Paula Onofre-Oliveira | The Hospital for Sick Children (original) (raw)

Papers by Paula Onofre-Oliveira

Revista Neurociências

As afecções neuromusculares humanas constituem um grupo heterogêneo de doenças genéticas caracter... more As afecções neuromusculares humanas constituem um grupo heterogêneo de doenças genéticas caracterizadas por degeneração muscular progressiva, levando ao desenvolvimento de fraqueza muscular e perda de capacidade motora. Na última década foram identificadas mutações em vários genes, resultando na deficiência ou perda de função de diversas proteínas musculares de importância significativa para o bom funcionamento do músculo. Estudos bioquímicos e imunohistológicos têm localizado estas proteínas nos diversos compartimentos da fibra muscular. Associadas à membrana sarcolemal, encontram-se a distrofina, as 4 sarcoglicanas, disferlina e caveolina 3; na matriz extracelular, a α2-laminina e colágeno VI; nos sarcômeros, a teletonina, miotilina, titina, actina e tropomiosina; no citosol muscular, canal de Cálcio (receptor de rianodina), a calpaina 3, FRPR, TRIM32, miotubularina; e nos núcleos, a emerina, lamina A/C, proteína SMN. Algumas das doenças associadas a alterações nestas proteínas são as distrofias musculares progressivas e as miopatias congênitas.

Disease Models & Mechanisms

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness o... more X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.

Scientific Reports

Satellite cells (Scs) are the main muscle stem cells responsible for its regenerative capacity. i... more Satellite cells (Scs) are the main muscle stem cells responsible for its regenerative capacity. in muscular dystrophies, however, a failure of the regenerative process results in muscle degeneration and weakness. To analyze the effect of different degrees of muscle degeneration in SCs behavior, we studied adult muscle of the dystrophic strains: DMD mdx , Large myd , DMD mdx /Large myd , with variable histopathological alterations. Similar results were observed in the dystrophic models, which maintained normal levels of PAX7 expression, retained the Pax7-positive SCs pool, and their proliferation capacity. Moreover, elevated expression of MYOG, an important myogenic factor, was also observed. The ability to form new fibers was verified by the presence of dMyHC positive regenerating fibers. However, those fibers had incomplete maturation characteristics, such as small and homogenous fiber caliber, which could contribute to their dysfunction. We concluded that dystrophic muscles, independently of their degeneration degree, retain their Scs pool with proliferating and regenerative capacities. nonetheless, the maturation of these new fibers is incomplete and do not prevent muscle degeneration. Taken together, these results suggest that the improvement of late muscle regeneration should better contribute to therapeutic approaches.

Dedico este trabalho a meus pais, que sempre acreditaram nas minhas escolhas;

Neuromuscular Disorders, 2015

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD),... more The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx 129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdx C57BL . Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdx C57BL presents three times more differentially expressed genes (DEGs) than the mdx 129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx 129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx 129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. 3 / 18 mdx 129 models. (B) Graphic representing the quantitative comparison between the two mdx strains.

Neuromuscular Disorders

The muscular dystrophies are a clinically and histologically well characterized group of genetic ... more The muscular dystrophies are a clinically and histologically well characterized group of genetic diseases, with predominantly monogenic etiologies. Many causative gene mutations have been already identified, but the underlying pathophysiological pathways and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, study the whole genome expression profile can significantly contribute to: identify altered biological functions which could contribute to the understanding of the disease; identify possible prognostic biomarkers and find points for therapeutic intervention. Here, we present the first global gene expression analysis of the muscle of MD mouse models: Largemyd (CMD1D), Dmdmdx/Largemyd (double mutant for DMD and CMD1D) compared to Dmdmdx (model for Duchenne MD) and normal C57BL, in three different ages: 21 days, 3 and 6 months. Five animals from each group were analyzed using DNA microarrays. The data were preprocessed a...

