RESUMEN
El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.
In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.
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Animales , Ratas , Apoptosis/genética , Regulación Enzimológica de la Expresión Génica/genética , Hemo/deficiencia , Degeneración Nerviosa/genética , Neuronas/metabolismo , Porfirias/complicaciones , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Colágeno Tipo XI/efectos de los fármacos , Colágeno Tipo XI/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemo/biosíntesis , Heptanoatos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Poli(ADP-Ribosa) Polimerasas , Porfirias/metabolismo , Porfirias/fisiopatología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoAsunto(s)
Humanos , Anticolesterolemiantes/uso terapéutico , Apolipoproteínas B/genética , Atrofia Muscular Espinal/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Empalme del ARN/genética , Proteínas del Complejo SMN/genética , Exones/genética , Modelos Biológicos , Atrofia Muscular Espinal/genética , Oligonucleótidos Antisentido/genéticaRESUMEN
Introducción: La atrofia muscular espinal de la infancia (AMEi) es una enfermedad neurodegenerativa, causada principalmente por deleciones del gen SMN 1 en su locus 5q11.1-13.3. La severidad va desde el tipo I, que compromete la vida en edades tempranas, hasta el tipo IV. Objetivos: Se describen hallazgos moleculares en pacientes con AME, nacionalmente remitidos al Instituto de Neurología y Neurocirugía así como se reporta la frecuencia por 100.000 habitantes en cada una de las provincias de la isla. Pacientes y Métodos: 105 pacientes fueron estudiados, remitidos entre 1997 y 2011. Para la detección de las deleciones se utilizó la técnica de reacción en cadena de la polimerasa (PCR) con cebadores correspondientes a los exones 7 y 8 del gen SMN 1, y digestión con enzimas DraI y DdeI respectivamente y analizados en gel de agarosa al 2 por ciento. Resultados y Discusión: Se encontró un 59 por ciento diagnosticados como AME I, 28,6 por ciento AME II, 12,4 por ciento AME III. Del total de casos 36,2 por ciento resultaron tener deleción de los exones 7 y 8, 35,2 por ciento deleción del exón 7 solamente y 28,2 por ciento no presentaron deleción de los exones correspondientes. Se discuten dichos resultados de acuerdo a la literatura internacional. Se presentan los resultados de frecuencias por 100.000 habitantes en cada provincia del país y discuten dichas frecuencias de acuerdo a la diversidad ancestral de la población cubana. Se concluye lo novedoso del estudio que constituye el primer reporte en una población caribeña.
Introduction: Infantile Spinal Muscular Atrophy (infantile SMA) is a neurodegenerative disease caused primarily by the deletion of the SMN1 gene at 5q11.1 - 13.3. Its severity ranges from type I, in early childhood, to type IV. Objectives: To describe molecular findings in patients with SMA, who were nationally referred to the Institute of Neurology and Neurosurgery and to describe the frequency per 100,000 people in each province of the island. Patients and Methods: 105 patients, referred between 1997 and 2011, were studied. The polymerase chain reaction (PCR) technique was used to detect the deletions of exons 7 and 8 of the SMN1 gene, and DraI and DdeI enzymes, in 2 percent agarose gel, were used for digestion. Results: 59 percent of the patients were diagnosed with SMA I, 28.6 percent with SMA II and 12.4 percent with SMA III. 36.2 percent of total patients presented deletions of exons 7, 8, 35.2 percent only deletion of exon 7 and 28.2 percentdid not present deletion of exons. Conclusion: These results are discussed according to the international literature. The frequency per 100,000 inhabitants in each province is presented and discussed according to ancestral diversity of the Cuban population. Also, the originality of the study is mentioned as it is the first report of this type in a Caribbean population.
