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1.
J Clin Immunol ; 39(5): 476-485, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31144250

RESUMO

OBJECTIVES: Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib. METHODS: We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis. RESULTS: We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease. CONCLUSIONS: We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment.

2.
J Rheumatol ; 46(5): 523-526, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30647181

RESUMO

OBJECTIVE: An upregulation of type I interferon (IFN) stimulated genes [IFN score (IS)] was described in patients with adenosine deaminase 2 deficiency (DADA2). We describe the clinical course of 5 such patients and the role of IS as a marker of disease activity and severity. METHODS: Expression levels of IS were determined by quantitative real-time PCR. RESULTS: Five white patients were identified as carrying CECR1 mutations. The IS before treatment was elevated in 4 out of 5 patients and decreased after treatment. CONCLUSION: Our data confirm the high variability of DADA2 and suggest type I IS as a biomarker of disease activity.

3.
Biochim Biophys Acta Gene Regul Mech ; 1860(11): 1138-1147, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28867298

RESUMO

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis­DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.


Assuntos
Distrofina/genética , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Sequências Reguladoras de Ácido Nucleico , Adolescente , Adulto , Animais , Células Cultivadas , Criança , Pré-Escolar , Epigênese Genética/fisiologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Camundongos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mutação , Adulto Jovem
4.
Am J Med Genet A ; 173(11): 2912-2922, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28884922

RESUMO

The prevalence of congenital heart defects (CHD) in Kabuki syndrome ranges from 28% to 80%. Between January 2012 and December 2015, 28 patients had a molecularly proven diagnosis of Kabuki syndrome. Pathogenic variants in KMT2D (MLL2) were detected in 27 patients, and in KDM6A gene in one. CHD was diagnosed in 19/27 (70%) patients with KMT2D (MLL2) variant, while the single patient with KDM6A change had a normal heart. The anatomic types among patients with CHD included aortic coarctation (4/19 = 21%) alone or associated with an additional CHD, bicuspid aortic valve (4/19 = 21%) alone or associated with an additional CHD, perimembranous subaortic ventricular septal defect (3/19 = 16%), atrial septal defect ostium secundum type (3/19 = 16%), conotruncal heart defects (3/19 = 16%). Additional CHDs diagnosed in single patients included aortic dilatation with mitral anomaly and hypoplastic left heart syndrome. We also reviewed CHDs in patients with a molecular diagnosis of Kabuki syndrome reported in the literature. In conclusion, a CHD is detected in 70% of patients with KMT2D (MLL2) pathogenic variants, most commonly left-sided obstructive lesions, including multiple left-sided obstructions similar to those observed in the spectrum of the Shone complex, and septal defects. Clinical management of Kabuki syndrome should include echocardiogram at the time of diagnosis, with particular attention to left-sided obstructive lesions and mitral anomalies, and annual monitoring for aortic arch dilatation.


Assuntos
Anormalidades Múltiplas/genética , Estenose da Valva Aórtica/genética , Proteínas de Ligação a DNA/genética , Face/anormalidades , Cardiopatias Congênitas/genética , Doenças Hematológicas/genética , Proteínas de Neoplasias/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/fisiopatologia , Coartação Aórtica/complicações , Coartação Aórtica/genética , Coartação Aórtica/fisiopatologia , Valva Aórtica/anormalidades , Valva Aórtica/fisiopatologia , Estenose da Valva Aórtica/complicações , Estenose da Valva Aórtica/fisiopatologia , Face/fisiopatologia , Feminino , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/fisiopatologia , Comunicação Interatrial/genética , Comunicação Interatrial/fisiopatologia , Comunicação Interventricular/genética , Comunicação Interventricular/fisiopatologia , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/fisiopatologia , Doenças Hematológicas/complicações , Doenças Hematológicas/fisiopatologia , Histona Desmetilases/genética , Humanos , Masculino , Proteínas Nucleares/genética , Doenças Vestibulares/complicações , Doenças Vestibulares/fisiopatologia
5.
Clin Exp Rheumatol ; 35 Suppl 108(6): 113-115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28628471

