Myotonia congenita in a Greek cohort: Genotype spectrum and impact of the CLCN1:c.501C > G variant as a genetic modifier.

INTRODUCTION/AIMS: Myotonia congenita (MC) is the most common hereditary channelopathy in humans. Characterized by muscle stiffness, MC may be transmitted as either an autosomal dominant (Thomsen) or a recessive (Becker) disorder. MC is caused by variants in the voltage-gated chloride channel 1 (CLCN1) gene, important for the normal repolarization of the muscle action potential. More than 250 disease-causing variants in the CLCN1 gene have been reported. This study provides an MC genotype-phenotype spectrum in a large cohort of Greek patients and focuses on novel variants and disease epidemiology, including additional insights for the variant CLCN1:c.501C > G. METHODS: Sanger sequencing for the entire coding region of the CLCN1 gene was performed. Targeted segregation analysis of likely candidate variants in additional family members was performed. Variant classification was based on American College of Medical Genetics (ACMG) guidelines. RESULTS: Sixty-one patients from 47 unrelated families were identified, consisting of 51 probands with Becker MC (84%) and 10 with Thomsen MC (16%). Among the different variants detected, 11 were novel and 16 were previously reported. The three most prevalent variants were c.501C > G, c.2680C > T, and c.1649C > G. Additionally, c.501C > G was detected in seven Becker cases in-cis with the c.1649C > G. DISCUSSION: The large number of patients in whom a diagnosis was established allowed the characterization of genotype-phenotype correlations with respect to both previously reported and novel findings. For the c.501C > G (p.Phe167Leu) variant a likely nonpathogenic property is suggested, as it only seems to act as an aggravating modifying factor in cases in which a pathogenic variant triggers phenotypic expression.

Clinical and molecular characterization of myotonia congenita using whole-exome sequencing in Egyptian patients.

BACKGROUND: Myotonia Congenita (MC) is a rare disease classified into two major forms; Thomsen and Becker disease caused by mutations in the CLCN1 gene, which affects muscle excitability and encodes voltage-gated chloride channels (CLC-1). While, there are no data regarding the clinical and molecular characterization of myotonia in Egyptian patients. METHODS: Herein, we report seven Egyptian MC patients from six unrelated families. Following the clinical diagnosis, whole-exome sequencing (WES) was performed for genetic diagnosis. Various in silico prediction tools were utilized to interpret variant pathogenicity. The candidate variants were then validated using Sanger sequencing technique. RESULTS: In total, seven cases were recruited. The ages at the examination were ranged from eight months to nineteen years. Clinical manifestations included warm-up phenomenon, hand grip, and percussion myotonia. Electromyography was performed in all patients and revealed myotonic discharges. Molecular genetic analysis revealed five different variants. Of them, we identified two novel variants in the CLCN1 gene ( c.1583G > C; p.Gly528Ala and c.2203_2216del;p.Thr735ValfsTer57) and three known variants in the CLCN1 and SCN4A gene. According to in silico tools, the identified novel variants were predicted to have deleterious effects. CONCLUSIONS: As the first study to apply WES among Egyptian MC patients, our findings reported two novel heterozygous variants that expand the CLCN1 mutational spectrum for MC diagnosis. These results further confirm that genetic testing is essential for early diagnosis of MC, which affects follow-up treatment and prognostic assessment in clinical practice.

Clinical and genetic characteristics of myotonia congenita in Chinese population.

Myotonia congenita (MC) is a rare hereditary muscle disease caused by variants in the CLCN1 gene. Currently, the correlation of phenotype-genotype is still uncertain between dominant-type Thomsen (TMC) and recessive-type Becker (BMC). The clinical data and auxiliary examinations of MC patients in our clinic were retrospectively collected. Electromyography was performed in 11 patients and available family members. Whole exome sequencing was conducted in all patients. The clinical and laboratory data of Chinese MC patients reported from June 2004 to December 2022 were reviewed. A total of 11 MC patients were included in the study, with a mean onset age of 12.64 ± 2.73 years. The main symptom was muscle stiffness of limbs. Warm-up phenomenon and percussion myotonia were found in all patients. Electromyogram revealed significant myotonic charges in all patients and two asymptomatic carriers, while muscle MRI and biopsy showed normal or nonspecific changes. Fourteen genetic variants including 6 novel variants were found in CLCN1. Ninety-eight Chinese patients were re-analyzed and re-summarized in this study. There were no significant differences in the demographic data, clinical characteristics, and laboratory findings between 52 TMC and 46 BMC patients. Among the 145 variants in CLCN1, some variants, including the most common variant c.892 G>A, could cause TMC in some families and BMC in others. This study expanded the clinical and genetic spectrum of Chinese patients with MC. It was difficult to distinguish between TMC and BMC only based on the clinical, laboratory, and genetic characteristics.

