Raffaella Brugnoni

Dystrophin characterization in BMD patients: correlation of abnormal protein with clinical phenotype

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

A large cohort of myotonia congenita probands: novel mutations and a high-frequency mutation region in exons 4 and 5 of the CLCN1 gene

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.

Identification of Three Novel Mutations in the Major Human Skeletal Muscle Chloride Channel Gene (CLCN1), Causing Myotonia Congenita

Myotonia congenita (MC) is a genetic disease characterized by mutations in the CLCN1 gene (OMIM*118425) encoding the skeletal muscle voltage‐gated chloride channel (ClC‐1). Autosomal dominant and recessive forms are observed, characterized by impaired muscle relaxation after forceful contraction (myotonia), which is more pronounced after inactivity and improves with exercise. We report three novel and one known mutations of the CLCN1 gene in four unrelated MC families. In two families the mutations were missense: 803C>T (T268M) and 1272C>G (I424M) in exons 7 and 12, respectively. The third was a splice mutation in intron 5 (696+2T>A), which induced a frame shift with a stop codon in exon 6 (fs213X). In the fourth family the previously‐reported missense mutation 689G>A (G230E) was found. We also report two known polymorphisms: 261C>T (T87T) and 2154T>C (D718D) in exons 2 and 17 of two MC families; also found in 14 (33%) and 28 (67%) of 42 healthy controls, respectively. These findings expand our knowledge of mutations responsible for myotonia congenita, reducing the proportion of MC patients in whom genetic alterations have not been found. Hum Mutat 14:447, 1999.

ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype–phenotype correlation

Loss‐of‐function mutations of the skeletal muscle ClC‐1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right‐shifting the slow gating of ClC‐1 channel, without exerting a dominant‐negative effect on the wild‐type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H‐bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC‐1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT‐like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC‐1 function.

Marked phenotypic variability in two siblings with congenital myasthenic syndrome due to mutations in MUSK.

Congenital myasthenic syndromes (CMS) are rare genetic diseases of the neuromuscular junction with 18 causative genes identified to date [1]. Among many patients investigated worldwide, MUSK mutations were identified in only nine patients [2–5]. Here, we describe clinical, neurophysiological and molecular features of two Italian siblings who carry compound heterozygous mutations in MUSK, one of which is novel. The first sibling is a 27-year-old woman. At birth, she had laryngeal stridor and respiratory failure requiring mechanical ventilation and tube feeding during the first month of life. She had recurring respiratory failure at age 3 months and frequent pulmonary infections during infancy. Her subsequent clinical course was characterized by bilateral fluctuating eyelid ptosis, ophthalmoparesis, mild dysphagia and exertional dyspnoea, severe upper and lower limb weakness. Menstrual periods and fever worsened her bulbar symptoms and fatigability. At age 8 years, a right deltoid muscle biopsy done elsewhere showed muscle fiber atrophy and type I fiber predominance. At age 13 years, a biopsy of left biceps brachii was interpreted as normal. At age 18 years, 3-Hz repetitive stimulation of the axillary nerve elicited a 37 % decrement and needle EMG revealed a diffuse myopathic pattern. She was treated with 240 mg/day pyridostigmine but this soon had to be discontinued due to rapidly worsening symptoms and severe side effects. At age 26 years, she was readmitted with a waddling gait, bilateral eyelid ptosis, oculoparesis on horizontal and vertical gaze, double vision on extreme lateral gaze, mild tongue weakness and atrophy, mild nasal speech, severe orbicularis oculi and oris weakness, severe proximal and neck flexors muscle

A new non-radioactive method for the screening and prenatal diagnosis of myotonic dystrophy patients.

Abstract Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with an estimated incidence of 1 in 8000 and is the most common form of muscular dystrophy affecting adults. An unstable, untranslated part of the myotonic dystrophy protein kinase gene on the long arm of chromosome 19, composed of CTG repeats, is a genetic marker for DM. We have developed a fast non-radioactive polymerase chain reaction (PCR) procedure to detect the (CTG)n repeat expansion in DM patients and their relatives. Genomic DNA extracted from peripheral blood lymphocytes was amplified by PCR using specific primers to flank the region containing the triplets. To improve the amplification of this CG-rich region, either 10% glycerol or rTth DNA polymerase XL (extra long) was added to the reaction mixture, allowing amplification of huge expansions otherwise not polymerized by PCR. The PCR products were Southern blotted and the expansion revealed using a fluorescein-labelled (CTG)10 probe. We compared our results with those obtained in 24 patients and relatives using genomic digestion followed by radioactive Southern blot; in all cases the results overlapped. The same technique was used for prenatal diagnosis in pregnant DM mothers. We conclude that this new method is reliable for the genetic testing of DM patients.

Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies

Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss‐of‐function mutations in the skeletal muscle ClC‐1 channel. We report a novel ClC‐1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I‐J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch‐clamp electrophysiology on T335N and ClC‐1 wild‐type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC‐1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I‐J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K+ channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.—Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj, K., Caloiero, R., Alberga, D., Marsano, R.M., Ricci, G., Siciliano, G., Nicolotti, O., Mora, M., Bernasconi, P., Desaphy, J.‐F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new CIC‐1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies. FASEB J. 30, 3285–3295 (2016). www.fasebj.org

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.