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1.
Int J Mol Sci ; 23(8)2022 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-35457088

RESUMEN

Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.


Asunto(s)
Distrofia Muscular de Duchenne , Línea Celular , Distrofina/genética , Exones/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos/genética , Oligonucleótidos Antisentido/genética
2.
Molecules ; 26(24)2021 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-34946743

RESUMEN

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.


Asunto(s)
Exones , Distrofia Muscular de Duchenne , Mioblastos Esqueléticos/metabolismo , Oligonucleótidos Antisentido , Precursores del ARN , Ácido Ursodesoxicólico , Línea Celular Transformada , Humanos , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/farmacología , Precursores del ARN/genética , Precursores del ARN/metabolismo , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacocinética , Ácido Ursodesoxicólico/farmacología
3.
Hum Mol Genet ; 25(20): 4518-4532, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-28173117

RESUMEN

The giant protein titin is the third most abundant protein in striated muscle. Mutations in its gene are responsible for diseases affecting the cardiac and/or the skeletal muscle. Titin has been reported to be expressed in multiple isoforms with considerable variability in the I-band, ensuring the modulation of the passive mechanical properties of the sarcomere. In the M-line, only the penultimate Mex5 exon coding for the specific is7 domain has been reported to be subjected to alternative splicing. Using the CRISPR-Cas9 editing technology, we generated a mouse model where we stably prevent the expression of alternative spliced variant(s) carrying the corresponding domain. Interestingly, the suppression of the domain induces a phenotype mostly in tissues usually expressing the isoform that has been suppressed, indicating that it fulfills (a) specific function(s) in these tissues allowing a perfect adaptation of the M-line to physiological demands of different muscles.


Asunto(s)
Empalme Alternativo , Sistemas CRISPR-Cas , Edición Génica/métodos , Modelos Animales , Proteínas Quinasas/metabolismo , Animales , Masculino , Ratones , Isoformas de Proteínas/genética , Proteínas Quinasas/genética , Sarcómeros/metabolismo
4.
J Cell Sci ; 129(8): 1671-84, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26945058

RESUMEN

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.


Asunto(s)
Factores de Transcripción ARNTL/genética , Relojes Circadianos/fisiología , Colágeno Tipo VI/genética , Contractura/genética , Mitocondrias/fisiología , Distrofias Musculares/congénito , Mutación/genética , Esclerosis/genética , Animales , Autofagia/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Análisis por Micromatrices , Distrofias Musculares/genética , ARN/análisis
5.
Biochim Biophys Acta Mol Basis Dis ; 1863(1): 15-20, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27639833

RESUMEN

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.


Asunto(s)
Enanismo/terapia , Exones , Vectores Genéticos/uso terapéutico , Microcefalia/terapia , Oligonucleótidos Antisentido/uso terapéutico , Empalme del ARN , ARN Nuclear Pequeño/uso terapéutico , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Secuencia de Bases , Línea Celular , Enanismo/genética , Facies , Terapia Genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Intrones , Lentivirus/genética , Ratones , Microcefalia/genética , Oligonucleótidos Antisentido/genética , Mutación Puntual , ARN Nuclear Pequeño/genética
6.
Sci Rep ; 14(1): 21238, 2024 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-39261505

RESUMEN

Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.


Asunto(s)
Sistemas CRISPR-Cas , Distrofina , Exones , Duplicación de Gen , Edición Génica , Distrofia Muscular de Duchenne , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Humanos , Exones/genética , Distrofina/genética , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Terapia Genética/métodos , Fibras Musculares Esqueléticas/metabolismo
7.
Pharmaceutics ; 16(8)2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39204368

RESUMEN

Delivery represents a major hurdle to the clinical advancement of oligonucleotide therapeutics for the treatment of disorders such as Duchenne muscular dystrophy (DMD). In this preliminary study, we explored the ability of 2'-O-methyl-phosphorothioate antisense oligonucleotides (ASOs) conjugated with lipophilic ursodeoxycholic acid (UDCA) to permeate across intestinal barriers in vitro by a co-culture system of non-contacting IEC-6 cells and DMD myotubes, either alone or encapsulated in exosomes. UDCA was used to enhance the lipophilicity and membrane permeability of ASOs, potentially improving oral bioavailability. Exosomes were employed due to their biocompatibility and ability to deliver therapeutic cargo across biological barriers. Exon skipping was evaluated in the DMD myotubes to reveal the targeting efficiency. Exosomes extracted from milk and wild-type myotubes loaded with 5'-UDC-3'Cy3-ASO and seeded directly on DMD myotubes appear able to fuse to myotubes and induce exon skipping, up to ~20%. Permeation studies using the co-culture system were performed with 5'-UDC-3'Cy3-ASO 51 alone or loaded in milk-derived exosomes. In this setting, only gymnotic delivery induced significant levels of exon skipping (almost 30%) implying a possible role of the intestinal cells in enhancing delivery of ASOs. These results warrant further investigations to elucidate the delivery of ASOs by gymnosis or exosomes.