Asunto(s)
Humanos , Atrofia Muscular Espinal/genética , Biología Molecular , Proteínas del Complejo SMN/genética , Atrofia Muscular Espinal/clasificación , Atrofia Muscular Espinal/epidemiología , Cuba/epidemiología , Eliminación de Gen , Enfermedades Neurodegenerativas/genética , Reacción en Cadena de la PolimerasaRESUMEN
<p><b>OBJECTIVE</b>To investigate a patient featuring a complex neuromuscular disease phenotype.</p><p><b>METHODS</b>A comprehensive analysis integrating clinical investigation, electrophysiological testing, pathological analysis and mutation screening was carried out.</p><p><b>RESULTS</b>The patient has presented clinical and pathological manifestations mimicking Duchenne muscular dystrophy. However, genetic analysis has identified no deletion in 21 exons of Dystrophin gene, no pathologic expansion of CTG repeats in DMPK gene or CCTG repeats in ZFN9 gene. Instead, a homozygous deletion of exons 7 and 8 in SMN gene was discovered.</p><p><b>CONCLUSION</b>A rare case of spinal muscular atrophy (SMA) was verified by genetic diagnosis. SMA is a group of neuromuscular disorders with great phenotypic heterogeneity and sometimes cannot be diagnosed by clinical manifestations, electrophysiological and pathological changes alone. Genetic diagnosis has become indispensable for accurate diagnosis for patients suspected to have the disease.</p>
Asunto(s)
Adulto , Humanos , Masculino , Adulto Joven , Diagnóstico Diferencial , Atrofia Muscular Espinal , Diagnóstico , Genética , Patología , Distrofia Miotónica , Diagnóstico , Genética , Patología , Proteína Quinasa de Distrofia Miotónica , Fenotipo , Proteínas Serina-Treonina Quinasas , Genética , Proteínas del Complejo SMN , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family.</p><p><b>METHODS</b>Deletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation.</p><p><b>RESULTS</b>In SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father.</p><p><b>CONCLUSION</b>A novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.</p>
Asunto(s)
Preescolar , Humanos , Masculino , Secuencia de Bases , Análisis Mutacional de ADN , Exones , Genética , Datos de Secuencia Molecular , Atrofia Muscular Espinal , Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas del Complejo SMN , Genética , Atrofias Musculares Espinales de la Infancia , Genética , Proteína 1 para la Supervivencia de la Neurona Motora , Genética , Proteínas Nucleares snRNP , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Twenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children.</p><p><b>RESULTS</b>Twenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP.</p><p><b>CONCLUSION</b>PCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.</p>
Asunto(s)
Niño , Femenino , Humanos , Masculino , Embarazo , Exones , Genética , Eliminación de Gen , Asesoramiento Genético , Atrofia Muscular Espinal , Diagnóstico , Genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Diagnóstico Prenatal , Proteínas del Complejo SMN , Genética , Atrofias Musculares Espinales de la Infancia , Diagnóstico , Genética , Proteína 1 para la Supervivencia de la Neurona Motora , GenéticaRESUMEN
<p><b>INTRODUCTION</b>Spinal muscular atrophy (SMA) is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The survival motor neuron (SMN) protein is encoded by 2 genes, SMN1 and SMN2. The most frequent mutation is the biallelic deletion of exon 7 of the SMN1 gene. In SMA, SMN2 cannot compensate for the loss of SMN1, due to the exclusion of exon 7. The aim of our study was to estimate the frequency of the common SMN1 exon 7 deletion in patients referred to our centre for carrier detection and prenatal diagnosis.</p><p><b>MATERIALS AND METHODS</b>We performed the detection of exon 7 deletion of the SMN1 gene for the affected patients and fetuses suspected to have SMA.</p><p><b>RESULTS</b>Of 243 families, 195 were classified as SMA type I, 30 as type II, and 18 as type III according to their family histories. The analysis of exon 7 deletion among living affected children showed that 94% of the patients with SMA type I, 95% with type II families and 100% with type III had homozygous deletions. Of the prenatal diagnoses, 21 (22.8%) of the 92 fetuses were found to be affected and these pregnancies were terminated.</p><p><b>CONCLUSIONS</b>The homozygosity frequency for the deletion of SMN1 exon 7 for all 3 types was (94%), similar to those of Western Europe, China, Japan and Kuwait.</p>
