Limb-Girdle Muscular Dystrophies In Northeastern China

Background:Limb-girdle muscular dystrophies (LGMD) are group of emerging dissimilar diseases that made some investigators dispute the term rationality, the mutual feature is limb-girdle affection. Herein, we reviewed the disease from historical, nosological, pathophysiological, diagnostic, preventive, therapeutic and future perspectives with specific attention to newly evolving techniques to bring these hereditary diseases from research bench to clinical zone and energies that made the incredible recovery from such group of diseases, now part of reality at least in preclinical sector. In conclusion, rapid progress was made in LGMD field in the foregoing decades, yet international patient registry, regular follow up, improvement of diagnostic standards for LGMD subtypes and more understanding pathophysiology of LGMD forms will feasibly improve disease outcome in subsequent years. We also added special chapter for LGMD in China and progression of molecular diagnosis and pathophysiological understanding.Historically, Walton and Nattrass (1954) first devised name’limb-girdle muscular dystrophies’to comprise cases of both sexes, beginning usually within the first three decades, with major involvement of scapular, pelvic girdle and trunk muscles and sparing of facial muscles, infrequent pseudo hypertrophy, moderately severe progression and usually an autosomal recessive mode of inheritance. In1995, the European Neuromuscular Centre Workshop established more precise criteria for diagnosis and classification of LGMD, grouping the different subtypes according to their genetic characteristics.As it is explicit from long nosological history of LGMD; it is not homogeneous disease, we can just consider LGMD like an ’umbrella’ term under which more than two dozens of gene defects were recognized.Mechanisms of action of LGMD’s involved proteins are diverse. In general we can classify LGMD subtypes according to site of their affected proteins into: sarcolemmal, sarcoplasmic, cytosolic proteins, sarcomeric, myofibrillar proteins and nuclear envelope proteins or according to their functions to:scaffolds, patch up, remodeling, assembling, glycosylating, preventing protein from irreversible aggregation or unknown function.Most of our knowledge on LGMD therapy came from animal models through reversal of etiopathogenic agents prompting the disease or through disrupting some steps believed to be downstream pathology of missing gene.To evaluate clinical outcome of pharmacological, cell or gene therapies, authors considered some markers that could reflect disease activity, regression and treatment efficacy without need to do multiple muscle biopsies e.g., contrast agent-enhanced MRI, secreted alkaline phosphatase level (Se AP) in blood, circulating monocyte, luciferase levels, neutralizing antibodies and INF-y to recombinant adeno associated virus (r AAV).The relative proportion of each LGMD form varied considerably among different populations. In general LGMD2A represents the most common type in different populations followed either by dysferlinopathies in some areas or by dystroglycanopathies in others or by sarcoglycanopathies in the third. LGMD made the second most common dystrophy after dystrophinopathies in several countries.Genotype-phenotype correlation is yet difficult to predict and this sector of genetic disorders represents the most obscure and confusing field for investigators and may interpret why most types of gene therapies nowadays attained suboptimal level of recovery.Clinical, electrophysiological, imaging, biochemical and genetic testing all should be recruited and tailored according to specific case not only to reach diagnosis and provide genetic counseling to probands and relatives but also to understand pathophysiology and to target therapy to precise pathway.Magnetic resonance imaging (MRI), perioral skin biopsies, blood based-assay, reverse-protein array, gene expression profiling, proteomic analysis, gene chips, exome and wide genome next generation sequencing are the most talented emerging techniques to facilitate LGMD diagnosis, whereas gene, cell and pharmaceutical therapies are the most important presumed therapies for this hereditary group of diseases.Genetic counseling is encouraged to reduce the burden of the disease in the community, especially in areas where founder mutation has been determined.Like other inherited disorders, no available therapy is provided to LGMD patients to date. Management is mainly supportive emotionally and physically.China contributed greatly to development of molecular genetic biology field; still many hurdles need to be passed in neuromuscular research area in general and LGMD especially. Rarity, heterogeneity, non-curability of LGMD made neuromuscular research committee in China pay less attention to this growing field of medicine. There still some obstacles in the way of LGMD project in China need to be paved in near future concerning national registry of patients, clinical outcomes measures and readiness to future clinical trials. We propose collaborative network among neuromuscular disease centers that help manage LGMD problem according to agreed national standards.More enigmas have been solved in recent years regarding diseases occurrence, founder effect in some locales and some aspects of pathophysiology and phenotype-genotype correlations. In addition, elucidation of hot spot mutations, disease biomarkers, general strategy of the diagnosis and treatments would point toward efficient and safe follow up and intervention. Yet, more emphasis should be put on pathogenesis, updating of diagnostic guidelines, with regular assessment and close follow up of disease progression to enrich translational research.Study1Background:Some hereditary myopathies can mimic acquired polymyositis (PM) in particular when they first