miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors

Background Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function. Methods To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells to determine the effect of miR-411 on myogenic markers. Results Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary (2.1 fold, p\u3c0.01) and immortalized (2.7 fold, p\u3c0.01) myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting (−3.7 fold, p\u3c0.01). C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411. Conclusions The study demonstrated that miR-411 was differentially expressed in FSHD myoblasts and may play a role in regulating myogenesis

Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells

Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

BACKGROUND: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-alpha2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-alpha2-deficiency. METHODS: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). RESULTS: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-alpha2-deficient mice. CONCLUSIONS: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-alpha2-deficiency

Generation of a human iPSC line from a patient with a defect of intergenomic communication

Human iPSC line PG64SV.2 was generated from fibroblasts of a patient with a defect of intergenomic communication. This patient harbored a homozygous mutation (c.2243G>C; p.Trp748Ser) in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma gene (POLG). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non integrative methodology that involves the use of Sendai virus.This work was supported by grants from the “Centro de Investigación Biomédica en Red en enfermedades raras” (CIBERER) (Grant 13-717/132.05 to RG), the “Instituto de Salud Carlos III” [Fondo de Investigación Sanitaria and Regional development fund (ERDF/FEDER) funds PI10/0703 and PI13/00556 to RG and PI15/00484 to MEG], “Comunidad Autónoma de Madrid” (Grant number S2010/BMD-2402 to RG); TG receives grant support from the Universidad Autónoma de Madrid (FPI-UAM) and FZD from the Ministerio de Educación, Cultura y Deporte (Grant FPU13/00544). MEG is staff scientist at the “Centro de Investigación Biomédica en Red en Enfermedades Raras” (CIBERER

Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery-Dreifuss muscular dystrophy

AbstractEmery–Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells – a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype

Facioscapulohumeral muscular dystrophy and Charcot-Marie-Tooth neuropathy 1A-evidence for "double trouble" overlapping syndromes

Background: We report on a patient with genetically confirmed overlapping diagnoses of CMT1A and FSHD. This case adds to the increasing number of unique patients presenting with atypical phenotypes, particularly in FSHD. Even if a mutation in one disease gene has been found, further genetic testing might be warranted in cases with unusual clinical presentation. Case presentation: The reported 53 years old male patient suffered from walking difficulties and foot deformities first noticed at age 20. Later on, he developed scapuloperoneal and truncal muscle weakness, along with atrophy of the intrinsic hand and foot muscles, pes cavus, claw toes and a distal symmetric hypoesthesia. Motor nerve conduction velocities were reduced to 20 m/s in the upper extremities, and not educible in the lower extremities, sensory nerve conduction velocities were not attainable. Electromyography showed both, myopathic and neurogenic changes. A muscle biopsy taken from the tibialis anterior muscle showed a mild myopathy with some neurogenic findings and hypertrophic type 1 fibers. Whole-body muscle MRI revealed severe changes in the lower leg muscles, tibialis anterior and gastrocnemius muscles were highly replaced by fatty tissue. Additionally, fatty degeneration of shoulder girdle and straight back muscles, and atrophy of dorsal upper leg muscles were seen. Taken together, the presenting features suggested both, a neuropathy and a myopathy. Patient's family history suggested an autosomal dominant inheritance. Molecular testing revealed both, a hereditary motor and sensory neuropathy type 1A (HMSN1A, also called Charcot-Marie-Tooth neuropathy 1A, CMT1A) due to a PMP22 gene duplication and facioscapulohumeral muscular dystrophy (FSHD) due to a partial deletion of the D4Z4 locus (19 kb). Conclusion: Molecular testing in hereditary neuromuscular disorders has led to the identification of an increasing number of atypical phenotypes. Nevertheless, finding the right diagnosis is crucial for the patient in order to obtain adequate medical care and appropriate genetic counseling, especially in the background of arising curative therapies

Flow cytometry for the analysis of α-dystroglycan glycosylation in fibroblasts from patients with dystroglycanopathies.

α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies

За кадры. 1975. № 51 (1886)

У знамени ПобедыВсесоюзная по ускорителям / В. Кононов"Начало поля" / А. НабатГод - на отлично! / [беседа с] И. И. КаляцкийРекомендовано к внедрениюАбитуриент становится студентом / М. ФальковскийСвоими руками. И блеск, и уют / И. ДмитриевНа балл ниже / Л. Марченко...Другие в стороне / О. СоловьеваТрудовой десантВсе остается людям / А. ГавриковВ память о павших / М. ПавлюшкевичНам любое дело по плечу / Т. БогорятскихПобедила бригада Петухова / Н. РогатинПервый, но не последний / Н. ПавлюченкоОсенние дары - на студенческий стол / В. РезвановЛюбимое дело / Р. ГорскаяНагражден дипломом / А. БатуринОранжерея в библиотеке / Л. ВолодинаЗдравствуй, осень! / В. СомовЕсли вы охотник... / С. Викторо

Summary of α-DG glycosylation as assessed by flow cytometry in 21 patient fibroblasts, three healthy controls, and seven pathological controls.

<p>The specific gene, mutation, phenotype, MFI of the IIH6 positive cells ± SEM, percentage of cells positive for the IIH6 epitope ± SEM, iMFI value ± SEM, N value, P value, and muscle α-DG IIH6 description is listed for each fibroblast cell line analysed. P values are the result of an unpaired t-test comparing the MFI, percentage of cells positive for the IIH6 epitope, or iMFI values of each fibroblast cell line to the respective values of the three healthy controls (C1, C2, C3). Muscle α-DG IIH6 description is the summary from the patient report of how the skeletal muscle α-DG glycosylation appeared by immunohistochemistry. iMFI is defined as iMFI = (MFI)(<i>P</i>), where P is the percentage of cells positive for the IIH6 epitope <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Darrah1" target="_blank">[43]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Shooshtari1" target="_blank">[44]</a>. Abbreviations are as follows: DMD, Duchenne muscular dystrophy; BMD, Becker muscular dystrophy; LGMD, Limb-girdle muscular dystrophy; MEB, Muscle-eye-brain disease; MDC1C, Muscular dystrophy type 1 C; CMD, Congenital muscular dystrophy; FCMD, Fukuyama congenital muscular dystrophy; LGMD-CRB, limb girdle muscular dystrophy with cerebellar involvement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068958#pone.0068958-Cirak1" target="_blank">[11]</a>; MR, mental retardation; NS, non-significant. * = Sections evaluated with the VIA4-1 antibody.</p