Frozen Muscle Biopsies Research Articles

PROGRESSIVE MUSCLE HYPOXIA RESEARCH: COULD PHARMACOLOGICAL AGENTS BE USED TO LIMIT REPERFUSION INJURY AT THE TIME OF FASCIOTOMY TO TREAT ACUTE COMPARTMENT SYNDROME?

AbstractBackgroundProgressive muscle ischaemia results in reduced aerobic respiration and increased anaerobic respiration, as cells attempt to survive in a hypoxic environment. Acute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency resulting in the production of Lactic acid by cells through anaerobic respiration. Our previous research has shown that it is possible to measure H+ ions concentration (pH) as a measure of progressive muscle ischaemia (in vivo) and hypoxia (in vitro). Our aim was to correlate intramuscular pH readings and cell viability techniques with the intramuscular concentration of key metabolic biomarkers [adenosine triphosphate (ATP), Phosphocreatine (PCr), lactate and pyruvate], to assess overall cell health in a hypoxic tissue model.MethodsNine euthanised Wistar rats were used in a non-circulatory model. A pH catheter was used to measure real-time pH levels from one of the exposed gluteus medius muscles, while muscle biopsies were taken from the contralateral gluteus medius at the start of the experiment and subsequently at every 0.1 of a pH unit decline. The metabolic biomarkers were extracted from the snap frozen muscle biopsies and analyzed with standard fluorimetric method. Another set of biopsies were stained with Hoechst 33342, Ethidium homodimer-1 and Calcein am and imaged with a Zeiss LSM880 confocal microscope.ResultsOur study shows that the direct pH electrode readings decrease with time and took an average of 69 minutes to drop to a pH of 6.0. The concentrations of ATP, pyruvate and PCr declined over time, and the concentration of lactate increased over time. At pH 6.0, both ATP and PCr concentrations had decreased by 20% and pyruvate has decreased by 50%, whereas lactate had increased 6-fold. The majority of cells were still viable at a pH of 6.0, suggesting that skeletal muscle cells are remarkably robust to hypoxic insult, although this was a hypoxic model where reperfusion was not possible.ConclusionsOur research suggests that histologically, skeletal muscle cells are remarkably robust to hypoxic insult despite the reduction in the total adenine nucleotide pool, but this may not reflect the full extent of cell injury and quite possibly irreversible injury. The timely restoration of blood flow in theory should halt the hypoxic insult, but late reperfusion results in cellular dysfunction and cell death due to localised free radical formation. Further research investigating the effects of reperfusion in vivo are warranted, as this may identify an optimal time for using pharmacological agents to limit reperfusion injury, around the time of fasciotomy to treat acute compartment syndrome.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Clinical Characteristics of Systemic Sclerosis-associated Myopathy Patients Comparing Different Subgroups of Inflammatory Myopathies

Background: Available data regarding clinical characteristics of systemic sclerosis-associated myopathy (SSc-M) patients comparing different subgroups of muscle pathology are limited. We aimed to compare clinical and laboratory findings among different subgroups of Thai patients with SSc-M. Methods: From January 2010 to December 2019, 27 patients with suspected SSc-M underwent a muscle biopsy. Twenty-three patients with available frozen muscle biopsy specimens for repeating immunohistochemical stained for reviewing were included. There were three subgroups of pathological findings, including immune-mediated necrotizing myopathy (IMNM), non-specific myopthy (NsM), and polymyositis (PM). No fibrosing myopathy was observed. Baseline clinical data and laboratory findings were compared within those three inflammatory myopathies. Results: Of the 23 SSc-M, there were 14 females and 19 DcSSc with a mean age and disease duration of SSc of 53.6±7.7 years and 16.4±23.6 months, respectively. Their mean duration from weakness to muscle biopsy was 3.6±6.0 months. There were 14 (60.9%) patients with IMNM, 6 (26.1%) with NsM, and 3 (13.0%) with PM. At the biopsy date, IMNM had a greater prevalence of severe muscle weakness (42.6% vs. 0% vs. 0%) and arthritis (87.5% vs. 50% vs. 0%) than the NsM and PM groups. There was no significant difference among the three inflammatory patterns regarding baseline clinical characteristics, including age, gender, SSc subtype, disease duration, other organ involvements and median values of CK and ESR levels. Conclusion: In this study, we found that the pathological findings of Thai SSc-M were IMNM, NsM, and PM. No fibrosing myopathy was observed. SSc with IMNM tended to have more severe baseline muscle weakness and arthritis than the other inflammatory patterns.

