Mutations In Dysferlin Gene Research Articles

Transcriptome analysis of skeletal muscle in dermatomyositis, polymyositis, and dysferlinopathy, using a bioinformatics approach.

Polymyositis (PM) and dermatomyositis (DM) are two distinct subgroups of idiopathic inflammatory myopathies. Dysferlinopathy, caused by a dysferlin gene mutation, usually presents in late adolescence with muscle weakness, degenerative muscle changes are often accompanied by inflammatory infiltrates, often resulting in a misdiagnosis as polymyositis. To identify differential biological pathways and hub genes related to polymyositis, dermatomyositis and dysferlinopathy using bioinformatics analysis for understanding the pathomechanisms and providing guidance for therapy development. We analyzed intramuscular ribonucleic acid (RNA) sequencing data from seven dermatomyositis, eight polymyositis, eight dysferlinopathy and five control subjects. Differentially expressed genes (DEGs) were identified by using DESeq2. Enrichment analyses were performed to understand the functions and enriched pathways of DEGs. A protein-protein interaction (PPI) network was constructed, and clarified the gene cluster using the molecular complex detection tool (MCODE) analysis to identify hub genes. A total of 1,048, 179 and 3,807 DEGs were detected in DM, PM and dysferlinopathy, respectively. Enrichment analyses revealed that upregulated DEGs were involved in type 1 interferon (IFN1) signaling pathway in DM, antigen processing and presentation of peptide antigen in PM, and cellular response to stimuli in dysferlinopathy. The PPI network and MCODE cluster identified 23 genes related to type 1 interferon signaling pathway in DM, 4 genes (PDIA3, HLA-C, B2M, and TAP1) related to MHC class 1 formation and quality control in PM, and 7 genes (HSPA9, RPTOR, MTOR, LAMTOR1, LAMTOR5, ATP6V0D1, and ATP6V0B) related to cellular response to stress in dysferliniopathy. Overexpression of genes related to the IFN1 signaling pathway and major histocompatibility complex (MHC) class I formation was identified in DM and PM, respectively. In dysferlinopathy, overexpression of HSPA9 and the mTORC1 signaling pathway genes was detected.

Generation and Analysis of Genetically Modified CD14+ Monocytes with Dysferlin Overexpression

IntroductionDysferlinopathies is a group of orphan neuromuscular diseases associated with impairment of mRNA and defect in dysferlin protein expression in skeletal muscle tissue due to mutations in DYSF (Dystrophy-associated fer-1-like) gene. This group of diseases is having autosomal recessive type of inheritance and the frequency of occurrence is 1/1,300-1/200,000 in different populations. There are three main clinical phenotypes associated with dysferlin gene mutations: Limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT) (Yakovlev et. al. Human Gene Therapy 2016). Currently there is no treatment for these diseases. Hence, the development of new methods of treating this group of diseases remains an urgent task, in particular, with the use of gene therapy. According to the literature, CD14+ cells play a key role in the pathogenesis of human dysferlinopathies and are involved in inflammatory reactions and pathological angiogenesis.MethodsIn the present work, by using previously created recombinant adenovirus Ad5-Dysf (Starostina et. al. Medicine 2016) that encodes human cDNA dysferlin gene and enables the expression of the recombinant dysferlin protein, genetic modification of CD14 + monocytes was carried out. Therefore, the following aims were set:1. Isolation of CD14+ cells from a patient with dysferlinopathy and a healthy donor.2. Genetic modification of CD14+ monocytes.3. Conduct western blot analysis of mRNA expression of recombinant dysferlin protein in modified cells after viral transduction. Evaluate the effectiveness of viral transduction by analyzing the expression of EGFP protein (adenovirus Ad5-EGFP) which was taken as a positive control by fluorescence microscopy.ResultsCD14+ cells were isolated from the freshly prepared peripheral blood leukocyte buffy coat layer of the patient with dysferlinopathy and a healthy donor by use of EasySep ™ Human Buffy Coat CD14 Positive Selection Kit. Since the correct assembly and effective DYSF gene delivery of the adenovirus Ad5-Dysf was confirmed in previous work, the viral transduction of human monocytes was carried out successfully. Ad5-EGFP adenovirus was used as a positive control to evaluate the effectiveness of viral transduction. EGFP protein expression analysis was conducted by fluorescence microscopy on an inverted fluorescent microscope of the research class AxioObserver.Z1 (Carl Zeiss) and demonstrated 100% of EGFP protein expression (Fig. 1). After genetic modification of the cells with resulting Ad5-Dysf adenovirus the evaluation of the recombinant dysferlin protein expression in cells was carried out by western blot analysis. Positive reaction was revealed with rabbit polyclonal antibodies to dysferlin (Fig. 2).ConclusionsIn this work we demonstrated that genetically modified CD14+ cells with DYSF overexpression can be successfully geberated and considered as one of the approaches for the therapy of human dysferlinopathies. Future studies are needed to analyze the functionality of the obtained genetic construct in animal models. SVV was supported by RFBR grant 16-34-00657.Fig. 1. CD14+ monocytes genetically modified with Ad5-EGFP. A - phase-contrast microscopy. B - fluorescence microscopy. 48 hours after genetic modification. Magnification 200X.Fig. 2. Western blot analysis of DYSF protein expression in CD14+ monocytes isolated from the peripheral blood of a patient with dysferlinopathy. Electrophoresis in 12% SDS-PAGE gel according to the Laemmli system. Antibodies to dysferlin (Abcam, ab15108) were used in a dilution of 1:250. The expected size of DYSF protein is 231 kDa. Well 1 - transduced CD14+ monocytes with Ad5-Dysf. Well 2 - native CD14+ monocytes. M - molecular weight marker. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

