Pompe’s disease is a progressive myopathy caused by mutations in the lysosomal enzyme acid alphaglucosidase gene (GAA). A wide clinical variability occurs also in patients sharing the same GAA mutations, even within the same family.
For a large series of GSDII patients we collected some clinical data as age of onset of the disease, presence or absence of muscular pain, Walton score, 6-Minute Walking Test, Vital Capacity, and Creatine Kinase. DNA was extracted and tested for GAA mutations and some genetic polymorphisms able to influence muscle properties (ACE, ACTN3, AGT and PPARα genes).
We compared the polymorphisms analyzed in groups of patients with Pompe disease clustered for their homogeneous genotype.
We have been able to identify four subgroups of patients completely homogeneous for their genotype, and two groups homogeneous as far as the second mutation is defined “very severe” or “potentially less severe”. When disease free life was studied we observed a high significant difference between groups. The DD genotype in the ACE gene and the XX genotype in the ACTN3 gene were significantly associated to an earlier age of onset of the disease. The ACE DD genotype was also associated to the presence of muscle pain.
We demonstrate that ACE and ACTN3 polymorphisms are genetic factors able to modulate the clinical phenotype of patients affected with Pompe disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-014-0102-z) contains supplementary material, which is available to authorized users.
Glycogen storage disease type II; Genetic polymorphisms; Modifier genes; GAA; ACE; ACTN3
A polymorphism (rs28357094) in the promoter region of the SPP1 gene coding for osteopontin (OPN) is a strong determinant of disease severity in Duchenne muscular dystrophy (DMD). The rare G allele of rs28357094 alters gene promoter function and reduces mRNA expression in transfected HeLa cells. To dissect the molecular mechanisms of increased disease severity associated with the G allele, we characterized SPP1 mRNA and protein in DMD muscle biopsies of patients with defined rs28357094 genotype. We did not find significant differences in osteopontin mRNA or protein expression between patients carrying the T (ancestral allele) or TG/GG genotypes at rs28357094. The G allele was significantly associated with reduced CD4+ and CD68+ cells on patient muscle biopsy. We also quantified transforming growth factor-β (TGFB) and TGFB receptor-2 (TGFBR2) mRNA in DMD muscle biopsies, given the ability of TGFB and TGFBR2 to activate SPP1 promoter region and their role in DMD pathogenesis. The amount of TGFB and TGFBR2 mRNA did not predict the amount of SPP1 mRNA or protein, while a polymorphism in the TGFBR2 gene (rs4522809) was found to be a strong predictor of SPP1 mRNA level. Our findings suggest that OPN mediates inflammatory changes in DMD and that TGFB signalling has a role in the complex regulation of osteopontin expression.
Duchenne muscular dystrophy; osteopontin; SPP1; TGFB; TGFBR2
Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognized (types I–III). All patients usually have 2–4 copies of a highly homologous gene (SMN2), which produces insufficient levels of functional survival motor neuron (SMN) protein due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-min walk test, myometry, forced vital capacity, and dual X-ray absorptiometry. Molecular assessments included SMN2 copy number, levels of full-length SMN2 (SMN2-fl) transcripts and those lacking exon 7 and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, whereas motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.
spinal muscular atrophy; SMN; biomarker; outcome measure; real-time PCR
Protein-o-mannosyl transferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan (α-DG) glycosylation. Clinical phenotype in POMT1-mutated patients ranges from congenital muscular dystrophy (CMD) with structural brain abnormalities, to limb-girdle muscular dystrophy (LGMD) with microcephaly and mental retardation, to mild LGMD. No cardiac involvement has until now been reported in POMT1-mutated patients. We report three patients who harbored compound heterozygous POMT1 mutations and showed left ventricular (LV) dilation and/or decrease in myocardial contractile force: two had a LGMD phenotype with a normal or close-to-normal cognitive profile and one had CMD with mental retardation and normal brain MRI. Reduced or absent α-DG immunolabeling in muscle biopsies were identified in all three patients. Bioinformatic tools were used to study the potential effect of POMT1-detected mutations. All the detected POMT1 mutations were predicted in silico to interfere with protein folding and/or glycosyltransferase function. The report on the patients described here has widened the clinical spectrum associated with POMT1 mutations to include cardiomyopathy. The functional impact of known and novel POMT1 mutations was predicted with a bioinformatics approach, and results were compared with previous in vitro studies of protein-o-mannosylase function.
