Deregulated microRNAs in myotonic dystrophy type 2

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.     

Oxidative stress in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults. The genetic basis of DM1 consists of a mutational expansion of a repetitive trinucleotide sequence (CTG). The number of triplets expansion divides patients in four categories related to the molecular changes (E1, E2, E3, E4). The pathogenic mechanisms of multi-systemic involvement of DM1 are still unclear. DM1 has been suspected to be due to premature aging, that is known to be sustained by increased free radicals levels and/or decreased antioxidants activities in neurodegenerative disorders. Recently, the gain-of-function at RNA level hypothesis has gained great attention, but oxidative stress might act in the disease progression. We have investigated 36 DM1 patients belonging to 22 unrelated families, 10 patients with other myotonic disorders (OMD) and 22 age-matched healthy controls from the clinical, biochemical and molecular point of view. Biochemical analysis detected blood levels of superoxide dismutase (SOD), malonilaldehyde (MDA), vitamin E (Vit E), hydroxyl radicals (OH) and total antioxidant system (TAS). Results revealed that DM1 patients showed significantly higher levels of SOD (+40%; MAL (+57%; RAD 2 (+106%; and TAS (+20%; than normal controls. Our data support the hypothesis of a pathogenic role of oxidative stress in DM1 and therefore confirm the detrimental role played by free radicals in this pathology and suggest the opportunity to undertake clinical trials with antioxidants in this disorder.     

Imbalanced oxidant and antioxidant ratio in myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy affecting adults and is due to trinucleotide sequence (CTG) in the 3′ UTR region of DMPK gene located at 19q13.3 chromosome. The pathogenic mechanisms of multisystemic involvement of DM1 are still unclear. The increased levels of reactive oxygen species/free radicals and lipid peroxides and decreased antioxidant levels play an important role in the pathogenesis of DM1. Present study includes 20 DM1 patients and 40 age- and sex-matched controls. Malonilaldehyde (MDA), superoxide dismutase (SOD), glutathione peroxidise (GPX), glutathione-S-transferase (GST), reduced glutathione (GSH), and TAS levels were measured and its association with clinical phenotype were evaluated. Results revealed significantly higher levels of MDA (p = 0.002), SOD (p = 0.006), and TAS p = 0.004) and lower level of GPX (p = 0.003), GST (P < 0.001) and GSH (P = 0.016) in DM1 patients. A significant negative correlation of MDA level with dyspepsia and CK-MB and GST level with serum SCK, CK-MB, and diabetes were observed. However, a significant positive correlation of SOD level with serum CK-MB, CK-MM, and diabetes and negative correlation with facial weakness were noted. Though, GSH level had significant positive correlation with learning and writing disability, speech, and languages disability yet found negative correlation with duration of disease. The GPX and TAS showed no correlation with any clinical findings. Our data further support the pathogenic role of oxidative stress in DM1 of Indian origin and support the opportunity to undertake clinical trials with antioxidants in this disorder.     

Decreased DMPK transcript levels in myotonic dystrophy 1 type IIA muscle fibers

Concentrations of free polyamines were investigated in Trypanosoma granulosum cultured in a semidefined medium containing traces of polyamines. Spermidine content peaked in early logarithmic growth while putrescine was not detectable. Unlike African trypanosomes and Leishmania, spermine was measured at equivalent amounts to spermidine in mid to late logarithmic stage cells. Addition of d,l-alpha-difluoromethylornithine to cultures did not decrease polyamine content nor was ornithine decarboxylase activity detected. In contrast, incubation of parasites with tritiated putrescine showed rapid uptake and subsequent conversion to spermidine and spermine. At late logarithmic growth, parasites contained glutathione (77% of total sulphydryl groups) and ovothiol A as major low molecular mass thiols with glutathionylpolyamine conjugates undetectable. However, the addition of exogenous putrescine elevated trypanothione and glutathionylspermidine content to 48% of total sulphydryl groups. Correspondingly, the addition of exogenous cadaverine increased homotrypanothione content. This first report of polyamines and low molecular mass thiols in Trypanosoma granulosum indicates intriguing similarities with the metabolism of the human pathogen Trypanosoma cruzi.     

