Exchange of subtelomeric regions between chromosomes 4q and 10q reverts the FSHD genotype and phenotype.

The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.

Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals.

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Analysis of genes regulated by DUX4 via oxidative stress reveals potential therapeutic targets for treatment of facioscapulohumeral dystrophy.

Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases.

Control of DUX4 Expression in Facioscapulohumeral Muscular Dystrophy and Cancer.

DUX4, a gene encoding a transcription factor involved in early embryogenesis, is located within the D4Z4 subtelomeric repeat on chromosome 4q35. In most healthy somatic tissues, DUX4 is heavily repressed by multiple genetic and epigenetic mechanisms, and its aberrant expression is linked to facioscapulohumeral muscular dystrophy (FSHD) where it has been extensively studied. Recently, DUX4 expression has been implicated in oncogenesis, although this is much less explored. In this review, we discuss multiple levels of control of DUX4 expression, including enhancer-promoter interactions, DNA methylation, histone modifications, noncoding RNAs, and telomere positioning effect. We also connect disparate data on intrachromosomal contacts involving DUX4 and emphasize the feedback loops in DUX4 regulation. Finally, we bridge data on DUX4 in FSHD and cancer and discuss prospective approaches for future FSHD therapies and the potential outcomes of DUX4 inhibition in cancer.

DUX4 Pathological Expression: Causes and Consequences in Cancer.

DUX4, a double homeobox transcription factor, has been mostly studied in facioscapulohumeral dystrophy (FSHD), a pathology linked to a deletion of subtelomeric repeats on chromosome 4q. More recently, however, the gene has been associated with various sarcomas and haematological malignancies. Drugs developed for FSHD could be tested on cancer cells to develop efficient treatment strategies for both pathologies.

Control of DNA integrity in skeletal muscle under physiological and pathological conditions.

Skeletal muscle is a highly oxygen-consuming tissue that ensures body support and movement, as well as nutrient and temperature regulation. DNA damage induced by reactive oxygen species is present in muscles and tends to accumulate with age. Here, we present a summary of data obtained on DNA damage and its implication in muscle homeostasis, myogenic differentiation and neuromuscular disorders. Controlled and transient DNA damage appears to be essential for muscular homeostasis and differentiation while uncontrolled and chronic DNA damage negatively affects muscle health.

DUX4-induced constitutive DNA damage and oxidative stress contribute to aberrant differentiation of myoblasts from FSHD patients.

Facioscapulohumeral dystrophy (FSHD) is one of the three most common muscular dystrophies in the Western world, however, its etiology remains only partially understood. Here, we provide evidence of constitutive DNA damage in in vitro cultured myoblasts isolated from FSHD patients and demonstrate oxidative DNA damage implication in the differentiation of these cells into phenotypically-aberrant myotubes. Double homeobox 4 (DUX4), the major actor in FSHD pathology induced DNA damage accumulation when overexpressed in normal human myoblasts, and RNAi-mediated DUX4 inhibition reduced the level of DNA damage in FSHD myoblasts. Addition of tempol, a powerful antioxidant, to the culture medium of proliferating DUX4-transfected myoblasts and FSHD myoblasts reduced the level of DNA damage, suggesting that DNA alterations are mainly due to oxidative stress. Antioxidant treatment during the myogenic differentiation of FSHD myoblasts significantly reduced morphological defects in myotube formation. We propose that the induction of DNA damage is a novel function of the DUX4 protein affecting myogenic differentiation of FSHD myoblasts.

Genome- and Cell-Based Strategies in Therapy of Muscular Dystrophies.

Muscular dystrophies are a group of heterogeneous genetic disorders characterized by progressive loss of skeletal muscle mass. Depending on the muscular dystrophy, the muscle weakness varies in degree of severity. The majority of myopathies are due to genetic events leading to a loss of function of key genes involved in muscle function. Although there is until now no curative treatment to stop the progression of most myopathies, a significant number of experimental gene- and cell-based strategies and approaches have been and are being tested in vitro and in animal models, aiming to restore gene function. Genome editing using programmable endonucleases is a powerful tool for modifying target genome sequences and has been extensively used over the last decade to correct in vitro genetic defects of many single-gene diseases. By inducing double-strand breaks (DSBs), the engineered endonucleases specifically target chosen sequences. These DSBs are spontaneously repaired either by homologous recombination in the presence of a sequence template, or by nonhomologous-end joining error prone repair. In this review, we highlight recent developments and challenges for genome-editing based strategies that hold great promise for muscular dystrophies and regenerative medicine.

Facioscapulohumeral dystrophy myoblasts efficiently repair moderate levels of oxidative DNA damage.

