Enrico Bugiardini

Co-segregation of DM2 with a recessive CLCN1 mutation in juvenile onset of myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is a common adult onset muscular dystrophy caused by a dominantly transmitted (CCTG)n expansion in intron 1 of the CNBP gene. In DM2 there is no obvious evidence for an intergenerational increase of expansion size, and no congenital cases have been confirmed. We describe the clinical and histopathological features, and provide the genetic and molecular explanation for juvenile onset of myotonia in a 14-year-old female with DM2 and her affected mother presenting with a more severe phenotype despite a later onset of symptoms. Histological and immunohistochemical findings correlated with disease severity or age at onset in both patients. Southern blot on both muscle and blood samples revealed only a small increase in the CCTG repeat number through maternal transmission. Fluorescence in situ hybridization, in combination with MBNL1 immunofluorescence on muscle sections, showed the presence of mutant mRNA and MBNL1 in nuclear foci; the fluorescence intensity and its area appeared to be similar in the two patients. Splicing analysis of the INSR, CLCN1 and MBNL1 genes in muscle tissue demonstrates that the level of aberrant splicing isoforms was lower in the daughter than in the mother. However, in the CLCN1 gene, a heterozygous mutation c.501C>G p.F167L was present in the daughter’s DNA and found to be maternally inherited. Biomolecular findings did not explain the unusual young onset in the daughter. The co-segregation of DM2 with a recessive CLCN1 mutation provided the explanation for the unusual clinical findings.

SCN4A mutation as modifying factor of myotonic dystrophy type 2 phenotype.

In myotonic dystrophy type 2 (DM2), an association has been reported between early and severe myotonia and recessive chloride channel (CLCN1) mutations. No DM2 cases have been described with sodium channel gene (SCN4A) mutations. The aim is to describe a DM2 patient with severe and early onset myotonia and co-occurrence of a novel missense mutation in SNC4A. A 26-year-old patient complaining of hand cramps and difficulty relaxing her hands after activity was evaluated at our department. Neurophysiology and genetic analysis for DM1, DM2, CLCN1 and SCN4A mutations were performed. Genetic testing was positive for DM2 (2650 CCTG repeat) and for a variant c.215C>T (p.Pro72Leu) in the SCN4A gene. The variation affects the cytoplasmic N terminus domain of Nav1.4, where mutations have never been reported. The biophysical properties of the mutant Nav1.4 channels were evaluated by whole-cell voltage-clamp analysis of heterologously expressed mutant channel in tsA201 cells. Electrophysiological studies of the P72L variant showed a hyperpolarizing shift (-5 mV) of the voltage dependence of activation that may increase cell excitability. This case suggests that SCN4A mutations may enhance the myotonic phenotype of DM2 patients and should be screened for atypical cases with severe myotonia.

Consensus on cerebral involvement in myotonic dystrophy: Workshop report: May 24–27, 2013, Ferrere (AT), Italy

Thirty-four clinicians, scientists and representatives from industries convened for a workshop on cerebral involvement in myotonic dystrophy (DM). The workshop was held in Ferrere (Asti) from May 24th to 27th 2013 and as the previous one [1] had the purpose to stimulate the research on CNS dysfunction in DM and to discuss major issues regarding CNS involvement. Classically viewed as a neuromuscular disease, for many years research on DM has been principally focused on muscular aspects. Therefore few data are available for CNS involvement. With new therapeutic developments with potential to affect pathophysiology across multiple tissues and organ systems it will be mandatory to increase our understanding of CNS pathophysiology in order to appropriately monitor whether or not there is beneficial effect in CNS. Indeed CNS dysfunction is one of the major issue affecting quality of life in DM patients thus it has to be appropriately considered in planning clinical trials. This workshop included different themes covering all the topics of DM CNS involvement. CNS research needs to proceed faster and consensus has to be established in the major themes of CNS study. The present report describes the major issues that emerged during the workshop, with the aim of updating and stimulating research in this critical field.

Overexpression of CUGBP1 in skeletal muscle from adult classic myotonic dystrophy type 1 but not from myotonic dystrophy type 2.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested.

Vitamin D, parathyroid hormone and muscle impairment in myotonic dystrophies

Parathyroid function in Myotonic Dystrophy (DM) patients has been poorly investigated. Parathyroid and muscle parameters were assessed in 31 male DM1 (44±2 years), 13 male DM2 (56±2 years) and 32 healthy controls. Hyperparathyroidism was diagnosed in 18% of patients without differences between DM types. In all DM patients, hyperparathyroidism was associated with normocalcemia but one with hypercalcemia. DM patients presented significantly higher PTH and lower vitamin D (25OHD) compared with controls, also considering seasonality. Severe vitamin D deficiency (25OHD<10 ng/ml) was diagnosed in 40% and hypovitaminosis D (25OHD<30 ng/ml) occurred in 88% of DM patients. About one-third of DM1 presented hypophosphatemia associated with elevated PTH levels. Serum 25OHD levels negatively correlated with PTH and with body fat mass. Considering DM1 patients, serum PTH levels positively correlated with CTG triplet repeats. Furthermore, PTH levels negatively correlated with total modified Medical Research Council (MRC) and positively with Muscular Impairment Rating Scale (MIRS). By contrast, in DM2 patients muscle assessment did not show any correlation with parathyroid function. In conclusion, we arrived at the following: 1) severe vitamin D deficiency is common in DM patients and it is associated with secondary hyperparathyroidism; 2) primary hyperparathyroidism, though rare, may occur; 3) increased adiposity in DM may be a risk factor for hypovitaminosis D; and 4) high serum PTH levels may indicate a muscle impairment, at least in DM1.