POMT1; LGMD; CMD; cardiomyopathy; α-dystroglycan glycosylation
This paper focuses on the psychological benefits of caregiving in key relatives of patients with muscular dystrophies (MD), a group of rare diseases characterized by progressive weakness and restriction of the patient’s functional abilities. We describe whether relatives perceived caregiving to be a positive experience and test whether relatives’ perceptions vary in relation to their view of the patient as a valued person, the degree of involvement in care, and the level of support provided by social network and professionals. The study sample included 502 key relatives of patients aged 4–25 years, suffering from Duchenne, Becker, or limb-girdle MD, in treatment for at least 6 months to one of the eight participating centers, living with at least one relative aged 18–80 years. Of key relatives, 88 % stated that they had gotten something positive out of the situation, 96 % considered their patients to be sensitive, and 94 % viewed their patients as talented. Positive aspects of caregiving were more recognized by key relatives who were more convinced that the patient was sensitive and who perceived that they received higher level of professional help and psychological social support. These results suggest that most key relatives consider that their caregiving experience has had a positive impact on their lives, despite the practical difficulties of caring for patients with MD. Professionals should help relatives to identify the benefits of caregiving without denying its difficulties. Clinicians themselves should develop positive attitudes towards family involvement in the care of patients with long-term diseases.
Muscular dystrophy; Psychological benefits; Caregiving; Social network; Professional support
The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid α-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.
atrophy; autophagy; muscle wasting; glycogen storage disease type II; Pompe disease; MURF-1; ATROGIN1
Glycogenosis type II (GSDII or Pompe disease) is an autosomal recessive disease, often characterized by a progressive accumulation of glycogen within lysosomes caused by a deficiency of α-1,4-glucosidase (GAA; acid maltase), a key enzyme of the glycogen degradation pathway. To date, more than 326 different mutations in the GAA gene have been identified in patients with GSDII but the course of the disease is difficult to be predicted on the basis of molecular genetic changes. Studies on large informative families are advisable to better define how genetics and non genetics factors like exercise and diet may influence the clinical phenotype.
Methods and results
In this study, we report on clinical, instrumental, and pathological features as well as on molecular analysis of a family with 10 out of 13 siblings affected by late-onset Pompe disease. Three mutations segregated in the family, two of which are novel mutations. Siblings showing a more severe phenotype were compound heterozygous for c.118C > T [p.R40X] and c.2647-7G > A [p.N882fs] on GAA, whereas, two patients showing a mild phenotype were compound heterozygous c.2647-7G > A [p.N882fs] and c.2276G > C [p.G759A] mutations. Quantitative expression analysis showed, in the patients carrying p.R40X/ p.N882fs, a significant (p 0.01) correlation between the levels of expression of the mutated allele and the age at onset of the disease.
As far as we know, this is the largest informative family with late-onset Pompe disease described in the literature showing a peculiar complex set of mutations of GAA gene that may partially elucidate the clinical heterogeneity of this family.