Facioscapulohumeral dystrophy (FSHD) is a progressive muscular dystrophy linked to a deletion of a subset of D4Z4 macrosatellite repeats accompanied by a chromatin relaxation of the D4Z4 array on chromosome 4q. In vitro, FSHD primary myoblasts show altered expression of oxidative-related genes and are more susceptible to oxidative stress. Double homeobox 4 (DUX4) gene, encoded within each D4Z4 unit, is normally transcriptionally silenced but is found aberrantly expressed in skeletal muscles of FSHD patients. Its expression leads to a deregulation of DUX4 target genes including those implicated in redox balance. Here, we assessed DNA repair efficiency of oxidative DNA damage in FSHD myoblasts and DUX4-transfected myoblasts. We have shown that the DNA repair activity is altered neither in FSHD myoblasts nor in immortalized human myoblasts transiently expressing DUX4. DNA damage caused by moderate doses of an oxidant is efficiently repaired while FSHD myoblasts exposed for 24 h to high levels of oxidative stress accumulated more DNA damage than normal myoblasts, suggesting that FSHD myoblasts remain more vulnerable to oxidative stress at high doses of oxidants.

Functional roles of HIV-1 Tat protein in the nucleus.

Human immunodeficiency virus-1 (HIV-1) Tat protein is one of the most important regulatory proteins for viral gene expression in the host cell and can modulate different cellular processes. In addition, Tat is secreted by the infected cell and can be internalized by neighboring cells; therefore, it affects both infected and uninfected cells. Tat can modulate cellular processes by interacting with different cellular structures and signaling pathways. In the nucleus, Tat might be localized either in the nucleoplasm or the nucleolus depending on its concentration. Here we review the distinct functions of Tat in the nucleoplasm and the nucleolus in connection with viral infection and HIV-induced oncogenesis.

Correction of the FSHD myoblast differentiation defect by fusion with healthy myoblasts.

Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context.

Lipoabdominoplastia. Sistematização para minimizar complicações / Lipoabdominoplasty. Systematization to minimize complications

Introdução: A abdominoplastia e a lipoaspiração estão entre as cirurgias plásticas estéticas mais realizadas em nosso país, inúmeros cirurgiões plásticos brasileiros contribuíram para o seu aperfeiçoamento, sempre visando um melhor resultado ao paciente. Devemos sempre estar atentos aos fenômenos tromboembólicos que podem ter um desfecho dramático. Métodos: Foram operadas 20 pacientes, com idade variando de 29 a 63 anos, idade média de 43 anos, todas do sexo feminino. As pacientes passaram por consulta pré-anestésica, onde foram classificadas segundo o risco cirúrgico utilizando tabela da ASA (American Society of Anesthesiologists), realizaram os exames de rotina, Ultrassonografia de parede abdominal, avaliação cardiológica e avaliação psicológica. Resultados: Em nossa casuística de 20 pacientes, tivemos um caso de seroma persistente, um caso de "dog ear" e um caso de deiscência de cicatriz umbilical, as complicações encontradas não influenciaram o resultado final da cirurgia e 15 pacientes se disseram muito satisfeitos após 6 meses de operados. Conclusão: A lipoabdominoplastia demonstrou ser uma técnica segura e que traz bons resultados ao paciente, aspectos psicológicos devem ser avaliados e uma rotina criteriosa desde o pré-operatório visa diminuir possíveis intercorrências.

Introduction: Abdominoplasty and liposuction are two of the most frequently performed plastic surgeries in Brazil. Many Brazilian plastic surgeons have contributed to their improvement and constantly seek the best result for their patients. Surgeons also must always be attentive to thromboembolic events that may have a tragic outcome. Methods: Twenty (20) female patients, aged 29 to 63 years, with a mean age of 43 years, underwent surgery. The patients attended a pre-anesthetic visit, where they were classified according to surgical risk using the American Society of Anesthesiologists classification, underwent routine testing, abdominal wall ultrasonography, cardiovascular assessment, and psychological assessment. Results: Among the 20 cases, there was one case of persistent seroma, one case of "dog-ear," and one case of umbilical wound dehiscence. The complications that occurred did not influence the final surgical outcome and 15 patients were very satisfied 6 months post-surgery. Conclusion: Lipoabdominoplasty proved to be a safe technique with good results. Psychological factors must be assessed and a necessarily rigorous routine starting from the preoperative period allows a reduction in the risk of complications.

Implantes de panturrilha: complicações, prevenção e tratamento / Calf implants: complications, prevent and treatment

A inclusão de implantes de panturrilha para correção de atrofias e distrofias de membros inferiores vem sendo cada vez mais realizada. Nosso objetivo é avaliar, prevenir e tratar as possíveis complicações deste procedimento. São 21 anos de acompanhamento,142 casos e sete complicações, sendo seis tratadas cirurgicamente, com remissão do problema.

The inclusion of calf implants to correct the atrophy and dystrophy of the inferior limb has been more achieve. Our objective is evaluated, prevent and treat the possible complications of this procedure. There are 21 years of fellow, 142 cases and seven complications, six of this were treated surgically with problem resolution.

Policização do quarto dedo: princípios, técnica e vantagens / Pollicization of the four finger: principles, technique and advantages

Introdução: O polegar é o dedo mais importante da mão e sua perda provoca grande incapacidade à mesma. O objetivo deste trabalho é preconizar a técnica de policização do quarto dedo, demonstrando seus princípios, técnica e vantagens. Método: Foram tratados seis pacientes em diferentes níveis de amputação do polegar. Resultados: Em todos os casos, obtivemos resultados satisfatórios, com retorno da capacidade funcional da mão.