[A pedigree of myotonia congenita with a novel mutation p.F343C of the CLCN1 gene].

A Japanese woman experienced slowness of movement in her early teens and difficulty in opening her hands during pregnancy. On admission to our hospital at 42 years of age, she showed grip myotonia with warm-up phenomenon. However, she had neither muscle weakness, muscle atrophy, cold-induced symptomatic worsening nor episodes of transient weakness of the extremities. Needle electromyography of the first dorsal interosseous and anterior tibial muscles demonstrated myotonic discharges. Whole exome sequencing of the patient revealed a heterozygous single-base substitution in the CLCN1 gene (c.1028T>G, p.F343C). The same substitution was identified in affected members of her family (mother and brother) by Sanger sequencing, but not in healthy family members (father and a different brother). We diagnosed myotonia congenita (Thomsen disease) with a novel CLCN1 mutation in this pedigree. This mutation causes a single amino acid substitution in the I-J extracellular loop region of CLCN1. Amino acid changes in the I-J loop region are rare in an autosomal-dominantly inherited form of myotonia congenita. We think that this pedigree is precious to understand the pathogenesis of myotonia congenita.

Improving Diagnostic Precision: Phenotype-Driven Analysis Uncovers a Maternal Mosaicism in an Individual with RYR1-Congenital Myopathy.

Congenital myopathies (CMs) are rare genetic disorders for which the diagnostic yield does not typically exceed 60% . We performed deep phenotyping, histopathological studies, clinical exome and trio genome sequencing and a phenotype-driven analysis of the genomic data, that led to the molecular diagnosis in a child with CM. We identified a heterozygous variant in RYR1 in the affected child, inherited from her asymptomatic mother. Given the alignment of the clinical and histopathological phenotype with RYR1-CM, we considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother, as subsequently demonstrated. Our study is an example of how heterozygous variants inherited from asymptomatic parents are frequently dismissed. When the genotype-phenotype correlation is strong, it is recommended to consider a parental mosaicism.

A c.1775C > T Point Mutation of Sodium Channel Alfa Subunit Gene (SCN4A) in a Three-Generation Sardinian Family with Sodium Channel Myotonia.

Background: The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. Methods: Next-generation sequencing including the CLCN1 and SCN4A genes was performed in patients with clinical neuromuscular disorders. Electromyography, Short Exercise Test, in vivo and in vitro electrophysiology, site-directed mutagenesis and heterologous expression were collected. Results: A heterozygous point mutation (c.1775C > T, p.Thr592Ile) of muscle voltage-gated sodium channel α subunit gene (SCN4A) has been identified in five female patients over three generations, in a family with non-dystrophic myotonia. The muscle stiffness and myotonia involve mainly the face and hands, but also affect walking and running, appearing early after birth and presenting a clear cold sensitivity. Very hot temperatures, menstruation and pregnancy also exacerbate the symptoms; muscle pain and a warm-up phenomenon are variable features. Neither paralytic attacks nor post-exercise weakness has been reported. Muscle hypertrophy with cramp-like pain and increased stiffness developed during pregnancy. The symptoms were controlled with both mexiletine and acetazolamide. The Short Exercise Test after muscle cooling revealed two different patterns, with moderate absolute changes of compound muscle action potential amplitude. Conclusions: The p.Thr592Ile mutation in the SCN4A gene identified in this Sardinian family was responsible of clinical phenotype of myotonia.

Novel TUBA4A variant causes congenital myopathy with focal myofibrillar disorganisation.

BACKGROUND: Congenital myopathies are a clinical, histopathological and genetic heterogeneous group of inherited muscle disorders that are defined on peculiar architectural abnormalities in the muscle fibres. Although there have been at least 33 different genetic causes of the disease, a significant percentage of congenital myopathies remain genetically unresolved. The present study aimed to report a novel TUBA4A variant in two unrelated Chinese patients with sporadic congenital myopathy. METHODS: A comprehensive strategy combining laser capture microdissection, proteomics and whole-exome sequencing was performed to identify the candidate genes. In addition, the available clinical data, myopathological changes, the findings of electrophysiological examinations and thigh muscle MRIs were also reviewed. A cellular model was established to assess the pathogenicity of the TUBA4A variant. RESULTS: We identified a recurrent novel heterozygous de novo c.679C>T (p.L227F) variant in the TUBA4A (NM_006000), encoding tubulin alpha-4A, in two unrelated patients with clinicopathologically diagnosed sporadic congenital myopathy. The prominent myopathological changes in both patients were muscle fibres with focal myofibrillar disorganisation and rimmed vacuoles. Immunofluorescence showed ubiquitin-positive TUBA4A protein aggregates in the muscle fibres with rimmed vacuoles. Overexpression of the L227F mutant TUBA4A resulted in cytoplasmic aggregates which colocalised with ubiquitin in cellular model. CONCLUSION: Our findings expanded the phenotypic and genetic manifestations of TUBA4A as well as tubulinopathies, and added a new type of congenital myopathy to be taken into consideration in the differential diagnosis.