Pompe disease; GAA gene; Mutation analysis; Genotype-phenotype correlations
Patients with muscle pathology are a challenge for anaesthesiologists because of possible life-threatening general anaesthesia complications. A review of the current medical literature on the issue clearly indicates that increasing awareness by anaesthesiologists in recent years has led to a reduction in the occurrence of adverse events in patients with diagnostically well-defined muscle disease. On the other hand, the current emerging aspect is that the great majority of complications concern subjects with clinically non-overt (silent to mildly symptomatic) and thus undiagnosed myopathy. With a view to improving prevention of possible critical anaesthesia complications in such patients, we present a "Safe Anaesthesia Table", listing both the anaesthetic drugs to be avoided and those considered harmless for myopathic patients, irrespective of age and type of pathology. In addition, a brief outline about the clinical aspects suggestive of a possible muscle pathology is also provided. Using "safe drugs" during routine surgical procedures in subjects with suspected undiagnosed myopathy will enable the anaesthesiologist to avoid delaying surgery, while protecting them from anaesthesia complications. By following this approach the presumed myopathy can be properly investigated after surgery.
anaesthesia complications; undiagnosed myopathy; safe anaesthesia; hyperCKemia
myotonic dystrophy; autonomic nervous system; heart rate; dystrophin; congenital disorders of glycosylation
Facioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of second- through fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1–3 repeats or 4–8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4–8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4–8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family’s genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families.
facioscapulohumeral muscular dystrophy; D4Z4 reduced allele; genotype–phenotype correlations; penetrance; disease expression
The world of metabolic myopathies has been dramatically modified by the advent of enzyme replacement therapy (ERT), the first causative treatment for glycogenosis type II (GSDII) or Pompe disease, which has given new impetus to research into that disease and also other pathologies. This article reviews new advances in the treatment of GSDII, the consensus about ERT, and its limitations. In addition, the most recent knowledge regarding the pathophysiology, phenotype, and genotype of the disease is discussed. Pharmacological, immunotherapy, nutritional, and physical/rehabilitative treatments for late-onset Pompe disease and other metabolic myopathies are covered, including treatments for defects in glycogen metabolism, such as glycogenosis type V (McArdle disease), and glycogenosis type III (debrancher enzyme deficiency), and defects in lipid metabolism, such as carnitine palmitoyltransferase II deficiency and electron transferring flavoprotein dehydrogenase deficiency, or riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.
Glycogenosis type II; McArdle disease; RR-MADD; glycogenosis type III; CPT2 deficiency
Several examples have always illustrated how access to large numbers of biospecimens and associated data plays a pivotal role in the identification of disease genes and the development of pharmaceuticals. Hence, allowing researchers to access to significant numbers of quality samples and data, genetic biobanks are a powerful tool in basic, translational and clinical research into rare diseases. Recently demand for well-annotated and properly-preserved specimens is growing at a high rate, and is expected to grow for years to come. The best effective solution to this issue is to enhance the potentialities of well-managed biobanks by building a network.
Here we report a 5-year experience of the Telethon Network of Genetic Biobanks (TNGB), a non-profit association of Italian repositories created in 2008 to form a virtually unique catalogue of biospecimens and associated data, which presently lists more than 750 rare genetic defects. The process of TNGB harmonisation has been mainly achieved through the adoption of a unique, centrally coordinated, IT infrastructure, which has enabled (i) standardisation of all the TNGB procedures and activities; (ii) creation of an updated TNGB online catalogue, based on minimal data set and controlled terminologies; (iii) sample access policy managed via a shared request control panel at web portal. TNGB has been engaged in disseminating information on its services into both scientific/biomedical - national and international - contexts, as well as associations of patients and families. Indeed, during the last 5-years national and international scientists extensively used the TNGB with different purposes resulting in more than 250 scientific publications. In addition, since its inception the TNGB is an associated member of the Biobanking and Biomolecular Resources Research Infrastructure and recently joined the EuroBioBank network. Moreover, the involvement of patients and families, leading to the formalization of various agreements between TNGB and Patients’ Associations, has demonstrated how promoting Biobank services can be instrumental in gaining a critical mass of samples essential for research, as well as, raising awareness, trust and interest of the general public in Biobanks. This article focuses on some fundamental aspects of networking and demonstrates how the translational research benefits from a sustained infrastructure.
