Homozygosity of a Founder Variant c.1508dupC in DOK7 Causes Congenital Myasthenia With Variable Severity.

Background and Objectives: Description of 15 patients with the same variant in DOK7 causing congenital myasthenic syndrome (CMS). Methods: Nine adult and 6 pediatric patients were studied with molecular genetic and clinical investigations. Results: All patients were identified with the c.1508dupC variant in DOK7, of whom 13 were homozygous and 2 patients compound heterozygous. Only 2 patients had limb girdle phenotype, while all adult patients also had ptosis, ophthalmoplegia, facial weakness, as well as inspiratory stridor. Pediatric patients had severe respiratory insufficiency and feeding difficulties at birth. Discussion: The disease severity in our patients varied extensively from ventilator or wheelchair dependence to mild facial weakness, ptosis, and ophthalmoparesis. Most of the patients had normal transmission in conventional 3 Hz stimulation electrophysiologic studies, making the diagnosis of CMS challenging. Our cohort of adult and pediatric patients expands the phenotype of DOK7 CMS and shows the importance of correct and early diagnosis.

Extension of the DNAJB2a isoform in a dominant neuromyopathy family.

Recessive mutations in the DNAJB2 gene, encoding the J-domain co-chaperones DNAJB2a and DNAJB2b, have previously been reported as the genetic cause of progressive peripheral neuropathies, rarely involving pyramidal signs, parkinsonism and myopathy. We describe here a family with the first dominantly acting DNAJB2 mutation resulting in a late-onset neuromyopathy phenotype. The c.832 T > G p.(*278Glyext*83) mutation abolishes the stop codon of the DNAJB2a isoform resulting in a C-terminal extension of the protein, with no direct effect predicted on the DNAJB2b isoform of the protein. Analysis of the muscle biopsy showed reduction of both protein isoforms. In functional studies, the mutant protein mislocalized to the endoplasmic reticulum due to a transmembrane helix in the C-terminal extension. The mutant protein underwent rapid proteasomal degradation and also increased the turnover of co-expressed wild-type DNAJB2a, potentially explaining the reduced protein amount in the patient muscle tissue. In line with this dominant negative effect, both wild-type and mutant DNAJB2a were shown to form polydisperse oligomers.

Improved Criteria for the Classification of Titin Variants in Inherited Skeletal Myopathies.

BACKGROUND: Extensive genetic screening results in the identification of thousands of rare variants that are difficult to interpret. Because of its sheer size, rare variants in the titin gene (TTN) are detected frequently in any individual. Unambiguous interpretation of molecular findings is almost impossible in many patients with myopathies or cardiomyopathies. OBJECTIVE: To refine the current classification framework for TTN-associated skeletal muscle disorders and standardize the interpretation of TTN variants. METHODS: We used the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) to re-analyze TTN genetic findings from our patient cohort. RESULTS: We identified in the classification guidelines three rules that are not applicable to titin-related skeletal muscle disorders; six rules that require disease-/gene-specific adjustments and four rules requiring quantitative thresholds for a proper use. In three cases, the rule strength need to be modified. CONCLUSIONS: We suggest adjustments are made to the guidelines. We provide frequency thresholds to facilitate filtering of candidate causative variants and guidance for the use and interpretation of functional data and co-segregation evidence. We expect that the variant classification framework for TTN-related skeletal muscle disorders will be further improved along with a better understanding of these diseases.

Novel mutation in TNPO3 causes congenital limb-girdle myopathy with slow progression.

OBJECTIVE: We report a second family with autosomal dominant transportinopathy presenting with congenital or early-onset myopathy and slow progression, causing proximal and less pronounced distal muscle weakness. METHODS: Patients had clinical examinations, muscle MRI, EMG, and muscle biopsy studies. The MYOcap gene panel was used to identify the gene defect in the family. Muscle biopsies were used for histopathologic and protein expression studies, and TNPO3 constructs were used to study the effect of the mutations in transfected cells. RESULTS: We identified a novel heterozygous mutation, c.2757delC, in the last part of the transportin-3 (TNPO3) gene in the affected family members. The mutation causes an almost identical frameshift affecting the stop codon and elongating the C-term protein product of the TNPO3 transcript, as was previously reported in the first large Spanish-Italian LGMD1F kindred. TNPO3 protein was increased in the patient muscle and accumulated in the subsarcolemmal and perinuclear areas. At least one of the cargo proteins, the splicing factor SRRM2 was normally located in the nucleus. Transiently transfected mutant TNPO3 constructs failed to localize to cytoplasmic annulate lamellae pore complexes in cells. CONCLUSIONS: We report the clinical, molecular genetic, and histopathologic features of the second transportinopathy family. The variability of the clinical phenotype together with histopathologic findings suggests that several molecular pathways may be involved in the disease pathomechanism, such as nucleocytoplasmic shuttling, protein aggregation, and defective protein turnover.

