Finland, and for comparison in a German myotonic dystrophy type 1 (DM1) patient cohort. The frequency of heterozygous recessive chloride channel (CLCN1) mutations is disproportionally higher (5%) in currently diagnosed DM2 patients compared to 1.6% in the control population (p = 0.037), while the frequency in DM1 patients was the same as in the controls. Because the two genes segregate independently, the prevalence of CLCN1 mutations in the total DM2 patient population is, by definition, the same as in the control population. Our findings are, however, not based on the total DM2 population but on the currently diagnosed DM2 patients and indicate a selection bias in molecular diagnostic referrals. DM2 patients with co-segregating CLCN1 mutation have an increased likelihood to be referred for molecular diagnostic testing compared to DM2 patients without co-segregating CLCN1 mutation.
myotonic dystrophy; co-segregation; CLCN1; genetic modifier; phenotype variation
Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, have been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca2+ plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca2+ signaling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca2+ metabolism in DM patients, including Ca2+ channels and Ca2+ binding proteins.
We used patient muscle biopsies to analyze mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting.
Most of the genes studied showed mRNA up-regulation in expression profiling. When analyzed by immunohistochemistry the Ca2+ release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca2+ storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1.
We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca2+ metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.
Myotonic dystrophy type 1 (DM1); myotonic dystrophy type 2 (DM2); skeletal muscle; calcium metabolism
Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.
Numerous natural or disease-related alterations occur in different tissues of the body with advancing age. Sarcopenia is defined as age-related decrease of muscle mass and strength beginning in mid-adulthood and accelerating in people older than 60 years. Pathophysiology of sarcopenia involves both neural and muscle dependent mechanisms and is enhanced by multiple factors. Aged muscles show loss in fiber number, fiber atrophy, and gradual increase in the number of ragged red fibers and cytochrome c oxidase-negative fibers. Generalized loss of muscle tissue and increased amount of intramuscular fat are seen on muscle imaging. However, the degree of these changes varies greatly between individuals, and the distinction between normal age-related weakening of muscle strength and clinically significant muscle disease is not always obvious. Because some of the genetic myopathies can present at a very old age and be mild in severity, the correct diagnosis is easily missed. We highlight this difficult borderline zone between sarcopenia and muscle disease by two examples: LGMD1D and myotonic dystrophy type 2. Muscle magnetic resonance imaging (MRI) is a useful tool to help differentiate myopathies from sarcopenia and to reach the correct diagnosis also in the elderly.
sarcopenia; myopathy; late-onset; genetic; muscle imaging
Spinal muscular atrophies (SMAs) are hereditary disorders characterized by degeneration
of lower motor neurons. Different SMA types are clinically and genetically heterogeneous
and many of them show significant phenotypic overlap. We recently described the clinical
phenotype of a new disease in two Finnish families with a unique autosomal dominant
late-onset lower motor neuronopathy. The studied families did not show linkage to any
known locus of hereditary motor neuron disease and thus seemed to represent a new disease
entity. For this study, we recruited two more family members and performed a more thorough
genome-wide scan. We obtained significant linkage on chromosome 22q, maximum LOD score
being 3.43 at marker D22S315. The linked area is defined by flanking markers D22S686 and
D22S276, comprising 18.9 Mb. The region harbours 402 genes, none of which is
previously known to be associated with SMAs. This study confirms that the disease in these
two families is a genetically distinct entity and also provides evidence for a founder
mutation segregating in both pedigrees.
motor neuron disease; spinal muscular atrophy; linkage analysis
Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinct type of myofibrillar myopathy (MFM).
We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes.
The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy.
We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.
filaminopathy; FLNC; myofibrillar myopathy; cerebellar ataxia; muscle MRI
In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.
hereditary myopathy with early respiratory failure; cytoplasmic body; titin; exome sequencing; distal myopathy
The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so.
An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies.
This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing.
We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.