Editorial introductions.

Current Opinion in Neurology was launched in 1988. It is one of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of neurology is divided into 14 sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Journal's Section Editors for this issue. SECTION EDITORS Luis QuerolLuis QuerolLuis Querol (Valencia, 1980) graduated in Medicine at the University of Oviedo, Spain, and completed his residency in Neurology at the Santa Creu i Sant Pau Hospital in Barcelona. In 2009, he joined the group of Prof. Isabel Illa where he developed his doctoral thesis on the role of autoantibodies in immune-mediated neuropathies. In 2012, he worked as an associate researcher in the group of Dr Kevin O’Connor, Department of Neurology, Yale University (USA), where he developed projects related to autoantibodies in multiple sclerosis and myasthenia gravis. He completed his PhD in Medicine at the Universidad Autónoma de Barcelona, Spain, in 2013 thanks to studies that identified anti-contactin antibodies in a specific subgroup of patients with CIDP, in what is the first clinically useful marker detected in CIDP. Since then, he has led the auoimmune neurology clinics at Hospital Sant Pau in Barcelona and has continued a fruitful scientific production in the field of autoantibodies and biomarkers in autoimmune neurological disorders. Ichizo NishinoIchizo NishinoDr Ichizo Nishino, FAAN, FANA, is Director of Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP). He obtained his M.D. in 1989 and Ph.D. in 1998 from Kyoto University, Japan. After five years of training in clinical neurology and six years of research fellowship, including two years at Columbia University, he was appointed to his current position in 2001. By now, he has published more than 600 PubMed-listed papers in the field, including the reports of gene discoveries for MNGIE and Danon disease, and the report of a new muscle disease – megaconial congenital muscular dystrophy (OMIM: 602541) whose causative was also later identified by his own group. His laboratory functions as a nationwide referral center for muscle disease, providing diagnostic analyses for muscle pathology and genetic diagnosis. His lab receives around 80% of muscle biopsies performed in Japan (1103 cases in 2021) and he signs out all cases. As a result, more than 22,000 frozen muscle biopsy samples have been accumulated in his muscle repository, which is one of the largest collections of the patient's muscles. Currently, Dr Nishino is a Visiting Professor at six universities: University of Yamanashi (Japan), Shinshu University (Japan), Kaohsiung Medical University (Taiwan), Fu Jen Catholic University (Taiwan), Peking University First Hospital (China), and Siriraj Hospital Mahidol University (Thailand). He is also serving as President of Asian-Oceanian Myology Center and Executive Board member in several societies including World Muscle Society. Albert LudolphAlbert LudolphAlbert Ludolph is currently Professor of Neurology, Chair and Director of the Department of Neurology, University of Ulm, Ulm, Germany and Speaker of the Site Ulm of the German Center for Neurodegenerative diseses (DZNE, Helmholtz Society). He obtained his M.D. from the University of Mainz, Germany, in 1981 and is a boarded neurologist and psychiatrist (education from University of Münster, Germany). His research focusses on translational work in neurodegenerative diseases; his work is mirrored by more than 600 PubMed-listed papers. His specific focus of interests are motor neuron diseases, Huntington's disease and frontotemporal dementias. The Department of Neurology in Ulm is an international referral center in particular for patients with ALS/MND. In the past his research focused on neurotoxicological and neuropharmacological research in humans, but also non-human primates. He was involved and initiated studies of neurogenetics of ALS, of the epidemiology of the disease (established registers in Southern Germany, Egypt and Mongolia), of the definition of the neuroanatomical sequence of events describing ALS and FTD, the development of biomarkers in ALS and FTD and beyond, and several drug trials, including investigator-initiated interventions. In recent years he tried to define the preclinical period of ALS including the role of the hypothalamus and its metabolic consequences.

