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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.
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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/genéticaRESUMEN
Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals' USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs' myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.
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Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008-2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.
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AIMS: We aimed to assess carpal tunnel syndrome (CTS) prevalence in transthyretin (TTR)-related and light-chain amyloidosis (AL), comparing it to the general population, adjusted for age and gender. In TTR-related amyloidosis (ATTR) we investigated (i) CTS prevalence in relation to genotype, cardiac amyloidosis (CA), age and gender; (ii) CTS role as an incremental risk factor for CA; (iii) temporal relationship between CTS and CA; and (iv) CTS prognostic role. METHODS AND RESULTS: Data from 538 subjects (166 hereditary ATTR, 107 wild-type ATTR, 196 AL amyloidosis, and 69 TTR mutation carriers; 64% male, median age 62.4 years), evaluated at our centre (Bologna, Italy), were analysed and compared to a published cohort of 14.9 million people, in which incidence rates of CTS had been estimated. CTS prevalence was highest in ATTR patients with CA (20.3% vs. 4.1% in the general population), while it was comparable to the general population when CA was absent and in AL patients. CTS standardized incidence rates were markedly elevated in ATTR males in the eighth decade of life (13.08 in hereditary ATTR, 15.5 in wild-type ATTR). The risk of developing CA was greater in ATTR patients with CTS; the probability of having CTS was highest 5-9 years prior to CA diagnosis. CTS was an independent mortality risk factor in ATTR. CONCLUSIONS: Compared to general population the adjusted prevalence of CTS is higher among elderly men with ATTR; CTS is a prognostic marker in ATTR, independently of cardiac involvement, and precedes CA diagnosis by 5-9 years. The awareness of this association and time delay offers the possibility of an early pre-clinical ATTR-CA diagnosis.
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Amiloidosis , Síndrome del Túnel Carpiano , Insuficiencia Cardíaca , Síndrome del Túnel Carpiano/diagnóstico , Síndrome del Túnel Carpiano/epidemiología , Síndrome del Túnel Carpiano/etiología , Diagnóstico Precoz , Femenino , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Emery Dreifuss muscular dystrophy (EDMD) is an inherited myopathy characterized by early contractures, slow progressive muscle weakness and cardiac involvement. To date at least seven genes have been associated to EDMD with different inheritance patterns, being emerin gene responsible for the X-linked form of the disease. We report a 40-year-old man who was referred for severe gait difficulty. At age 6 years the patient presented with a waddling gate, lumbar lordosis and heel contractures. Both electrophysiology and muscle biopsy were consistent with a neurogenic disorder and he received a diagnosis of spinal muscular atrophy type 3. At the age of 30 the patient developed heart involvement with junctional escape rhythm and, eight years later, had a spontaneous chordae tendinae rupture. A new clinical examination showed severe muscular weakness and atrophy in scapulohumeroperoneal pattern with significant involvement of the lower facial and intrinsic hand muscles and on a second muscle biopsy emerin was absent by immunohistochemistry and by immunoblot analysis. Sequence analysis of EMD gene revealed the presence of a novel mutation represented by an out-of-frame deletion spanning from the beginning of exon 1 to the half of intron 2 (p.Asp6Glyfs*27). Our study expands the clinical and molecular spectrum of X-linked EDMD.
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Cuerdas Tendinosas/lesiones , Proteínas de la Membrana/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutación/genética , Proteínas Nucleares/genética , Rotura Espontánea/genética , Adulto , Cuerdas Tendinosas/diagnóstico por imagen , Electrocardiografía/métodos , Humanos , Masculino , Distrofia Muscular de Emery-Dreifuss/complicaciones , Distrofia Muscular de Emery-Dreifuss/diagnóstico por imagen , Linaje , Rotura Espontánea/complicaciones , Rotura Espontánea/diagnóstico por imagenRESUMEN
Bethlem myopathy represents the milder form of the spectrum of Collagen VI-related dystrophies, which are characterized by a clinical continuum between the two extremities, the Bethlem myopathy and the Ullrich congenital muscular dystrophy, and include less defined intermediate phenotypes. Bethlem myopathy is mainly an autosomal dominant disorder and the causing mutations occur in the COL6A genes encoding for the α1 (COL6A1), α2 (COL6A2) and α3 (COL6A3) chains. However, few cases of recessive inheritance have been also reported. We here describe clinical, genetic and functional findings in a recessive Bethlem myopathy family harbouring two novel pathogenic mutations in the COL6A2 gene. Two adult siblings presented with muscle weakness and wasting, elbows and Achilles tendon retractions, lumbar hyperlordosis, waddling gait and positive Gowers' sign. Muscle biopsy showed a dystrophic pattern. Molecular analysis of the COL6A2 gene revealed the novel paternally-inherited nonsense p.Gln889* mutation and the maternally-inherited p.Pro260_Lys261insProPro small insertion. Fibroblast studies in both affected patients showed the concomitant reduction in the amount of normal Collagen VI (p.Gln889*) and impairment of Collagen VI secretion and assembly (p.Pro260_Lys261insProPro). Each of the two variants behave as a recessive mutation as shown by the asymptomatic heterozygous parents, while their concomitant effects determined a relatively mild Bethlem myopathy phenotype. This study confirms the occurrence of recessive inherited Bethlem myopathy and expands the genetic heterogeneity of this group of muscle diseases.
