Down syndrome (DS), the principal cause for intellectual disability, is also associated with hormonal, immunological, and gastrointestinal abnormalities. Muscle hypotonia (MH) and congenital heart diseases (CHD) are also frequently observed. Collagen molecules are essential components for maintaining muscle integrity and are formed by the assembly of three chains, alpha 1-3. The type VI collagen is crucial for cardiac as well as skeletal muscles. The COL α1 (VI) and α2 (VI) chains are encoded by genes located at the 21st chromosome and are expected to have higher dosage in individuals with DS. The α 3 (VI) chain is encoded by the COL6A3 located at the chromosome 2. We hypothesized that apart from COL6A1 and COL6A2, COL6A3 may also have some role in the MH of subjects with DS. To find out the relevance of COL6A3 in DS associated MH and CHD, we genotyped two SNPs in COL6A3, rs2270669 and rs2270668, in individuals with DS. Subjects with DS were recruited based on the Diagnostic and Statistical Manual for Mental Disorders-IV and having trisomy of the 21st chromosome. Parents of individuals with DS and ethnically matched controls were enrolled for comparison. Informed written consent was obtained for participation. Peripheral blood was used for isolation of genomic DNA. Target genetic loci were studied by DNA sequence analysis. Data obtained was subjected to population - as well as family-based statistical analysis. rs2270668 was found to be non-polymorphic in the studied population. rs2270669 showed significant association of the "C" allele and "CC" genotype with DS probands having MH (P = 0.02). Computational analysis showed that rs2270669 may induce structural and functional alterations in the COL α3 (VI). Interaction of COLα3 (VI) with different proteins, crucial for muscle integrity, was also noticed by computational methods. This pioneering study on COL6A3 with DS related MH thus indicates that rs2270669 "C" could be considered as a risk factor for DS related MH.

Phelan-McDermid syndrome (PMS) is a syndromic form of Autism Spectrum Disorders (ASD) classified as a rare genetic neurodevelopmental disorder featuring global developmental delay, absent or delayed speech, ASD-like behaviour and neonatal skeletal muscle hypotonia. PMS is caused by a heterozygous deletion of the distal end of chromosome 22q13.3 or SHANK3 mutations. We analyzed striated muscles of newborn Shank3Δ11(-/-) animals and found a significant enlargement of the sarcoplasmic reticulum as previously seen in adult Shank3Δ11(-/-) mice, indicative of a Shank3-dependent and not compensatory mechanism for this structural alteration. We analyzed transcriptional differences by RNA-sequencing of muscle tissue of neonatal Shank3Δ11(-/-) mice and compared those to Shank3(+/+) controls. We found significant differences in gene expression of ion channels crucial for muscle contraction and for molecules involved in calcium ion regulation. In addition, calcium storage- [i.e., Calsequestrin (CSQ)], calcium secretion- and calcium-related signaling-proteins were found to be affected. By immunostainings and Western blot analyses we could confirm these findings both in Shank3Δ11(-/-) mice and PMS patient muscle tissue. Moreover, alterations could be induced in vitro by the selective downregulation of Shank3 in C2C12 myotubes. Our results emphasize that SHANK3 levels directly or indirectly regulate calcium homeostasis in a cell autonomous manner that might contribute to muscular hypotonia especially seen in the newborn.

Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.

Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.

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.

Neonatal hypotonia is extremely challenging to diagnose because numerous disorders present similar clinical manifestations. Two panels for diagnosing neonatal hypotonia were developed, which enriches 35 genes corresponding to 61 neonatal hypotonia-related disorders. A cohort of 214 neonates with hypotonia was recruited from 2012 to 2014 in China for this study. Of these subjects, twenty-eight neonates with hypotonia were eliminated according to exclusion criteria and 97 were confirmed using traditional detection methods. The clinical diagnoses of the remaining 89 neonates with hypotonia were approached by targeted next-generation sequencing (NGS). Among the 89 tested neonates, 25 potentially pathogenic variants in nine genes (RYR1, MECP2, MUT, CDKL5, MPZ, PMM2, MTM1, LAMA2 and DMPK) were identified in 22 patients. Six of these pathogenic variants were novel. Of the 186 neonates with hypotonia, we identified the genetic causes for 117 neonates by the traditional detection methods and targeted NGS, achieving a high solving rate of 62.9%. In addition, we found seven neonates with RETT syndrome carrying five mutations, thus expanding the mutation profiles in Chinese neonates with hypotonia. Our study highlights the utility of comprehensive molecular genetic testing, which provides the advantage of speed and diagnostic specificity without invasive procedures.

Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gβ. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gβ binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gβγ interaction (resulting in a constitutively active Gβγ) or through the disruption of residues relevant for interaction between Gβγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues.

Striated muscle preferentially expressed protein kinase (SPEG) variants have been reported to cause centronuclear myopathy associated with cardiac diseases. The severity of skeletal muscle symptoms and cardiac symptoms are presumably related to the location of the variant. Here, we report novel SPEG compound heterozygous pathological variants in a neonate with severe dilated cardiomyopathy and relatively mild hypotonia. This report expands the genotype-phenotype correlations of patients with SPEG variants.

Children with developmental central hypotonia have reduced muscle tone secondary to non-progressive damage to the brain or brainstem. Children may have transient delays, mild or global functional impairments, and the lack of a clear understanding of this diagnosis makes evaluating appropriate interventions challenging. This overview aimed to systematically describe the best available evidence for tools to identify and evaluate children with developmental central hypotonia aged 2 months to 6 years. A systematic review of systematic reviews or syntheses was conducted with electronic searches in PubMed, Medline, CINAHL, Scopus, Cochrane Database of Systematic Reviews, Google Scholar, and PEDro and supplemented with hand-searching. Methodological quality and risk-of-bias were evaluated, and included reviews and tools were compared and contrasted. Three systematic reviews, an evidence-based clinical assessment algorithm, three measurement protocols, and two additional measurement tools were identified. For children aged 2 months to 2 years, the Hammersmith Infant Neurological Examination has the strongest measurement properties and contains a subset of items that may be useful for quantifying the severity of hypotonia. For children aged 2-6 years, a clinical algorithm and individual tools provide guidance. Further research is required to develop and validate all evaluative tools for children with developmental central hypotonia.

Congenital hyperinsulinemic hypoglycemia is the most frequent cause of persistent and recurrent hypoglycemia in the first years of life and in many patients rare genetic variants can be identified. Recently a case of congenital hyperinsulinemic hypoglycemia and a severe neurodevelopmental syndrome due to a mutation in the voltage-gated Cav1.3 Ca2+ channel CACNA1D gene has been reported which required long-term treatment with diazoxide. This suggested CACNA1D variants as a potential cause for this condition.Here we support this observation by presenting the case of a female child with congential hyperinsulinemic hypoglycemia and primary hyperaldosteronism, aortic insufficiency, pronounced developmental delay, muscle hypotonia, and facial dysmorphias but without seizures. Sequencing of the exome of the child and its parents identified a novel de novo CACNA1D missense mutation p.L271 H, replacing a highly conserved residue in a functionally relevant region of the voltage-gated Cav1.3 Ca2+ channel. The patient was treated with diazoxide and nifedipine with adequate control of glucose metabolism and blood pressure, and with improvement in muscle tone.Our findings further confirm the pathogenic role of CACNA1D for congentital hyperinsulinemic hypoglycemia and primary aldosteronism. Moreover, we provide evidence that the dihydropyridine Ca2+ channel blocker nifedipine, although not considered a first-line treatment for congenital hyperinsulinism, may be beneficial to control blood pressure and neurological symptoms in patients with CACNA1D mutations.

Obstructive sleep apnea (OSA), where the upper airway collapses repeatedly during sleep due to inadequate dilator muscle tone, is challenging to treat as current therapies are poorly tolerated or have variable and unpredictable efficacy. We propose a novel, optogenetics-based therapy, that stimulates upper airway dilator muscle contractions in response to light. To determine the feasibility of a novel optogenetics-based OSA therapy, we developed a rodent model of human sleep-related upper airway muscle atonia. Using this model, we evaluated intralingual delivery of candidate optogenetic constructs, notably a muscle-targeted approach that will likely have a favorable safety profile.