present in adulthood with a limb-girdle distribution of weakness. The diagnosis is made yet more complex when inflammatory infiltrates attend muscle biopsy. This is common in certain dystrophies in particular limb-girdle muscular dystrophies.The clinical picture of LGMD and PM is rather similar, e.g., muscle weakness and atrophy, and often confuses clinicians. Muscle biopsy from both patients, as aforementioned, may demonstrate inflammatory infiltrates. Dramatic progress in biochemical and molecular biology in the last decade had made pathologist somewhat entirely dependent on immunohistochemical, immunoblot and genetic screening results in differentiating the two diseases. However, in some undeveloped areas in the world where such advanced approaches are unobtainable, muscle biopsy and routine histochemical stain are often used to help diagnose muscle diseases. Present literature focuses little on morphological findings of muscular diseases, hence a comprehensive study including various parameters of myopathy and dystrophy, comparing between the most confusing myopathic diseases represents necessity.Methods:For better distinction between the two diseases, we performed histochemical morphometric analysis for twenty LGMD biopsies confirmed by protein testing and eight PM biopsies met Dalakas and Hohlfeld2003definite criteria for polymyositis. Each parameter of myopathy was evaluated in five or ten randomly selected fields, using Image-Pro plus bio-analytical software. Comparison between groups was tested using independent samples Student’s t-test analysis.Results:The muscle of LGMD patients revealed more splitting fibers and larger connective tissue surface area in comparison to PM patients (P=0.02,0.01respectively). On the opposite the number of regenerating fibers was higher in myositis biopsies (P=0.04). Other parameters were akin to those of PM patients. We also described different patterns of major histocompatibility complex I (MHC-I) up regulation on LGMD biopsies.Conclusion: We reported some tips of difference between LGMD biopsies and polymyositis. The results attained in the present study have a considerable diagnostic application: wide area of connective tissue combined with high number of splitting fibers and less regenerating fibers should alarm pathologist about dystrophic process. This quantitation study represents the first, involving many parameters of dystrophy comparing them with those of inflammatory myopathy.Study2:Background:The relative frequency of different limb-girdle muscular dystrophies subtypes varies widely among different populations. While the most common LGMD subtype in Italy, Spain, Turkey, Russia, Brazil and Australia is calpainopathy (LGMD2A), the prevalence of Dystroglycanopathy (LGMD2I) in Norway, Denmark and North England ranks the first among LGMD. In India, sarcoglycanopthies (LGMD2C-2F) quoted the highest figures. Yet USA, Mexico and Japan reported dysferlinopathy as the most frequent type.Though much is known about clinical features discerning calpainopathy from dysferlinopathy, little is known concerning their pathological findings. Many reports described clinical features and pathological findings of each disease separately and correlated genotype to phenotype in order to outline each subtype in a well characterized fashion, nevertheless comparable pathological consequences of most dystrophic diseases made the field of morphology vacant except for unique findings. Though, wide array of disease activities ranging from slowly progressive to malignant LGMD may possibly be reflected on morphological features. A rigorous comparison between both LGMD2A and LGMD2B biopsy sections was warranted. In China to date, to our acquaintance, only two cohorts were separately published regarding LGMD; each considered only one LGMD form. Low incidence rate, rarity of consanguineous marriage and one child policy, all hampered wide statistics of this group of diseases. To our knowledge, no cohort has been conducted in China, rating the relative frequency of different LGMD subtypes.Purpose, material and methods:The purpose of the experiment is to:●Summarize the main clinical features and pathological characteristics of the different LGMD forms.●Compare the morphological findings between the two major LGMD subgroups.Authors ascertained retrospectively all patients with limb-girdle phenotype from the Muscle Center of First Hospital of Jilin University, Changchun, China, for the period from august2007to January2012. Total number of patients was68for which the following proteins were tested:calpain3, dysferlin, a-sarcoglycan and caveoin3. Here in, we also made pathological correlations between the most common subtypes and reported some odd clinical and pathological features. Besides, we utilized acid phosphatase reaction to label macrophages on biopsies with cellular infiltrates.Results: Whilst the diagnosis of calpainopathy was mounded to twelve cases (17%), dysferlin deficit was detected in ten (15%). Two biopsies revealed a-sarcoglycan deficiency (3%) and other two showed caveolin3lack (3%). The remaining patients stayed with the diagnosis of unclassified LGMD (62%). In general, our results coincide with those in Italy, Turkey, Russia, Brazil and Australia with parallel percentages of various LGMD forms. Now it is accepted that calpainopathy is the most frequent LGMD subtypes worldwide followed by dysferlinopathy whereas LGMD1C represents2-3%in most conducted cohorts. Whether our sample is representative of Chinese population, still needs to be elucidated by collaborated multicenter LGMD research project throughout China.We made use of acid phosphatase stain as an alternate of anti-macrophage antibodies to quantify and scan distribution of macrophages on DYSF mutant muscles. Authors could benefit from this easy and cost-effective method with comparable specificity to monoclonal antibodies.Many