Neuromuscular Disorders, 2008

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by lack of dystr... more Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by lack of dystrophin, leading to progressive muscle weakness, functional impairment and premature death. The therapeutic approach closest to clinical application is reading frame restoration by antisense oligonucleotide (AON) mediated exon skipping. Both in cultured DMD cells and in the mdx mouse model AON treatment results in the synthesis of dystrophin proteins similar to those seen in Becker muscular dystrophy patients who display a milder phenotype than DMD patients. Recently, local restoration of dystrophin expression after intramuscular administration of an exon 51 specific 2 0 -O-methyl phosphorothioate AON has been shown effective in four Duchenne patients in our first-in-man clinical trial. We are now optimizing systemic AON delivery for future clinical studies on full body treatment by comparing different doses and treatment periods with murin exon 23 specific AONs in mdx mice using subcutaneous injections. In order to assess the therapeutic effects of AON treatment in more detail and to determine which levels of dystrophin restoration are required for improved muscle function in mdx mice, we have set up several functional tests with which muscle strength and condition can be monitored. These tests consist of gripstrength analysis, rota-rod and treadmill running and 2 and 4 limb hanging wire tests. Creatine kinase levels are examined over time and previously identified fibrotic and immunologic biomarkers after sacrifice. Since there is some debate on the effect of functional tests due to exercise on disease progression, we are also assessing whether there is a difference on histological and biomarker levels between mice that do or do not perform functional tests. Preliminary tests reveal more fibrosis in skeletal muscle from mice starting functional tests at 2 months compared to 5 months old mice. No histological differences were present in the heart of these mice.

Neuromuscular Disorders, 2008

could not be detected in the heart until assay conditions were biased towards generation of short... more could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.

Stem Cells in Clinic and Research, 2011

Disease Models & Mechanisms, 2013

Neuromuscular Disorders, 2008

weakness. The onset of the disease was in both cases during early childhood, followed by a mild p... more weakness. The onset of the disease was in both cases during early childhood, followed by a mild progressive deterioration during adulthood. Physical examination of the patients showed elongated face, typical skeletal abnormalities like congenital hip dislocation, foot deformities, thoraco-lumbar scoliosis, bilateral atrophy of calf and tibialis anterior, scapular winging, Gowers' sign, mild facial weakness, normal strength of the upper limbs, marked weakness of ileo-psoas, moderate weakness of quadriceps, tibialis anterior, extensors and flexors digitorum. Serum CK was normal and an electromyographic study showed myopathic findings. Muscle biopsy showed fibre size variation with hypertrophic and angular hypotrophic fibres and a large number of internally located nuclei. Oxidative enzyme stains showed central and eccentric cores in the majority of fibres mildly positive to esterase reaction. An increase in lysosomial activity especially around the cores was also observed. Analysis of RYR1 gene showed a mutation in exon 85 (c.11708G>A; p.R3903Q): in the two affected daughters the mutation was present in homozygous form, the parents were heterozygotic. Our data suggest the hypothesis that the same mutation present in different state (homozygous versus heterozygous) can give rise to a different phenotype (CCD versus MH) implying that mutations in homozygous state can severely affect the tetrameric RyR1 protein.

Neuromuscular Disorders, 2010

As no effective treatment options are still available for muscular dystrophies, cell therapy is a... more As no effective treatment options are still available for muscular dystrophies, cell therapy is a strong hope. Mouse models for these diseases are an important tool for testing putative therapies. Mesenchymal cells derived from bone marrow (bMSC) and from adipose tissue (aMSC) are multipotent and can lead to other tissues such as bone, cartilage, connective and muscle tissues, in vivo and in vitro.

Neuromuscular Disorders, 2010

As no effective treatment options are still available for muscular dystrophies, cell therapy is a... more As no effective treatment options are still available for muscular dystrophies, cell therapy is a strong hope. Mouse models for these diseases are an important tool for testing putative therapies. Mesenchymal cells derived from bone marrow (bMSC) and from adipose tissue (aMSC) are multipotent and can lead to other tissues such as bone, cartilage, connective and muscle tissues, in vivo and in vitro.