Genome Wide Identification of Aberrant Alternative Splicing Events in Myotonic Dystrophy Type 2

Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.

Progression of muscle histopathology but not of spliceopathy in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is an autosomal dominant progressive disease involving skeletal and cardiac muscle and brain. It is caused by a tetranucleotide repeat within the first intron of the CNBP gene that leads to an alteration of the alternative splicing of several genes. To understand the molecular mechanisms that play a role in DM2 progression, the evolution of skeletal muscle histopathology and biomolecular findings in successive biopsies have been studied. Biceps brachii biopsies from 5 DM2 patients who underwent two successive biopsies at different years of age have been used. Muscle histopathology has been assessed on sections immunostained with fast or slow myosin. FISH in combination with MBNL1-immunofluorescence has been performed to evaluate ribonuclear inclusion and MBNL1 foci dimensions in myonuclei. Gene and protein expression and alteration of alternative splicing of several genes have been evaluated over time. All DM2 patients examined show a worsening of muscle histopathology and an increase of foci dimensions over time. The progressive worsening of myotonia in DM2 patients may be due to the decrease of CLCN1 mRNA observed in all patients examined. However, a worsening of alternative splicing alterations has not been evidenced over time. The data obtained in this study confirm that DM2 is a slow progression disease since histological and biomolecular alterations observed in skeletal muscle are minimal even after 10-year interval. The data indicate that muscle morphological alterations evolve more rapidly over time than the molecular changes thus indicating that muscle biopsy is a more sensitive tool than biomolecular markers to assess disease progression at muscle level.

Gonadal failure is associated with visceral adiposity in myotonic dystrophies

Hypogonadism occurs in myotonic dystrophies type 1 (MD1) and type 2 (MD2). Sertoli and Leydig cell secretions, including insulin‐like peptide‐3 (INSL3), anti‐Müllerian hormone (AMH) and inhibin B, were evaluated in male patients with MD.

Muscle histopathology in a large cohor of 65 italian DM2 patients: diagnostic role and pitfalls of ribonuclear inclusions

The abstracts have been reviewed by: F. Antonaci, Z. Argov, I. Arnulf, A. Arzimanoglou, T. Back, O. Bajenaru, E. Bartels, P.D. Berlit, K. Bhatia, P. Boon, T. Brandt, B. Brochet, M.J. Brodie, A. Bronstein, H. Cock, G. Comi, J. de Keyser, M. de Visser, L. Deecke, R. Dengler, S. Di Donato, H.C. Diener, M. Dieterich, V. Dietz, M. Donaghy, M. Eraksoy, T. Ettlin, F. Fazekas, L. Ferini-Strambi, J. Ferro, M. Filippi, D. Galimberti, A. Grau, W. Grisold, O. Hardiman, H.P. Hartung, W. Heide, C. Helmchen, D.M. Hermann, G. Ickenstein, L. Kappos, R. Khatami, B. Kieseier, T. Klopstock, C. Krarup, G. Lammers, G. Lauria, A. Luft, P. Lyrer, Z. Martinovic, G. Mayer, S.I. Mellgren, G. Meola, R. Milo, I. Milonas, C. Möller, X. Montalban, G. Moonen, M. Mumenthaler, N. Nardocci, O. Nascimento, E. Nobile-Orazio, W.H. Oertel, M. Onofrj, D. Pareyson, Y. Parman, H.W. Pfister, D. Pohl, P. Portegies, J. Rees, H. Reichmann, P.F. Reyes, A. Rossetti, M. Rousseaux, E. Ruzicka, G. Said, J. Santamaria, E. Scarpini, N. Schaeren-Wiemers, B. Schalke, P. Schestatsky, E. Schmutzhard, J. Schoenen, M. Seeck, A. Sena, S. Sergay, V. Silani, M. Sinnreich, A. Siva, R. Soffietti, C. Sommer, A. Steck, G. Stoll, D. Straumann, E. Tolosa, A. Toscano, K.V. Toyka, H. Tumani, J. Valls-Solé, J. van Gijn, P. Vermersch, M.J. Vidailhet, R.D. Voltz, J. Wokke We would like to thank the reviewers for their precious help and asistance.Children with “pure” receptive hearing impairment (RHI) and with specific language impairment (SLI) are not supposed to have additional developmental disabilities. However, mental health problems (MHP) may be associated with communicative disorders, and should be detected. Also motor coordination disorder (DCD) has an overlap of 50% with language developmental disorders. Screening and early detection of mental health and motor problems might have considerable health benefits for children with SLI. Aim of this study was to assess MHP and DCD in pure RHI and SLI. Methods: In a sample of 104 children (mean age 6.6 (SD 0.9) years; boys 71%) with well established SLI and of 51 RHI children (5-8 years old), we applied the parental strengths and difficulties questionnaire (SDQ) to detect mental health problems and DCD-criteria. DCD was defined as not only severe difficulties on the Movement-ABC-test (total score P5) and 5 neurological disorders (M-ABC