Generation of two iPSC lines from adult central core disease patients with dominant missense variants in the RYR1 gene.

RYR1 variants are a common cause of congenital myopathies, including multi-minicore disease (MmD) and central core disease (CCD). Here, we generated iPSC lines from two CCD patients with dominant RYR1 missense variants that affect the transmembrane (pore) and SPRY3 protein domains (p.His4813Tyr and p.Asn1346Lys, respectively). Both lines had typical iPSC morphology, expressed canonical pluripotency markers, exhibited trilineage differentiation potential, and had normal karyotypes. Together with existing RYR1 iPSC lines, these represent important tools to study and develop treatments for RYR1-related myopathies.

Generation of two iPSC lines from patients with inherited central core disease and concurrent malignant hyperthermia caused by dominant missense variants in the RYR1 gene.

RYR1 variants are the most common genetic cause of congenital myopathies, and typically cause central core disease (CCD) and/or malignant hyperthermia (MH). Here, we generated iPSC lines from two patients with CCD and MH caused by dominant RYR1 variants within the central region of the protein (p.Val2168Met and p.Arg2508Cys). Both lines displayed typical iPSC morphology, uniform expression of pluripotency markers, trilineage differentiation potential, and had normal karyotypes. These are the first RYR1 iPSC lines from patients with both CCD and MH. As these are common CCD/MH variants, these lines should be useful to study these conditions and test therapeutics.

Pathogenic DPAGT1 variants in limb-girdle congenital myasthenic syndrome (LG-CMS) associated with tubular aggregates and ORAI1 hypoglycosylation.

AIMS: Limb-girdle congenital myasthenic syndrome (LG-CMS) is a genetically heterogeneous disorder characterized by muscle weakness and fatigability. The LG-CMS gene DPAGT1 codes for an essential enzyme of the glycosylation pathway, a posttranslational modification mechanism shaping the structure and function of proteins. In DPAGT1-related LG-CMS, reduced glycosylation of the acetylcholine receptor (AChR) reduces its localization at the neuromuscular junction (NMJ), and results in diminished neuromuscular transmission. LG-CMS patients also show tubular aggregates on muscle biopsy, but the origin and potential contribution of the aggregates to disease development are not understood. Here, we describe two LG-CMS patients with the aim of providing a molecular diagnosis and to shed light on the pathways implicated in tubular aggregate formation. METHODS: Following clinical examination of the patients, we performed next-generation sequencing (NGS) to identify the genetic causes, analysed the biopsies at the histological and ultrastructural levels, investigated the composition of the tubular aggregates, and performed experiments on protein glycosylation. RESULTS: We identified novel pathogenic DPAGT1 variants in both patients, and pyridostigmine treatment quantitatively improved muscle force and function. The tubular aggregates contained proteins of the sarcoplasmic reticulum (SR) and structurally conformed to the aggregates observed in tubular aggregate myopathy (TAM). TAM arises from overactivation of the plasma membrane calcium channel ORAI1, and functional studies on muscle extracts from our LG-CMS patients evidenced abnormal ORAI1 glycosylation. CONCLUSIONS: We expand the genetic variant spectrum of LG-CMS and provide a genotype/phenotype correlation for pathogenic DPAGT1 variants. The discovery of ORAI1 hypoglycosylation in our patients highlights a physiopathological link between LG-CMS and TAM.

Generation of two induced pluripotent stem cell lines from a 33-year-old central core disease patient with a heterozygous dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene.

Central core disease (CCD) is a congenital disorder that results in hypotonia, delayed motor development, and areas of reduced oxidative activity in the muscle fibre. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 33-year-old male with CCD, caused by a previously unreported dominant c.14145_14156delCTACTGGGACA (p.Asn4715_Asp4718del) deletion in the RYR1 gene. Both lines demonstrated typical morphology, pluripotency, trilineage differentiation, and had a normal karyotype. As the first published iPSC model of CCD caused by an RYR1 variant these lines are a potential resource for further investigation of RYR1-related myopathies in a human context.

Human skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation.

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant.

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.

LAMA2-Related Muscular Dystrophy: The Importance of Accurate Phenotyping and Brain Imaging in the Diagnosis of LGMD.

We report three siblings from a non-consanguineous family presenting with contractural limb-girdle phenotype with intrafamilial variability. Muscle MRI showed posterior thigh and quadriceps involvement with a sandwich-like sign. Whole-exome sequencing identified two compound heterozygous missense TTN variants and one heterozygous LAMA2 variant. Brain MRI performed because of concentration difficulties in one of the siblings evidenced white-matter abnormalities, subsequently found in the others. The genetic analysis was re-oriented, revealing a novel pathogenic intronic LAMA2 variant which confirmed the LAMA2-RD diagnosis. This work highlights the importance of a thorough clinical phenotyping and the importance of brain imaging, in order to orientate and interpret the genetic analysis.