Neuromuscular Disorders, 2009

Neuromuscular Disorders, 2009

Neuromuscular Disorders, 2012

ABSTRACT Dystroglycanopathies are genetic muscular dystrophies caused by defects in the glycosyla... more ABSTRACT Dystroglycanopathies are genetic muscular dystrophies caused by defects in the glycosylation of α-dystroglycan (DG), an important component of the dystrophin-associated glycoprotein complex (DAG). There are at least six proven or putative glycosyltransferases enzymes, related to muscle diseases, which play a role in O-mannosyl-linked glycosylation of α-DG. In order to address the roles of α-dystroglycan glycosylation in distinct pathways of muscle degeneration and during muscle development we investigated the relative expression of four of them: POMT1, POMGnT1, LARGE and FKRP, in the gastrocnemius muscle from four mouse models for muscular dystrophies: Dmdmdx, Lama2dy2J/J, Largemyd, SJL/J, as compared to normal C57Black6 lineage. The study was done through real time PCR quantification, in three different ages: new born, 3 and 6 months of age. In normal mice, a decreased expression with the age was observed for all genes, mainly for Fkrp and Large. In dystrophic lineages, we observed a large variation in the expression of the four glycosylation genes, as compared to normal age-matched mice. In new born, these differences were more significant and an upregulation was observed in Dmdmdx and Largemyd strains, while a downregulation, was detected in Lama2dy2J/J and SJL/J strains. In adult animals, the pattern was more close to normal mice of the same age. Our results suggest that an increase in α-dystroglycan glycosylation occurs during muscle development, and possibly also during the process of muscle regeneration. However, this process is variable in the diverse dystrophic strains. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

Neuromuscular Disorders, 2011

Journal of Molecular Neuroscience, 2008

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in... more The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for δ-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy 2J /dy 2J mouse, both presenting significant reduction of α2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large myd ) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of α-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Revista Neurociências

As afecções neuromusculares humanas constituem um grupo heterogêneo de doenças genéticas caracter... more As afecções neuromusculares humanas constituem um grupo heterogêneo de doenças genéticas caracterizadas por degeneração muscular progressiva, levando ao desenvolvimento de fraqueza muscular e perda de capacidade motora. Na última década foram identificadas mutações em vários genes, resultando na deficiência ou perda de função de diversas proteínas musculares de importância significativa para o bom funcionamento do músculo. Estudos bioquímicos e imunohistológicos têm localizado estas proteínas nos diversos compartimentos da fibra muscular. Associadas à membrana sarcolemal, encontram-se a distrofina, as 4 sarcoglicanas, disferlina e caveolina 3; na matriz extracelular, a α2-laminina e colágeno VI; nos sarcômeros, a teletonina, miotilina, titina, actina e tropomiosina; no citosol muscular, canal de Cálcio (receptor de rianodina), a calpaina 3, FRPR, TRIM32, miotubularina; e nos núcleos, a emerina, lamina A/C, proteína SMN. Algumas das doenças associadas a alterações nestas proteínas são as distrofias musculares progressivas e as miopatias congênitas.

Disease Models & Mechanisms

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness o... more X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.

Scientific Reports

Satellite cells (Scs) are the main muscle stem cells responsible for its regenerative capacity. i... more Satellite cells (Scs) are the main muscle stem cells responsible for its regenerative capacity. in muscular dystrophies, however, a failure of the regenerative process results in muscle degeneration and weakness. To analyze the effect of different degrees of muscle degeneration in SCs behavior, we studied adult muscle of the dystrophic strains: DMD mdx , Large myd , DMD mdx /Large myd , with variable histopathological alterations. Similar results were observed in the dystrophic models, which maintained normal levels of PAX7 expression, retained the Pax7-positive SCs pool, and their proliferation capacity. Moreover, elevated expression of MYOG, an important myogenic factor, was also observed. The ability to form new fibers was verified by the presence of dMyHC positive regenerating fibers. However, those fibers had incomplete maturation characteristics, such as small and homogenous fiber caliber, which could contribute to their dysfunction. We concluded that dystrophic muscles, independently of their degeneration degree, retain their Scs pool with proliferating and regenerative capacities. nonetheless, the maturation of these new fibers is incomplete and do not prevent muscle degeneration. Taken together, these results suggest that the improvement of late muscle regeneration should better contribute to therapeutic approaches.