RESUMO

OBJECTIVES: Hyperzincaemia/hypercalprotectinemia (Hz/Hc) syndrome is a recently described condition caused by a specific de novo mutation (E250K) affecting PSTPIP1 gene. It has a phenotype distinct from classical pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome that includes severe systemic and cutaneous inflammation, hepatosplenomegaly, arthritis without sequelae, pancytopenia and failure to thrive. METHODS: We describe an 8-year-old boy who presented recurrent right knee swelling mimicking septic arthritis and persistent bone marrow involvement, without cutaneous involvement. RESULTS: Molecular analysis of the PSTPIP1 gene revealed the presence of a heterozygous E250K mutation. No growth failure was detected nor in the patient neither in his mother, carrying the same variant. Blood zinc and calprotectin MRP8/14 concentrations of the patient were found to be markedly increased. Therapy with anakinra was started with rapid disappearance of clinical symptoms and normalization of CRP levels in 24 hours, but persistence of bone marrow involvement. CONCLUSIONS: The patient described has a milder phenotype, with no skin features, minor episodes of arthritis with no sequelae and normal growth. Compared to the patients with de novo mutations described in the literature, familial cases seem to have a milder phenotype. Our case further confirms the lack of efficacy of anakinra on bone marrow involvement.


Assuntos
Acne Vulgar/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Infecciosa/genética , Proteínas do Citoesqueleto/genética , Erros Inatos do Metabolismo dos Metais/genética , Mutação/genética , Pioderma Gangrenoso/genética , Transportadores de Cassetes de Ligação de ATP/sangue , Acne Vulgar/sangue , Acne Vulgar/diagnóstico , Acne Vulgar/tratamento farmacológico , Antirreumáticos/uso terapêutico , Artrite Infecciosa/sangue , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/tratamento farmacológico , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Calgranulina B/sangue , Criança , Análise Mutacional de DNA , Predisposição Genética para Doença , Heterozigoto , Humanos , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Masculino , Erros Inatos do Metabolismo dos Metais/sangue , Erros Inatos do Metabolismo dos Metais/diagnóstico , Erros Inatos do Metabolismo dos Metais/tratamento farmacológico , Fenótipo , Valor Preditivo dos Testes , Pioderma Gangrenoso/sangue , Pioderma Gangrenoso/diagnóstico , Pioderma Gangrenoso/tratamento farmacológico , Fatores de Risco , Resultado do Tratamento , Zinco/sangue
6.
Ann Rheum Dis ; 76(10): 1648-1656, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28522451

RESUMO

OBJECTIVES: To analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis nodosa (PAN). Forty-eight patients from 43 families were included in the study. METHODS: Direct sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor. RESULTS: Biallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect. CONCLUSIONS: DADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.


Assuntos
Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Livedo Reticular/genética , Poliarterite Nodosa/genética , Acidente Vascular Cerebral/genética , Adolescente , Idade de Início , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Heterozigoto , Homozigoto , Humanos , Imunoglobulinas/sangue , Imunossupressores/uso terapêutico , Lactente , Itália , Livedo Reticular/tratamento farmacológico , Livedo Reticular/enzimologia , Masculino , Linhagem , Poliarterite Nodosa/tratamento farmacológico , Poliarterite Nodosa/enzimologia , Acidente Vascular Cerebral/enzimologia , Talidomida/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto Jovem
7.
J Cell Sci ; 129(8): 1671-84, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26945058

RESUMO

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.


Assuntos
Fatores de Transcrição ARNTL/genética , Relógios Circadianos/fisiologia , Colágeno Tipo VI/genética , Contratura/genética , Mitocôndrias/fisiologia , Distrofias Musculares/congênito , Mutação/genética , Esclerose/genética , Animais , Autofagia/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Análise em Microsséries , Distrofias Musculares/genética , RNA/análise
8.
J Clin Invest ; 126(1): 239-53, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26642364

RESUMO

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.


Assuntos
Arritmias Cardíacas/etiologia , Proteínas de Membrana/genética , Distrofia Muscular do Cíngulo dos Membros/etiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Moléculas de Adesão Celular , Criança , AMP Cíclico/metabolismo , Humanos , Masculino , Potenciais da Membrana , Proteínas de Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas Musculares , Mutação , Canais de Potássio de Domínios Poros em Tandem/análise , Transporte Proteico , Peixe-Zebra
9.
Molecules ; 20(10): 18168-84, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26457695