Asunto(s)
Femenino , Humanos , Masculino , Embarazo , ADN , Genética , Exones , Eliminación de Gen , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Irán , Epidemiología , Atrofia Muscular Espinal , Diagnóstico , Epidemiología , Genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Diagnóstico Prenatal , Métodos , Prevalencia , Pronóstico , Estudios Retrospectivos , Proteínas del Complejo SMN , Genética , Proteína 1 para la Supervivencia de la Neurona Motora , Genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>INTRODUCTION</b>Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuropathies characterised by the selective degeneration of anterior horn cells. SMA has an estimated incidence of 1 in 10,000 live births. The causative genes are survival motor neuron (SMN) gene and neuronal apoptosis inhibitory protein (NAIP) gene. Deletions of the telomeric copy of SMN gene (SMN1) have been reported in 88.5% to 95% of SMA cases, whereas the deletion rate for NAIP gene (NAIP) is between 20% and 50% depending on the disease severity. The main objective of this study was to genetically characterise the childhood onset of SMA in Iran.</p><p><b>MATERIALS AND METHODS</b>Molecular analysis was performed on a total of 75 patients with a clinical diagnosis of SMA. In addition to common PCR analysis for SMN1 exons 7 and 8, we analysed NAIP exons 4 and 5, along with exon 13, as a internal control, by bi-plex PCR.</p><p><b>RESULTS</b>The homozygous-deletion frequency rate for the telomeric copy of SMN exons 7 and 8 in all types of SMA was 97%. Moreover, exons 5 and 6 of NAIP gene were deleted in approximately 83% of all SMA types. Three deletion haplotypes were constructed by using SMN and NAIP genotypes. Haplotype A, in which both genes are deleted, was seen in approximately 83% of SMA types I and II but not type III. It was also found predominantly in phenotypically severe group with an early age of onset (i.e., less than 6-month-old). We also report 34 of our prenatal diagnosis.</p><p><b>CONCLUSIONS</b>To our knowledge, the present study is the first one giving detailed information on SMN and NAIP deletion rates in Iranian SMA patients. Our results show that the frequency of SMN1 homozygous deletions in Iran is in agreement with previous studies in other countries. The molecular analysis of SMA-related gene deletion/s will be a useful tool for pre- and postnatal diagnostic.</p>
Asunto(s)
Preescolar , Femenino , Humanos , Masculino , Edad de Inicio , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Genética , Eliminación de Gen , Irán , Atrofia Muscular Espinal , Genética , Proteínas del Tejido Nervioso , Genética , Proteína Inhibidora de la Apoptosis Neuronal , Genética , Proteínas de Unión al ARN , Genética , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>OBJECTIVE</b>To detect the correlation between the clinical phenotype of spinal muscular atrophy (SMA) and survival motor neuron gene (SMN2) copy number.</p><p><b>METHODS</b>The SMN2 gene copy numbers of 57 different types of SMA were detected by real-time fluorescence quantitative PCR method with TaqMan technique.</p><p><b>RESULTS</b>Average SMN2 copy number was 1.017 +/- 0.090, 2.019+/- 0.080, 3.104+/- 0.170 in predicting one, two, three copy numbers, respectively, and CV was 8.9%, 3.9%, 5.4%, respectively. Average SMN2 copy number was 1.926+/- 0.460, 2.508+/- 0.460, 2.876+/- 0.270, in type I, II and III SMA, respectively. The SMN2 gene copy number in type II and III SMA were higher than that of type I SMA (P < 0.01). The SMN2 gene copy number in type III SMA was higher than that of type II SMA (P < 0.01). 