Features Of Dystrophy Research Articles

Clinical analysis of Duchenne/Becker muscular dystrophy with transaminase elevation as the initial presentation

Objective To analyze the clinical characteristics of Duchenne/Becker muscular dystrophy (DMD/BMD)with the initial presentation of transaminase elevation, in order to improve the clinician′s understanding of this disease, and reduce misdiagnosis and missed diagnosis.To investigate the relationship between the elevation of transaminase and the early stage of DMD/BMD, and to provide the strategy for early diagnosis. Methods Twenty-four patients admitted to the hospital with elevated serum transaminase as the initial presentation from January 2012 to December 2014, who were diagnosed as DMD/BMD by genetic testing or muscle biopsy, were enrolled.Their clinical data and laboratory examinations were retrospectively analyzed, including clinical features, diagnostic steps, serum muscle enzymes, genetic analysis, electromyography and muscle pathological changes. Results The 24 patients were all male without family history of DMD/BMD prior to birth.The average visiting age was (3.4±1.2)years (ranging from 0.8 to 6.1 years), and 87.5% (21/24 cases)of cases were preschool children aged 2-6 years.Hypertransaminasemia was found in 21 cases (87.5%)during the kindergarten physical examination, 1 case during pre-operative investigation and 2 cases during respiratory infection.Due to its insidious onset, the time interval between incidental finding of elevated transaminase and definitive diagnosis was between 0.6 and 20.4 months.Among them, 16 cases (66.7%)had obvious pseudohypertrophy of calf muscles, and 18 cases (81.8%)showed different degrees of movement disorder, such as unable to jump, easy to fall, and difficulty in climbing stairs.In addition, 18.2% cases (4/22 cases)had a delay in language development.The serum alanine aminotransferase and aspartate aminotransferase levels were 120.3-761.7 U/L and 83.3-675.5 U/L, respectively.Serum creatine kinase (CK)was found to be markedly elevated (ranging from 3 940 to 27 510 U/L)in all patients.Electromyography showed myogenic damage in 13/23 cases (56.5%). DMD gene deletions were found in 18 cases (75.0%), and duplications in 4 cases (16.7%). The muscle biopsy performed in 2 cases (8.3%)multiplex ligation-dependent probe amplification (MLPA)-negative cases showed evidence of dystrophic features on routine histology.Immunohistochemistry showed absent dystrophin staining in all 2 MLPA-negative cases. Conclusion The clinical manifestation of DMD/BMD is not typical in the early stage, so it is easy to be neglected or misdiagnosed.Elevated ALT and AST are important clues in the early diagnosis of DMD/BMD.Children with elevated transaminase, in the absence of other signs and symptoms of hepatic injury, may have occult muscular di-sease, most frequently DMD/BMD.A thorough physical examination and history taking as well as the measurement of serum CK are helpful in the differential diagnosis.Genetic testing or muscle biopsy should be done early for correct diagnosis of DMD/BMD. Key words: Duchenne/Becker muscular dystrophy; Transaminase; Creatine kinase

Limb-girdle muscular dystrophy type 2I: two Chinese families and a review in Asian patients

ABSTRACTBackground: Limb-girdle muscular dystrophy type 2I (LGMD2I) is an autosomal recessive hereditary disorder caused by mutations in the fukutin-related protein (FKRP) gene. Although the features of the disorder in European patients have been summarized, Asian patients with LGMD2I have rarely been reported. Thus, the clinical differences in LGMD2I between Asian and European patients and the associated genetic changes remain unclear.Methods: We reported detailed clinical data as well as results from muscle biopsy, muscle MRI and genetic analysis of the FKRP gene in two unrelated Chinese families with LGMD2I. Additionally, a review of the literature focusing on the clinical and mutational features of LGMD2I in Asian patients was performed.Results: The muscle biopsy results showed dystrophic features. Immunohistochemical staining revealed decreased glycosylations on α-dystroglycan. The muscle MRI results showed that the gluteus maximus, adductor, biceps femoris, vastus intermedius and vastus lateralis were severely affected. The patients in the two families harbored the same compound heterozygous mutations (c.545A>G and c.948delC). One patient showed significant clinical improvement after corticosteroid treatment.Conclusion: Our study expanded the reported spectrum of Asian LGMD2I patients. Our literature review revealed that pathogenic mutations in the FKRP gene in Asian LGMD2I patients are compound heterozygous rather than homozygous. Compound heterozygous Asian patients have a mild phenotype but frequently show respiratory and cardiac impairments. Corticosteroids may be beneficial for the treatment of LGMD2I and should be further investigated.

P.441 - Tubular aggregate myopathy with dystrophic features

P.441 - Tubular aggregate myopathy with dystrophic features

P.241 - Comparative high resolution proteomic analysis of dystrophic mouse models reveals a core dystrophic proteome and the impact of aging

The most common form of childhood muscular dystrophy is Duchenne muscular dystrophy (DMD), which is a severe, X-linked muscle wasting disorder. The disease is caused by mutations in the DMD gene, leading to loss of the dystrophin protein which in turn causes progressive muscle degeneration and premature death. Various mouse models have been developed to study the disease, such as the commonly used mdx mouse, which has a nonsense mutation in exon 23, though this mutation is not characteristically present in humans. Another model is the mdx52 mouse, which has an exon 52 deletion in the mutation “hotspot” region of the DMD gene. To better understand the dystrophic phenotype and its progression with aging, we undertook for the first-time comparative proteomic analysis of both mdx and mdx52 models, using high-resolution isoelectric Focusing Liquid Chromatography-tandem Mass Spectrometry (HiRIEF-LC-MS/MS) with Tandem Mass Tag (TMT) isobaric labelling of tibialis anterior (TA) skeletal muscle to identify proteomic characteristics at 8, 16 and 80 weeks of age. Comparative proteomic analysis of mdx, mdx52 and C57BL/6 control mice identified 4,974 proteins in 27plex format and revealed a core set of proteomic changes reflective of the dystrophic phenotype. Proteomic analysis with age identified core proteomic features of normal muscle aging, overlap of these with progression of the dystrophic phenotype and features specific to the dystrophic phenotype with age. This study provides novel insight into specific proteomic changes characteristic of muscle aging and the relationship of these to dystrophic pathology over time.

P.236 - Establishment of a panel for the evaluation of the dystrophic process by quantitative RT-PCR

Muscular dystrophies (MD) are rare genetic diseases affecting the skeletal and/or cardiorespiratory muscles. Histological and functional evaluations are usual procedures to assess severity and progression of muscle impairment in MD. However, quantitative and early detection of the disease onset and follow-up would be valuable especially when the phenotype is moderate or the progression is slow. Pathological features (degeneration/regeneration cycles, immune response, fibrosis, oxidative stress …) triggered by the genetic defect are often associated with gene and microRNA dysregulations that could be used as biomolecular markers. In this study, several genes and microRNAs involved in these different pathways were selected and measured in mouse models of Duchenne MD and Limb-Girdle MD type 2I. Three genetic backgrounds were tested: 1) C57BL/6, 2) CMAH background, characterized by deletion of the gene encoding CMAH, an enzyme found to be inactive in humans and 3) DBA2 background, characterized by a polymorphism in the gene encoding LTBP4, a protein involved in the TGFβ pathway. Based on histological and functional evaluations, these two backgrounds have already been shown to significantly affect the phenotype of some MD mouse models. Therefore, these different models represent different levels of impairment. Correlation of our results obtained by quantitative RT-PCR with the level of dystrophic features allows us assessing quantitatively the different aspects of the dystrophic process.

P.296 - Congenital myopathy with protein aggregates and nemaline bodies related to CFL2 mutations

Congenital myopathy (CM) caused by mutations in cofilin-2 gene (CFL2) is a rare neuromuscular disorder. The few reported cases show phenotypic heterogeneity ranging from early onset and rapid progressive form to milder myopathy characterized by limb girdle and axial muscles weakness. Muscle histology is also heterogeneous showing features of nemaline or myofibrillar myopathy or the coexistence of both histopathological changes. CFL2- null mutations result in more severe disease variants than those related to missense mutations although the precise mechanism through which cofilin-2 abnormality results in nemaline myopathy remains elusive. We report on three new cases, from two unrelated families, of severe CMs related to novel homozygous (p.D86H) or compound heterozygous (p.D79Y and p.S94LfsX6) mutations in CFL2.All babies presented severe neonatal generalized muscle weakness needing continuous respiratory and nutritional support since birth. One baby died at the age of 3 months; his sister and the other proband are still alive at the age of 1 year and 7 years respectively. Muscle biopsies showed severe myopathic changes, with fiber splitting, internal nuclei, many rod bodies and sarcoplasmic protein aggregates that mainly look like actin filaments. In the older baby dystrophic features such as fatty infiltration and fibrosis were also observed and CFL2 protein is reduced thus suggesting that the p.D86H mutationresults in a misfolding or destabilization of the protein's tertiarystructure, which leads to cofilin-2 degradation.

HANAC Col4a1 Mutation in Mice Leads to Skeletal Muscle Alterations due to a Primary Vascular Defect

Collagen IV is a major component of basement membranes (BMs). The α1(IV) chain, encoded by the COL4A1 gene, is expressed ubiquitously and associates with the α2(IV) chain to form the α1α1α2(IV) heterotrimer. Several COL4A1 mutations affecting a conformational domain containing integrin-binding sites are responsible for the systemic syndrome of hereditary angiopathy, nephropathy, aneurysms, and cramps (HANAC). To analyze the pathophysiology of HANAC, Col4a1 mutant mice bearing the p.Gly498Val mutation were generated. Analysis of the skeletal muscles of Col4a1G498V mutant animals showed morphologic characteristics of a muscular dystrophy phenotype with myofiber atrophy, centronucleation, focal inflammatory infiltrates, and fibrosis. Abnormal ultrastructural aspects of muscle BMs was associated with reduced extracellular secretion of the mutant α1α1α2(IV) trimer. In addition to muscular dystrophic features, endothelial cell defects of the muscle capillaries were observed, with intracytoplasmic accumulation of the mutant α1α1α2(IV) molecules, endoplasmic reticulum cisternae dilation, and up-regulation of endoplasmic reticulum stress markers. Induction of the unfolded protein response in Col4a1 mutant muscle tissue resulted in an excess of apoptosis in endothelial cells. HANAC mutant animals also presented with a muscular functional impairment and increased serum creatine kinase levels reflecting altered muscle fiber sarcolemma. This extensive description of the muscular phenotype of the Col4a1 HANAC murine model suggests a potential contribution of primary endothelial cell defects, together with muscle BM alterations, to the development of COL4A1-related myopathy.

Management of scoliosis associated with neurofibromatosis

Scoliosis is the most common skeletal deformity seen in patients with neurofibromatosis type 1. It is classified based on dystrophic features seen on imaging. Classification is important for deciding on the management options. Nondystrophic curves can be treated similar to idiopathic curves. Conservatively treated curves should be monitored for curve modulation. Dystrophic curves should be managed surgically with 360-degree fusion. Special attention should be focused on fusion as the rate of pseudoarthrosis is higher in these curves. To get good results and avoid neurologic complications, screening of the whole spine is mandatory to identify associated bony and intraspinal anomalies that may be missed when focusing on the main curve.

Clinical features of the myasthenic syndrome arising from mutations in GMPPB

BackgroundCongenital myasthenic syndrome (CMS) due to mutations in GMPPB has recently been reported confirming the importance of glycosylation for the integrity of neuromuscular transmission.MethodsReview of case notes of patients with...

Sarcolemmal deficiency of sarcoglycan complex in an 18-month-old Turkish boy with a large deletion in the beta sarcoglycan gene.

Limb-girdle muscular dystrophy type 2E (LGMD-2E) is caused by autosomal recessive defects in the beta sarcoglycan (SGCB) gene located on chromosome 4q12. In this case report, the clinical findings, histopathological features and molecular genetic data in a boy with β sarcoglycanopathy are presented. An 18-month-old boy had a very high serum creatinine phosphokinase (CPK) level that was accidentally determined. The results of molecular analyses for the dystrophin gene was found to be normal. He underwent a muscle biopsy which showed dystrophic features. Immunohistochemistry showed that there was a total loss of sarcolemmal sarcoglycan complex. DNA analysis revealed a large homozygous deletion in the SCGB gene. During 4 years of follow-up, there was no evidence to predict a severe clinical course except the muscle enzyme elevation and myopathic electromyography (EMG) finding. The presented milder phenotype of LGMD-2E with a large deletion in the SGCB gene provided additional support for the clinical heterogeneity and pathogenic complexity of the disease.

Management of congenital muscular dystrophies related to defects in the LMNA gene

Congenital Muscular Dystrophies are a heterogeneous group of muscular disorders defined as early onset muscle weakness and progressive course associated to dystrophic features at muscle biopsy. CMDs related to lamina A/C gene (LMNA) defect include different phenotypes that can be classified as i) severe phenotype with generalized muscular weakness and contractures by birth, ii) ‘dropped head’ phenotype with prominent involvement of axial muscles that generally evolves to rigid spine phenotype and iii) early Emery-Dreifuss phenotype. All these condition generally lead in the first 2 decades to cardiac disturbances, respiratory insufficiency, orthopedic complication and metabolic disorders. The clinical management requires a multidisciplinary and rigorous approach focused on early medical and rehabilitative interventions with the main aims to prevent ‘fatal heart event’, to cure co-morbidities (pulmonary insufficiency and spinal and joint contractures) and to improve the quality of life of these children.

Choline Kinase Beta–Related Muscular Dystrophy, Appearance of Muscle Involvement on Magnetic Resonance Imaging

BackgroundClinical presentation with motor delay, proximal weakness, and learning difficulties raise the possibility of a dystrophinopathy, dystroglycanopathy, or myotonic dystrophy. This differential should also include the more recently described choline kinase beta–related muscular dystrophy. This condition is typically characterized by large and abnormally distributed mitochondria on muscle biopsy, which can distinguish this condition from the other muscle conditions in the differential. MethodsWe present a boy with choline kinase beta mutations with relatively mild clinical manifestations, including proximal weakness, learning difficulties and elevated creatine kinase. Investigations included muscle magnetic resonance imaging (MRI) with T1 axial sequences through thigh and calves, and needle muscle biopsy of the left vastus lateralis muscle. ResultsMRI showed involvement mainly of the quadriceps femoris, sartorius, and adductor magnus, with selective sparing of the gracilis, hamstrings, and adductor longus and brevis. Muscle biopsy revealed chronic dystrophic features. Oxidative stains demonstrated enlarged mitochondria accentuated peripherally or present diffusely in a few fibres giving a coarsely stippled appearance. A homozygous C.722A>G (p.Asn241Ser) mutation was detected in exon 6 of the CHKB gene. ConclusionThis selective pattern of skeletal muscle involvement might be helpful for identifying other patients with this condition, even in the absence of diagnostic muscle pathology.

COLQ variant associated with Devon Rex and Sphynx feline hereditary myopathy.

SummarySome Devon Rex and Sphynx cats have a variably progressive myopathy characterized by appendicular and axial muscle weakness, megaesophagus, pharyngeal weakness and fatigability with exercise. Muscle biopsies from affected cats demonstrated variable pathological changes ranging from dystrophic features to minimal abnormalities. Affected cats have exacerbation of weakness following anticholinesterase dosing, a clue that there is an underlying congenital myasthenic syndrome (CMS). A genome‐wide association study and whole‐genome sequencing suggested a causal variant for this entity was a c.1190G>A variant causing a cysteine to tyrosine substitution (p.Cys397Tyr) within the C‐terminal domain of collagen‐like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase (COLQ). Alpha‐dystroglycan expression, which is associated with COLQ anchorage at the motor end‐plate, has been shown to be deficient in affected cats. Eighteen affected cats were identified by genotyping, including cats from the original clinical descriptions in 1993 and subsequent publications. Eight Devon Rex and one Sphynx not associated with the study were identified as carriers, suggesting an allele frequency of ~2.0% in Devon Rex. Over 350 tested cats from other breeds did not have the variant. Characteristic clinical features and variant presence in all affected cats suggest a model for COLQ CMS. The association between the COLQ variant and this CMS affords clinicians the opportunity to confirm diagnosis via genetic testing and permits owners and breeders to identify carriers in the population. Moreover, accurate diagnosis increases available therapeutic options for affected cats based on an understanding of the pathophysiology and experience from human CMS associated with COLQ variants.

β-Sarcoglycan gene transfer decreases fibrosis and restores force in LGMD2E mice.

Limb-girdle muscular dystrophy type 2E (LGMD2E) results from mutations in the β-sarcoglycan (SGCB) gene causing loss of functional protein and concomitant loss of dystrophin-associated proteins. The disease phenotype is characterized by muscle weakness and wasting, and dystrophic features including muscle fiber necrosis, inflammation and fibrosis. The Sgcb-null mouse recapitulates the clinical phenotype with significant endomysial fibrosis providing a relevant model to test whether gene replacement will be efficacious. We directly addressed this question using a codon optimized human β-sarcoglycan gene (hSGCB) driven by a muscle-specific tMCK promoter (scAAVrh74.tMCK.hSGCB). Following isolated limb delivery (5 × 10(11) vector genome (vg)), 91.2% of muscle fibers in the lower limb expressed β-sarcoglycan, restoring assembly of the sarcoglycan complex and protecting the membrane from Evans blue dye leakage. Histological outcomes were significantly improved including decreased central nucleation, normalization of muscle fiber size, decreased macrophages and inflammatory mononuclear cells, and an average of a 43% reduction in collagen deposition in treated muscle compared with untreated muscle at end point. These measures correlated with improvement of tetanic force and resistance to eccentric contraction. In 6-month-old mice, as indicated by collagen staining, scAAVrh74.tMCK.hSGCB treatment reduced fibrosis by 42%. This study demonstrates the potential for gene replacement to reverse debilitating fibrosis, typical of muscular dystrophy, thereby providing compelling evidence for movement to clinical gene replacement for LGMD2E.

Mechanical Overloading Increases Maximal Force and Reduces Fragility in Hind Limb Skeletal Muscle from Mdx Mouse

There is fear that mechanical overloading (OVL; ie, high-force contractions) accelerates Duchenne muscular dystrophy. Herein, we determined whether short-term OVL combined with wheel running, short-term OVL combined with irradiation, and long-term OVL are detrimental for hind limb mdx mouse muscle, a murine model of Duchene muscular dystrophy exhibiting milder dystrophic features. OVL was induced by the surgical ablation of the synergic muscles of the plantaris muscle, a fast muscle susceptible to contraction-induced muscle damage in mdx mice. We found that short-term OVL combined with wheel and long-term OVL did not worsen the deficit in specific maximal force (ie, absolute maximal force normalized to muscle size) and histological markers of muscle damage (percentage of regenerating fibers and fibrosis) in mdx mice. Moreover, long-term OVL did not increase the alteration in calcium homeostasis and did not deplete muscle cell progenitors expressing Pax 7 in mdx mice. Irradiation before short-term OVL, which is believed to inhibit muscle regeneration, was not more detrimental to mdx than control mice. Interestingly, short-term OVL combined with wheel and long-term OVL markedly improved the susceptibility to contraction-induced damage, increased absolute maximal force, induced hypertrophy, and promoted a slower, more oxidative phenotype. Together, these findings indicate that OVL is beneficial to mdx muscle, and muscle regeneration does not mask the potentially detrimental effect of OVL.

P91 – 2939: A novel mutation in the TK2 gene and clinical phenotype in Spanish patients

Objective The aim of this study is to describe the phenotype of 4 Spanish patients and the finding of a novel mutation in TK2 gene. Methods We report 4 unrelated male patients from non-consanguineous families. Details of the clinically features, biochemical, anatomy pathology and genetic findings are presented. Results 3/4 showed a similar phenotype with normal early development until a progressive hypotonia and muscular weakness with a fast motor regression started between 14 and 24 months old. The other patient showed a mild late onset phenotype, starting the clinical symptoms at 4 years old with facial and hand-feet distal muscle weakness. Currently he is able to walk independently with severe hyperlordosis, facial and scapular muscle weakness. All patients presented a mild increase of serum CK and lactate levels. Electromyography showed a myopathy pattern. Muscle biopsy showed dystrophic features and COX negative fibres, lipid vacuoles and ragged red fibres (RRF) in 4/4 patients, but the patient with the mild-late onset phenotype had a less severe pattern. Genetic studies showed more than 80% of depletion in mtDNA in the muscles of all of them. The one with late-onset mild phenotype showed a less reduced percentage of depletion in mtDNA in muscle (50–60%). Sequence analysis of the TK2 gene found two heterozygous pathogenic mutations as previously described in the literature. A novel mutation (c.623A>G; p.Tyr208Cys) was found in one patient with a significant reduction of TK2 activity studied in fibroblast. Conclusion Severe and fast progressive muscle weakness in early childhood should lead the clinicians to search for evidence of mitochondria dysfunction and alert about a possible TK2 deficiency diagnosis. The new approach with dTMP–dCMP in early stages of this disorder should be considered.

Distrofias musculares, miopatías metabólicas y tóxicas

Muscular dystrophies are genetically determined conditions that manifest with progressive muscular weakness and show dystrophic features in the muscle biopsy. It is important to reach a genetic diagnosis when possible due to implications in the management, prevention of complications, genetic counselling and treatment. Next generation sequencing has provided a valuable tool for genetic diagnosis which is becoming otherwise challenging, time-consuming and expensive when based on traditional methods, due to the increasing number of entities described. Metabolic myopathies are caused by abnormalities in the utilization of energy by skeletal muscle. They can manifest as a fixed myopathy with progressive weakness or only with transient symptoms during high energy-demanding situations, such as exercise. Biochemical determinations in muscle, blood or urine may be useful but the definitive diagnosis is usually genetic.

A mitochondrial tRNAMet mutation causing developmental delay, exercise intolerance and limb girdle phenotype with onset in early childhood

A 10-year-old girl presented with exercise intolerance, learning difficulty, and muscle weakness in a limb girdle distribution. She had delayed achievement of motor milestones and difficulties with social interaction at pre-school age. Muscle biopsy showed no myopathic or dystrophic features, but 90% COX negative fibres and ragged blue fibres. Respiratory chain enzyme analysis in muscle showed a combined deficiency and mitochondrial DNA sequencing revealed the presence of an m.4450G>A mutation in the MT-TM gene encoding the tRNA for methionine. The mutation was only detected in mtDNA extracted from muscle and skin fibroblast, and could not be found in other tissues or in the mother. This is the second patient reported in the literature with a mitochondrial myopathy due to a mt-tRNAMet mutation. The first patient, a 30-year-old woman, presented with exercise intolerance, limb girdle muscle weakness, lactic acidosis, learning difficulty, and growth retardation in early childhood. Thus, the two patients exhibit strikingly overlapping phenotypes.

Muscle proteomics reveals novel insights into the pathophysiological mechanisms of collagen VI myopathies.

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Recessive ACTA1 variant causes congenital muscular dystrophy with rigid spine.

Variants in ACTA1, which encodes α-skeletal actin, cause several congenital myopathies, most commonly nemaline myopathy. Autosomal recessive variants comprise approximately 10% of ACTA1 myopathy. All recessive variants reported to date have resulted in loss of skeletal α-actin expression from muscle and severe weakness from birth. Targeted next-generation sequencing in two brothers with congenital muscular dystrophy with rigid spine revealed homozygous missense variants in ACTA1. Skeletal α-actin expression was preserved in these patients. This report expands the clinical and histological phenotype of ACTA1 disease to include congenital muscular dystrophy with rigid spine and dystrophic features on muscle biopsy. This represents a new class of recessive ACTA1 variants, which do not abolish protein expression.