Expanding the importance of HMERF titinopathy: new mutations and clinical aspects.

OBJECTIVE: Hereditary myopathy with early respiratory failure (HMERF) is caused by titin A-band mutations in exon 344 and considered quite rare. Respiratory insufficiency is an early symptom. A collection of families and patients with muscle disease suggestive of HMERF was clinically and genetically studied. METHODS: Altogether 12 new families with 19 affected patients and diverse nationalities were studied. Most of the patients were investigated using targeted next-generation sequencing; Sanger sequencing was applied in some of the patients and available family members. Histological data and muscle MRI findings were evaluated. RESULTS: Three families had several family members studied while the rest were single patients. Most patients had distal and proximal muscle weakness together with respiratory insufficiency. Five heterozygous TTN A-band mutations were identified of which two were novel. Also with the novel mutations the muscle pathology and imaging findings were compatible with the previous reports of HMERF. CONCLUSIONS: Our collection of 12 new families expands mutational spectrum with two new mutations identified. HMERF is not that rare and can be found worldwide, but maybe underdiagnosed. Diagnostic process seems to be complex as this study shows with mostly single patients without clear dominant family history.

Homozygous Nonsense Mutation p.Q274X in TRIM63 (MuRF1) in a Patient with Mild Skeletal Myopathy and Cardiac Hypertrophy.

TRIM63 mutations have been described as a potential cause for cardiac and skeletal myopathy in only one family so far. We describe a new patient carrying the same homozygous TRIM63 nonsense mutation c.739 C>T p.Q247X, that was originally reported in two members of a Spanish family manifesting cardiac hypertrophy. One of these original patients also had an additional heterozygous mutation in TRIM54 and a much more severe phenotype also involving skeletal muscles, and a digenic inheritance was therefore suggested. Our case report confirms the role of TRIM63 as a new cardiac myopathy gene, although it is unclear whether the homozygous p.Q247X mutation alone is sufficient to cause an additional skeletal myopathy.

TIA1 variant drives myodegeneration in multisystem proteinopathy with SQSTM1 mutations.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

Late-onset limb-girdle muscular dystrophy caused by GMPPB mutations.

Mutations in GMPPB gene have been reported in patients with early-onset disease ranging from severe congenital muscular dystrophies to limb-girdle muscular dystrophy (LGMD) with mental retardation. More recently mutations in GMPPB have been identified with congenital myasthenic syndromes as well as milder phenotypes. We report two unrelated cases with LGMD that underwent clinical, histopathological and genetic studies. In both cases, we found identical compound heterozygous GMPPB mutations c.79G>C p.D27H and c.859C>T p.R287W, leading to a glycosylation defect of alpha-dystroglycan. The onset of muscle weakness was 30-40 years and the progression rate mild to moderate. Case 2 became wheelchair-bound at the age of 60. No cognitive or behavioral symptoms were noted. These cases provide further evidence that GMPPB mutations can also cause late-onset recessive LGMD with milder phenotypes than previously reported, and thus should be considered in the differential diagnosis of patients with adult-onset muscular dystrophies.

Predominantly myalgic phenotype caused by the c.3466G>A p.A1156T mutation in SCN4A gene.