85.72% of type I SMA patients usually had 2 SMN2 copies; 40% and 60% of type II SMA patients had 2 and 3 SMN2 copies, respectively; 82% of type III SMA patients had 3 SMN2 copies.</p><p><b>CONCLUSION</b>There is significant correlation between the change of SMA clinical phenotype and SMN2 cope number. The distributions of the SMN2 gene copy number are various in different types of SMA patients. All types of SMA patients have at least one copy SMN2. The SMN2 gene copy numbers in type II, III SMA are higher than that of type I. All of these findings suggest that the severity of SMA patients depend on the change of the SMN2 copy numbers.</p>
Asunto(s)
Humanos , Dosificación de Gen , Predisposición Genética a la Enfermedad , Genética , Atrofia Muscular Espinal , Genética , Patología , Fenotipo , Reacción en Cadena de la Polimerasa , Proteínas del Complejo SMN , Genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>OBJECTIVE</b>To perform prenatal diagnosis for 5 pregnant women who had given birth to children with spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Thirty to forty mililiters of amniotic fluid was obtained by amniocentesis under ultrasonic monitoring. DNA was extracted directly from sediment of amniotic fluid. Short tandem repeat (STR) profiling was carried out to evaluate the contamination of amniotic DNA by maternal genomic DNA. Two methods, PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific PCR, were used to analyze exon 7 of SMN gene from amniotic DNA.</p><p><b>RESULTS</b>Comparing the 16 STR sites of each fetus with those of his/her parents, there was no or little contamination of amniotic DNA by maternal genomic DNA. In conventional PCR-RFLP, part of the PCR product (189 bp) from amniotic DNA of fetus A, C, or D remained intact after digestion with Dra I, while the PCR product from amniotic DNA of fetus B or E was completely digested by Dra I. In allele-specific PCR, exon 7 of both SMN1 and SMN2 gene could be seen when amniotic DNA of fetuses A, C, or D was analyzed, while only exon 7 of SMN2 could be seen when amniotic DNA of fetuses B or E was analyzed.</p><p><b>CONCLUSION</b>Homozygous deletion of SMN1 is not detected in fetuses A, C, and D, predicting that the risk of developing SMA after birth would be extremely low. Homozygous deletion of SMN1 was present in fetuses B and E suggesting high risk of developing SMA after birth.</p>
Asunto(s)
Femenino , Humanos , Masculino , Embarazo , Exones , Genética , Salud de la Familia , Homocigoto , Repeticiones de Microsatélite , Genética , Atrofia Muscular Espinal , Diagnóstico , Genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Diagnóstico Prenatal , Métodos , Proteínas del Complejo SMN , Genética , Proteína 1 para la Supervivencia de la Neurona Motora , Genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>OBJECTIVE</b>Spinal muscular atrophy (SMA) is one of common autosomal recessive diseases and is characterized by degeneration of the anterior horn cells of the spinal cord. The reported prevalence is 1/10,000 live births with a carrier rate of one in 50. It is important in genetic counseling to identify the carriers with one copy deletion for the survival motor neuron (SMN(1)) gene. However, the duplication of the SMA locus makes the detection of SMA carriers difficult. This study aimed to determine the potential of the quantitative PCR analysis in the identification of SMA carriers.</p><p><b>METHODS</b>The SMN(1) gene copy number was detected by realdouble ended arrowtime PCR with TaqMan technology in 109 SMA parents of affected children and 40 normal controls.</p><p><b>RESULTS</b>The average copy numbers of SMN(1) in the individuals with known one copy of the SMN(1) gene and with the two copies were 0.777 +/-0.035 (CV=4.5%) and 2.064 +/-0.120 (CV= 5.8%) respectively. The average copy number of SMN(1) in all of the parents with affected individuals was 0.798 +/-0.108 (CV=13.5%), and that of normal controls was 2.106 +/-0.18 (CV=8.5%). About 98% of SMA patients' parents carried 1 copy SMN(1), and 95% of normal controls carried 2 copies.</p><p><b>CONCLUSIONS</b>The gene copy numbers for SMN(1) were one and two for SMA carriers and non-carriers, respectively. Our results suggested that the quantitative PCR analysis can distinguish the SMN(1) deletion carriers from non-carriers.</p>