Dedico este trabalho a meus pais, que sempre acreditaram nas minhas escolhas;

Neuromuscular Disorders, 2015

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD),... more The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx 129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdx C57BL . Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdx C57BL presents three times more differentially expressed genes (DEGs) than the mdx 129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx 129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx 129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process. 3 / 18 mdx 129 models. (B) Graphic representing the quantitative comparison between the two mdx strains.

Neuromuscular Disorders

The muscular dystrophies are a clinically and histologically well characterized group of genetic ... more The muscular dystrophies are a clinically and histologically well characterized group of genetic diseases, with predominantly monogenic etiologies. Many causative gene mutations have been already identified, but the underlying pathophysiological pathways and phenotypic variability in each form are much more complex, suggesting the involvement of many other genes. Thus, study the whole genome expression profile can significantly contribute to: identify altered biological functions which could contribute to the understanding of the disease; identify possible prognostic biomarkers and find points for therapeutic intervention. Here, we present the first global gene expression analysis of the muscle of MD mouse models: Largemyd (CMD1D), Dmdmdx/Largemyd (double mutant for DMD and CMD1D) compared to Dmdmdx (model for Duchenne MD) and normal C57BL, in three different ages: 21 days, 3 and 6 months. Five animals from each group were analyzed using DNA microarrays. The data were preprocessed a...

Neuromuscular Disorders, 2008

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by lack of dystr... more Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by lack of dystrophin, leading to progressive muscle weakness, functional impairment and premature death. The therapeutic approach closest to clinical application is reading frame restoration by antisense oligonucleotide (AON) mediated exon skipping. Both in cultured DMD cells and in the mdx mouse model AON treatment results in the synthesis of dystrophin proteins similar to those seen in Becker muscular dystrophy patients who display a milder phenotype than DMD patients. Recently, local restoration of dystrophin expression after intramuscular administration of an exon 51 specific 2 0 -O-methyl phosphorothioate AON has been shown effective in four Duchenne patients in our first-in-man clinical trial. We are now optimizing systemic AON delivery for future clinical studies on full body treatment by comparing different doses and treatment periods with murin exon 23 specific AONs in mdx mice using subcutaneous injections. In order to assess the therapeutic effects of AON treatment in more detail and to determine which levels of dystrophin restoration are required for improved muscle function in mdx mice, we have set up several functional tests with which muscle strength and condition can be monitored. These tests consist of gripstrength analysis, rota-rod and treadmill running and 2 and 4 limb hanging wire tests. Creatine kinase levels are examined over time and previously identified fibrotic and immunologic biomarkers after sacrifice. Since there is some debate on the effect of functional tests due to exercise on disease progression, we are also assessing whether there is a difference on histological and biomarker levels between mice that do or do not perform functional tests. Preliminary tests reveal more fibrosis in skeletal muscle from mice starting functional tests at 2 months compared to 5 months old mice. No histological differences were present in the heart of these mice.

Neuromuscular Disorders, 2008

could not be detected in the heart until assay conditions were biased towards generation of short... more could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.