NEW INSIGHTS INTO CELLULAR OR MUSCLE FUNCTION: EP.308 Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies

NEW INSIGHTS INTO CELLULAR OR MUSCLE FUNCTION: EP.308 Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies.

Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss.

Anatomopathological Assessment of the Diaphragm in Formalin-Fixed, Paraffin-Embedded Sections

Introduction The analysis of frozen muscle biopsies has become a routine method in the evaluation of muscle structure in health and disease. However, the technique for frozen muscle specimens is not widely available in countries with limited medical facilities. The present study aimed to elucidate a reproducible formalin-fixed and paraffin-embedded (FFPE) method for this type of analysis in postmortem muscles. Methods Diaphragm muscle was obtained within 1 hour of sudden death. Diaphragm strips were washed in saline solution, fixed in 10% formalin, frozen at 4°C in a refrigerator, and stored for 24 hours. Then, the tissue samples were processed into paraffin-embedded blocks. Transversal sections were cut from each paraffin block and stained with hematoxylin and eosin, Picrosirius red, Verhoeff-Van Gieson, and Congo red for the qualitative analysis. Results Our analysis indicated a well-preserved muscle. Conclusion In summary, we demonstrate a simple technique for a reproducible FFPE method in postmortem muscle tissues.

Validation of a Muscle-Specific Tissue Image Analysis Tool for Quantitative Assessment of Dystrophin Staining in Frozen Muscle Biopsies.

Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.

Establishment of primary myoblast cell cultures from cryopreserved skeletal muscle biopsies to serve as a tool in related research & development studies

BackgroundPrimary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. Methods and resultsThis protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at −80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. ConclusionsThis protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

The expression of gene ANKRD1 correlates with hypoxia status in muscle biopsies of treatment-näive adult dermatomyositis

OBJECTIVES. The ANKRD1 gene codes for the ankyrin repeat domain containing protein 1 and has an important role in myogenesis and possibly also in angiogenesis. Microvasculopathy is a cornerstone and an early pathological marker of change in dermatomyositis, leading to hypoxia and muscle perifascicular atrophy. These alterations could upregulate genes involved in myogenesis and angiogenesis such as ANKRD1. Therefore, we analyzed ANKRD1 expression in muscle biopsies of dermatomyositis and correlated with other hypoxia parameters and with histological changes. METHODS. Total RNA was extracted from frozen muscle biopsies samples of 29 dermatomyositis patients. A control group consisted of 20 muscle biopsies from adult patients with non-inflammatory myopathy diseases. The gene coding for hypoxia-inducible factor 1, alpha subunit (HIF1A), was analyzed to estimate the degree of hypoxia. ANKRD1 and HIF1A transcript expression levels were determined by quantitative real time PCR. RESULTS. Significantly higher ANKRD1 and HIF1A expression levels were observed in dermatomyositis relative to the control group (P<0.001, both genes). In addition, ANKRD1 and HIF1A were coexpressed (r=0.703, P=0.001) and their expression levels correlated positively to perifascicular atrophy (r=0.420, P=0.023 and r=0.404, P=0.030, respectively). CONCLUSIONS. Our results demonstrate ANKRD1 overexpression in dermatomyositis correlated to HIF1A expression and perifascicular atrophy. ANKRD1 involvement in myogenesis and angiogenesis mechanisms indicates that further investigation is worthwhile.

P.403 - Muscle biopsies reprocessed for electron microscopy from paraffin blocks and frozen tissue produce material of sufficient quality for diagnostic use