studies delivered inflammatory infiltrates in various LGMD subtypes, yet to our information, inflammatory cells in adhalin deficient cases, had never been hitherto described. We identified extensive infiltrates in LGMD2D biopsies, mainly macrophages (stained by acid phosphatase reaction) with little eosinophils. Calpain3deficient biopsies were insignificantly different from dysferlinopathies (χ2, P>0.05) from morphological perspectives.Creatine kinase (CK) level was highest in LGMD2B>LGMD2D>LGMD1C>LGMD2A in decreasing order of their values. The results quite indicate that dysferlinopathies are the most active disease followed by sarcoglycanopthies and these data were parallel to figures depicted from other geographic areas. neurogenic muscular diseases and myotonic dystrophy. Yet, they have not been determined in LGMD biopsies.To date, few reports have been published of genetically confirmed dysferlinopathy in China. In this report, we described three LGMD2B and one MM patients with their clinical, physiological, biochemical and molecular findings.Purpose, Material and methods:We followed genetically four biochemically proved dysferlinopathic Chinese individuals with atypical morphological and physiological features using direct gene sequencing to decode secretes behind these phenomena.Results:We reported genetically confirmed dysferlinopathic patients with their clinical, biochemical and molecular features. Three unrelated patients were presented with pelvic muscles weakness and wasting while calf atrophy brought the fourth for medical attention. Asymptomatic cardiac problem was discovered incidentally in one patient. Biopsy specimens showed variable dystrophic, myopathic and inflammatory patterns. Rimmed vacuoles were identified in another patient’ s biopsy and mononuclear inflammatory infiltrates of non-necrotic fibers (MNN) usually seen in polymyositis were detected in the third, whereas the fourth expressed pyknotic nuclear clumps. Seven different mutations were recognized, four were novel. One mutation shared the hot-spot mutation seen in Japanese and Koreans.Conclusions: Our findings emphasize dysferlinopathy should be considered in differential diagnosis of dystrophy with cardiac disorders, rimmed vacuoles-myopathy, mononuclear inflammatory infiltrates of non-necrotic fibers or pyknotic nuclear clumps. These features might enhance our present knowledge of pathophysiology of dysferlin and might have influence on future therapeutic promises. The study might also point out that the hot-spot mutation seen in northeastern Chinese and other eastern Asians could feasibly and cost-effectively be screened before running whole gene sequencing. To our information, this is the first report of genetically confirmed LGMD2B from northeastern China.In our view, inflammosome up-regulation and complement activation mechanisms are not working on cardiomyocytes given no detected infiltrates and normal gene expression of complement inhibitor decay-accelerating factor/CD55in cardiac muscles. Understanding the protective mechanism working against dysferlin deficiency in heart muscles may have impact on future therapeutic choice.We documented mononuclear inflammatory infiltrates of non-necrotic fibers (MNN) in a genetically confirmed Miyoshi myopathy (MM) patient. The case might emphasize that mechanism involved in polymyositis could also be active in dysferlin deficient muscles. The hypothesis has been substantiated by recent work of Cohen et al which revealed that pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) was up-regulated in dysferlin deficient myoblasts and anti-inflammatory drug’celastrol’reduced the NFκB triggering and improved myoblasts myogenesis.Pathological significance of rimmed vacuoles is yet unresolved, however, LGMD2B must be added to the list of ’proximal myopathy with rimmed vacuoles’ and DYSF should be one of candidate genes to be tested if vacuoles were challenged histologically.Protein-genotype correlation is hard to be drawn given small number of cases, nevertheless patients with only missense mutations showed marked dysferlin reduction, but not complete absence, while entire protein paucity was obtained in the null-mutation verified biopsy.Based on our current knowledge, it is random to call the novel missense mutations in cardiac affected patient as’cardiopathic’. Yet, more understanding of dysferlin physiology in health and disease and its interactions with other proteins and with external stressor would feasibly solve the mystery of dysferlin gene defect.It is priori that child born to indigenous marriage, theoretically should have mutation in homozygous state given common ancestor confers the same mutation. Though, this is not always the case and the child might be affected with two heterozygous mutations inherited separately from uncommon ancestors. Or denovo mutations ensued in that child. We described MM patient with two heterozygous mutations born to consanguineous marriage. Unfortunately, parents of our patient were not assessed genetically to see the specific mechanism working in that case. Nonetheless, many papers described heterozygous mutations in children born to related parents.The detected missense mutation c.2997G> T (P. T999C) was frequently reported in Japanese and Koreans. The age of onset, the severity of symptoms and protein expression was in accordance to what have been previously described by Tagawa et al. It was surprising that the mutation was associated with different phenotypes (LGMD2B, MM and rigid spine syndrome) in Japanese population. This confirms the view that no genotype-phenotype relationship can be outlined in dysferlinopathies and rather some epigenetic influences and other modulating genes, still to be detected, may perhaps decide the final form of distribution of weakness. The case was considered as one of few reported symptomatic DYSF mutation carriers.