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked inherited neuromuscular disorder due to mutations in the dystrophin gene. It is characterized by progressive muscle weakness and wasting due to the absence of dystrophin protein that causes degeneration of skeletal and cardiac muscle. The molecular diagnostic of DMD involves a deletions/duplications analysis performed by quantitative technique such as microarray-based comparative genomic hybridization (array-CGH), Multiple Ligation Probe Assay MLPA. Since traditional methods for detection of point mutations and other sequence variants require high cost and are time consuming, especially for a large gene like dystrophin, the use of next-generation sequencing (NGS) has become a useful tool available for clinical diagnosis. The dystrophin gene is large and finely regulated in terms of tissue expression, and RNA processing and editing includes a variety of fine tuned processes. At present, there are no effective treatments and the steroids are the only fully approved drugs used in DMD therapy able to slow disease progression. In the last years, an increasing variety of strategies have been studied as a possible therapeutic approach aimed to restore dystrophin production and to preserve muscle mass, ameliorating the DMD phenotype. RNA is the most studied target for the development of clinical strategies and Antisense Oligonucleotides (AONs) are the most used molecules for RNA modulation. The identification of delivery system to enhance the efficacy and to reduce the toxicity of AON is the main purpose in this area and nanomaterials are a very promising model as DNA/RNA molecules vectors. Dystrophinopathies therefore represent a pivotal field of investigation, which has opened novel avenues in molecular biology, medical genetics and novel therapeutic options.


Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/tratamento farmacológico , Mutação , Animais , Hibridização Genômica Comparativa , Terapia Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Oligonucleotídeos Antissenso/uso terapêutico , Análise de Sequência de DNA
12.
J Neurol Neurosurg Psychiatry ; 86(10): 1060-5, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25476005

RESUMO

OBJECTIVE: Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials. METHODS: Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates. RESULTS: We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss. CONCLUSIONS: This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.


Assuntos
Proteínas de Ligação a TGF-beta Latente/genética , Distrofia Muscular de Duchenne/genética , Osteopontina/genética , Fatores Etários , Criança , Estudos de Coortes , Progressão da Doença , Europa (Continente) , Feminino , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Prognóstico , Reprodutibilidade dos Testes , Esteroides/uso terapêutico , Caminhada , Cadeiras de Rodas
13.
Hum Gene Ther ; 25(11): 927-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25244215

RESUMO

Abstract Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.


Assuntos
Oligorribonucleotídeos Antissenso/farmacocinética , Ácidos Polimetacrílicos/farmacocinética , Animais , Terapia Genética , Humanos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/terapia , Nanopartículas/administração & dosagem , Oligorribonucleotídeos Antissenso/administração & dosagem , Ácidos Polimetacrílicos/administração & dosagem , Distribuição Tecidual
14.
Nat Med ; 20(9): 992-1000, 2014 09.
Artigo em Inglês | MEDLINE | ID: mdl-25108525

RESUMO

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.


Assuntos
Distrofina/genética , Éxons , Distrofia Muscular de Duchenne/genética , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Sequência de Aminoácidos , Animais , Distrofina/química , Humanos , Camundongos , Dados de Sequência Molecular , Distrofia Muscular de Duchenne/patologia , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Homologia de Sequência de Aminoácidos
15.
Nucleic Acid Ther ; 24(1): 87-100, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24506782

RESUMO

Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy.


Assuntos
Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Reparo Gênico Alvo-Dirigido/métodos , Animais , Cães , Sistemas de Liberação de Medicamentos , Distrofina/genética , Éxons , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Nanomedicina , Nanopartículas/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico
16.
Exp Cell Res ; 325(1): 44-9, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24389168

RESUMO

Neuromuscular diseases (NMDs) comprise a range of rare disorders that include both hereditary peripheral neuropathies and myopathies. The heterogeneity and rarity of neuromuscular disorders are challenges for researchers seeking to develop effective diagnosis and treatment strategies. In particular, clinical trials of new therapies are made more difficult due to lack of reliable and monitorable clinical outcome measures. Biomarkers could be a way to speed up research in this field, shedding light on the pathophysiological mechanisms behind such diseases and providing invaluable tools for monitoring their progression, prognosis and response to drug treatment. Furthermore, biomarkers could represent a surrogate endpoint for clinical trials, enabling better stratification of patient cohorts through more accurate diagnosis and prognosis prediction. This review summarizes the types, applications, characteristics and best strategies for biomarker discovery to date.


Assuntos
Doenças Neuromusculares/metabolismo , Animais , Biomarcadores/metabolismo , Marcadores Genéticos , Humanos , Doenças Neuromusculares/genética , Proteoma/metabolismo
17.
Neurogenetics ; 14(3-4): 247-50, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23975261

RESUMO

Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.


Assuntos
Cerebelo/patologia , Exoma , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas de Ligação a RNA/genética , Adolescente , Atrofia , Bangladesh , Análise Mutacional de DNA , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Espasticidade Muscular/genética , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/genética , Ataxias Espinocerebelares/genética , Adulto Jovem
18.
Biomed Res Int ; 2013: 527418, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24392452

RESUMO

We have previously demonstrated that intraperitoneal injections of 2'-O-methyl-phosphorothioate (2'OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles of mdx mice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were tracked in vivo as well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, in mdx mice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.


