Xavière Lornage

Dihydropyridine receptor (DHPR, CACNA1S) congenital myopathy

Muscle contraction upon nerve stimulation relies on excitation–contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.

RecessiveMYPNmutations cause cap myopathy with occasional nemaline rods: MYPNMutations

Congenital myopathies are phenotypically and genetically heterogeneous. We describe homozygous truncating mutations in MYPN in 2 unrelated families with a slowly progressive congenital cap myopathy. MYPN encodes the Z‐line protein myopalladin implicated in sarcomere integrity. Functional experiments demonstrate that the mutations lead to mRNA defects and to a strong reduction in full‐length protein expression. Myopalladin signals accumulate in the caps together with alpha‐actinin. Dominant MYPN mutations were previously reported in cardiomyopathies. Our data uncover that mutations in MYPN cause either a cardiac or a congenital skeletal muscle disorder through different modes of inheritance. Ann Neurol 2017;81:467–473

CASQ1 mutations impair calsequestrin polymerization and cause tubular aggregate myopathy

involving the proximal muscles in the lower limbs for Family 1, and early 50s with post-exercise myalgia in the lower limbs for Family 2 (Supplementary Table 1). Histological and ultrastructural analyses of the muscle biopsies displayed tubular aggregates as the main histopathological hallmark in both families (Fig. 1a). Exome sequencing identified the heterozygous CASQ1 missense mutations c.166A>T (N56Y) in exon 1 in Family 1, and c.308G>A (G103D) in exon 2 in Family 2. Both mutations affect highly conserved amino acids (Supp. Figure 1), none was found in the available healthy family members, and none was listed in the public or internal SNP databases. A single CASQ1 missense mutation (D244G) has previously been associated with vacuolar myopathy involving protein aggregates [9]. CASQ1 is primarily expressed in skeletal muscle and encodes calsequestrin, the major Ca2+ storage protein in the sarcoplasmic reticulum. Calsequestrin binds Ca2+ with moderate affinity and high capacity, and forms higher order polymers with increasing Ca2+-binding capacities [4]. Immunohistochemistry on a muscle biopsy from Family 2 revealed strong signals for calsequestrin, STIM1, and RyR1 in aggregated structures most likely corresponding to the tubular aggregates, while ORAI1 was not trapped (Fig. 1b). This conforms to the observations made on biopsies from STIM1 and ORAI1 patients and demonstrates that the trapped proteins are primarily of sarcoplasmic reticulum origin [1, 2]. These findings on a single muscle biopsy also suggest that aggregation of STIM1 appears to be a consequence of CASQ1 mutations, providing a pathological link between STIM1and CASQ1-related TAM. In transfected C2C12 myoblasts, WT and both TAM N56Y and G103D mutants formed calsequestrin networks of comparable complexity, while the vacuolar myopathy D244G mutant induced major calsequestrin aggregation (Fig. 1c). Calsequestrin polymerization and depolymerization are dynamic Tubular aggregate myopathy (TAM) is a rare muscle disorder characterized by abnormal accumulations of membrane tubules in muscle fibers, and marked by progressive muscle weakness, cramps, and myalgia [3]. Genetically, TAM has been assigned to mutations in STIM1 [2] and ORAI1 [7], both encoding key regulators of Ca2+ homeostasis. Through exome sequencing of molecularly undiagnosed TAM cases, we now identified CASQ1 as the third TAM gene, and we support our findings by clinical, histological, genetic, and functional data. Family 1 has an ancestral history of a muscle phenotype segregating as a dominant disease, and a partial clinical and histological description was reported earlier [8]. Patient 103901 from Family 2 is a singleton. Birth, early childhood, and motor milestones were normal for all affected members from both families. Disease onset was between early 20s and mid-40s with a slowly progressive muscle weakness mainly

Common and variable clinical, histological, and imaging findings of recessive RYR1-related centronuclear myopathy patients.

Mutations in RYR1 give rise to diverse skeletal muscle phenotypes, ranging from classical central core disease to susceptibility to malignant hyperthermia. Next-generation sequencing has recently shown that RYR1 is implicated in a wide variety of additional myopathies, including centronuclear myopathy. In this work, we established an international cohort of 21 patients from 18 families with autosomal recessive RYR1-related centronuclear myopathy, to better define the clinical, imaging, and histological spectrum of this disorder. Early onset of symptoms with hypotonia, motor developmental delay, proximal muscle weakness, and a stable course were common clinical features in the cohort. Ptosis and/or ophthalmoparesis, facial weakness, thoracic deformities, and spinal involvement were also frequent but variable. A common imaging pattern consisted of selective involvement of the vastus lateralis, adductor magnus, and biceps brachii in comparison to adjacent muscles. In addition to a variable prominence of central nuclei, muscle biopsy from 20 patients showed type 1 fiber predominance and a wide range of intermyofibrillary architecture abnormalities. All families harbored compound heterozygous mutations, most commonly a truncating mutation combined with a missense mutation. This work expands the phenotypic characterization of patients with recessive RYR1-related centronuclear myopathy by highlighting common and variable clinical, histological, and imaging findings in these patients.

Expanding the spectrum of congenital myopathy linked to recessive mutations in SCN4A

Congenital myopathies are phenotypically and genetically heterogeneous.1 While SCN4A mutations were previously described in hypokalemic or hyperkalemic periodic paralysis, paramyotonia, myotonia congenita, or myasthenic syndrome,2–6 loss-of-function recessive mutations in SCN4A were recently identified in 11 individuals with severe fetal hypokinesia or congenital myopathies.7 Among them, 7 died in the perinatal period and 4 had congenital hypotonia, significant respiratory and swallowing difficulties, and spinal deformities.

Sarcomeric disorganization and nemaline bodies in muscle biopsies of patients with EXOSC3-related type 1 pontocerebellar hypoplasia: Nemaline bodies in EXOSC3 PCH1

Mutations in the EXOSC3 gene are responsible for type 1 pontocerebellar hypoplasia, an autosomal recessive congenital disorder characterized by cerebellar atrophy, developmental delay, and anterior horn motor neuron degeneration. Muscle biopsies of these patients often show characteristics resembling classic spinal muscle atrophy, but to date, no distinct features have been identified.

Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies

Titin-related myopathies are heterogeneous clinical conditions associated with mutations in TTN. To define their histopathologic boundaries and try to overcome the difficulty in assessing the pathogenic role of TTN variants, we performed a thorough morphological skeletal muscle analysis including light and electron microscopy in 23 patients with different clinical phenotypes presenting pathogenic autosomal dominant or autosomal recessive (AR) mutations located in different TTN domains. We identified a consistent pattern characterized by diverse defects in oxidative staining with prominent nuclear internalization in congenital phenotypes (AR-CM) (n = 10), ± necrotic/regenerative fibers, associated with endomysial fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Dreifuss-like (AR-ED) (n = 4) and AR adult-onset distal myopathies (n = 4), and cytoplasmic bodies (CBs) as predominant finding in hereditary myopathy with early respiratory failure (HMERF) patients (n = 5). Ultrastructurally, the most significant abnormalities, particularly in AR-CM, were multiple narrow core lesions and/or clear small areas of disorganizations affecting one or a few sarcomeres with M-band and sometimes A-band disruption and loss of thick filaments. CBs were noted in some AR-CM and associated with RVs in HMERF and some AR-ED cases. As a whole, we described recognizable histopathological patterns and structural alterations that could point toward considering the pathogenicity of TTN mutations.