RESUMEN
BACKGROUND: Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy. METHODS: A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2'deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells. RESULTS: In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS. CONCLUSION: Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neuropéptidos/genética , Receptores de Superficie Celular/genética , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Biopsia , Línea Celular Tumoral , Análisis por Conglomerados , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/farmacología , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Humanos , Ácidos Hidroxámicos/farmacología , Metástasis de la Neoplasia , Regiones Promotoras Genéticas , Protocadherinas , TranscriptomaRESUMEN
OBJECTIVE: Duchenne muscular dystrophy (DMD) is the most common single-gene lethal disorder. Substantial patient-patient variability in disease onset and progression and response to glucocorticoids is seen, suggesting genetic or environmental modifiers. METHODS: Two DMD cohorts were used as test and validation groups to define genetic modifiers: a Padova longitudinal cohort (n = 106) and the Cooperative International Neuromuscular Research Group (CINRG) cross-sectional natural history cohort (n = 156). Single nucleotide polymorphisms to be genotyped were selected from mRNA profiling in patients with severe vs mild DMD, and genome-wide association studies in metabolism and polymorphisms influencing muscle phenotypes in normal volunteers were studied. RESULTS: Effects on both disease progression and response to glucocorticoids were observed with polymorphism rs28357094 in the gene promoter of SPP1 (osteopontin). The G allele (dominant model; 35% of subjects) was associated with more rapid progression (Padova cohort log rank p = 0.003), and 12%-19% less grip strength (CINRG cohort p = 0.0003). CONCLUSIONS: Osteopontin genotype is a genetic modifier of disease severity in Duchenne dystrophy. Inclusion of genotype data as a covariate or in inclusion criteria in DMD clinical trials would reduce intersubject variance, and increase sensitivity of the trials, particularly in older subjects.
Asunto(s)
Distrofia Muscular de Duchenne/genética , Osteopontina/genética , Polimorfismo de Nucleótido Simple , Niño , Preescolar , Estudios Transversales , Progresión de la Enfermedad , Femenino , Genotipo , Glucocorticoides/administración & dosificación , Humanos , Cooperación Internacional , Italia , Estimación de Kaplan-Meier , Masculino , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Oportunidad Relativa , Valor Predictivo de las Pruebas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la EnfermedadRESUMEN
Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Regiones Promotoras Genéticas , Animales , Repetición de Anquirina , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas Musculares/análisis , Músculo Esquelético/citología , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Proteínas Nucleares , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Represoras , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , TATA Box , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
In this paper we describe a novel 19 kDa sarcomeric protein named telethonin. The cDNA sequence discloses an open reading frame of 167 amino acids that does not resemble any known protein. Antibodies against a recombinant telethonin fragment were used for Western blot analysis, confirming the presence of this 19 kDa protein in heart and skeletal muscle and revealing an immunofluorescence pattern typical of sarcomeric proteins, overlapping myosin. The frequency of specific cDNA clones in different libraries indicates that the telethonin transcript is amongst the most abundant in skeletal muscle. In human, telethonin maps at 17q12, adjacent to the phenylethanolamine N-methyltransferase gene.
Asunto(s)
Proteínas Musculares/química , Músculo Esquelético/química , Miocardio/química , Sarcómeros/química , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Western Blotting , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , Clonación Molecular , Conectina , ADN Complementario/química , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculo Esquelético/citología , Miocardio/citología , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia , Transcripción Genética/genéticaRESUMEN
The systematic sequencing of cDNA libraries is an efficient approach for the identification of new genes, but the presence of abundant mRNAs is often a major problem. This paper describes a very simple method of "semi-multiplex PCR" that allows specific identification of such abundant transcripts before DNA sequencing without using nonrepresentative subtracted libraries. The PCR utilizes a series of forward primers specific for abundant transcripts with a pair of universal primers used for template generation. cDNA clones corresponding to abundant mRNAs are then revealed by double bands in agarose gel.
Asunto(s)
ADN Complementario/química , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Secuencia de Bases , Humanos , Datos de Secuencia MolecularRESUMEN
A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for > 45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.