8.
Hum Mutat ; 33(3): 572-81, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22223181

RESUMEN

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan(®) low-density array technology and is able to define the full-exon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies.


Asunto(s)
Distrofina/genética , Exoma/genética , Humanos , Masculino , Mutación , ARN/genética
9.
BMC Med Genet ; 13: 20, 2012 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-22455600

RESUMEN

BACKGROUND: In X-linked dilated cardiomyopathy due to dystrophin mutations which abolish the expression of the M isoform (5'-XLDC), the skeletal muscle is spared through the up-regulation of the Brain (B) isoform, a compensatory mechanism that does not appear to occur in the heart of affected individuals. METHODS: We quantitatively studied the expression topography of both B and M isoforms in various human heart regions through in-situ RNA hybridization, Reverse-Transcriptase and Real-Time PCR experiments. We also investigated the methylation profile of the B promoter region in the heart and quantified the B isoform up regulation in the skeletal muscle of two 5'-XLDC patients. RESULTS: Unlike the M isoform, consistently detectable in all the heart regions, the B isoform was selectively expressed in atrial cardiomyocytes, but absent in ventricles and in conduction system structures. Although the level of B isoform messenger in the skeletal muscle of 5'-XLDC patients was lower that of the M messenger present in control muscle, it seems sufficient to avoid an overt muscle pathology. This result is consistent with the protein level in XLDC patients muscles we previously quantified. Methylation studies revealed that the B promoter shows an overall low level of methylation at the CG dinucleotides in both atria and ventricles, suggesting a methylation-independent regulation of the B promoter activity. CONCLUSIONS: The ventricular dilatation seen in 5'-XLDC patients appears to be functionally related to loss of the M isoform, the only isoform transcribed in human ventricles; in contrast, the B isoform is well expressed in heart but confined to the atria. Since the B isoform can functionally replace the M isoform in the skeletal muscle, its expression in the heart could potentially exert the same rescue function. Methylation status does not seem to play a role in the differential B promoter activity in atria and ventricles, which may be governed by other regulatory mechanisms. If these mechanisms could be deduced, de-silencing of the B isoform may represent a therapeutic strategy in 5'-XLDC patients.


Asunto(s)
Cardiomiopatía Dilatada/genética , Distrofina/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Ventrículos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Metilación de ADN , Humanos , Hibridación in Situ , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
10.
BMC Med Genet ; 13: 73, 2012 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-22894145

RESUMEN

BACKGROUND: Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. METHODS: Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. RESULTS: The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. CONCLUSIONS: This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype.


Asunto(s)
Compensación de Dosificación (Genética) , Distrofina/genética , Debilidad Muscular/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Mutación/genética , Transcripción Genética , Inactivación del Cromosoma X/genética , Adolescente , Adulto , Alelos , Western Blotting , Niño , Preescolar , Hibridación Genómica Comparativa , Distrofina/metabolismo , Femenino , Heterocigoto , Humanos , Persona de Mediana Edad , Debilidad Muscular/patología , Osteopontina/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Adulto Joven
11.
Muscle Nerve ; 44(1): 80-4, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21488057

RESUMEN

INTRODUCTION: Collagen VI expression was tested in peripheral blood macrophages from patients with collagen VI-related myopathies and compared with muscle biopsy. METHODS: RNA and protein studies were performed in blood macrophages from 5 patients previously diagnosed with either Ullrich congenital muscular dystrophy (UCMD) or Bethlem myopathy (BM). The full spectrum of possible genotypes was considered, including both dominant and recessive UCMD and BM cases. RESULTS: In the dominant BM patient, no collagen VI alterations were detectable in macrophages or muscle biopsy. In the remaining patients, the protein defect caused by the selected mutations, as well as the transcriptional abnormalities, were readily detectable in macrophages, at levels comparable to those observed in muscle biopsy samples and cultured skin fibroblasts. CONCLUSIONS: Our data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing muscle/skin biopsies in the diagnosis and monitoring of collagen VI-related myopathies.


