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.

Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Anti-Angiogenic Activity of Drugs in Multiple Myeloma.

Angiogenesis represents a pivotal hallmark of multiple myeloma (MM) that correlates to patients' prognosis, overall survival, and drug resistance. Hence, several anti-angiogenic drugs that directly target angiogenic cytokines (i.e., monoclonal antibodies, recombinant molecules) or their cognate receptors (i.e., tyrosine kinase inhibitors) have been developed. Additionally, many standard antimyeloma drugs currently used in clinical practice (i.e., immunomodulatory drugs, bisphosphonates, proteasome inhibitors, alkylating agents, glucocorticoids) show anti-angiogenic effects further supporting the importance of inhibiting angiogenesis from potentiating the antimyeloma activity. Here, we review the most important anti-angiogenic therapies used for the management of MM patients with a particular focus on their pharmacological profile and on their anti-angiogenic effect in vitro and in vivo. Despite the promising perspective, the direct targeting of angiogenic cytokines/receptors did not show a great efficacy in MM patients, suggesting the need to a deeper knowledge of the BM angiogenic niche for the design of novel multi-targeting anti-angiogenic therapies.

Coexistence of SCN4A and CLCN1 mutations in a family with atypical myotonic features: A clinical and functional study.

Non-dystrophic myotonias include several entities with possible clinical overlap, i.e. myotonia congenita caused by CLCN1 gene mutations, as well as paramyotonia congenita and sodium channel myotonia caused by SCN4A gene mutations. Herein, we describe the clinical features of five relatives affected by clinical and neurophysiological myotonia, with an aspecific and mixed phenotype. Next-generation sequencing identified the novel p.K1302R variant in SCN4A and the p.H838P variant in CLCN1. Segregation of the two mutations with the disease was confirmed by genotyping affected and non-affected family members. Patch-clamp experiments showed that sodium currents generated by p.K1302R and WT hNav1.4 were very similar. Mutant channel showed a small negative shift (5 mV) in the voltage-dependence of activation, which increased the likelihood of the channel to open at more negative voltages. The p.H838P mutation caused a reduction in chloride current density and a small voltage-dependence shift towards less negative potentials, in agreement with its position into the CBS2 domain of the C-terminus. Our results demonstrated that the mild functional alterations induced by p.K1302R and p.H838P in combination may be responsible for the mixed myotonic phenotypes. The K1302R mutant was sensitive to mexiletine and lamotrigine, suggesting that both drugs might be useful for the K1302R carriers.

Drug repurposing in skeletal muscle ion channelopathies.

Skeletal muscle ion channelopathies are rare genetic diseases mainly characterized by myotonia (muscle stiffness) or periodic paralysis (muscle weakness). Here, we reviewed the available therapeutic options in non-dystrophic myotonias (NDM) and periodic paralyses (PP), which consists essentially in drug repositioning to address stiffness or weakness attacks. Empirical use followed by successful randomized clinical trials eventually led to the orphan drug designation and marketing authorization granting of mexiletine for NDM and dichlorphenamide for PP. Yet, these treatments neither consider the genetic cause of the diseases nor address the individual variability in drug response. Thus, ongoing research aims at the identification of repurposed drugs alternative to mexiletine and dichlorphenamide to allow personalization of treatment. This review highlights how drug repurposing may represent an efficient strategy in rare diseases, allowing reduction of drug development time and costs in a context in which the return on investment may be particularly challenging.

Chaperone activity of niflumic acid on ClC-1 chloride channel mutants causing myotonia congenita.

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in "gating-defective" or "expression-defective" mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.

Integrated approach for legionellosis risk analysis in touristic-recreational facilities.

Legionellosis is a severe pneumonia caused by the inhalation of aerosols containing Legionella, Gram-negative bacteria present in the water systems of touristic-recreational facilities. The purpose of this study was to develop a scoring tool to predict the risk of both environmental contamination and Legionnaires' disease cases in such facilities in the Apulia region of southern Italy. We analyzed 47 structural and management parameters/risk factors related to the buildings, water systems, and air conditioning at the facilities. A Poisson regression model was used to compute an overall risk score for each facility with respect to three outcomes: water samples positive for Legionella (risk score range: 7-54), water samples positive for Legionella with an average load exceeding 1000 colony-forming units per liter (CFU/L) (risk score range: 22-179,871), and clinical cases of Legionnaire's disease (risk score range: 6-31). The cut-off values for three outcomes were determined by receiver operating characteristic curves (first outcome, samples positive for Legionella in a touristic-recreational facility: 19; second outcome, samples positive for Legionella in a touristic-recreational facility with an average load exceeding 1000 CFU/L: 2062; third outcome, clinical cases of Legionnaire's disease in a touristic-recreational facility: 22). Above these values, there was a significant probability of observing the outcome. We constructed this predictive model using 70% of a large dataset (18 years of clinical and environmental surveillance) and tested the model on the remaining 30% of the dataset to demonstrate its reliability. Our model enables the assessment of risk for a touristic facility and the creation of a conceptual framework to link the risk analysis with prevention measures.

First Detection of Severe Acute Respiratory Syndrome Coronavirus 2 on the Surfaces of Tourist-Recreational Facilities in Italy.

A Coronavirus disease (COVID-19), caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spreads via direct contact through droplets produced by infected individuals. The transmission of this virus can also occur via indirect contact if objects and surfaces are contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. Environmental contamination with SARS-CoV-2 is high in hospital settings; on the contrary, surface contamination in non-healthcare settings is still poorly studied. In this study, the presence of SARS-CoV-2 on the surfaces of 20 tourist-recreational facilities was investigated by performing a total of 100 swabs on surfaces, including refrigerator handles, handrails, counters, tables, and bathroom access doors. Six (6%) swabs from four (20%) tourist-recreational facilities tested positive for SARS-CoV-2; the surfaces that were involved were toilet door handles, refrigerator handles, handrails, and bar counters. This study highlights that SARS-CoV-2 is also present in non-healthcare environments; therefore, in order to limit this worrying pandemic, compliance with behavioral rules and the adoption of preventive and protective measures are of fundamental importance not only in healthcare or work environments but also in life environments.

Occurrence of Fungi in the Potable Water of Hospitals: A Public Health Threat.

Since the last decade, attention towards the occurrence of fungi in potable water has increased. Commensal and saprophytic microorganisms widely distributed in nature are also responsible for causing public health problems. Fungi can contaminate hospital environments, surviving and proliferating in moist and unsterile conditions. According to Italian regulations, the absence of fungi is not a mandatory parameter to define potable water, as a threshold value for the fungal occurrence has not been defined. This study evaluated the occurrence of fungi in potable water distribution systems in hospitals. The frequency of samples positive for the presence of fungi was 56.9%; among them, filamentous fungi and yeasts were isolated from 94.2% and 9.2% of the samples, respectively. The intensive care unit (87.1%) had the highest frequency of positive samples. Multivariable model (p < 0.0001), the variables of the period of the year (p < 0.0001) and type of department (p = 0.0002) were found to be statistically significant, suggesting a high distribution of filamentous fungi in the potable water of hospitals. Further studies are necessary to validate these results and identify the threshold values of fungi levels for different types of water used for various purposes to ensure the water is safe for consumption and protect public health.

Legionella and legionellosis in touristic-recreational facilities: Influence of climate factors and geostatistical analysis in Southern Italy (2001-2017).

Legionella is the causative agent of Legionnaires' disease, a flu-like illness normally acquired following inhalation or aspiration of contaminated water aerosols. Our recent studies revealed that climatic parameters can increase the number of reported cases of community-acquired Legionnaires' disease. Here, we evaluated the presence of Legionella in water networks and the distribution of Legionnaires' disease cases associated with touristic-recreational facilities in the Apulia region (southern Italy) during the period 2001-2017 using geostatistical and climatic analyses. Geostatistical analysis data revealed that the area with the highest concentration of Legionella in water systems also had the greatest number of cases of Legionnaires' disease associated with touristic-recreational facilities. Climatic analysis showed that higher daily temperature excursion (difference between maximum and minimum temperature) on the day of sampling was more often associated with Legionella-positive samples than Legionella-negative samples. In addition, our data highlighted an increased risk of Legionnaires' disease with increases in precipitation and average temperature and with decreases in daily temperature excursion (difference between maximum and minimum temperature over the course of 24 h in the days of incubation period of disease) and minimum temperature. Healthcare professionals should be aware of this phenomenon and be particularly vigilant for cases of community-acquired pneumonia during such climatic conditions and among the tourist population. The innovative geo-statistical approach used in this study could be applied in other contexts when evaluating the effects of climatic conditions on the incidence of Legionella infections.