REPARATIVE RHABDOMYOGENESIS IN MICE WITH DYSF MUTATION

Dysferlinopathies are a group of muscular dystrophies with autosomal-recessive inheritance caused by mutations in DYSF gene. Dysferlin is a 237 kDa transmembrane protein responsible for reparation of the sarcolemma. It has calcium-sensitive C2 domains and after dysferlin binding with calcium ions the first one activates vesicles fusion and patch mechanism repair. There are number of knockout animal strains with dysferlin gene mutations. Bla/J mice have the ETn retrotransposon inserted in intron 4 of the DYSF gene of wild-type mice - C57Bl/6. The pathogenesis ascertainment of dysferlinopathies is important not only for revealing of physiological function of dysferlin, but its deficiency influence on reparative regeneration of skeletal muscles. In this paper the description of main pathohistological processes in skeletal muscle that take place in mice with dysferlinopathy after acute myotoxic injury is present. This article reviews quantitative evaluation of main pathomorphological processes in reparative regeneration: alteration (necrotized muscle fibers ratio), proliferation (Ki-67-positive myonuclei ratio), differentiation (mean cros-sectional area, percentage of centrinucleated muscle fibers, myo-genin-positive nuclei ratio, slow/fast muscle fibers ratio). It was identified that dysferlin-deficient mice have increased alteration level with more necrotized muscle fibers (35,1% (29,4%; 42,9%) in Bla/J vs. 25,8% (17,9%; 37,4%) in C57Bl/6 on 2 day after alteration, p

Gene co-expression network analysis of dysferlinopathy: Altered cellular processes and functional prediction of TOR1AIP1, a novel muscular dystrophy gene

Dysferlinopathy, caused by a dysferlin gene mutation, is a clinically heterogeneous autosomal recessive muscle disease characterized by progressive muscle degeneration. The dysferlin protein's functions and dysferlinopathy disease pathogenesis are not fully explored, and there is no specific treatment available that can alter the disease progression. This study uses publicly available dysferlinopathy patient microarray data to construct a gene co-expression network and investigates significant cellular pathways and their key players in dysferlinopathy pathogenesis. Extracellular matrix deposition, inflammation, mitochondrial abnormalities and protein degradation were found to be important in dysferlinopathy. Out of the hub genes, OXR1 and TIMP1 were selected through literature search as candidate genes for possible biomarker and molecular therapeutic target studies. A recently identified muscular dystrophy gene TOR1AIP1 was detected as a hub gene in dysferlinopathy. Co-expression and protein sequence feature analysis were adopted to predict TOR1AIP1's function. Our results suggest that LAP1 protein encoded by TOR1AIP1 may play a role in protein degradation possibly through transcriptional regulation in muscle tissue. These findings extend dysferlinopathy pathogenesis by presenting key genes and also suggest a novel function for a poorly characterized gene.

Dysferlin Gene Mutation Spectrum in a Large Cohort of Chinese Patients with Dysferlinopathy.

Background:Dysferlinopathy is caused by mutations in the dysferlin (DYSF) gene. Here, we described the genetic features of a large cohort of Chinese patients with this disease.Methods:Eighty-nine index patients were included in the study. DYSF gene analysis was performed by Sanger sequencing in 41 patients and targeted next generation sequencing (NGS) in 48 patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect exon duplication/deletion in patients with only one pathogenic mutation.Results:Among the 89 index patients, 79 patients were demonstrated to carry two disease-causing (73 cases) or possibly disease-causing mutations (6 cases), including 26 patients with homozygous mutations. We identified 105 different mutations, including 59 novel ones. Notably, in 13 patients in whom only one pathogenic mutation was initially found by Sanger sequencing or NGS, 3 were further identified to carry exon deletions by MLPA. The mutations identified in this study appeared to cluster in the N-terminal region. Mutation types included missense mutations (30.06%), nonsense mutations (17.18%), frameshift mutations (30.67%), in-frame deletions (2.45%), intronic mutations (17.79%), and exonic rearrangement (1.84%). No genotype-phenotype correlation was identified.Conclusions:DYSF mutations in Chinese patients clustered in the N-terminal region of the gene. Exonic rearrangements were found in 23% of patients with only one pathogenic mutation identified by Sanger sequencing or NGS. The novel mutations found in this study greatly expanded the mutational spectrum of dysferlinopathy.

A.O.7 - Research for the pathogenesis and therapy of dysferlinopathy using proteomics approach

Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in dysferlin gene (DYSF). There are two main phenotypes including Miyoshi muscular dystrophy and limb-girdle muscular dystrophy type 2B. DYSF is composed of 55 exons coding for 2,080 amino acid residues. DYSF is expressed in the plasma membrane of skeletal muscles and is involved in calcium-mediated membrane fusion events and membrane repair. We previously identified over 50 different mutations across the entire DYSF in approximately 60% of the patients. Our research theme is focusing on these DYSF and DYSF-related proteins. Recent advances have identified some components of the cell membrane repair process, particularly those involved in a putative pathway specific to striated muscles. Two important members are DYSF and Mitsugumin 53 (MG53). Compared to DYSF, molecular size of MG53 is rather small and can be expressed and work as membrane repair molecules. To find the binding partner of DYSF, which can be working as the similar functional role, would be a good clue to find the therapeutic strategy of dysferlinopathy. Using recombinant proteins and affinity column binding methods combined with LC/MS/MS, we identified novel DYSF-binding proteins. Protein X, one of the novel proteins, is considered to be an essential component of the membrane repair process. To directly evaluate the membrane repair capacity of the X knockdown C2C12 cells, we measured FM fluorescent dye entry after laser-induced damage to cells. The wild-type cells could effectively reseal sarcolemmal membranes, as they showed only minimal FM dye entry after laser damage. In contrast, significant entry of FM dye into the X knockdown cells was observed after laser-induced damage, suggesting insufficient membrane repair in X-deficient cells. To elucidate the molecular function of protein X in dysferlinopathy condition, we will also use several kinds of myoblasts originated from dysferlinopathy patients.

Robust genotyping tool for autosomal recessive type of limb-girdle muscular dystrophies.

BackgroundLimb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap; therefore, advanced molecular techniques are required to achieve differential diagnosis.MethodsWe investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 (CAPN3), anoctamin 5 (ANO5) and fukutin related protein (FKRP) genes.ResultsAnalysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin (DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T. Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound – c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene – c.2680C > T p.Arg894Ter. ANO5 c.191dupA was not present.ConclusionsGenetic diagnosis was possible in 12 of 60 patients (20 %). The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-1058-z) contains supplementary material, which is available to authorized users.

404. Development of Human Artificial Chromosomes for Gene Cell Therapy of Muscular Dystrophies

Muscular dystrophies are clinically heterogeneous group of muscle diseases related to different genes defects. The results of defects impair myogenic differentiation and histophysiology of skeletal muscles. One of major forms of disease – limb-girdle muscular dystrophy 2B (LGMD2B), caused by dysferlin gene mutations. Both big size of gene and wide variety of mutations complicate development of genetically engineered drugs. Human artificial chromosomes (HAC) could be used as alternative approach. This vector can contain unlimitedly large DNA region for transfer, is retained during cell division, and does not have carcinogenic and immunogenic properties.HAC matrix was developed earlier by Larionov V. et al. (http://www.ncbi.nlm.nih.gov/pubmed/22123967). Optimized DYSF gene sequence (optDYSF) from pAd/CMV/V5-DEST vector (kindly provided by A.A. Rizvanov) was transferred into backbone vector also containing loxP sites for further transfer into HAC. Transfer correctness confirmed by sequencing. After that Chinese hamster ovary cells carrying HAC were transfected by backbone vector containing optDYSF. Selection performed in HAT medium. 7 clones were selected and analyzed at day 10. Expression of optDYSF observed in all obtained clones. Episomal HAC localization determined using fluorescent hybridization in sity by colocalization of label at optDYSF and a-satellite DNA. Episomal localization of HAC determined only in 2 clones from 7. Dysferlin production observed in all clones.On other hand described vector requires somatic myogenic vector cell. Human myoblasts could be successfully used for this purpose. The major source of myoblasts – skeletal muscle tissue is not optimal due to limited proliferative potential, especially in patients with muscular dystrophies. Our team revealed presence of myoblasts in gingival mucosa specimens and developed new method of myoblasts obtainment from it. Primary cultures of multipotent mesenchymal stromal cells (MSC) were derived from gingiva biopsy samples. The cells expressed typical mesenchymal stem cells markers and had ability for 3-way differentiation. Myogenic induction of gingiva derived MSC in culture was demonstrated. Gingiva derived MSC are capable to express markers of myogenic differentiation (skeletal actin, sceletal myosin, MyoD1) and form multicellular elongated fibers – myotubes. Ability of gingiva derived MSC cultures to differentiate in myogenic direction was preserved during up to 10th passage. Such features make this cell source attractive for using in treatment of inherited muscular degenerative diseases.In our future research we are planning to transfer HAC containing optDYSF gene into clonogenic gingiva derived MSC using microcell-mediated chromosome transfer. This method could be used for further development of gene cell therapy of LGMD2B.

Limb girdle muscular dystrophy type 2B masquerading as inflammatory myopathy: case report

Limb girdle muscular dystrophy type 2B is a rare subtype of muscular dystrophy, the predominant feature of which is muscle weakness. The disease is caused by an autosomal recessively inherited reduction/absence of muscle dysferlin due to a mutation in dysferlin gene at 2p12-14. We report a 10 year old boy who presented with severe non-transient right knee pain and swelling, which later became bilateral. His pain was worst in the morning and during rest. Blood tests revealed markedly raised creatine kinase values (highest 22, 297 U/l), raising the possibility of an inflammatory myositis. MRI showed bilateral asymmetrical muscle involvement of thighs and calves with oedematous changes mimicking the imaging appearances of inflammatory myositis. CRP and ESR levels were consistently within normal limits. Over several months his knee pain worsened and limited walking. Muscle biopsy revealed a severe reduction of dysferlin immunostaining, indicating the diagnosis, which was confirmed by 2 compound heterozygous pathogenic mutations in the dysferlin gene. It is not unusual for this subtype of the disease to mimic myositis: however, significant pain is a rare presenting symptom. Given the significant overlap between this form of muscular dystrophy and inflammatory myopathies, a high index of suspicion is needed to ensure an accurate and timely diagnosis. Furthermore, characteristic inflammatory-related morning pain should not rule out consideration of non-inflammatory causes.

Inhibition of muscle fibrosis and improvement of muscle histopathology in dysferlin knock-out mice treated with halofuginone.

Absence of, or loss-of-function mutations in the dysferlin gene (dysf) result in dysferlinopathy, characterized by increased muscle inflammation, collagen deposition and deterioration in muscle function. We evaluated halofuginone efficacy in improving muscle histopathology in mice with deleted dysf transmembrane domain. Quadriceps sublumbar and longissimus muscles of 9-month-old dysf-/- mice treated with halofuginone for 4 months exhibited a reduction in centrally-nucleated myofibers, inflammatory infiltrates and collagen content. Late onset of dysferlinopathy makes it ideal for evaluating the efficacy of early treatments on late outcome. The dysf-/- mice were treated with halofuginone for 3 to 4 months starting at 1, 5 or 9 months of age, and quadricep muscle histopathology was evaluated at 12 months. Collagen content and number of centrally nucleated myofibers decreased after early halofuginone treatment, administered when myofibers with central nuclei and inflammatory infiltrates are evident, but there was almost no fibrosis. When administered at the beginning of fibrosis it resulted in a further decrease in the number of centrally-nucleated myofibers with no additional decrease in collagen levels. Cardiac fibrosis was almost completely abolished following early halofuginone treatment. Halofuginone inhibited Smad3 phosphorylation and its translocation to the nucleus and increased the activity of matrix metalloproteinases 9 and 2 responsible for resolution of pre-existing collagen. Macrophage and myofibroblast invasion into the dystrophic muscle at the site of myofibers with central nuclei was inhibited by halofuginone. These results suggest that early halofuginone treatment can prevent the late outcome of dysferlinopathy and can cause resolution of the established fibrosis when administered at later stages.

Dysferlin-deficient immortalized human myoblasts and myotubes as a useful tool to study dysferlinopathy.

Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well as to test therapeutic strategies at the cellular level.

Two common mutations (p.Gln832X and c.663+1G>C) account for about a third of the DYSF mutations in Korean patients with dysferlinopathy

Dysferlinopathy refers to autosomal recessive muscular dystrophies caused by mutations in dysferlin gene (DYSF). It includes two major distinct disorders, Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Twenty-three Korean patients were recruited. Full sequence analysis of DYSF detected 10 novel and 9 known mutations. The p.Gln832X showed the highest allele frequency (10/46) as a unique recurrent mutation among Korean population, and two common mutations (p.Gln832X and c.663+1G>C) accounted for 34.8% of the identified mutations. Korean DYSF mutations appeared to cluster in the N-terminal region. Notably, none of homozygous mutations was found in this study. Clinical features were similar to previous reports showing onset in early adulthood, high serum CK and inflammatory reactions on muscle pathology. In Miyoshi myopathy, gastrocnemius muscle was first affected on muscle CT scans, and anterior lower legs and thigh muscles were then affected with disease progression. Despite the genetic variety of DYSF mutations, clinical features were rather invariable among the patients.

Muscular dystrophy with marked Dysferlin deficiency is consistently caused by primary dysferlin gene mutations.

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.

Progressive dysphagia in limb‐girdle muscular dystrophy type 2B

Dysphagia has not been reported in genetically confirmed limb-girdle muscular dystrophy type 2B (LGMD2B). A 40-year-old woman reported exercise-induced calf pain at age 34, followed by progressive lower and upper limb weakness. At age 38, progressive dysphagia for solids, and subsequently liquids, ensued. Endoscopic and videofluoroscopic-radiological findings indicated a myopathic swallowing disorder. Molecular genetic analysis confirmed two dysferlin gene mutations consistent with a compound heterozygote state. Progressive dysphagia should be considered as part of the expanding dysferlinopathy phenotype.

Dysferlin‐deficient muscular dystrophy features amyloidosis

Dysferlin (DYSF) gene mutations cause limb girdle muscular dystrophy type 2B and Miyoshi's myopathy. The consequences of DYSF mutations on protein structure are poorly understood. The gene encoding dysferlin was sequenced in patients with suspected dysferlin-deficient muscular dystrophy. Muscle biopsy specimens were analyzed by histochemistry, immunohistochemistry, and electron microscopy. Antibodies against N-terminal dysferlin-peptides were raised. We found three families with muscular dystrophy caused by homozygous or compound heterozygous DYSF mutations featuring sarcolemmal and interstitial amyloid deposits. These mutations were all located in the N-terminal region of the protein. Dysferlin was a constituent of the amyloid deposits. Limb girdle muscular dystrophy type 2B is the first muscular dystrophy associated with amyloidosis. Molecular treatment strategies will necessarily have to consider the presence of amyloidogenesis.

Dysferlinopathy: Clinical aspects in India

Mutations in the dysferlin gene on Chromosome 2p cause dysferlinopathies which include two distinct clinical entities, Miyoshi myopathy (MM) (OMIM no. 254130) and limb girdle muscular dystrophy 2B (LGMD2B) (OMIM no. 253601). They have been diagnosed as different diseases clinically depending on the affected muscles at the onset. In this issue of Neurology India, Nalini and Gayathri reported the differences in their clinical findings in India,[1] while the histopathological characteristics of their muscle specimens were not any different. The authors have presented the largest study of dysferlinopathy in India which focused on clinical aspects, according to our knowledge . MM and LGMD2B are autosomal recessive, slow progressive muscular dystrophies recognized by primary deficiency of dysferlin expression on their patients’ muscles. The patients who have dysferlin deficiency are affected in the teens or early adulthood, and show dramatic elevation of serum creatine kinase (CK).[2] Liu and colleagues discovered dysferlin, a skeletal muscle gene, which is found to be mutated in both MM and LGMD2B.[3] The study employing dysferlin-null mice showed that the mice developed progressive muscular dystrophy and exhibited elevated serum CK like human dysferlinopathy. [4] Dysferlin-deficient muscle fibers were defective in Ca2+-dependent sarcolemma resealing and it suggested the membrane repair system did not work during contraction-induced injuries. We reported that dantrolene, a well-known Ca2+ release inhibitor during muscle contraction, was effective for some Japanese MM siblings.[5] Altogether these facts might support that both dysferlinopathies had the same pathological background. Although both diseases have the same genetic and pathological background, they are distinguishable clinically. Basically, the calf muscles are affected at the initial stage in MM, but LGMD2B begins with proximal muscle weakness. The investigators focused on the clinical differences between MM and LGMD2B employing 28 Indian patients who had been diagnosed as dysferlinopathy by immunohistochemical analysis since 6.4±4.2 years. They found that the occurrence of distal muscle weakness in upper limbs was seen specifically in MM patients. It was pretty interesting, because originally Miyoshi and colleagues focused on the lower limb weakness.[2] Another Japanese group focused on the clinical differences between patients with and without G3370T or G3510A mutation in dysferlin gene, but in only MM. They found that the clinical onset was delayed in the patients with G3370T mutation and higher serum CK was seen in the patients with G3510A mutation.[6] The same group has recently reported that a 53-year-old man with LGMD2B (homozygous G3370T) mutation had chorea in addition to the myopathy. This is perhaps explained by the expression of smaller brain isoform of dysferlin in putamen.[7] These findings imply that the future genetic analysis might be able to provide new correlation between dysferlin mutation and clinical phenotype and much longer follow-up could uncover other interesting central nervous system-derived symptoms. As the authors noticed, they had to still exclude calpainopathy (LGMD2A) (OMIM no.253600) and caveolinopathy (LGMD1C) (OMIM no.607801). Three cases of LGMD2A showed a double defect of both calpain-3 and dysferlin in dystrophy muscles[8] and dysferlin expression was patchy in LGMD1C muscles.[9] Further genetic analysis or caveolin-3 immunostaining for LGMD1C might be helpful for differential diagnosis in the future. Nevertheless the present paper has outlined clinical details about both forms of dysfelinopathy in India preciously, and it will be further informative to uncover the geographical and ethnical differences of dysferlinopathy in the world.

Phenotypic Study in 40 Patients With Dysferlin Gene Mutations

To describe the phenotypic spectrum of dysferlin (DYSF) gene mutations (which cause dysferlinopathies, autosomal recessive muscular dystrophies) in patients with a dysferlin protein deficiency. Clinical, biological, and pathological data from 40 patients were reviewed. The diagnosis of dysferlinopathy was based on the absence or strong reduction of dysferlin in muscle, and confirmed by mutational screening of the DYSF gene. Two French neuromuscular diseases centers (in Paris and Marseilles). Two main dysferlinopathy phenotypes are well recognized: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B were found in 20 (50%) patients only. Unusual phenotypes included a mixed phenotype, referred to as "proximodistal," combining distal and proximal onset in 14 (35%) patients, pseudometabolic myopathy in 4 (10%), and asymptomatic hyperCKemia (an increased serum creatine kinase level) in 2 (5%). The disease may worsen rapidly, and 10 (25%) patients were initially misdiagnosed as having polymyositis. We suggest a relationship between proximodistal phenotype, inflammation, and severity. In addition to typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, dysferlinopathies are a clinically heterogeneous group of disorders ranging from asymptomatism to severe functional disability.

Symptomatic dysferlin gene mutation carriers: Characterization of two cases

To describe two symptomatic dysferlin gene mutation carriers. One patient had limb girdle weakness. His brother was diagnosed with limb girdle muscular dystrophy 2B with two mutations in the dysferlin gene (D625Y and E1734G). The second patient had distal weakness. He had two sons with Miyoshi myopathy with a homozygous mutation (G519R). We performed immunofluorescence (dystrophin, DAG proteins, dysferlin, caveolin-3), Western blot (dysferlin, caveolin-3, calpain-3), and real-time PCR (dysferlin) using skeletal muscle samples. We also studied dysferlin in peripheral blood monocytes (PBMs) by Western blot. In addition to the muscle weakness, both patients showed elevated creatine kinase and abnormal muscle MRI. They presented a mutation in only one allele after screening of the whole gene (skeletal muscle and monocyte mRNA and genomic DNA). A muscle biopsy specimen showed moderate dystrophic changes and patchy dysferlin expression in the sarcolemma. Western blot of both PBMs and skeletal muscle demonstrated a significant reduction in dysferlin. All the other proteins including caveolin-3 and calpain-3 were normal. Real-time PCR showed normal levels of dysferlin mRNA vs the patients' affected relatives. The diagnosis of symptomatic carriers of dysferlin mutations should be considered when a pathologic pattern of dysferlin protein is observed.

Patients with a Non‐dysferlin Miyoshi Myopathy have a Novel Membrane Repair Defect

Two autosomal recessive muscle diseases, limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), are caused by mutations in the dysferlin gene. These mutations result in poor ability to repair cell membrane damage, which is suggested to be the cause for this disease. However, many patients who share clinical features with MM-type muscular dystrophy do not carry mutations in dysferlin gene. To understand the basis of MM that is not due to mutations in dysferlin gene, we analyzed cells from patients in one such family. In these patients, we found no defects in several potential candidates - annexin A2, caveolin-3, myoferlin and the MMD2 locus on chromosome 10p. Similar to dysferlinopathy, these cells also exhibit membrane repair defects and the severity of the defect correlated with severity of their disease. However, unlike dysferlinopathy, none of the conventional membrane repair pathways are defective in these patient cells. These results add to the existing evidence that cell membrane repair defect may be responsible for MM-type muscular dystrophy and indicate that a previously unsuspected genetic lesion that affects cell membrane repair pathway is responsible for the disease in the non-dysferlin MM patients.

Proteomic investigation of the molecular pathophysiology of dysferlinopathy

Mutations in dysferlin gene cause several types of muscular dystrophy in humans, including the limb-girdle muscular dystrophy type 2B and the distal muscular dystrophy of Miyoshi. The dysferlin gene product is a membrane-associated protein belonging to the ferlins family of proteins. The function of the dysferlin protein and the cause of deterioration and regression of muscle fibres in its absence, are incompletely known. A functional clue may be the presence of six hydrophilic domains, C2, that bind calcium and mediate the interaction of proteins with cellular membranes. Dysferlin seems to be involved in the membrane fusion or repair. Molecular diagnosis of dysferlinopathies is now possible and the types of gene alterations that have been characterized so far include missense mutations, deletions and insertions.