Reprocessed frozen tissue (FT) or formalin-fixed paraffin-embedded (FFPE) tissue for transmission electron microscopy (TEM) has been used in general pathology but there is little information on its use in diagnostic muscle pathology. A recent study of re-processed stored frozen muscle biopsies from healthy volunteers showed morphology almost comparable to that of freshly fixed muscle (FFM). This is a retrospective study of 8 muscle biopsies referred to the DNC pathology service where TEM was required to confirm/exclude diagnosis and only FFPE/FT blocks were available. A small strip parallel to the fibres was cut from each of the FT blocks in the cryo-cabinet at −25°C and immediately placed into cold 4% glutaraldehyde in cacodylate buffer and some also into a 2% glutaraldehyde/2% paraformaldehyde mixture, allowed to thaw at room temperature, then cut into smaller pieces approximately 1.5 × 0.5 mm2. After 1.5 hours the samples were thoroughly rinsed, dehydrated through an ethanol series to propylene oxide and embedded in Araldite resin. Stained slides were reviewed to select an area of interest for FFPE blocks; these were cut and de-waxed overnight in chloroform, then re-hydrated, fixed and processed as above. Resin semi-/ultra-thin sections were cut and stained as per standard procedures and viewed with a Phillips CM10 or CM12 electron microscope. Reprocessed FT/FFPE tissue morphology was somewhat poor compared to FFM, but there were better-preserved foci giving good diagnostic yield. Mitochondrial morphology was poorer in reprocessed cases. Diagnostic pathology identified included reducing bodies, granulofilamentous aggregates, nemaline rods, polyglucosan aggregates and abnormal mitochondria (5/9), non-specific pathology (2/9) and exclusion of macrophagic myofascitis (1/9). In conclusion, in instances where FFM processed tissue for TEM is not available, it is possible to produce adequate results using re-processed FT/FFPE material for diagnostic muscle biopsies.

Oral Essential Amino Acids, Sprint Exercise And Collagens In Human Skeletal Muscle

PURPOSE: To investigate the effects of oral ingestion of essential amino acids (EAA exercise on the global gene expression in skeletal muscle after sprint exercise. METHODS: Twelve healthy physically active subjects (age 20-30 years) performed three 30-s cycle sprints with maximal voluntary pedaling rate on a mechanically braked cycle ergometer with 20 min rest between the sprints. The braking force was set at 0.75 N/kg body weight. The subjects consumed either EAA + maltodextrin solution or flavored water (placebo) in a randomized order. A post exercise biopsy (m. vastus lateralis) was taken 200 minutes after the last cycle sprint. RNA was isolated from the frozen muscle biopsies using TRIzol protocol. Purification was executed using RNeasy Mini Kit (Qiagen). The RNA concentration was determined using the NanoDrop (Thermo Fischer Scientific) and quality assessed by Agilent (Agilent Technologies). The Affymetrix Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual was used for complementary DNA (cDNA) generation, hybridization and array processing (GeneChip® Human Transcriptome Array 2.0). Differentially expressed genes in response to EEA in combination with the sprint exercise were analyzed using the Ingenuity Pathways Analysis (IPA) software application. To validate microarray data total RNA was reverse transcribed using random hexamer primers. Real-time PCR was applied to measure mRNA expression using RPS18 as reference to correct for potential variation in RNA loading. RESULTS: The IPA Network analysis showed an enrichment of activated genes related to collagen metabolism. Some of the genes in the pathway analysis were validated by the PCR analysis. The expression of COL15A1, COL1A1, COL2A1 and BGN was higher in the EEA condition compared to the placebo condition (P<0.05). CONCLUSION: Oral ingestion of EAA after sprint exercise increases expression of genes related to collagen metabolism. The functional role of an activation of collagen metabolism is not known, although it could be speculated that an altered collagen metabolism reflects an extracellular remodeling after exercise and that this process is enhanced by oral ingestion of EEA. SUPPORT: The study was supported by grants from the Swedish National Center for Research in Sports.

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris).

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 μm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was "type 9", 1-2 μm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7-9 μm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were "type 1", thin walled (< 0.5 μm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin-fixed paraffin-embedded sections. Dogs were either singly infected with S. caninum or multiply co-infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).

Assessing dystrophies and other muscle diseases at the nanometer scale by atomic force microscopy.