Muscle MRI as a Diagnostic Challenge in Emery-Dreifuss Muscular Dystrophy.

 Emery-Dreifuss Muscular Dystrophy (EDMD) is an early-onset, slowly-progressive group of myopathies, presenting with joint contractures, muscle weakness and cardiac abnormalities. Variants in the EMD gene cause an X-linked recessive form (EDMD1). The scarce EDMD1 muscle MRI accounts in the literature describe fatty replacement of posterior thigh and leg muscles.We report a 22-year-old patient with early-onset bilateral joint contractures, slowly progressive muscle weakness and minor cardiac rhythm abnormalities. A novel loss-of-function variant of EMD was identified and deemed probably pathogenic in the absence of emerin detection by immunofluorescence and Western Blot. MRI revealed fatty replacement of the lumbar spinal erectors and the posterior compartment of lower limbs. Interestingly, Short Tau Inversion Recovery (STIR) sequences showed a heterogenous hyper signal on the vasti, hamstrings and left lateral gastrocnemius muscles.Oedema-like abnormalities were previously reported in early stages of other muscular dystrophies, preceding fatty replacement and muscle atrophy, but not in EDMD1 patients. We hypothesize that these oedema-like changes may be a marker of early muscle pathology in EDMD1. Further studies focusing on these abnormalities in the early phase of EDMD1 are required to test our hypothesis.

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Novel autosomal dominant TPM3 mutation causes a combined congenital fibre type disproportion-cap disease histological pattern.

Tropomyosin 3 (TPM3) gene mutations associate with autosomal dominant and recessive nemaline myopathy 1 (NEM1), congenital fiber type disproportion myopathy (CFTD) and cap myopathy (CAPM1), and a combination of caps and nemaline bodies. We report on a 47-year-old man with polyglobulia, restricted vital capacity and mild apnea hypopnea syndrome, requiring noninvasive ventilation. Physical assessment revealed bilateral ptosis and facial paresis, with high arched palate and retrognathia; global hypotonia and diffuse axial weakness, including neck and upper and lower limb girdle and foot dorsiflexion weakness. Whole body MRI showed a diffuse fatty replacement with an unspecific pattern. A 122 gene NGS neuromuscular disorders panel revealed the heterozygous VUS c.709G>A (p.Glu237Lys) on exon 8 of TMP3. A deltoid muscle biopsy showed a novel histological pattern combining fiber type disproportion and caps. Our findings support the pathogenicity of the novel TPM3 variant and widen the phenotypic gamut of TMP3-related congenital myopathy.

Generation of an induced pluripotent stem cell line from a 3-month-old nemaline myopathy patient with a heterozygous dominant c.515C > A (p.Ala172Glu) variant in the ACTA1 gene.

Variants in the ACTA1 gene are a common cause of nemaline myopathy (NM); a muscle disease that typically presents at birth or early childhood with hypotonia and muscle weakness. Here, we generated an induced pluripotent stem cell line (iPSC) from lymphoblastoid cells of a 3-month-old female patient with intermediate NM caused by a dominant ACTA1 variant (c.515C > A (p.Ala172Glu)). iPSCs showed typical morphology, expressed pluripotency markers, demonstrated trilineage differentiation potential, and had a normal karyotype. This line complements our previously published ACTA1 iPSC lines derived from patients with typical and severe NM.

Generation of two isogenic induced pluripotent stem cell lines from a 1-month-old nemaline myopathy patient harbouring a homozygous recessive c.121C > T (p.Arg39Ter) variant in the ACTA1 gene.

Nemaline myopathy (NM) is a congenital skeletal muscle disorder that typically results in muscle weakness and the presence of rod-like structures (nemaline bodies) in the sarcoplasma and/or in the nuclei of myofibres. Two induced pluripotent stem cell (iPSC) lines were generated from the lymphoblastoid cells of a 1-month-old male with severe NM caused by a homozygous recessive mutation in the ACTA1 gene (c.121C > T, p.Arg39Ter). The iPSC lines demonstrated typical morphology, expressed pluripotency markers, exhibited trilineage differentiation potential and displayed a normal karyotype. These isogenic lines represent a potential resource to investigate and model recessive ACTA1 disease in a human context.

Diagnostic interest of whole-body MRI in early- and late-onset LAMA2 muscular dystrophies: a large international cohort.