Biobanking; Networking; Biological resources centre; IT infrastructure; Biological material; Biospecimens; Cryopreservation; Rare diseases; Patients’ associations
To test the effect of the single nucleotide polymorphism −66 T>G (rs28357094) in the osteopontin gene (SPP1) on functional measures over 12 months in Duchenne muscular dystrophy (DMD).
This study was conducted on a cohort of ambulatory patients with DMD from a network of Italian neuromuscular centers, evaluated longitudinally with the North Star Ambulatory Assessment (NSAA) and the 6-Minute Walk Test (6MWT) at study entry and after 12 months. Genotype at rs28357094 was determined after completion of the clinical evaluations. Patients were stratified in 2 groups according to a dominant model (TT homozygotes vs TG heterozygotes and GG homozygotes) and clinical data were retrospectively compared between groups.
Eighty patients were selected (age 4.1–19.3 years; mean 8.3 ± 2.7 SD). There were no differences in age or steroid treatment between the 2 subgroups. Paired t test showed a significant difference in both NSAA (p = 0.013) and 6MWT (p = 0.03) between baseline and follow-up after 12 months in patients with DMD carrying the G allele. The difference was not significant in the T subgroup. The analysis of covariance using age and baseline values as covariate and SPP1 genotype as fixed effect showed that these parameters are significantly correlated with the 12-month values.
These data provide evidence of the role of SPP1 genotype as a disease modifier in DMD and support its relevance in the selection of homogeneous groups of patients for future clinical trials.
Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.
Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (GSDII) disease, characterised by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to GAA maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.
atrophy; autophagy; glycogen storage disease; Pompe disease; MuRF1
Autoimmune myasthenia gravis (MG) is a neuromuscular disorder caused by autoantibodies directed against the acetylcholine receptor (AChR). Current symptomatic therapy is based on acetylcholinesterase (AChE) drugs. The available long-term current therapy includes steroids and other immunomodulatory agents. MG is associated with the production of a soluble, rare isoform of AChE, also referred as the “read-through” transcript (AChE-R). Monarsen (EN101) is a synthetic antisense compound directed against the AChE gene. Monarsen was administered in 16 patients with MG and 14 patients achieved a clinically significant response. The drug is now in a Phase II study. Further investigations are required to confirm its long-term effects.
myasthenia gravis; antisense oligonucleotides; acetyl cholinesterase; EN101
Fatigue is a frequent complaint in muscular dystrophies but it is yet not well defined or studied. We have examined the issue of muscle fatigue in a series of molecularly defined muscular dystrophies. A greater fatigability is seen in muscular dystrophy patients and can be an acute or chronic status. In Duchenne Muscular Dystrophy and beta-sarcoglycanopathy besides the alteration of dystrophin and/or sarcoglycan complex, a neuronal nitric oxide synthase depletion is frequently found and might correlate with post-exercise fatigability as well as with cardiac involvement. Therefore, it might be an important modulating factor of the severity of myopathy. In myotonic dystrophy, fatigue is a common complaint: muscle is involved and type 1 atrophy is a frequent feature; brain involvement and depressed mood might likely explain the extent of fatigue and daytime sleepiness commonly observed in these patients. Furthermore, in our observation in a series of 24 cases, muscle and brain can be independently involved in DM1 patients. These observations have profound impact on the type of physical therapy to be prescribed in such patients.