Asunto(s)
Femenino , Humanos , Masculino , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Genética , Dosificación de Gen , Tamización de Portadores Genéticos , Métodos , Atrofia Muscular Espinal , Genética , Proteínas del Tejido Nervioso , Genética , Reacción en Cadena de la Polimerasa , Métodos , Proteínas de Unión al ARN , Genética , Análisis de Regresión , Proteínas del Complejo SMNRESUMEN
BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting in progressive muscle weakness and atrophy. AIMS: The molecular analysis of two marker genes for spinal muscular atrophy (SMA) i.e, the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP) was conducted in 39 Indian patients with clinical symptoms of SMA. Out of these, 28 showed homozygous deletions and the phenotypic features of these SMA patients were compared with the corresponding genotypes. SETTINGS: A tertiary care teaching Hospital. DESIGN: This is a prospective hospital based study. MATERIALS AND METHODS: Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene. RESULTS: Exons 7 and 8 of SMN and NAIP (exon 5) were homozygously deleted in 73% of SMA I and 27% of SMA II patients. SMN exon 7 and 8 deletions without NAIP deletions were seen in 27% of type I SMA and 46% of SMA type II patients. Two patients of type III SMA showed single deletion of SMN exon 7 along with 27% of SMA type II patients. CONCLUSION: With the advent of molecular biology techniques, SMN gene deletion studies have become the first line of investigation for confirmation of a clinical diagnosis of SMA. The findings of homozygous deletions of exons 7 and/or 8 of SMN1 gene confirms the diagnosis of SMA, even in patients with atypical clinical features. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful for predicting the prognosis.
Asunto(s)
Adolescente , Adulto , Niño , Preescolar , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Análisis Mutacional de ADN/métodos , Exones , Femenino , Eliminación de Gen , Humanos , India/epidemiología , Lactante , Masculino , Datos de Secuencia Molecular , Atrofia Muscular Espinal/clasificación , Proteínas del Tejido Nervioso/genética , Proteína Inhibidora de la Apoptosis Neuronal/genética , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Prospectivos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>OBJECTIVE</b>To introduce the application of denaturing high-performance liquid chromatography (DHPLC) in the diagnosis of childhood type spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Exon 7 and flanking area of survival motor neuron (SMN) gene were amplified by PCR in 1 standard sample, 25 normal individuals and 25 patients with SMA. The PCR products were then directly loaded onto the DHPLC system after denaturing and annealing. Different DNA segments were separated by changing the concentration of buffer A relative to that of buffer B.</p><p><b>RESULTS</b>Different DNA segments were separable on the DHPLC chromatogram. Three peaks including SMN1/SMN2 heteroduplex peak, SMN2 homoduplex peak and SMN1 homoduplex peak were detected in 23 out of 25 normal individuals. Only SMN1 homoduplex peak was detected in 2 normal individuals and the standard sample, indicating the deletion of SMN2 On the contrary, only the SMN2 homoduplex peak was detected in 22 out of 25 patients with SMA, indicating deletion of SMN1. The three peaks as those of normal individuals were detected in the other 3 patients, indicating no SMN1 or SMN2 deletion.</p><p><b>CONCLUSION</b>As a new technology for diagnosing SMA, DHPLC is sensitive, accurate, rapid and convenient.</p>
Asunto(s)