Brody Disease, an Early-Onset Myopathy With Delayed Relaxation and Abnormal Gait: A Case Series of 9 Children.

Brody disease is a rare autosomal recessive myopathy, caused by pathogenic variants in the ATP2A1 gene. It is characterized by an exercise-induced delay in muscle relaxation, often reported as muscle stiffness. Children may manifest with an abnormal gait and difficulty running. Delayed relaxation is commonly undetected, resulting in a long diagnostic delay. Almost all published cases so far were adults with childhood onset and adult diagnosis. With diagnostic next-generation sequencing, an increasing number of patients are diagnosed in childhood. We describe the clinical and genetic features of 9 children from 6 families with Brody disease. All presented with exercise-induced delayed relaxation, reported as difficulty running and performing sports. Muscle strength and mass was normal, and several children even had an athletic appearance. However, the walking and running patterns were abnormal. The diagnostic delay ranged between 2 and 7 years. Uniformly, a wide range of other disorders were considered before genetic testing was performed, revealing pathogenic genetic variants in ATP2A1. To conclude, this case series is expected to improve clinical recognition and timely diagnosis of Brody disease in children. We propose that ATP2A1 should be added to gene panels for congenital myopathies, developmental and movement disorders, and muscle channelopathies.

Contractile properties and magnetic resonance imaging-assessed fat replacement of muscles in myotonia congenita.

BACKGROUND AND PURPOSE: Myotonia congenita (MC) is a muscle channelopathy in which pathogenic variants in a key sarcolemmal chloride channel Gene (CLCN1) cause myotonia. This study used muscle magnetic resonance imaging (MRI) to quantify contractile properties and fat replacement of muscles in a Danish cohort of MC patients. METHODS: Individuals with the Thomsen (dominant) and Becker (recessive) variants of MC were studied. Isometric muscle strength, whole-body MRI, and clinical data were collected. The degree of muscle fat replacement of thigh, calf, and forearm muscles was quantitively calculated on Dixon MRI as fat fractions (FFs). Contractility was evaluated as the muscle strength per contractile muscle cross-sectional area (PT/CCSA). Muscle contractility was compared with clinical data. RESULTS: Intramuscular FF was increased and contractility reduced in calf and in forearm muscles compared with controls (FF = 7.0-14.3% vs. 5.3-9.6%, PT/CCSA = 1.1-4.9 Nm/cm2 vs. 1.9-5.8 Nm/cm2 [p < 0.05]). Becker individuals also showed increased intramuscular FF and reduced contractility of thigh muscles (FF = 11.9% vs. 9.2%, PT/CCSA = 1.9 Nm/cm2 vs. 3.2 Nm/cm2 [p < 0.05]). Individual muscle analysis showed that increased FF was limited to seven of 18 examined muscles (p < 0.05). There was a weak correlation between reduced contractility and severity of symptoms. CONCLUSIONS: Individuals with MC have increased fat replacement and reduced contractile properties of muscles. Nonetheless, changes were small and likely did not impact clinically on their myotonic symptoms.

Morphological and functional alterations of neuromuscular synapses in a mouse model of ACTA1 congenital myopathy.

Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ.

Function of a mutant ryanodine receptor (T4709M) linked to congenital myopathy.