Stem Cells in Clinic and Research, 2011

Disease Models & Mechanisms, 2013

Neuromuscular Disorders, 2008

weakness. The onset of the disease was in both cases during early childhood, followed by a mild p... more weakness. The onset of the disease was in both cases during early childhood, followed by a mild progressive deterioration during adulthood. Physical examination of the patients showed elongated face, typical skeletal abnormalities like congenital hip dislocation, foot deformities, thoraco-lumbar scoliosis, bilateral atrophy of calf and tibialis anterior, scapular winging, Gowers' sign, mild facial weakness, normal strength of the upper limbs, marked weakness of ileo-psoas, moderate weakness of quadriceps, tibialis anterior, extensors and flexors digitorum. Serum CK was normal and an electromyographic study showed myopathic findings. Muscle biopsy showed fibre size variation with hypertrophic and angular hypotrophic fibres and a large number of internally located nuclei. Oxidative enzyme stains showed central and eccentric cores in the majority of fibres mildly positive to esterase reaction. An increase in lysosomial activity especially around the cores was also observed. Analysis of RYR1 gene showed a mutation in exon 85 (c.11708G>A; p.R3903Q): in the two affected daughters the mutation was present in homozygous form, the parents were heterozygotic. Our data suggest the hypothesis that the same mutation present in different state (homozygous versus heterozygous) can give rise to a different phenotype (CCD versus MH) implying that mutations in homozygous state can severely affect the tetrameric RyR1 protein.

Neuromuscular Disorders, 2010

As no effective treatment options are still available for muscular dystrophies, cell therapy is a... more As no effective treatment options are still available for muscular dystrophies, cell therapy is a strong hope. Mouse models for these diseases are an important tool for testing putative therapies. Mesenchymal cells derived from bone marrow (bMSC) and from adipose tissue (aMSC) are multipotent and can lead to other tissues such as bone, cartilage, connective and muscle tissues, in vivo and in vitro.

Neuromuscular Disorders, 2010

As no effective treatment options are still available for muscular dystrophies, cell therapy is a... more As no effective treatment options are still available for muscular dystrophies, cell therapy is a strong hope. Mouse models for these diseases are an important tool for testing putative therapies. Mesenchymal cells derived from bone marrow (bMSC) and from adipose tissue (aMSC) are multipotent and can lead to other tissues such as bone, cartilage, connective and muscle tissues, in vivo and in vitro.

Neuromuscular Disorders, 2009

Neuromuscular Disorders, 2009

Neuromuscular Disorders, 2012

ABSTRACT Dystroglycanopathies are genetic muscular dystrophies caused by defects in the glycosyla... more ABSTRACT Dystroglycanopathies are genetic muscular dystrophies caused by defects in the glycosylation of α-dystroglycan (DG), an important component of the dystrophin-associated glycoprotein complex (DAG). There are at least six proven or putative glycosyltransferases enzymes, related to muscle diseases, which play a role in O-mannosyl-linked glycosylation of α-DG. In order to address the roles of α-dystroglycan glycosylation in distinct pathways of muscle degeneration and during muscle development we investigated the relative expression of four of them: POMT1, POMGnT1, LARGE and FKRP, in the gastrocnemius muscle from four mouse models for muscular dystrophies: Dmdmdx, Lama2dy2J/J, Largemyd, SJL/J, as compared to normal C57Black6 lineage. The study was done through real time PCR quantification, in three different ages: new born, 3 and 6 months of age. In normal mice, a decreased expression with the age was observed for all genes, mainly for Fkrp and Large. In dystrophic lineages, we observed a large variation in the expression of the four glycosylation genes, as compared to normal age-matched mice. In new born, these differences were more significant and an upregulation was observed in Dmdmdx and Largemyd strains, while a downregulation, was detected in Lama2dy2J/J and SJL/J strains. In adult animals, the pattern was more close to normal mice of the same age. Our results suggest that an increase in α-dystroglycan glycosylation occurs during muscle development, and possibly also during the process of muscle regeneration. However, this process is variable in the diverse dystrophic strains. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

Neuromuscular Disorders, 2011

Journal of Molecular Neuroscience, 2008

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in... more The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for δ-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy 2J /dy 2J mouse, both presenting significant reduction of α2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Large myd ) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of α-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.