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Colágeno Tipo VI/metabolismo , Contractura/genética , Patrón de Herencia , Distrofias Musculares/congénito , Adulto , Codón sin Sentido , Colágeno Tipo VI/genética , Contractura/metabolismo , Contractura/patología , Contractura/fisiopatología , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Músculo Esquelético/patología , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofias Musculares/fisiopatología , Mutagénesis Insercional , Cultivo Primario de Células , Multimerización de Proteína/genética , Hermanos , Adulto JovenRESUMEN
Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory.
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Pruebas Genéticas/estadística & datos numéricos , Laboratorios/estadística & datos numéricos , Encuestas y Cuestionarios/estadística & datos numéricos , Carga de Trabajo/estadística & datos numéricos , Pruebas Genéticas/economía , Pruebas Genéticas/tendencias , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Italia , Laboratorios/economía , Laboratorios/tendencias , Sistemas de Información Administrativa , Factores de Tiempo , Carga de Trabajo/economíaRESUMEN
We describe a family carrying a γ-globin gene deletion associated with an increase of Hb A2 level beyond the normal range. The family included the proband, his sister and their father, all with increased Hb A2 and normal Hb F levels. The proband and his sister showed borderline values of mean corpuscular volume (MCV) and reduced values of mean corpuscular hemoglobin (Hb) (MCH). The proband was referred to our Medical Genetics Service for preconception counseling together with his partner, a typical ß-thalassemia (ß-thal) carrier. The results were negative for the most frequent α-thalassemia (α-thal) mutations, and had no significant sequence variations of the coding sequences and promoter of the ß- and δ-globin genes. Quantitative analysis by multiplex ligation-dependent probe amplification (MPLA) of the ß-globin gene cluster detected a heterozygous deletion, ranging between 2.1 and 4.7 kb, in the proband, his sister and the father. The deletion involved the (G)γ gene and (G)γ-(A)γ intergenic region, whereas the 3' region of the (A)γ gene was preserved. A subsequent gap-polymerase chain reaction (gap-PCR) showed that a hybrid (GA)γ fusion gene was present. The deletion segregated with the elevation of Hb A2. The MLPA analysis of the ß-globin gene cluster in 150 control alleles excluded a common polymorphism. Despite stronger evidence being needed, the described family suggests a possible role of this γ-globin gene deletion in contributing to Hb A2 elevation, possibly by altering the transcription regulation of the cluster. We propose γ-globin gene dosage analysis to be performed in patients with unexplained elevated Hb A2 levels.