Asunto(s)
Colágeno Tipo VI/biosíntesis , Macrófagos/metabolismo , Macrófagos/patología , Enfermedades Musculares/sangre , Enfermedades Musculares/patología , Adulto , Células Cultivadas , Niño , Colágeno Tipo VI/sangre , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación/genética , Proyectos Piloto
12.
Cell Rep ; 35(2): 108983, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33852870

RESUMEN

Preclinical models of ischemia/reperfusion injury (RI) demonstrate the deleterious effects of permeability transition pore complex (PTPC) opening in the first minutes upon revascularization of the occluded vessel. The ATP synthase c subunit (Csub) influences PTPC activity in cells, thus impacting tissue injury. A conserved glycine-rich domain in Csub is classified as critical because, when mutated, it modifies ATP synthase properties, protein interaction with the mitochondrial calcium (Ca2+) uniporter complex, and the conductance of the PTPC. Here, we document the role of a naturally occurring mutation in the Csub-encoding ATP5G1 gene at the G87 position found in two ST-segment elevation myocardial infarction (STEMI) patients and how PTPC opening is related to RI in patients affected by the same disease. We report a link between the expression of ATP5G1G87E and the response to hypoxia/reoxygenation of human cardiomyocytes, which worsen when compared to those expressing the wild-type protein, and a positive correlation between PTPC and RI.


Asunto(s)
Hipoxia/genética , Mitocondrias/genética , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , Miocitos Cardíacos/metabolismo , Infarto del Miocardio con Elevación del ST/genética , Anciano , Animales , Secuencia de Bases , Canales de Calcio/genética , Canales de Calcio/metabolismo , Exones , Femenino , Expresión Génica , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Intrones , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/patología , ATPasas de Translocación de Protón Mitocondriales/deficiencia , Mutación , Miocitos Cardíacos/patología , Oxígeno/efectos adversos , Estudios Prospectivos , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Infarto del Miocardio con Elevación del ST/metabolismo , Infarto del Miocardio con Elevación del ST/patología
13.
BMC Med Genet ; 11: 64, 2010 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-20426824

RESUMEN

BACKGROUND: Multiple sclerosis (MS) is a complex disorder thought to result from an interaction between environmental and genetic predisposing factors which have not yet been characterised, although it is known to be associated with the HLA region on 6p21.32. Recently, a picture of chronic cerebrospinal venous insufficiency (CCSVI), consequent to stenosing venous malformation of the main extra-cranial outflow routes (VM), has been described in patients affected with MS, introducing an additional phenotype with possible pathogenic significance. METHODS: In order to explore the presence of copy number variations (CNVs) within the HLA locus, a custom CGH array was designed to cover 7 Mb of the HLA locus region (6,899,999 bp; chr6:29,900,001-36,800,000). Genomic DNA of the 15 patients with CCSVI/VM and MS was hybridised in duplicate. RESULTS: In total, 322 CNVs, of which 225 were extragenic and 97 intragenic, were identified in 15 patients. 234 known polymorphic CNVs were detected, the majority of these being situated in non-coding or extragenic regions. The overall number of CNVs (both extra- and intragenic) showed a robust and significant correlation with the number of stenosing VMs (Spearman: r = 0.6590, p = 0.0104; linear regression analysis r = 0.6577, p = 0.0106). The region we analysed contains 211 known genes. By using pathway analysis focused on angiogenesis and venous development, MS, and immunity, we tentatively highlight several genes as possible susceptibility factor candidates involved in this peculiar phenotype. CONCLUSIONS: The CNVs contained in the HLA locus region in patients with the novel phenotype of CCSVI/VM and MS were mapped in detail, demonstrating a significant correlation between the number of known CNVs found in the HLA region and the number of CCSVI-VMs identified in patients. Pathway analysis revealed common routes of interaction of several of the genes involved in angiogenesis and immunity contained within this region. Despite the small sample size in this pilot study, it does suggest that the number of multiple polymorphic CNVs in the HLA locus deserves further study, owing to their possible involvement in susceptibility to this novel MS/VM plus phenotype, and perhaps even other types of the disease.