Physiological muscle contraction requires an intact ligand gating mechanism of the ryanodine receptor 1 (RyR1), the Ca2+-release channel of the sarcoplasmic reticulum. Some mutations impair the gating and thus cause muscle disease. The RyR1 mutation T4706M is linked to a myopathy characterized by muscle weakness. Although, low expression of the T4706M RyR1 protein can explain in part the symptoms, little is known about the function RyR1 channels with this mutation. In order to learn whether this mutation alters channel function in a manner that can account for the observed symptoms, we examined RyR1 channels isolated from mice homozygous for the T4709M (TM) mutation at the single channel level. Ligands, including Ca2+, ATP, Mg2+ and the RyR inhibitor dantrolene were tested. The full conductance of the TM channel was the same as that of wild type (wt) channels and a population of partial open (subconductive) states were not observed. However, two unique sub-populations of TM RyRs were identified. One half of the TM channels exhibited high open probability at low (100 nM) and high (50 µM) cytoplasmic [Ca2+], resulting in Ca2+-insensitive, constitutively high Po channels. The rest of the TM channels exhibited significantly lower activity within the physiologically relevant range of cytoplasmic [Ca2+], compared to wt. TM channels retained normal Mg2+ block, modulation by ATP, and inhibition by dantrolene. Together, these results suggest that the TM mutation results in a combination of primary and secondary RyR1 dysfunctions that contribute to disease pathogenesis.

Effects of AFQ056 on language learning in fragile X syndrome.

BACKGROUNDFXLEARN, the first-ever large multisite trial of effects of disease-targeted pharmacotherapy on learning, was designed to explore a paradigm for measuring effects of mechanism-targeted treatment in fragile X syndrome (FXS). In FXLEARN, the effects of metabotropic glutamate receptor type 5 (mGluR5) negative allosteric modulator (NAM) AFQ056 on language learning were evaluated in 3- to 6-year-old children with FXS, expected to have more learning plasticity than adults, for whom prior trials of mGluR5 NAMs have failed.METHODSAfter a 4-month single-blind placebo lead-in, participants were randomized 1:1 to AFQ056 or placebo, with 2 months of dose optimization to the maximum tolerated dose, then 6 months of treatment during which a language-learning intervention was implemented for both groups. The primary outcome was a centrally scored videotaped communication measure, the Weighted Communication Scale (WCS). Secondary outcomes were objective performance-based and parent-reported cognitive and language measures.RESULTSFXLEARN enrolled 110 participants, randomized 99, and had 91 who completed the placebo-controlled period. Although both groups made language progress and there were no safety issues, the change in WCS score during the placebo-controlled period was not significantly different between the AFQ056 and placebo-treated groups, nor were there any significant between-group differences in change in any secondary measures.CONCLUSIONDespite the large body of evidence supporting use of mGluR5 NAMs in animal models of FXS, this study suggests that this mechanism of action does not translate into benefit for the human FXS population and that better strategies are needed to determine which mechanisms will translate from preclinical models to humans in genetic neurodevelopmental disorders.TRIAL REGISTRATIONClincalTrials.gov NCT02920892.FUNDING SOURCESNeuroNEXT network NIH grants U01NS096767, U24NS107200, U24NS107209, U01NS077323, U24NS107183, U24NS107168, U24NS107128, U24NS107199, U24NS107198, U24NS107166, U10NS077368, U01NS077366, U24NS107205, U01NS077179, and U01NS077352; NIH grant P50HD103526; and Novartis IIT grant AFQ056X2201T for provision of AFQ056.

Congenital Myopathy as a Phenotypic Expression of CACNA1S Gene Mutation: Case Report and Systematic Review of the Literature.

BACKGROUND: Congenital myopathies are a group of clinically, genetically, and histologically heterogeneous diseases caused by mutations in a large group of genes. One of these is CACNA1S, which is recognized as the cause of Dihydropyridine Receptor Congenital Myopathy. METHODS: To better characterize the phenotypic spectrum of CACNA1S myopathy, we conducted a systematic review of cases in the literature through three electronic databases following the PRISMA guidelines. We selected nine articles describing 23 patients with heterozygous, homozygous, or compound heterozygous mutations in CACNA1S and we added one patient with a compound heterozygous mutation in CACNA1S (c.1394-2A>G; c.1724T>C, p.L575P) followed at our Institute. We collected clinical and genetic data, muscle biopsies, and muscle MRIs when available. RESULTS: The phenotype of this myopathy is heterogeneous, ranging from more severe forms with a lethal early onset and mild-moderate forms with a better clinical course. CONCLUSIONS: Our patient presented a phenotype compatible with the mild-moderate form, although she presented peculiar features such as a short stature, myopia, mild sensorineural hearing loss, psychiatric symptoms, and posterior-anterior impairment gradient on thigh muscle MRI.

Emigration of Scientists From Czechoslovakia During the Soviet Domination.