OBJECTIVE: To characterize the clinical phenotype in patients with p.A1156T sodium channel mutation. METHODS: Twenty-nine Finnish patients identified with the c.3466G>A p.A1156T mutation in the SCN4A gene were extensively examined. In a subsequent study, 63 patients with similar myalgic phenotype and with negative results in myotonic dystrophy type 2 genetic screening (DM2-neg group) and 93 patients diagnosed with fibromyalgia were screened for the mutation. Functional consequences of the p.A1156T mutation were studied in HEK293 cells with whole-cell patch clamp. RESULTS: The main clinical manifestation in p.A1156T patients was not myotonia or periodic paralysis but exercise- and cold-induced muscle cramps, muscle stiffness, and myalgia. EMG myotonic discharges were detected in most but not all. Electrophysiologic compound muscle action potentials exercise test showed variable results. The p.A1156T mutation was identified in one patient in the DM2-neg group but not in the fibromyalgia group, making a total of 30 patients so far identified. Functional studies of the p.A1156T mutation showed mild attenuation of channel fast inactivation. CONCLUSIONS: The unspecific symptoms of myalgia stiffness and exercise intolerance without clinical myotonia or periodic paralysis in p.A1156T patients make the diagnosis challenging. The symptoms of milder SCN4A mutations may be confused with other similar myalgic syndromes, including fibromyalgia and myotonic dystrophy type 2.

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.

Novel compound heterozygous mutation in SACS gene leads to a milder autosomal recessive spastic ataxia of Charlevoix-Saguenay, ARSACS, in a Finnish family.

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.

Homozygosity of the Dominant Myotilin c.179C>T (p.Ser60Phe) Mutation Causes a More Severe and Proximal Muscular Dystrophy.

Most myotilinopathy patients present with a dominant late onset distal phenotype and myofibrillar pathology, although the first MYOT mutation in a family reported to have LGMD phenotype. We report here a French family affected with a late onset proximal and distal muscle weakness and myofibrillar myopathy on muscle pathology, in which the siblings known to be clinically affected were homozygous for the c.179C>T (p.Ser60Phe) myotilin gene mutation. One subjectively asymptomatic member of the family was heterozygous for this mutation. This is the first report of a family with patients being homozygous for a known dominant MYOT mutation. Dominant negative mutations are generally considered not to cause a more severe disease in homozygosity, but our data clearly demonstrate the existence of dominant MYOT mutations with a possible dose effect causing a more severe disease phenotype in homozygosity in the spectrum of myofibrillar myopathies (MFM).

Epidermolysis bullosa simplex with muscular dystrophy associated with PLEC deletion mutation.

Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD; OMIM #226670) is an autosomal recessive disorder characterized by neonatal blistering and later-onset muscle weakness.

Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders.

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.

Mutations in HSPB8 causing a new phenotype of distal myopathy and motor neuropathy.

OBJECTIVE: To report novel disease and pathology due to HSPB8 mutations in 2 families with autosomal dominant distal neuromuscular disease showing both myofibrillar and rimmed vacuolar myopathy together with neurogenic changes. METHODS: We performed whole-exome sequencing (WES) in tandem with linkage analysis and candidate gene approach as well as targeted next-generation sequencing (tNGS) to identify causative mutations in 2 families with dominant rimmed vacuolar myopathy and a motor neuropathy. Pathogenic variants and familial segregation were confirmed using Sanger sequencing. RESULTS: WES and tNGS identified a heterozygous change in HSPB8 in both families: c.421A > G p.K141E in family 1 and c.151insC p.P173SfsX43 in family 2. Affected patients had a distal myopathy that showed myofibrillar aggregates and rimmed vacuoles combined with a clear neurogenic component both on biopsy and neurophysiologic studies. MRI of lower limb muscles demonstrated diffuse tissue changes early in the disease stage progressing later to fatty replacement typical of a myopathy. CONCLUSION: We expand the understanding of disease mechanisms, tissue involvement, and phenotypic outcome of HSPB8 mutations. HSPB8 is part of the chaperone-assisted selective autophagy (CASA) complex previously only associated with Charcot-Marie-Tooth type 2L (OMIM 60673) and distal hereditary motor neuronopathy type IIa. However, we now demonstrate that patients can develop a myopathy with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles, similar to the pathology in myopathies due to gene defects in other compounds of the CASA complex such as BAG3 and DNAJB6 after developing the early neurogenic effects.

Distal myopathies in Finnish patients.

Distal myopathies are a group of rare muscular dystrophies comprising more than 20 different genetic entities. The first distal myopathy in Finland, tibial muscular dystrophy, was identified more than 20 years ago. Muscle weakness predominantly affects the feet and hands, although variable weakness can be detected clinically and on muscle MRI in the proximal muscles in the later stages of the disease. Advanced molecular genetic techniques have enabled identification of several distinct distal myopathies in Finland. The clinical findings of different distal myopathies overlap, but there are also distinguishable differences that might help final genetic diagnostics.

Differential isoform expression and selective muscle involvement in muscular dystrophies.

Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies.