Phenotypic Variability of Andersen-Tawil Syndrome Due to Allelic Mutation c.652C>T in the KCNJ2 Gene-A New Family Case Report.

Andersen-Tawil syndrome (ATS) is a multisystem channelopathy characterized by periodic paralysis, ventricular arrhythmias, prolonged QT interval, and facial dysmorphisms occurring in the first/second decade of life. High phenotypic variability and incomplete penetrance of the genes causing the disease make its diagnosis still a challenge. We describe a three-generation family with six living individuals affected by ATS. The proband is a 37-year-old woman presenting since age 16, with episodes of muscle weakness and cramps in the pre-menstrual period. The father, two brothers, one paternal uncle and one cousin also complained of cramps, muscle stiffness, and weakness. Despite normal serum potassium concentration, treatment with potassium, magnesium, and acetazolamide alleviated paralysis attacks suggesting a dyskalemic syndrome. Dysmorphic features were noted in the proband, only later. On the ECG, all but one had normal QT intervals. The affected males developed metabolic syndrome or obesity. The father had two myocardial infarctions and was implanted with an intracardiac cardioverter defibrillator (ICD). A genetic investigation by WES analysis detected the heterozygous pathogenic variant (NM_000891.2: c.652C>T, p. Arg218Trp) in the KCNJ2 gene related to ATS, confirmed by segregation studies in all affected members. Furthermore, we performed a review of cases with the same mutation in the literature, looking for similarities and divergences with our family case.

Digital health and Clinical Patient Management System (CPMS) platform utility for data sharing of neuromuscular patients: the Italian EURO-NMD experience.

BACKGROUND: The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients' data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients' data and an effective tool for data sharing; it facilitates specialists' consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. RESULTS: Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion-amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. CONCLUSIONS: In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a "map" of neuromuscular patients across Europe that might be improved by a wider use of CPMS.

Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement.

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16-29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a "non-carrier" status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1-15 and 30-34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16-29 deletion, while the second allele showed a 1-34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16-29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

CLCN1 Molecular Characterization in 19 South-Italian Patients With Dominant and Recessive Type of Myotonia Congenita.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia). It is caused by mutations in the CLCN1 gene, encoding the voltage-gated chloride channel of skeletal muscle, ClC-1. According to the pattern of inheritance, two distinct clinical forms have been described, Thomsen disease, inherited as an autosomal dominant trait and Becker disease inherited as an autosomal recessive trait. We report genetic and clinical data concerning 19 patients-13 familial and six isolated cases-all but one originating from the Campania Region, in southern Italy. Twelve patients (63.2%) present Becker type myotonia and 7 (36.8%) Thomsen type. Sex ratio M:F in Becker type is 6:6, while in Thomsen myotonia 4:3. The age of onset of the disease ranged from 2 to 15 years in Becker patients, and from 4 to 20 years in Thomsen. Overall 18 mutations were identified, 10 located in the coding part of the gene (exons 1, 3, 4, 5, 7, 8, 13, 15, 21, 22), and four in the intron part (introns 1, 2, 10, 18). All the exon mutations but two were missense mutations. Some of them, such as c.2551 G > A, c.817G > A and c.86A > C recurred more frequently. About 70% of mutations was inherited with an autosomal recessive pattern, two (c.86A and c.817G>A) with both mechanisms. Three novel mutations were identified, never described in the literature: p.Gly276Ser, p.Phe486Ser, and p.Gln812*, associated with Becker phenotype. Furthermore, we identified three CLCN1 mutations-c.86A>C + c.2551G > A, c.313C > T + c.501C > G and 899G > A + c.2284+5C > T, two of them inherited in cis on the same allele, in three unrelated families. The concomitant occurrence of both clinical pictures-Thomsen and Becker-was observed in one family. Intra-familial phenotypic variability was observed in two families, one with Becker phenotype, and one with Thomsen disease. In the latter an incomplete penetrance was hypothesized.

Cardiac diseases as a predictor warning of hereditary muscle diseases. The case of laminopathies.

Mutations in the LMNA gene are associated with a wide spectrum of disease phenotypes, ranging from neuromuscular, cardiac and metabolic disorders to premature aging syndromes. Skeletal muscle involvement may present with different phenotypes: limb-girdle muscular dystrophy type 1B or LMNA-related dystrophy; autosomal dominant Emery-Dreifuss muscular dystrophy; and a congenital form of muscular dystrophy, frequently associated with early onset of arrhythmias. Heart involvement may occur as part of the muscle involvement or independently, regardless of the presence of the myopathy. Notably conduction defects and dilated cardiomyopathy may exist without a muscle disease. This paper will focus on cardiac diseases presenting as the first manifestation of skeletal muscle hereditary disorders such as laminopathies, inspired by two large families with cardiovascular problems long followed by conventional cardiologists who did not suspect a genetic muscle disorder underlying these events. Furthermore it underlines the need for a multidisciplinary approach in these disorders and how the figure of the cardio-myo-geneticist may play a key role in facilitating the diagnostic process, and addressing the adoption of appropriate prevention measures.

The analysis of myotonia congenita mutations discloses functional clusters of amino acids within the CBS2 domain and the C-terminal peptide of the ClC-1 channel.

Myotonia congenita (MC) is a skeletal-muscle hyperexcitability disorder caused by loss-of-function mutations in the ClC-1 chloride channel. Mutations are scattered over the entire sequence of the channel protein, with more than 30 mutations located in the poorly characterized cytosolic C-terminal domain. In this study, we characterized, through patch clamp, seven ClC-1 mutations identified in patients affected by MC of various severities and located in the C-terminal region. The p.Val829Met, p.Thr832Ile, p.Val851Met, p.Gly859Val, and p.Leu861Pro mutations reside in the CBS2 domain, while p.Pro883Thr and p.Val947Glu are in the C-terminal peptide. We showed that the functional properties of mutant channels correlated with the clinical phenotypes of affected individuals. In addition, we defined clusters of ClC-1 mutations within CBS2 and C-terminal peptide subdomains that share the same functional defect: mutations between 829 and 835 residues and in residue 883 induced an alteration of voltage dependence, mutations between 851 and 859 residues, and in residue 947 induced a reduction of chloride currents, whereas mutations on 861 residue showed no obvious change in ClC-1 function. This study improves our understanding of the mechanisms underlying MC, sheds light on the role of the C-terminal region in ClC-1 function, and provides information to develop new antimyotonic drugs.

Therapeutic approach with Ataluren in Duchenne symptomatic carriers with nonsense mutations in dystrophin gene. Results of a 9-month follow-up in a case report.

Duchenne muscular Dystrophy (DMD) is a X-linked degenerative disorder affecting skeletal muscles and myocardium caused by mutations in the dystrophin gene, mainly deletions and duplications. Point-mutations account for 13% and stop codon mutations are even more unfrequent. A drug treatment for patients with DMD caused by stop codon gene mutations and still ambulant, has become recently available, based on the clear demonstration of its efficacy in slowing the course of the disease. The drug is able to read through the stop codon; furthermore it has the advantage of an oral administration and a better patient's compliance. We report a case of a still ambulant 27 year-old DMD symptomatic carrier with a stop-codon mutation in exon 53 (c.7792C > T; p.Gln2598Stop), who started the treatment with Ataluren at a dosage of 2,250 mg/die, reporting a prompt subjective improvement in muscle strength. Unfortunately two months after, the patient discontinued taking the drug for a traumatic femur fracture requiring surgical repair and prolonged rehabilitation. With the resumption of the drug intake in February 2018, the patient reported almost immediately an improvement in motor skills, including the possibility of recovering walking, first with support and then unsupported. These results seem even more encouraging, as Duchenne patients hardly recover the ability to walk following a fracture at this age and extend the possibility to treat with ataluren also the symptomatic Duchenne carriers who have nonsense dystrophin gene mutations. Furthermore the case here reported supports the concept that symptomatic DMD female carriers must enjoy the same therapeutic opportunities offered to males.

Heart transplantation in patients with dystrophinopathic cardiomyopathy: Review of the literature and personal series.

Cardiomyopathy associated with dystrophinopathies [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy (XL-dCM) and cardiomyopathy of Duchenne/Becker (DMD/BMD) carriers] is an increasing recognized manifestation of these neuromuscular disorders and notably contributes to their morbidity and mortality. Dystrophinopathic cardiomyopathy (DCM) is the result of the dystrophin protein deficiency at the myocardium level, parallel to the deficiency occurring at the skeletal muscle level. It begins as a "presymptomatic" stage in the first decade of life and evolves in a stepwise manner toward pictures of overt cardiomyopathy (hypertrophic stage, arrhythmogenic stage and dilated cardiomyopathy). The final stage caused by the extensive loss of cardiomyocytes results in an irreversible cardiac failure, characterized by frequent episodes of acute congestive heart failure (CHF), despite a correct pharmacological treatment. The picture of a severe dilated cardiomyopathy with intractable heart failure is typical of BMD, XL-dCM and cardiomyopathy of DMD/BMD carriers, while it is less frequently observed in patients with DMD. Heart transplantation (HT) is the only curative therapy for patients with dystrophinopathic end-stage heart failure who remain symptomatic despite an optimal medical therapy. However, no definitive figures exist in literature concerning the number of patients with DCM transplanted, and their outcome. This overview is to summarize the clinical outcomes so far published on the topic, to report the personal series of dystrophinopathic patients receiving heart transplantation and finally to provide evidence that heart transplantation is a safe and effective treatment for selected patients with end-stage DCM.

Diagnosis of Duchenne Muscular Dystrophy in Italy in the last decade: Critical issues and areas for improvements.

Despite all the advances in diagnosis and management of Duchenne muscular dystrophy over the past 50 years, the average age at diagnosis in most countries in the world around is still around 4-5 years. This retrospective study investigates the age at diagnosis in Italy in the past 10 years. We report findings from 384 boys who were diagnosed with DMD from 2005 to 2014. The mean age at first medical contact, which raised the suspicion of DMD, was 31 months. The mean age at diagnosis was 41 months. The finding that more frequently brought to suspect a DMD was the incidental finding of consistent elevated creatine kinase serum level detected during routine assessments in children undergoing general anesthesia or with intercurrent illness. This was followed by motor delay and signs of muscle weakness. Initial concerns were raised by general pediatricians (29%), specialists at tertiary centers (35%) or first level hospitals (23%). In children presenting incidental elevated creatine kinase values the diagnosis was achieved earlier than in children presenting a developmental delay. The mean age at diagnosis in our cohort was about 10-12 months lower than that reported in other countries.

Brachydactyly type E in an Italian family with 6p25 trisomy.

Brachydactyly type E is a congenital limb malformation characterized by small hands and feet as a result of shortened metacarpals and metatarsals. Genetic causes of this anomaly are heterogeneous and only partially characterized. In this report we describe an Italian family in which four subjects share brachydactyly type E and a 3 Mb microduplication in region 6p25. The duplication involves the gene FOXC1, expressed during the osteoblast differentiation, which appears a potential candidate gene for brachydactyly.

Is the epicardial left ventricular lead implantation an alternative approach to percutaneous attempt in patients with Steinert disease? A case report.

Steinert's disease or Myotonic Dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder characterized by myotonia, muscle and facial weakness, cataracts, cognitive, endocrine and gastrointestinal involvement, and cardiac conduction abnormalities. Although mild myocardial dysfunction may be detected in this syndrome with age, overt myocardial dysfunction with heart failure is not frequent. Cardiac resynchronization therapy is an effective treatment to improve morbidity and reduce mortality in patients with DM1 showing intra-ventricular conduction delay and/or congestive heart failure. We report the case of a patient with Steinert disease showing an early onset ventricular dysfunction due to chronic right ventricular apical pacing, in which an epicardial left ventricular lead implantation was performed following the failure of the percutaneous attempt. As no relief in symptoms of heart failure, nor an improvement of left ventricular ejection fraction and reverse remodelling was observed six months later, the patient was addressed to the heart transplantation.

The genetic basis of undiagnosed muscular dystrophies and myopathies: Results from 504 patients.

OBJECTIVE: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. METHODS: We applied an NGS-based platform named MotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. RESULTS: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30% of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. CONCLUSIONS: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.

TMEM5-associated dystroglycanopathy presenting with CMD and mild limb-girdle muscle involvement.

The dystroglycanopathies, which are caused by reduced glycosylation of alpha-dystroglycan, are a heterogeneous group of neurodegenerative disorders characterized by variable brain and skeletal muscle involvement. Recently, mutations in TMEM5 have been described in severe dystroglycanopathies. We present the clinical, molecular and neuroimaging features of an Italian boy who had delayed developmental milestones with mild limb-girdle muscle involvement, bilateral frontotemporal polymicrogyria, moderate intellectual disability, and no cerebellar involvement. He also presented a cochlear dysplasia and harbored a reported mutation (p.A47Rfs*42) in TMEM5, detected using targeted next-generation sequencing. The relatively milder muscular phenotype and associated structural brain abnormalities distinguish this case from previously reported patients with severe dystroglycanopathies and expand the spectrum of TMEM5-associated disorders.

Timed Rise from Floor as a Predictor of Disease Progression in Duchenne Muscular Dystrophy: An Observational Study.

BACKGROUND: The role of timed items, and more specifically, of the time to rise from the floor, has been reported as an early prognostic factor for disease progression and loss of ambulation. The aim of our study was to investigate the possible effect of the time to rise from the floor test on the changes observed on the 6MWT over 12 months in a cohort of ambulant Duchenne boys. SUBJECTS AND METHODS: A total of 487 12-month data points were collected from 215 ambulant Duchenne boys. The age ranged between 5.0 and 20.0 years (mean 8.48 ±2.48 DS). RESULTS: The results of the time to rise from the floor at baseline ranged from 1.2 to 29.4 seconds in the boys who could perform the test. 49 patients were unable to perform the test at baseline and 87 at 12 month The 6MWT values ranged from 82 to 567 meters at baseline. 3 patients lost the ability to perform the 6mwt at 12 months. The correlation between time to rise from the floor and 6MWT at baseline was high (r = 0.6, p<0.01). CONCLUSIONS: Both time to rise from the floor and baseline 6MWT were relevant for predicting 6MWT changes in the group above the age of 7 years, with no interaction between the two measures, as the impact of time to rise from the floor on 6MWT change was similar in the patients below and above 350 m. Our results suggest that, time to rise from the floor can be considered an additional important prognostic factor of 12 month changes on the 6MWT and, more generally, of disease progression.

Management of cardiac involvement in muscular dystrophies: paediatric versus adult forms.

Muscular dystrophies are a group of genetic disorders characterized by muscle degeneration and consequent substitution by fat and fibrous tissue. Cardiac involvement is an almost constant feature in a great part of these diseases, as both primary myocardial involvement and secondary involvement due to respiratory insufficiency, pulmonary hypertension or reduced mobility. Primary myocardial involvement usually begins more precociously compared to the secondary involvement. In fact the first signs of cardiomyopathy can be observed in the first decade of life in muscular dystrophies with childhood onset and later in adult form of muscular dystrophies as myotonic dystrophy type 1. At least an annual cardiac follow-up is recommended in these patients including clinical and instrumental examination (ECG, 24h Holter monitoring, ECHO), to detect cardiac involvement. A more frequent monitoring may be required according to the type of cardiomyopathy and the patient's needs. In this short review practical guide-lines are shown for physicians routinely involved in the management of these patients.

Increased heterogeneity of ventricular repolarization in myotonic dystrophy type 1 population.

Sudden cardiac death in myotonic dystrophy type I (DM1) patients can be attributed to atrioventricular blocks as far as to the development of life-threatening arrhythmias which occur even in hearts with normal left ventricular systolic and diastolic function. Heterogeneity of ventricular repolarization is considered to provide an electrophysiological substrate for malignant arrhythmias. QTc dispersion (QTc-D), JTc dispersion (JTc-D) and transmural dispersion of repolarization (TDR) could reflect the physiological variability of regional and transmural ventricular repolarization. Aim of the present study was to investigate the heterogeneity of ventricular repolarization in patients with DM1 and preserved diastolic and systolic cardiac function. The study enrolled 50 DM1 patients (mean age 44 ± 5 years; M:F: 29:21) with preserved systolic and diastolic function of left ventricle among 247 DM1 patients followed at Cardiomyology and Medical Genetics of Second University of Naples, and 50 sexand age-matched healthy controls. The electrocardiographic parameters investigated were the following: Heart Rate, QRS duration, maximum and minimum QT and JT intervals, QTc- D, JTc-D and TDR. Compared to the controls, the DM1 group presented increased values of QTc-D (86.7 ± 40.1 vs 52.3 ± 11.9 ms; p = 0.03), JTc-D (78.6 ± 31.3 vs 61.3 ± 10.2 ms; p = 0.001) and TDR (101.6 ± 18.06 vs 90.1 ± 14.3 ms; p = 0.004) suggesting a significant increase in regional and transmural heterogeneity of the ventricular repolarization in these patients, despite a preserved systolic and diastolic cardiac function.

Clinical features of patients with dystrophinopathy sharing the 45-55 exon deletion of DMD gene.

Becker muscular dystrophy (BMD) was first described in 1953 by Emile Becker as a benign variant of Duchenne muscular Dystrophy (DMD). Compared with DMD, BMD is clinically more heterogeneous, with initial presentation in the teenage years and loss of ambulation beyond the age of 16 and a wide spectrum of clinical presentations, ranging from only myalgias and muscle cramps to exercise intolerance and myoglobinuria, asymptomatic elevation of serum creatin-kinase, or mild limb-girdle weakness and quadriceps myopathy. About 50% of patients become symptomatic by the age of 10 and the most part by the age of 20 years. However few patients can be free of symptoms till their fifties and cases of late-onset Becker Muscular Dystrophy have also been described. In this report we describe the clinical features of patients with dystrophinopathy sharing a deletion of exons 45-55, occasionally or retrospectively diagnosed. These data are important for both the prognostic aspects of children presenting this dystrophin gene mutation, and for the genetic counseling in these families (reassuring them on the benign course of the disease), and last but not least to keep in mind a diagnosis of BMD in asymptomatic adults with mild hyperckemia.

Clinical and genetic spectrum in limb-girdle muscular dystrophy type 2E.

OBJECTIVE: To determine the clinical spectrum of limb-girdle muscular dystrophy 2E (LGMD2E) and to investigate whether genetic or biochemical features can predict the phenotype of the disease. METHODS: All LGMD2E patients followed in participating centers were included. A specific clinical protocol was created, including quantitative evaluation of motor, respiratory, and cardiac function. Phenotype was defined as severe or mild if the age at loss of ambulation occurred before or after 18 years. Molecular analysis of SGCB gene and biochemical features of muscle biopsies were reviewed. RESULTS: Thirty-two patients were included (16 male, 16 female; age 7-67 years; 15 severe, 12 mild, and 5 unknown). Neurologic examination showed proximal muscle weakness in all patients, but distal involvement was also observed in patients with severe disease early in the disease course. Cardiac involvement was observed in 20 patients (63%) even before overt muscle involvement. Six patients had restrictive respiratory insufficiency requiring assisted ventilation (19%). Seventeen different mutations were identified, and 3 were recurrent. The c.377_384dup (13 alleles) was associated with the severe form, the c.-22_10dup (10) with the milder form, and the c.341C>T (9) with both. The entire sarcoglycan complex was undetectable by muscle immunohistochemistry or Western blot in 9/10 severe cases and reduced in 7/7 mild cases. The residual amount of sarcoglycan in muscle resulted a predictor of age at loss of ambulation. CONCLUSIONS: This study expands the spectrum of phenotype in ß-sarcoglycanopathy and provides strong evidence that severity of clinical involvement may be predicted by SGCB gene mutation and sarcoglycan protein expression.

Novel mutations in LMNA A/C gene and associated phenotypes.

Mutations in the lamin A/C gene (LMNA) have been associated with several phenotypes ranging from systemic to prevalent of muscle, heart, skin, nerve etc. More recently they have been associated with dilated cardiomyopathy (DCM) and severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). We report four novel mutations - 3 missense and 1 deletion - in 4 unrelated patients showing different phenotypes, ranging from the early onset congenital form of laminopathy to classical LGMD phenotype, to LGMD and heart involvement. All these newly identified variants were not found in 300 ethnicallymatched control subjects. The variant c.103-105del CTG was described post-mortem in a young patient with congenital muscular dystrophy who presented at the age of 9 a first degree A-V block and subsequently several episodes of supraventricular parossystic tachycardia. Two patients presented as onset symptom lower limbs muscle weakness, and developed heart conduction defects requiring pacemaker implantation at the age of 26 and 38 years, respectively. One of them who carried the mutation c.1339G>C died at the age of 40 by intractable heart failure; the second one carrying the mutation 265C>T died at the age of 30, for a trmboembolic event. A classical LGMD phenotype without heart involvement was observed in the patient with the mutation 1579C>T, who died at the age of 68 years for respiratory insufficiency.