Humanos , Cromatografía Líquida de Alta Presión , Métodos , Exones , Genética , Atrofia Muscular Espinal , Diagnóstico , Genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Proteínas del Complejo SMN , Genética , Sensibilidad y Especificidad , Proteína 1 para la Supervivencia de la Neurona Motora , Genética , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
<p><b>BACKGROUND</b>Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of anterior horn cells of the spinal cord. The survival motor neuron gene is SMA-determining gene deleted in approximately 95% of SMA patients. This study was undertaken to predict prenatal SMA efficiently and rapidly in families with previously affected child.</p><p><b>METHODS</b>Prenatal diagnosis was made in 8 fetuses with a family history of SMA. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used for the detection of the survival motor neuron gene.</p><p><b>RESULTS</b>The survival motor neuron gene was not found in 6 fetuses, ruling out the diagnosis of SMA. Two fetuses were detected positive and the pregnancies were terminated.</p><p><b>CONCLUSION</b>Our method is effective and convenient in prenatal diagnosis of SMA.</p>
Asunto(s)
Adulto , Femenino , Humanos , Embarazo , Líquido Amniótico , Biología Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Genética , Exones , Sangre Fetal , Biología Celular , Proteínas del Tejido Nervioso , Genética , Reacción en Cadena de la Polimerasa , Diagnóstico Prenatal , Métodos , Proteínas de Unión al ARN , Genética , Proteínas del Complejo SMN , Análisis de Secuencia de ADN , Atrofias Musculares Espinales de la Infancia , Diagnóstico , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To study the genetic basis in the patients with clinical diagnosis of spinal muscular atrophy(SMA) but without survival motor neuron telomeric copy (SMN-T) deletion; the relationship between the SMN-C (centromeric) copies and the phenotype; and the distribution of SMN-C and SMN-T copies in the SMA patients, the carriers and the controls.</p><p><b>METHODS</b>Quantitative PCR analysis of SMN-T and SMN-C copies were carried out in 45 patients, 25 consanguineous and 33 control individuals. The patients were identified by clinical manifestation and muscular pathology. Two internal standards of SMN-T and cystic fibrosis transmembrane conductance regulator (CFTR) were constructed. Nonradioactive and nonfluorescence-labelling competitive PCR were used. The numbers of SMN-T and SMN-C copies were determined by calculating the ratios of SMN-T/CFTR and SMN-C/CFTR.</p><p><b>RESULTS</b>Quantitation of SMN-T gene copies in SMA patients revealed that nine cases of type I-III were homozygously deleted. Two cases of type III had only one copy and four cases of type III had two copies. SMA IV and other type cases had two copies. Nine cases of consanguineous individuals had one copy, but other 16 had two copies. All of the normal individuals had two copies. Analysis of SMN-C copies showed that SMA I had < or = 2 copies, II-III had < or = 3 copies, SMA IV and others had 0-3 copies, the consanguineous individuals and normal individuals had 0-3 copies.</p><p><b>CONCLUSION</b>The number of copies determined by PCR quantitative assay of SMN-T is in accordance with the result of PCR qualitative assay of homozygous deletion. Quantitative assay of the number of copies can find out the cases and the carriers of heterozygous deletion. The SMA phenotype is related to the number of copies of SMN-C; the smaller the number of copies the patient has, the severer the patient's phenotype will be. The pathogenesis of SMA IV and other types of SMA may not relate to SMN gene.</p>
Asunto(s)
Humanos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Dosificación de Gen , Atrofia Muscular Espinal , Genética , Proteínas del Tejido Nervioso , Genética , Proteínas de Unión al ARN , Proteínas del Complejo SMNRESUMEN
Survival of motor neurons(SMN) protein is the product of spinal muscular atrophy(SMA) gene. Now the function researching of SMN protein has become hotspot field to discuss the pathogenic mechanism of SMA. The construction, distribution and function of SMN protein are reviewed in this paper.