Brain-specific change in alternative splicing of Tau exon 6 in myotonic dystrophy type 1

Alternative splicing is altered in myotonic dystrophy of type 1 (DM1), a syndrome caused by an increase of CTG triplet repeats in the 3' untranslated region of the myotonic dystrophy protein kinase gene. Previously, we reported the preferential skipping of Tau exon 2 in DM1 brains. In this study, we analyze the alternative splicing of Tau exon 6 which can be inserted in three different forms (c, p and d) depending on the 3' splice site used. In fact, inclusion of exon 6c decreases in DM1 brains compared to control brains whereas inclusion of 6d increases. Alteration of exon 6 splicing was not observed in DM1 muscle although this exon was inserted in RNAs from normal muscle and DM1 splicing alterations were first described in this organ. In contrast, alteration of exon 2 of Tau mRNA was observed in both muscle and brain. However, co-transfections of a minigene containing exon 6 with CELF or MBNL1 cDNAs, two splicing factor families suspected to be involved in DM1, showed that they influence exon 6 splicing. Altogether, these results show the importance of determining all the exons and organs targeted by mis-splicing to determine the dysregulation mechanisms of mis-splicing in DM1.     

Altered signal transduction pathways and induction of autophagy in human myotonic dystrophy type 1 myoblasts

Congenital myotonic dystrophy type 1 (CDM1) affects patients from birth and is associated with mental retardation and impaired muscle development. CDM1 patients carry 1000–3000 CTG repeats in the DMPK gene and display defective skeletal muscles differentiation, resulting in reduced size of myotubes and decreased number of satellite cells. In this study, human myoblasts in culture deriving from control and DM1 embryos (3200 CTG repeats) were analyzed using both a biochemical and electron microscopic approach, in order to provide new insights into the molecular mechanisms underlying such alteration. Interestingly, electron microscopy analysis showed not only ultrastructural features of abnormal differentiation but also revealed the presence of autophagic vacuoles in DM1 myoblasts not undergoing differentiation. In accordance with the electron microscopic findings, the autophagic markers LC3 and ATG5, but not apoptotic markers, were significantly up regulated in DM1 myoblasts after differentiating medium addition. The induction of autophagic processes in DM1 myoblasts was concomitant to p53 over-expression and inhibition of the mTOR–S6K1 pathway, causatively involved in autophagy. Moreover biochemical alterations of the two main signal transduction pathways involved in differentiation were observed in DM1 myoblasts, in particular decreased activation of p38MAPK and persistent activation of the MEK–ERK pathway. This work, while demonstrating that major signaling pathways regulating myoblasts differentiation are profoundly deranged in DM1 myoblasts, for the first time provides evidence of autophagy induction, possibly mediated by p53 activation in response to metabolic stress which might contribute to the dystrophic alterations observed in the muscles of congenital DM1 patients.     

The myotonic dystrophy type 1 triplet repeat sequence induces gross deletions and inversions

The capacity of (CTG.CAG)n and (GAA.TTC)n repeat tracts in plasmids to induce mutations in DNA flanking regions was evaluated in Escherichia coli. Long repeats of these sequences are involved in the etiology of myotonic dystrophy type 1 and Friedreich's ataxia, respectively. Long (CTG.CAG)n (where n = 98 and 175) caused the deletion of most, or all, of the repeats and the flanking GFP gene. Deletions of 0.6-1.8 kbp were found as well as inversions. Shorter repeat tracts (where n = 0 or 17) were essentially inert, as observed for the (GAA.TTC)176-containing plasmid. The orientation of the triplet repeat sequence (TRS) relative to the unidirectional origin of replication had a pronounced effect, signaling the participation of replication and/or repair systems. Also, when the TRS was transcribed, the level of deletions was greatly elevated. Under certain conditions, 30-50% of the products contained gross deletions. DNA sequence analyses of the breakpoint junctions in 47 deletions revealed the presence of 1-8-bp direct or inverted homologies in all cases. Also, the presence of non-B folded conformations (i.e. slipped structures, cruciforms, or triplexes) at or near the breakpoints was predicted in all cases. This genetic behavior, which was previously unrecognized for a TRS, may provide the basis for a new type of instability of the myotonic dystrophy protein kinase (DMPK) gene in patients with a full mutation.     

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.     

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.     