Introduction: The thumb is the more important finger of the hand and his lost cause a big incapacity. The purpose of this trial is precognize the pollicization of the four finger technique showing your principles, techniques and advantages. Methods: Six patients were treated in different levels of thumb amputation. Results: In all of the cases were obtained satisfactory results, with return of the functional capacity of the hand.

Contraturas em flexão da mão queimada: avaliação do tratamento por meio da abertura cutânea e cicatrização por segunda intenção / Flexion contracture in burned hand: treatment evaluation through open

Objetivos: Avaliar a evolução dos pacientes com contratura na mão queimada submetidosao tratamento cirúrgico por meio da abertura cutânea e cicatrização por segunda intenção.Métodos: Em um período de dez meses, os autores avaliaram cinco pacientes com contraturasna mão em decorrência de queimaduras. Três eram do sexo masculino e dois do sexofeminino, com média de idade de 10 anos. Todos os pacientes foram submetidos à aberturadas lesões através de incisões transversas e permitida a cicatrização por segunda intenção.Também fizeram uso de imobilização e fisioterapia. Resultados: Os cinco casos avaliadosapresentaram evolução funcional considerada satisfatória pelos médicos, fisioterapeutas epelos próprios pacientes.

Objectives: Evaluate the evolution of patients with contracture burn hand submitted tosurgical treatment across cutaneous open and second intention scare. Methods: In a periodof ten months, the authors evaluated five patients with hand contracture because of burn.Three of them were male and two female, with middle ages 10 years. All the patients weresubmitted to open lesions using transverse incision with second intention scare. They useimmobilization and physiotherapy. Results: All five operated cases show satisfactoryfunctional evolution.

Estudo comparativo do retorno da força da pinça após o tratamento da síndrome do túnel do carpo crônica associado a oponentoplastia / Comparative study of pinch power return after chronic carpal tunnel release associated with opponensplasty

A oposição do polegar é um importante e complexo movimento da mão, realizado por meio da musculatura tênar, que recebe inervação do nervo mediano, permitindo a realização do movimento de pinça, força e sensibilidade. O objetivo deste trabalho é comparar a força de pinça e grau de oponência do polegar, no pré e pós-operatório em 10 pacientes com síndrome do túnel do carpo crônica, hipotrofia da musculatura tênar e diminuição da força de pinça, que foram selecionados no nosso ambulatório de cirurgia da mão, e submetidos a cirurgia de oponentoplastia pela técnica de Camitz. Todos os pacientes evoluíram satisfatoriamente com rápido retorno as suas atividades cotidianas.

Thumb opponency is a complex and important hand’s movement, realized by the thenar muscle wich is innervated by the median nerve, providing it of sensibility, strength and the ability to do the pinch movement. The goal of this work is to compare the pinch strength and the degrees of opponency before and after the surgery in 10 patients with chronical carpal tunnel’s syndrome, hypotrophic thenar muscle and weakness of pinch’s movement, wich were selected in our hand’s surgery ambulatory center and submitted to an opponensplasty Thumb surgery using the Camitz technique. All the patients had a faster and satisfactory evolution with an amazing comeback to their quotidian activities.

Sindactilia: técnica de Marumo modificada / Syndactyly: Marumo’s modify technique

Sindactilia é um defeito congênito dos dedos das mãos e pés, caracterizado por um defeitode separação das partes. Seu tratamento é essencialmente cirúrgico. O objetivo deste trabalhoé relatar a experiência da equipe com a técnica de Marumo modificada, em doze mãosoperadas. Em todos os casos, obtivemos bons resultados, com retorno da funcionalidade eestética das mãos.

Syndactyly is a finger congenital disease of the hand and foot, characterized by a separateddefect of parts. Their treatment is essential surgery. The objective of this study is showingthe experience with the Marumo’s modified technique in twelve operated hands. In all caseswe have good results, with return of aesthetic and functionality of hand.

Doença de Dupuytren: nossa conduta / Dupuytren’s disease: our conduct

Introdução: A doença de Dupuytren é caracterizada pela contratura em flexão progressiva dosdígitos, o que pode levar à incapacidade funcional da mão. O objetivo deste trabalho épreconizar uma técnica operatória eficaz e menos traumática para conduzir a doença. Método:Foram tratados cirurgicamente 55 pacientes em vários estágios da doença. Resultados:Em todos os casos, foram obtidos resultados satisfatórios, com bom retorno dos pacientesàs suas atividades.

Introduction: The Dupuytren’s disease is characterized by the progressive contracture inflexion of the digits, which could carry the hand to a full functional incapacity. The purposeof this study is precognize an efficient operative technique less traumatic for treating thedisease. Methods: Fifty-five patients underwent to surgery, in different levels of the disease,were analyzed. Results: In all of the cases were obtained satisfactory results, returning thepatients to their activities.