Atomic force microscopy nanoindentation of myofibers was used to assess and quantitatively diagnose muscular dystrophies from human patients. Myofibers were probed from fresh or frozen muscle biopsies from human dystrophic patients and healthy volunteers, as well as mice models, and Young's modulus stiffness values were determined. Fibers displaying abnormally low mechanical stability were detected in biopsies from patients affected by 11 distinct muscle diseases, and Young's modulus values were commensurate to the severity of the disease. Abnormal myofiber resistance was also observed from consulting patients whose muscle condition could not be detected or unambiguously diagnosed otherwise. This study provides a proof-of-concept that atomic force microscopy yields a quantitative read-out of human muscle function from clinical biopsies, and that it may thereby complement current muscular dystrophy diagnosis.

C.P.4 Congenital fiber type disproportion with myofibrillar disorganization and altered internal nuclei is caused by RYR1 mutation

Mutations in the gene encoding skeletal muscle ryanodine receptor (RYR1) have been implicated in various forms of neuromuscular diseases including congenital myopathy. There is a broad range of clinical and pathological features including central core disease, congenital neuromuscular disease with uniform type 1, multiminicore disease, core-rod myopathy, and malignant hyperthermia susceptibility. Here we describe a group of patients with RYR1 mutations showing a unique muscle pathology. We chose seven unrelated Japanese patients among 12,000 frozen muscle biopsies in NCNP, based on the characteristic pathological findings including fiber type disproportion), marked type 1 fiber predominance, and scattered fibers with abnormal internal nuclei. On NADH-TR stain, myofibrillar disorganization was commonly seen. Unique fibers characterized by darkly-stained central portion with multiple small vacuoles were seen in three patients. On electron microscopy, center of muscle fibers were frequently occupied by aggregations of enlarged, vacuolated sarcoplasmic reticulum and abnormal shaped nuclei. Clinically, all seven patients showed axial and proximal dominant muscle weakness of variable severity from neonatal period, and often required respiratory supports. Muscle weakness was stable or rather improved during childhood. In addition, some patients revealed ptosis and/or ophthalmoparesis. All patients had compound heterozygous mutations in the central portion of RYR1 distributed between 18 and 66. In conclusion, mutations in RYR1 gene are associated with a unique myopathy characterized as congenital fiber type disproportion associated with myofibrillar disorganization and altered internal nuclei. These pathological changes may be closely related to degeneration of sarcoplasmic reticulum.

G.P.13 Merosin-deficient congenital muscular dystrophy: A case with immunohistochemical analysis

Congenital muscular dystrophies (CMDs) are an autosomal recessive and heterogeneous diseases. The CMDs are subclassified into two major categories: merosin positive and deficient. Primary merosin (laminin α2 chain)-deficient congenital muscular dystrophy (CMD) is a uncommon and severe form of CMD, which is caused by the mutations in the laminin α2 chain gene. In European studies reported to approximately a half of CMDs are merosin-deficient congenital muscular dystrophy (MDCMD), but it is rarely reported in asian. There were few case reports of MDCMD in Korea, we report a case of MDCMD confirmed by the immunohistochemical analysis of the frozen muscle biopsy. A 7-months-year old female was evaluated for floppy infant syndrome. She was born healthy at IUP 38weeks without perinatal distress. She had neonatal hypotonia, a high serum creatine kinase level of 3130IU/L. Her brain MRI showed a diffuse white matter change on T2WI. There was no family history of muscle weakness or developmental delay. The muscle biopsy showed a marked dystrophic pattern with many endomysial inflammatory cell infiltration. Immunohistochemical staining for dystrophin-C, α-sarcoglycan, β-sarcoglycan and α-dystroglycan proteins were decreased, and for dystrophin-R, dystrophin-N, γ-sarcoglycan, δ-sarcoglycan, dysferlin, caveolin-3 proteins were normal. We report a case of merosin-deficiency congenital muscular dystrophy confirmed by muscle biopsy.