Czechoslovakia was created after the First World War in 1918 as a common state of Czechs, Moravians, and Slovaks. After several transformations, 2 separate republics were established from Czechoslovakia in 1993: the Czech Republic and the Slovak Republic. The objective of this article was to analyze the Prague Spring (1968), the period after the invasion into Czechoslovakia by Warsaw Pact Troops (1968), the period of cruel normalization (1968-1989), and the influence of Soviet domination in the Czechoslovak Republic on people with higher education. The invasion of the Warsaw Pact Troops into Czechoslovakia and the period of normalization had a highly negative impact on the life and work of the Czechoslovak people. Many eminent scientists left the Republic. The reason for this was persecution for their attitude to the situation behind the Iron Curtain. Professor Jan Brod, a world-renowned nephrologist and cardiologist, one of the signatories of the Two Thousand Words Manifesto, emigrated to the Federal Republic of Germany in 1968. Professor William Ganz, a world-renowned cardiologist of Slovak origin, emigrated to the United States in 1966. With Jeremy Swan, he was a coinventor of the Swan-Ganz balloon flotation catheter. Primary reasons for the emigration of scientists from Czechoslovakia was the suppression of the nascent democracy (the Prague Spring in 1968 by the invasion of Warsaw Pact Troops and the continuation of Soviet rule).

Clinical and Genetic Spectrum of Myotonia Congenita in Turkish Children.

BACKGROUND: Myotonia congenita is the most common form of nondystrophic myotonia and is caused by Mendelian inherited mutations in the CLCN1 gene encoding the voltage-gated chloride channel of skeletal muscle. OBJECTIVE: The study aimed to describe the clinical and genetic spectrum of Myotonia congenita in a large pediatric cohort. METHODS: Demographic, genetic, and clinical data of the patients aged under 18 years at time of first clinical attendance from 11 centers in different geographical regions of Türkiye were retrospectively investigated. RESULTS: Fifty-four patients (mean age:15.2 years (±5.5), 76% males, with 85% Becker, 15% Thomsen form) from 40 families were included. Consanguineous marriage rate was 67%. 70.5% of patients had a family member with Myotonia congenita. The mean age of disease onset was 5.7 (±4.9) years. Overall 23 different mutations (2/23 were novel) were detected in 52 patients, and large exon deletions were identified in two siblings. Thomsen and Becker forms were observed concomitantly in one family. Carbamazepine (46.3%), mexiletine (27.8%), phenytoin (9.3%) were preferred for treatment. CONCLUSIONS: The clinical and genetic heterogeneity, as well as the limited response to current treatment options, constitutes an ongoing challenge. In our cohort, recessive Myotonia congenita was more frequent and novel mutations will contribute to the literature.

A complex CLCN1 variant associated with hereditary myotonia in a mixed-breed dog.

Hereditary myotonia (HM) is characterized by delayed muscle relaxation after contraction as a result of a mutation in the CLCN1 gene. We describe here a complex CLCN1 variant in a mixed-breed dog with clinical and electromyographic signs of HM. Blood samples from the myotonic dog, as well as from his male littermate and parents, were analyzed via amplification of the 23 exons encoding CLCN1. After sequencing the CLCN1 gene, a complex variant was found in exon 6 c.[705T>G; 708del; 712_732del], resulting in a premature stop codon in exon 7 and a protein that was 717 amino acids shorter than the normal CLC protein. The myotonic dog was identified as homozygous recessive for the complex CLCN1 variant; its parents were heterozygous, and its male littermate was homozygous wild-type. Knowledge of the CLCN1 mutations responsible for the development of hereditary myotonia allows greater clarification of this condition.

Neutral lipid storage disease with myopathy and myotonia associated to pathogenic variants on PNPLA2 and CLCN1 genes: case report.

BACKGROUND: Neutral lipid storage disease with myopathy (NLSD-M) is an autosomal recessive disease that manifests itself around the 3rd to 4th decade with chronic myopathy predominantly proximal in the shoulder girdle. Clinical myotonia is uncommon. We will report a rare case of association of pathogenic variants on PNPLA2 and CLCN1 genes with a mixed phenotype of NLSD-M and a subclinical form of Thomsen's congenital myotonia. CASE PRESENTATION: We describe a patient with chronic proximal myopathy, subtle clinical myotonia and electrical myotonia on electromyography (EMG). Serum laboratory analysis disclosure hyperCKemia (CK 1280 mg/dL). A blood smear analysis showed Jordan's anomaly, a hallmark of NLSD-M. A genetic panel was collected using next-generation sequencing (NGS) technique, which identified two pathogenic variants on genes supporting two different diagnosis: NLSD-M and Thomsen congenital myotonia, whose association has not been previously described. CONCLUSIONS: Although uncommon, it is important to remember the possibility of association of pathogenic variants to explain a specific neuromuscular disease phenotype. The use of a range of complementary methods, including myopathy genetic panels, may be essential to diagnostic definition in such cases.