BACKGROUND: LAMA2-related muscular dystrophy (LAMA2-RD) encompasses a group of recessive muscular dystrophies caused by mutations in the LAMA2 gene, which codes for the alpha-2 chain of laminin-211 (merosin). Diagnosis is straightforward in the classic congenital presentation with no ambulation and complete merosin deficiency in muscle biopsy, but is far more difficult in milder ambulant individuals with partial merosin deficiency. OBJECTIVE: To investigate the diagnostic utility of muscle imaging in LAMA2-RD using whole-body magnetic resonance imaging (WBMRI). RESULTS: 27 patients (2-62 years, 21-80% with acquisition of walking ability and 6 never ambulant) were included in an international collaborative study. All carried two pathogenic mutations, mostly private missense changes. An intronic variant (c.909 + 7A > G) was identified in all the Chilean cases. Three patients (two ambulant) showed intellectual disability, epilepsy, and brain structural abnormalities. WBMRI T1w sequences or T2 fat-saturated images (Dixon) revealed abnormal muscle fat replacement predominantly in subscapularis, lumbar paraspinals, gluteus minimus and medius, posterior thigh (adductor magnus, biceps femoris, hamstrings) and soleus. This involvement pattern was consistent for both ambulant and non-ambulant patients. The degree of replacement was predominantly correlated to the disease duration, rather than to the onset or the clinical severity. A "COL6-like sandwich sign" was observed in several muscles in ambulant adults, but different involvement of subscapularis, gluteus minimus, and medius changes allowed distinguishing LAMA2-RD from collagenopathies. The thigh muscles seem to be the best ones to assess disease progression. CONCLUSION: WBMRI in LAMA2-RD shows a homogeneous pattern of brain and muscle imaging, representing a supportive diagnostic tool.

Diagnostic work-up and phenotypic characteristics of a family with variable severity of distal arthrogryposis type 2B (Sheldon-Hall syndrome) and TNNT3 pathogenic variant.

Background: Sheldon-Hall syndrome (SHS) or distal arthrogryposis 2B (DA2B) is a rare clinically and genetically heterogeneous multiple congenital contracture syndrome characterized by contractures of the distal joints of the limbs and mild facial involvement, due to pathogenic variants in genes encoding the fast-twitch skeletal muscle contractile myofiber complex (TNNT3, TNNI2, TMP2, and MYH3 genes). Patients and methods: A 16-year-old boy with a history of congenital distal arthrogryposis developed severe kyphoscoliosis and respiratory insufficiency. His mother and younger sister had phenotypes compatible with SHS but to a much lesser extent. Diagnostic work-up included physical examination and whole-body muscular MRI (WBMRI) in all three patients and electroneuromyography (ENMG) and paravertebral muscle biopsy in the proband. DNA sequencing was used to confirm the diagnosis. Results: Physical examination suggested the diagnosis of SHS. No muscle signal abnormalities were found in WBMRI. Large motor unit potentials and reduced recruitment suggestive of neurogenic changes were observed on needle EMG in distal and paravertebral muscles in the proband. DNA sequencing revealed a pathogenic variant in TNNT3 (c.187C>T), which segregated as a dominant trait with the phenotype. Discussion: This is the first report on neurogenic features in a patient with DA2B and a pathogenic variant in TNNT3 encoding the fast-twitch skeletal muscle contractile myofiber complex. A superimposed length-dependent motor nerve involvement was unexpected. Whether developmental disarrangements in number, distribution, or innervation of the motor unit in fetal life might lead to pseudo-neurogenic EMG features warrants further studies, as well as the role of genetic modifiers in SHS variability.

[JAG2-related muscular dystrophy: When differential diagnosis matters]. / Dystrophie musculaire liée à des mutations du gène JAG2 - L'importance du diagnostic différentiel.

JAG2 has recently been involved in autosomal recessive forms of muscular dystrophy as illustrated in this clinical vignette. In many ways, this disease can mimick a COL6-related retractile myopathy including at the imaging level.

A recurrent RYR1 mutation associated with early-onset hypotonia and benign disease course.

The ryanodine receptor RyR1 is the main sarcoplasmic reticulum Ca2+ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca2+-dependent muscle contraction. Abnormal RyR1 activity compromises normal muscle function and results in various human disorders including malignant hyperthermia, central core disease, and centronuclear myopathy. However, RYR1 is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe a recurrent RYR1 mutation previously classified as VUS, and we provide clinical, histological, and genetic data supporting its pathogenicity. The heterozygous c.12083C>T (p.Ser4028Leu) mutation was found in thirteen patients from nine unrelated congenital myopathy families with consistent clinical presentation, and either segregated with the disease in the dominant families or occurred de novo. The affected individuals essentially manifested neonatal or infancy-onset hypotonia, delayed motor milestones, and a benign disease course differing from classical RYR1-related muscle disorders. Muscle biopsies showed unspecific histological and ultrastructural findings, while RYR1-typical cores and internal nuclei were seen only in single patients. In conclusion, our data evidence the causality of the RYR1 c.12083C>T (p.Ser4028Leu) mutation in the development of an atypical congenital myopathy with gradually improving motor function over the first decades of life, and may direct molecular diagnosis for patients with comparable clinical presentation and unspecific histopathological features on the muscle biopsy.