Assuntos
Distrofina/metabolismo , Músculos/metabolismo , Distrofias Musculares/genética , Nanopartículas/administração & dosagem , Oligorribonucleotídeos Antissenso/administração & dosagem , Administração Oral , Animais , Modelos Animais de Doenças , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculos/efeitos dos fármacos , Distrofias Musculares/metabolismo , Distrofias Musculares/terapia , Nanopartículas/química , Oligorribonucleotídeos Antissenso/química , Distribuição Tecidual
19.
Ann Neurol ; 72(4): 550-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23109149

RESUMO

OBJECTIVE: Congenital disorders of glycosylation (CDG) are a group of metabolic diseases due to defects in protein and lipid glycosylation. We searched for the primary defect in 3 children from 2 families with a severe neurological phenotype, including profound developmental delay, intractable epilepsy, progressive microcephaly, severe hypotonia with elevated blood creatine kinase levels, and early fatal outcome. There was clinical evidence of a muscular dystrophy-dystroglycanopathy syndrome, supported by deficient O-mannosylation by muscle immunohistochemistry. METHODS: Biochemical and molecular methods were combined to pinpoint the defect in the glycosylation pathway in the endoplasmic reticulum. RESULTS: Metabolic investigations revealed CDG-I, pointing to a defect in protein N-glycosylation in the endoplasmic reticulum. Analysis of lipid-linked oligosaccharides in fibroblasts showed accumulation of Dol-PP-GlcNAc(2) -Man(5) . DNA analysis revealed mutations in DPM2, 1 of the subunits of the dolichol-phosphate-mannose (DPM) synthase; the patient in the first family is compound heterozygous for 2 mutations (c.68A>G, predicting a missense mutation p.Y23C and c.4-1G>C, a splice mutation), whereas the patients in the second family are homozygous for the same missense mutation (c.68A>G, p.Y23C). INTERPRETATION: We describe a new CDG, due to a deficiency of DPM2. Hence, mutations have now been described in the genes for the 3 subunits of DPM: DPM1, DPM2, and DPM3, whereby DPM2-CDG links the congenital disorders of glycosylation to the congenital muscular dystrophies.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Epilepsia/genética , Manosiltransferases/genética , Distrofias Musculares/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Transtornos de Proteínas de Coagulação/genética , Defeitos Congênitos da Glicosilação/complicações , Análise Mutacional de DNA , Resistência a Medicamentos , Distroglicanas/metabolismo , Eletromiografia , Retículo Endoplasmático , Epilepsia/etiologia , Feminino , Fibroblastos/metabolismo , Glicosilação , Humanos , Lactente , Focalização Isoelétrica , Hepatopatias/complicações , Hepatopatias/genética , Masculino , Manose/metabolismo , Microcefalia/genética , Microcefalia/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Distrofias Musculares/complicações , Mutação/genética , Mutação/fisiologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Gravidez , Transtornos da Visão/genética , Transtornos da Visão/patologia , Adulto Jovem
20.
Hum Gene Ther ; 23(12): 1313-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22992134

RESUMO

Collagen VI gene mutations cause Ullrich and Bethlem muscular dystrophies. Pathogenic mutations frequently have a dominant negative effect, with defects in collagen VI chain secretion and assembly. It is agreed that, conversely, collagen VI haploinsufficiency has no pathological consequences. Thus, RNA-targeting approaches aimed at preferentially inactivating the mutated COL6 messenger may represent a promising therapeutic strategy. By in vitro studies we obtained the preferential depletion of the mutated COL6A2 messenger, by targeting a common single-nucleotide polymorphism (SNP), cistronic with a dominant COL6A2 mutation. We used a 2'-O-methyl phosphorothioate (2'OMePS) antisense oligonucleotide covering the SNP within exon 3, which is out of frame. Exon 3 skipping has the effect of depleting the mutated transcript via RNA nonsense-mediated decay, recovering the correct collagen VI secretion and restoring the ability to form an interconnected microfilament network into the extracellular matrix. This novel RNA modulation approach to correcting dominant mutations may represent a therapeutic strategy potentially applicable to a great variety of mutations and diseases.


Assuntos
Colágeno Tipo VI/genética , Distrofias Musculares/genética , Mutação , Oligorribonucleotídeos Antissenso/farmacologia , Células Cultivadas , Colágeno Tipo VI/metabolismo , Éxons , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/patologia , Genes Dominantes , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , Polimorfismo de Nucleotídeo Único
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