Fatigue; Duchenne dystrophy; Limb girdle dystrophy; Myotonic dystrophy; Nitric oxide synthase
Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy with an estimated prevalence of 1/8000. There are two genetically distinct types, DM1 and DM2. DM2 is generally milder with more phenotypic variability than the classic DM1. Our previous data on co-segregation of heterozygous recessive CLCN1 mutations in DM2 patients indicated a higher than expected DM2 prevalence. The aim of this study was to determine the DM2 and DM1 frequency in the general population, and to explore whether the DM2 mutation functions as a modifier in other neuromuscular diseases (NMD) to account for unexplained phenotypic variability. We genotyped 5535 Finnish individuals: 4532 normal blood donors, 606 patients with various non-myotonic NMD, 221 tibial muscular dystrophy patients and their 176 healthy relatives for the DM2 and DM1 mutations. We also genotyped an Italian idiopathic non-myotonic proximal myopathy cohort (n=93) for the DM2 mutation. In 5496 samples analyzed for DM2, we found three DM2 mutations and two premutations. In 5511 samples analyzed for DM1, we found two DM1 mutations and two premutations. In the Italian cohort, we identified one patient with a DM2 mutation. We conclude that the DM2 mutation frequency is significantly higher in the general population (1/1830; P-value=0.0326) than previously estimated. The identification of DM2 mutations in NMD patients with clinical phenotypes not previously associated with DM2 is of particular interest and is in accord with the high overall prevalence. On the basis of our results, DM2 appears more frequent than DM1, with most DM2 patients currently undiagnosed with symptoms frequently occurring in the elderly population.
myotonic dystrophy; mutation frequency; prevalence; population
We report the first case of a heterozygous T78M mutation in the caveolin-3 gene (CAV3) associated with rippling muscle disease and proximal myopathy. The patient displayed also bilateral winged scapula with limited abduction of upper arms and marked asymmetric atrophy of leg muscles shown by magnetic resonance imaging. Immunohistochemistry on the patient’s muscle biopsy demonstrated a reduction of caveolin-3 staining, compatible with the diagnosis of caveolinopathy. Interestingly, consistent with the possible diagnosis of FSHD, the patient carried a 35 kb D4Z4 allele on chromosome 4q35. We discuss the hypothesis that the two genetic mutations may exert a synergistic effect in determining the phenotype observed in this patient.
Rippling muscle disease; Caveolinopathy; Facioscapulohumeral dystrophy; Limb girdle muscular dystrophy type 1C
Steroids have been used since two decades and several trials were conducted to establish their efficacy in DMD patients with various regimens. The clinical outcomes showed increased function in the treated boys, and in a single trial with deflazacort, prolongation of ambulation but with different side effects. Steroids clinical efficacy is now established. The main concern is to increase steroid efficacy and decrease side effect and toxicity. A trial comparing daily prednisone, deflazacort and intermittent glucocorticoids (prednisone 10 days on/10 days off) (FOR-DMD) is starting under NIH grant. The primary outcomes will be muscle strength, forced vital capacity and patient/parents satisfaction.
Steroids; Duchenne; DMD; side effect; quality of life
Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in the CAPN3 gene and complete lack of functional calpain 3 leads to the most severe muscle wasting. Calpain 3 is suggested to be involved in maturation of contractile elements after muscle degeneration. The aim of this study was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration.
We studied muscle regeneration in 22 patients with LGMD2A with calpain 3 deficiency, in five patients with LGMD2I, with a secondary reduction in calpain 3, and in five patients with Becker muscular dystrophy (BMD) with normal calpain 3 levels. Regeneration was assessed by using the developmental markers neonatal myosin heavy chain (nMHC), vimentin, MyoD and myogenin and counting internally nucleated fibers.
We found that the recent regeneration as determined by the number of nMHC/vimentin-positive fibers was greatly diminished in severely affected LGMD2A patients compared to similarly affected patients with LGMD2I and BMD. Whorled fibers, a sign of aberrant regeneration, was highly elevated in patients with a complete lack of calpain 3 compared to patients with residual calpain 3. Regeneration is not affected by location of the mutation in the CAPN3 gene.
Our findings suggest that calpain 3 is needed for the regenerative process probably during sarcomere remodeling as the complete lack of functional calpain 3 leads to the most severe phenotypes.
Limb girdle muscular dystrophy; Calpain 3; Muscle regeneration; INF; Neonatal myosin heavy chain; Vimentin
We investigated the clinical and molecular pattern of two young men affected by dysferlinopathy, that was first diagnosed as polymyositis. We show that their symptoms and clinical course although progressive were peculiar, as well as their biopsy suggesting a subsequent analysis of dysferlin protein by western blotting. Molecular analysis of dysferlin gene revealed pathogenetic mutations in both cases.