Six individuals of consanguineous Bedouin kindred presented at infancy with an autosomal recessive syndrome of severe global developmental delay, positive pyramidal signs, unique dysmorphism, skeletal abnormalities, and severe failure to thrive with normal birth weights. Patients had a profound intellectual disability and cognitive impairment with almost no acquired developmental milestones by 12 months. Early-onset axial hypotonia evolved with progressive muscle weakness, reduced muscle tone, and hyporeflexia. Craniofacial dysmorphism consisted of a triangular face with a prominent forehead and midface hypoplasia. Magnetic resonance imaging (MRI) demonstrated thinning of the corpus callosum and paucity of white matter. Genome-wide linkage analysis identified a single ~4 Mbp disease-associated locus on chromosome 7q21.13-q21.3 (LOD score>5). Whole-exome and genome sequencing identified no nonsynonymous pathogenic biallelic variants in any of the genes within this locus. Following the exclusion of partially resembling syndromes, we now describe a novel autosomal recessive syndrome mapped to a ~4Mbp locus on chromosome 7.

Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue.

Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2) have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies.

Down syndrome (DS) is a genetic disorder caused by presence of extra copy of human chromosome 21. It is characterised by several clinical phenotypes. Motor dysfunction due to hypotonia is commonly seen in individuals with DS and its etiology is yet unknown. Ts1Cje, which has a partial trisomy (Mmu16) homologous to Hsa21, is well reported to exhibit various typical neuropathological features seen in individuals with DS. This study investigated the role of skeletal muscles and peripheral nerve defects in contributing to muscle weakness in Ts1Cje mice.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor.

ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.

Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients.

Peroxisomes are ubiquitous organelles that perform lipid and reactive oxygen species metabolism. Defects in peroxisome biogenesis cause peroxisome biogenesis disorders (PBDs). The most severe PBD, Zellweger syndrome, is characterized in part by neuronal dysfunction, craniofacial malformations, and low muscle tone (hypotonia). These devastating diseases lack effective therapies and the development of animal models may reveal new drug targets. We have generated Drosophila mutants with impaired peroxisome biogenesis by disrupting the early peroxin gene pex3, which participates in budding of pre-peroxisomes from the ER and peroxisomal membrane protein localization. pex3 deletion mutants lack detectible peroxisomes and die before or during pupariation. At earlier stages of development, larvae lacking Pex3 display reduced size and impaired lipid metabolism. Selective loss of peroxisomes in muscles impairs muscle function and results in flightless animals. Although, hypotonia in PBD patients is thought to be a secondary effect of neuronal dysfunction, our results suggest that peroxisome loss directly affects muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in Drosophila physiology, specifically in muscle cells may reveal novel aspects of PBD etiology.

Rett syndrome (RTT) is an autism spectrum disorder mainly caused by mutations in the X-linked MECP2 gene and affecting roughly 1 out of 10.000 born girls. Symptoms range in severity and include stereotypical movement, lack of spoken language, seizures, ataxia and severe intellectual disability. Notably, muscle tone is generally abnormal in RTT girls and women and the Mecp2-null mouse model constitutively reflects this disease feature. We hypothesized that MeCP2 in muscle might physiologically contribute to its development and/or homeostasis, and conversely its defects in RTT might alter the tissue integrity or function. We show here that a disorganized architecture, with hypotrophic fibres and tissue fibrosis, characterizes skeletal muscles retrieved from Mecp2-null mice. Alterations of the IGF-1/Akt/mTOR pathway accompany the muscle phenotype. A conditional mouse model selectively depleted of Mecp2 in skeletal muscles is characterized by healthy muscles that are morphologically and molecularly indistinguishable from those of wild-type mice raising the possibility that hypotonia in RTT is mainly, if not exclusively, mediated by non-cell autonomous effects. Our results suggest that defects in paracrine/endocrine signaling and, in particular, in the GH/IGF axis appear as the major cause of the observed muscular defects. Remarkably, this is the first study describing the selective deletion of Mecp2 outside the brain. Similar future studies will permit to unambiguously define the direct impact of MeCP2 on tissue dysfunctions.