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

Mutagenic stress modulates the dynamics of CTG repeat instability associated with myotonic dystrophy type 1

The molecular basis of the myotonic dystrophy type 1 is the expansion of a CTG repeat at the DMPK locus. The expanded disease-associated repeats are unstable in both somatic and germ lines, with a high tendency towards expansion. The rate of expansion is directly related to the size of the pathogenic allele, increasing the size heterogeneity with age. It has also been suggested that additional factors, including as yet unidentified environmental factors, might affect the instability of the expanded CTG repeats to account for the observed CTG size dynamics over time. To investigate the effect of environmental factors in the CTG repeat instability, three lymphoblastoid cell lines were established from two myotonic dystrophy patients and one healthy individual, and parallel cultures were concurrently expanded in the presence or absence of the mutagenic chemical mitomycin C for a total of 12 population doublings. The new alleles arising along the passages were analysed by radioactive small pool PCR and sequencing gels. An expansion bias of the stepwise mutation was observed in a (CTG)₁₂₄ allele of a cell line harbouring two modal alleles of 28 and 124 CTG repeats. Interestingly, this expansion bias was clearly enhanced in the presence of mitomycin C. The effect of mitomycin C was also evident in the normal size alleles in two cell lines with alleles of 13/13 and 12/69 repeats, where treated cultures showed new longer alleles. In conclusion, our results indicate that mitomycin C modulates the dynamics of myotonic dystrophy-associated CTG repeats in LBCLs, enhancing the expansion bias of long-pathogenic repeats and promoting the expansion of normal length repeats.     

Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1

Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in D...     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting a variety of organs, including the central nervous system. By using neuronal progeny derived from human embryonic stem cells carrying the causal DM1 mutation, we have identified an early developmental defect in genes involved in neurite formation and the establishment of neuromuscular connections. Differential gene expression profiling and quantitative RT-PCR revealed decreased expression of two members of the SLITRK family in DM1 neural cells and in DM1 brain biopsies. In addition, DM1 motoneuron/muscle cell cocultures showed alterations that are consistent with the known role of SLITRK genes in neurite outgrowth, neuritogenesis, and synaptogenesis. Rescue and knockdown experiments suggested that the functional defects can be directly attributed to SLITRK misexpression. These neuropathological mechanisms may be clinically significant for the functional changes in neuromuscular connections associated with DM1.     

Dynamic docking of small molecules targeting RNA CUG repeats causing myotonic dystrophy type 1

Expansion of RNA CUG repeats causes myotonic dystrophy type 1 (DM1). Once transcribed, the expanded CUG repeats strongly attract muscleblind-like 1 (MBNL1) proteins and disturb their functions in cells. Because of its unique structural form, expanded RNA CUG repeats are prospective drug targets, where small molecules can be utilized to target RNA CUG repeats to inhibit MBNL1 binding and ameliorate DM1-associated defects. In this contribution, we developed two physics-based dynamic docking approaches (DynaD and DynaD/Auto) and applied them to nine small molecules known to specifically target RNA CUG repeats. While DynaD uses a distance-based reaction coordinate to study the binding phenomenon, DynaD/Auto combines results of umbrella sampling calculations performed on 1 × 1 UU internal loops and AutoDock calculations to efficiently sample the energy landscape of binding. Predictions are compared with experimental data, displaying a positive correlation with correlation coefficient (R) values of 0.70 and 0.81 for DynaD and DynaD/Auto, respectively. Furthermore, we found that the best correlation was achieved with MM/3D-RISM calculations, highlighting the importance of solvation in binding calculations. Moreover, we detected that DynaD/Auto performed better than DynaD because of the use of prior knowledge about the binding site arising from umbrella sampling calculations. Finally, we developed dendrograms to present how bound states are connected to each other in a binding process. Results are exciting, as DynaD and DynaD/Auto will allow researchers to utilize two novel physics-based and computer-aided drug-design methodologies to perform in silico calculations on drug-like molecules aiming to target complex RNA loops.     

Risk prediction for clinical phenotype in myotonic dystrophy type 1: data from 2,650 patients

Myotonic dystrophy type 1 (DM1) is a multisystem disorder that affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system. DM1 is caused by expansion of a CTG trinucleotidedaggerrepeat in the gene DMPK. Clinical findings in DM1 span a continuum from mild to severe. Although the CTG repeat correlates with the disease phenotype, caution is used in predicting disease severity on the basis of CTG repeat number. This study reports an extensive genotype-phenotype study to evaluate the clinical validity and clinical utility of the molecular genetic test. Data were analyzed by multiple logistic regression, used to estimate the odds ratio (OR) and correlation coefficients for patients phenotype in respect to the categorical variables expansion class, gender, familiarity, and the continuous variables age and disease duration. We assessed disease expression by clinical evaluation and the molecular genetic test in 2,650 patients identified by accurate clinical diagnosis and family segregation. We were able to estimate OR and correlation coefficients for patients phenotype according to CTG number. A genotype-phenotype correlation was established to derivate a clinical predictive risk on the basis of molecular data. This study demonstrates that measurement of triplet expansions in patients' DNA can be considered as a useful tool for DM1 phenotype assessment and presymptomatic testing.