Asunto(s)
Cromosomas Humanos Par 6 , Variación Genética , Antígenos HLA/genética , Antígenos HLA-DR/genética , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple/genética , Venas/anomalías , Mapeo Cromosómico , Hibridación Genómica Comparativa , Exones/genética , Genotipo , Cadenas HLA-DRB1 , Humanos , Intrones/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/fisiopatología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Fenotipo , Polimorfismo Genético , Índice de Severidad de la Enfermedad
14.
BMC Med Genet ; 11: 44, 2010 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-20302629

RESUMEN

BACKGROUND: Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. METHODS: We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. RESULTS: A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. CONCLUSIONS: Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes.


Asunto(s)
Colágeno Tipo VI/genética , Enfermedades Musculares/diagnóstico , Adolescente , Adulto , Alelos , Niño , Preescolar , Estudios de Cohortes , Hibridación Genómica Comparativa , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Eliminación de Gen , Dosificación de Gen , Heterogeneidad Genética , Genotipo , Heterocigoto , Humanos , Inmunohistoquímica , Intrones , Masculino , Persona de Mediana Edad , Enfermedades Musculares/genética , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo
15.
Mol Ther ; 17(5): 820-7, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19240694

RESUMEN

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.


Asunto(s)
Distrofina/metabolismo , Nanopartículas/química , Oligorribonucleótidos Antisentido/fisiología , Polimetil Metacrilato/química , Animales , Western Blotting , Distrofina/genética , Electroforesis en Gel de Poliacrilamida , Exones/genética , Terapia Genética/métodos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Mutantes , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/metabolismo , Polimetil Metacrilato/síntesis química
16.
Hum Mutat ; 30(5): E662-72, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19309692

RESUMEN

Splicing mutations occurring outside the invariant GT and AG dinucleotides are frequent in disease genes and the definition of their pathogenic potential is often challenging. We have identified four patients affected by Ullrich congenital muscular dystrophy and carrying unusual mutations of COL6 genes affecting RNA splicing. In three cases the mutations occurred in the COL6A2 gene and consisted of nucleotide substitutions within the degenerated sequences flanking the canonical dinucleotides. In the fourth case, a genomic deletion occurred which removed the exon8-intron8 junction of the COL6A1 gene. These mutations induced variable splicing phenotypes, consisting of exon skipping, intron retention and cryptic splice site activation/usage. A quantitative RNA assay revealed a reduced level of transcription of the mutated in-frame mRNA originating from a COL6A2 point mutation at intronic position +3. At variance, the transcription level of the mutated in-frame mRNA originating from a genomic deletion which removed the splicing sequences of COL6A1 exon 8 was normal. These findings suggest a different transcriptional efficiency of a regulatory splicing mutation compared to a genomic deletion causing a splicing defect.


Asunto(s)
Colágeno Tipo VI/genética , Distrofias Musculares/congénito , Distrofias Musculares/genética , Mutación/genética , Empalme del ARN/genética , Adolescente , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Codón sin Sentido/genética , Colágeno Tipo VI/metabolismo , Exones/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Heterocigoto , Humanos , Patrón de Herencia/genética , Intrones/genética , Masculino , Distrofias Musculares/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia
17.
Hum Mutat ; 30(11): 1527-34, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19760747

RESUMEN

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.


Asunto(s)
Codón sin Sentido , Distrofina/genética , Exones , Mutación del Sistema de Lectura , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/uso terapéutico , Sistemas de Lectura , Células Cultivadas , Análisis Mutacional de ADN , Humanos , Distrofia Muscular de Duchenne/terapia , Mutación Puntual , Empalme del ARN , Transcripción Genética
18.
Biochim Biophys Acta Gene Regul Mech ; 1862(6): 619-624, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31005673

RESUMEN

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.


Asunto(s)
Sistemas CRISPR-Cas , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Sitios de Unión , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular , Células Endoteliales , Factor VII , Factor VIII/genética , Expresión Génica , Genes Reporteros , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Humanos , Mutación , ARN Guía de Kinetoplastida , Receptores Inmunológicos/genética
19.
Front Genet ; 10: 573, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31297130

RESUMEN

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10-6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10-7 and p < 1 × 10-20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.

20.
BMC Genomics ; 9: 572, 2008 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-19040728

RESUMEN

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.


Asunto(s)
Hibridación Genómica Comparativa , Distrofina/genética , Reordenamiento Génico , Intrones/genética , Distrofias Musculares/genética , Mutación , Regiones no Traducidas 3'/genética , Dosificación de Gen , Humanos
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