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Humanos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Galectina 1 , Genética , Metabolismo , Galectina 3 , Genética , Metabolismo , Antígenos de Histocompatibilidad Menor , Proteínas del Tejido Nervioso , Genética , Metabolismo , Proteínas Nucleares , Genética , Metabolismo , Unión Proteica , Proteínas de Unión al ARN , Investigación , Proyectos de Investigación , Ribonucleoproteínas Nucleares Pequeñas , Proteínas del Complejo SMN , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: To study the clinical profile of paralytic floppy infants undertaking available investigations and detect the frequency of exon7 of survival motor neuron (SMNT) gene deletion among the spinal muscular atrophy (SMA) cases. DESIGN: Descriptive study. SETTING: Tertiary care teaching hospital. SUBJECTS: 70 paralytic floppy infants (40 males/30 females) with age less than 13 years were included in the study. Exclusion criteria included central hypotonia of any cause. Detailed clinical evaluation was done followed by serum creatine phosphokinase levels, electrophysiological studies, muscle biopsy including immunohistochemistry and electron microscopy. Exon7 of SMNT gene deletion studies was done by PCR. RESULTS: Final diagnosis of SMA was assigned to 37 patients followed by congenital myopathy (n = 7), cogenital muscular dystrophy (n = 5), mitochondrial myopathy (n = 4), neuropathies (n = 5) and diaphragmatic SMA (n = 1). Only 15.7% of cases remained unclassified. When EMG was correlated with final diagnosis, it was 80.6% and 75% sensitive and 68.8% and 87.5% specific for neurogenic and muscle disease, respectively. Muscle biopsy revealed neurogenic atrophy in 47.8% cases followed by normal in 37.3% and myopathic pattern in 14.97% cases. Exon7 of SMNT gene was deleted in only 50% of SMA cases. CONCLUSIONS: Spinal muscular atrophy was the commonest cause of floppy children. The low rate of SMNT gene deletion detected needs confirmation with further studies.
Asunto(s)
Niño , Preescolar , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Electromiografía , Exones , Femenino , Eliminación de Gen , Humanos , Lactante , Masculino , Hipotonía Muscular/diagnóstico , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN , Proteínas del Complejo SMN , Atrofias Musculares Espinales de la Infancia/diagnóstico , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
BACKGROUND & OBJECTIVES: Distal upper limb spinal muscular atrophy (SMA) is an uncommon segmental variant of SMA. The condition is usually sporadic, affects males more often than females, and manifests late in the second decade of life, remaining confined to the upper limbs. We examined four patients with this form of SMA in order to determine if they carried homozygous deletion mutations in the survival motor neuron (SMN) or neuronal apoptosis inhibitory protein (NAIP) genes that underlie proximal SMA. METHODS: The four patients with distal upper limb SMA were analysed clinically, electrophysiologically and biochemically. Genomic DNA from each of the patients was analysed by restriction enzyme digestion of polymerase chain reaction (PCR) amplification products, as well single stranded conformation polymorphism (SSCP), to detect deletion events of selected exons of the SMN and NAIP genes. RESULTS: The clinical phenotype of the four patients, together with the biochemical and electrophysiological studies, confirmed a diagnosis of distal upper limb SMA. The molecular studies excluded homozygous deletion mutations in these patients as causative of their phenotype. INTERPRETATION & CONCLUSION: The genetic component underlying distal upper limb SMA appears not to involve mutations that are common in proximal SMA patients. It is possible that genes other than SMN and NAIP may be involved, while somatic mosaicism of SMN gene mutations could be implicated in the segmental nature of distal upper limb SMA.
Asunto(s)
Adolescente , Brazo/fisiopatología , Secuencia de Bases , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Cartilla de ADN , Femenino , Amplificación de Genes , Eliminación de Gen , Humanos , Masculino , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Polimorfismo Conformacional Retorcido-Simple , Proteínas de Unión al ARN , Proteínas del Complejo SMNRESUMEN
The objective of this study was to provide prenatal prediction of spinal muscular atrophy (SMA) by survival motor neuron (SMN) gene deletion analysis and genetic counseling in families with previous child affected with SMA. The SMN gene is absent or interrupted in approximately 95% of SMA patients independence of clinical severity. We study four families with one previous child affected in each by performing the SMN deletion analysis in the index case. When a homozygous deletion in exon 7 or exon 8 is found, we offer prenatal prediction to the family. All four index cases had homozygous deletions of the SMN gene. Prenatal diagnosis by amniocentesis was performed in all pregnancies. Two pregnancies were positive for the homozygous deletion of the SMN gene, non-directive counseling was given and the two pregnancies were terminated. The other two pregnancies showed no deletion of the SMN gene. The unborn child is yet to be followed up. The prenatal prediction of SMA shows considerable requirements and potential effectiveness in prevention of the SMA in families at risk which cut the cost of care in this incurable disease.