Muscle biopsy

Muscle biopsy is required to provide a definitive diagnosis in many neuromuscular disorders. It can be performed through an open or needle technique under local anesthesia. The major limitations of the needle biopsy technique are the sample size, which is smaller than that obtained with open biopsy, and the impossibility of direct visualization of the sampling site. However, needle biopsy is a less invasive procedure than open biopsy and is particularly indicated for diagnosis of neuromuscular disease in infancy and childhood. The biopsied muscle should be one affected by the disease but not be too weak or too atrophic. Usually, in case of proximal muscle involvement, the quadriceps and the biceps are biopsied, while under suspicion of mitochondrial disorder, the deltoid is preferred. The samples must be immediately frozen or fixed after excision to prevent loss of enzymatic reactivity, DNA depletion or RNA degradation. A battery of stainings is performed on muscle sections from every frozen muscle biopsy arriving in the pathology laboratory. Histological, histochemical, and histoenzymatic stainings are performed to evaluate fiber atrophy, morphological, and structural changes and metabolic disorders. Moreover, immunohistochemistry and Western blotting analysis may be used for expression analysis of muscle proteins to obtain a specific diagnosis. There are myopathies that do not need muscle biopsy since a genetic test performed on a blood sample is enough for definitive diagnosis. Muscle biopsy is a useful technique which can make an enormous contribution in the field of neuromuscular disorders but should be considered and interpreted together with the patient's family and clinical history.

Using complementary DNA from MyoD‐transduced fibroblasts to sequence large muscle genes

Large muscle genes are often sequenced using complementary DNA (cDNA) made from muscle messenger RNA (mRNA) to reduce the cost and workload associated with sequencing from genomic DNA. Two potential barriers are the availability of a frozen muscle biopsy, and difficulties in detecting nonsense mutations due to nonsense-mediated mRNA decay (NMD). We present patient examples showing that use of MyoD-transduced fibroblasts as a source of muscle-specific mRNA overcomes these potential difficulties in sequencing large muscle-related genes.

Pseudoexon Activation Caused by Intronic Double‐deletion Events in the DMD Gene

Gross genomic rearrangements (GGRs) including copy number variations are increasingly being recognized as an important source of the “missing heritability” for both monogenic and complex diseases. The functional consequences of GGRs involving coding sequences or splice sites are often self-evident. By contrast, the functional consequences of GGRs involving deep intronic sequences often need to be sought by laborious experimental testing. In this issue, Khelifi et al. (Hum Mutat 32:467–475, 2011) characterized two unusual and complex intronic GGRs in the DMD gene at the nucleotide level through array-CGH and direct genomic sequencing. Both events comprise two deletions within single introns, the intervening sequence in the first event being also inverted. Each event was shown to give rise to pseudoexon activation by RT-PCR analysis using total RNA isolated from frozen muscle biopsy samples from the patients, suggesting pathogenic relevance. The authors performed a series of deletion experiments using a splicing reporter minigene assay, providing compelling evidence that an upstream intronic 592-bp sequence plays a crucial role in repressing pseudoexon activation in the wild-type allele. However, a combination of in silico analysis and experimental validation failed to identify any splicing regulatory elements that could have accounted for the inclusion/exclusion of the 166-bp pseudoexon. In addition, MFold prediction analysis of possible RNA secondary structure involvement in splicing regulation was inconclusive. Other factors such as nucleosome positioning and specific histone modifications may well turn out to play a role in regulating exon recognition. In reporting these two cases of DMD pseudoexon activation resulting from pure intronic double-deletion events, Khelifi et al. remind us that we still have some way to go before we are able to decode all the regulatory elements lurking in our own genome.

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO4 fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.

Dysferlinopathy in Chile: Evidence of Two Novel Mutations in the First Reported Cases

We describe two Chilean patients with dysferlinopathy, a 32-year-old man with Miyoshi's distal myopathy and a 29-year-old woman with a proximodistal phenotype. Absence of dysferlin in frozen muscle biopsy allowed diagnostic confirmation. In these two patients, two mutations not previously identified in other populations were found: a homozygous c.1948delC (p.Leu650TyrfsX6) was found in the male patient; the heterozygous mutation c.1276G>A (p.Gly426Arg) was found in the female patient in association with the previously reported c.2858dupT (p.Phe954ValfsX2). To our knowledge, this is the first time that mutations in DYSF are identified in native Chileans. Our findings suggest the possibility that mutations in the DYSF gene were present in the Native American population before colonization.