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Hemoglobina A2/genética , Talasemia/genética , gamma-Globinas/genética , Adulto , Femenino , Dosificación de Gen , Regulación de la Expresión Génica/genética , Hemoglobina A2/análisis , Humanos , Masculino , Linaje , Eliminación de SecuenciaRESUMEN
AIMS: Hereditary transthyretin (TTR)-related amyloidosis (ATTR) is mainly considered a neurologic disease. We assessed the phenotypic and genotypic spectra of ATTR in a Caucasian area and evaluated the prevalence, genetic background, and disease profile of cases with an exclusively cardiac phenotype, highlighting possible hints for the differential diagnosis with hypertrophic cardiomyopathy (HCM) and senile systemic amyloidosis (SSA). METHODS AND RESULTS: In this Italian multicentre study, 186 patients with ATTR were characterized at presentation. Thirty patients with SSA and 30 age-gender-matched HCM patients were used for comparison. Phenotype was classified as exclusively cardiac (n = 31, 17%), exclusively neurologic (n = 46, 25%), and mixed cardiac/neurologic (n = 109, 58%). Among the eight different mutations responsible for an exclusively cardiac phenotype, Ile68Leu was the most frequent. Five patients with an exclusively cardiac phenotype developed mild abnormalities at neurological examination, but no symptoms during a 36-month follow-up (range: 14-50). Exclusively cardiac phenotype was characterized by male gender, age >65 years, heart failure symptoms, symmetric left ventricular (LV) 'hypertrophy', and moderately depressed LV ejection fraction. This profile was similar to SSA, but relatively distinct from HCM. Compared with patients with a mixed phenotype, patients with an exclusively cardiac phenotype showed a more pronounced cardiac involvement on both echocardiogram and electrocardiogram (ECG). CONCLUSION: A clinically relevant subset of Caucasian ATTR patients present with an exclusively cardiac phenotype, mimicking HCM or SSA. Echocardiographic and ECG findings are useful to differentiate ATTR from HCM but not from SSA. The role of liver transplantation in these patients should be reconsidered.
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Neuropatías Amiloides Familiares/genética , Cardiomiopatía Hipertrófica/genética , Mutación/genética , Adulto , Anciano , Neuropatías Amiloides Familiares/diagnóstico , Cardiomiopatía Hipertrófica/diagnóstico , Estudios de Casos y Controles , Diagnóstico Diferencial , Ecocardiografía , Electrocardiografía , Femenino , Genotipo , Humanos , Italia , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
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.
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Distrofina/metabolismo , Músculos/metabolismo , Distrofias Musculares/genética , Nanopartículas/administración & dosificación , Oligorribonucleótidos Antisentido/administración & dosificación , Administración Oral , Animales , Modelos Animales de Enfermedad , Terapia Genética , Humanos , Ratones , Ratones Endogámicos mdx , Músculos/efectos de los fármacos , Distrofias Musculares/metabolismo , Distrofias Musculares/terapia , Nanopartículas/química , Oligorribonucleótidos Antisentido/química , Distribución TisularRESUMEN
The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.
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Distrofina/genética , Músculo Esquelético/metabolismo , Isoformas de ARN/genética , ARN Largo no Codificante/genética , Femenino , Humanos , Masculino , Transcripción Genética/genéticaRESUMEN
In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2'-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.
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Distrofina/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Nanocápsulas/administración & dosificación , Oligonucleótidos Antisentido/administración & dosificación , Animales , Distrofina/genética , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos mdx , Resultado del Tratamiento , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach for subsets of Duchenne muscular dystrophy (DMD) patients to ameliorate the severe DMD phenotype. Several groups have successfully induced exon skipping by AONs to reframe the mRNA in various patients carrying deletions, and phase I/II clinical trials are ongoing. The approach is based on targeting specific splicing motifs, both exonic and located on the exon borders, thus interfering with the spliceosome assembly by steric hindrance. Evaluation of the effectiveness of treatment with AONs in cells, animal models, and humans requires a sensitive, specific, and highly reproducible method. We have developed a real-time PCR-based protocol that uses the probe-based approach to recognize specific sequences internal to the target exon (exon-specific real-time assay). The methods for this protocol are described in this chapter.
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Distrofina/genética , Exones , Distrofia Muscular de Duchenne/genética , Oligonucleótidos Antisentido/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Colorantes Fluorescentes/análisis , Humanos , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
Taxanes represent a group of anticancer drugs with a wide range of activity against breast cancer. Therapy side effects include haematologic toxicity (neutropenia, leucopenia), peripheral neuropathy and hypersensitivity, and demonstrate inter-individual variations. Since it is known that three genes are implicated in taxane turnover, namely ABCB1 in the transport, CYP2C8 in the metabolism and CYP1B1 in the activity, we explored the association among polymorphisms (single nucleotide polymorphisms, SNPs) in these three genes and the occurrence of taxane-induced toxicity. We studied 95 patients affected by breast cancer and under treatment with taxanes as adjuvant, metastatic or neo-adjuvant therapy. We genotyped them for SNPs in the CYP2C8 (alleles *1, *2, *3 and *4), CYP1B1 (alleles *1 and *3) and ABCB1 (1236 C>T; 2677 G>T/A; 3435 C>T) genes by real-time PCR assay. We observed a significant association between the CYP1B1*3 allele and a lower occurrence of hypersensitivity reactions to taxane treatment. We speculate that the highest production of 4-hydroxyestradiol (4-OHE2) metabolite by CYP1B1*3 allele could increase the formation of the 4-OHE2-taxane adduct and possibly inhibit taxane toxicity. We suggest that CYP1B1 might affect taxane hypersensitivity therefore representing, if confirmed in a large cohort of patients, an exploratory hypersensitivity predictive biomarker.