In such cases a screening with Western blot followed by DNA analysis of dysferlin gene is therefore recommended. We present a diagnostic algorythm for patients with suspected myositis but presenting signs of disease progression and poor response to steroids.
Dysferlin; LGMD2B; Western blot
Dysferlin is a 237-kDa transmembrane protein involved in calcium-mediated sarcolemma resealing. Dysferlin gene mutations cause limb-girdle muscular dystrophy (LGMD) 2B, Miyoshi myopathy (MM) and distal myopathy of the anterior tibialis. Considering that a secondary Dysferlin reduction has also been described in other myopathies, our original goal was to identify cases with a Dysferlin deficiency without dysferlin gene mutations. The dysferlin gene is huge, composed of 55 exons that span 233 140 bp of genomic DNA. We performed a thorough mutation analysis in 65 LGMD/MM patients with ≤20% Dysferlin. The screening was exhaustive, as we sequenced both genomic DNA and cDNA. When required, we used other methods, including real-time PCR, long PCR and array CGH. In all patients, we were able to recognize the primary involvement of the dysferlin gene. We identified 38 novel mutation types. Some of these, such as a dysferlin gene duplication, could have been missed by conventional screening strategies. Nonsense-mediated mRNA decay was evident in six cases, in three of which both alleles were only detectable in the genomic DNA but not in the mRNA. Among a wide spectrum of novel gene defects, we found the first example of a ‘nonstop' mutation causing a dysferlinopathy. This study presents the first direct and conclusive evidence that an amount of Dysferlin ≤20% is pathogenic and always caused by primary dysferlin gene mutations. This demonstrates the high specificity of a marked reduction of Dysferlin on western blot and the value of a comprehensive molecular approach for LGMD2B/MM diagnosis.
dysferlin; limb-girdle muscular dystrophy; Miyoshi myopathy; nonsense-mediated mRNA decay; comparative genomic hybridization
MicroRNAs are highly conserved, noncoding RNAs involved in post-transcriptional gene silencing. They have been shown to participate in a wide range of biological processes, including myogenesis and muscle regeneration. The goal of this study is to test the hypothesis that myo-miRs (myo = muscle + miR = miRNA) expression is altered in muscle from patients affected by myotonic dystrophy type 1 (DM1), the most frequently inherited neuromuscular disease in adults. In order to gain better insights about the role of miRNAs in the DM1 pathogenesis, we have also analyzed the muscular expression of miR-103 and miR-107, which have been identified in silico as attractive candidates for binding to the DMPK mRNA.
To this aim, we have profiled the expression of miR-133 (miR-133a, miR-133b), miR-1, miR-181 (miR-181a, miR-181b, miR-181c) and miR-206, that are specifically induced during myogenesis in cardiac and skeletal muscle tissues. miR-103 and miR-107, highly expressed in brain, heart and muscle have also been included in this study. QRT-PCR experiments have been performed on RNA from vastus lateralis biopsies of DM1 patients (n = 7) and control subjects (n = 4). Results of miRNAs expression have been confirmed by Northern blot, whereas in situ hybridization technique have been performed to localize misexpressed miRNAs on muscle sections from DM1 and control individuals.
Only miR-206 showed an over-expression in 5 of 7 DM1 patients (threshold = 2, fold change between 1.20 and 13.22, average = 5.37) compared to the control group. This result has been further confirmed by Northern blot analysis (3.37-fold overexpression, R2 = 0.89). In situ hybridization localized miR-206 to nuclear site both in normal and DM1 tissues. Cellular distribution in DM1 tissues includes also the nuclear regions of centralized nuclei, with a strong signal corresponding to nuclear clumps.
This work provides, for the first time, evidences about miRNAs misexpression in DM1 muscle tissues, adding a new element in the pathogenesis of this complex genetic disease.