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Antineoplásicos Fitogénicos/efectos adversos , Neoplasias de la Mama Masculina/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Sistema Enzimático del Citocromo P-450/genética , Hipersensibilidad a las Drogas/genética , Paclitaxel/efectos adversos , Polimorfismo de Nucleótido Simple , Taxoides/efectos adversos , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Anciano , Antineoplásicos Fitogénicos/farmacocinética , Hidrocarburo de Aril Hidroxilasas/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama Masculina/enzimología , Neoplasias de la Mama Masculina/genética , Quimioterapia Adyuvante , Distribución de Chi-Cuadrado , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP2C8 , Sistema Enzimático del Citocromo P-450/metabolismo , Docetaxel , Hipersensibilidad a las Drogas/enzimología , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Italia , Modelos Logísticos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Oportunidad Relativa , Paclitaxel/farmacocinética , Fenotipo , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Taxoides/farmacocinética , Resultado del TratamientoRESUMEN
Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.
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Cardiomiopatía Dilatada/genética , Distrofina/genética , Exones/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Intrones/genética , Oligonucleótidos Antisentido/farmacología , Empalme del ARN/genética , Secuencia de Bases , Bioensayo , Sistema Libre de Células , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/patología , Proteína MioD/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transformación Genética/efectos de los fármacosRESUMEN
Antisense oligonucleotides (AONs) are being developed as RNA therapeutic molecules for Duchenne muscular dystrophy. For oligonucleotides with the 2'-O-methyl-phosphorothioate (2OMePS) RNA chemistry, proof of concept has been obtained in patient-specific muscle cell cultures, the mouse and dog disease models, and recently by local administration in Duchenne patients. To further explore the pharmacokinetic (PK)/pharmacodynamic (PD) properties of this chemical class of oligonucleotides, we performed a series of preclinical studies in mice. The results demonstrate that the levels of oligonucleotides in dystrophin-deficient muscle fibers are much higher than in healthy fibers, leading to higher exon-skipping levels. Oligonucleotide levels and half-life differed for specific muscle groups, with heart muscle showing the lowest levels but longest half-life (approximately 46 days). Intravenous (i.v.), subcutaneous (s.c.), and intraperitoneal (i.p.) delivery methods were directly compared. For each method, exon-skipping and novel dystrophin expression were observed in all muscles, including arrector pili smooth muscle in skin biopsies. After i.v. administration, the oligonucleotide peak levels in plasma, liver, and kidney were higher than after s.c. or i.p. injections. However, as the bioavailability was similar, and the levels of oligonucleotide, exon-skipping, and dystrophin steadily accumulated overtime after s.c. administration, we selected this patient-convenient delivery method for future clinical study protocols.
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Oligonucleótidos Fosforotioatos/farmacología , ARN sin Sentido/farmacología , Animales , Western Blotting , Modelos Animales de Enfermedad , Distrofina/administración & dosificación , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos mdx , Oligonucleótidos Fosforotioatos/farmacocinética , ARN sin Sentido/farmacocinética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Mutación/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Biopsia , Señalización del Calcio/genética , Análisis Mutacional de ADN , Regulación hacia Abajo/genética , Activación Enzimática/genética , Tolerancia al Ejercicio/genética , Femenino , Marcadores Genéticos/genética , Genotipo , Humanos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Hipertonía Muscular/genética , Hipertonía Muscular/metabolismo , Hipertonía Muscular/fisiopatología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Enfermedades Musculares/fisiopatología , Fenotipo , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patología , Adulto JovenRESUMEN
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 GenéticaRESUMEN
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.