Download - Atrofia Muscular Espinal
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TEN YEARS OF EXPERIENCE IN MOLECULAR PRENATAL DIAGNOSIS AND CARRIER TESTING FOR SPINAL MUSCULAR ATROPHY AMONG FAMILIES FROM SERBIAMarijana Miskovic , Tanja Lalic , Danijela Radivojevic , Sanja Cirkovic,Slavica Ostojic , Marija Guc-Scekic.
María Alejandra Gómez OtálvaroDaniela Hincapié Tabares
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1. INTRODUCTION
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MUSCULAR ATROPHY
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SPINAL MUSCULAR ATROPHY
Affects Anterior Horn Cells Of The
Spinal Cord
Autosomal Recessive Hereditary
Disease
Neuromuscular Disease
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SPINAL MUSCULAR ATROPHY
Alteration in SMN1 gene
Homologous gene SMN2Locus: 5q13
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SMA TYPES
I
In neonatal period or
early infancy
-Muscle weakness -Arreflexia
-Flared thorax
-Globular belly
They are unable to sit
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SMA TYPES
II
6-18 Months
-Some walk -Most wheelchair
-Quake in hands -Scoliosis
Respiratory complications
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SMA TYPES
Tremor Weakness
Inability to run Frequent falls
IIIFrom 18 months Usually reaches adulthood
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SMA TYPES
IV
Recurrent falls
In the second or
third decade of life
Symptoms worsen
over time Tremor
Hypotonia
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Set of techniques available to know the proper training and correct development of the fetus before its birth.
PRENATAL DIAGNOSIS
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PRENATAL DIAGNOSIS
NON-INVASIVE TECHNIQUES
CLINIC
DNA TEST MATERNAL FETAL
BLOOD
ANALYTICAL
ULTRASOUND
INVASIVE TECHNIQUES
BIOPSY*
AMNIOCENTESIS*
FUNICULOCENTESIS
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RELATIONSHIP
SMA
SMN1 genePRENATAL DIAGNOSIS
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2. GENERAL OBJECTIVE
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GENERAL OBJECTIVE To describe ten years of experience of prenatal analysis of
spinal muscular atrophy (SMA).
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3. MATERIALES Y MÉTODOS
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DATOS GENERALES
The Mother and Child
Health Care Institute of Serbia Dr.
Vukan Cupic
44 Familias Serbias con 1 individuo con
SMA
Enero 1 de 2003-
Diciembre 31 de 2013
Análisis prenatal en 66 embarazos de
alto riesgo
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Los estudios familiares se dividieron en:
Grupo 1: 36 familias con al menos un niño afectado con deleción homocigótica de los exones 7 y 8 del gen SMN1.
Grupo 2: 5 familias con al menos un niño afectado sin confirmación molecular genética de la enfermedad.
Grupo 3: 3 familias en donde uno de los padres debía tener historia familiar de un individuo afectado con deleción homocigótica del gen SMN1.
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EXTRACCIÓN DEL DNA
1. Lisis celular
2. Tratamiento con RNAsa
3. Precipitación de proteínas
4. Precipitación
de DNA
5. Almacenamiento en TE
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PCR
• Es una técnica desarrollada por Kary Mullis.
• Amplificación directa de un gen o un fragmento de DNA o indirecta de un RNA.
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REACCIÓN EN CADENA DE LA POLIMERASA-PCR
Desnaturalización: calentamiento 90-97°.
Alineamiento: hibridación de DNA con primer´s y DNTp´s (37-65°).
Síntesis: amplificación (72-75°). Replicación en dirección 5´-3´.
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1. Conocer la secuencia que rodea al sitio de restricción.
2. La PCR amplifica la secuencia con el sitio de restricción.
3. Se corta con enzimas de restricción.
PCR-RFLP
Los productos se ven con electroforesis en gel de agarosa con LUV.
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PCR-RFLP
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BAYESIAN ANALYSIS
• Is a method of combining probabilities and which is used to calculate the probability of having or not having a disease causing mutation after a negative test is obtained.
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MAPAS GENÉTICOS
Representación gráfica de la ordenación y ubicación de los genes en cada cromosoma.
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MAPEO/RFLP
Se basa en la detección de aquellas variaciones de la secuencia del DNA que tienen como consecuencia un cambio en un sitio de restricción.
RFLP- polimorfismos en la longitud de los fragmentos de restricción.
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MLPA
• Detección de variación en el número de copias de múltiples secuencias de ADN en una única reacción (más de 50 secuencias génicas o genes)
Multiplex Ligation
Dependent Probe
Amplification
Técnica descrita por
Schouten JP et al en 2002.
Procedimiento de
cuantificación relativa basado
en la PCR.
Detección de variación en el
número de copias de
secuencias de DNA.
Detección de deleciones o inserciones
que afectan un exón o más.
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4. RESULTADOS
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66 MUESTRAS
FETALES
13 CON DELECIÓN HOMOCIGÓTICA EN GEN
SMN1
12 CASOS:
DELECIÓN EN AMBOS EXONES
1 CASO:
ELIMINACIÓN DE UN SÓLO EXÓN
RESULTADOS
7 8 7
1 caso Embarazo dicigótico->ambos hijos afectados
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5 Control (-)6 Control (+)
Gen homocigoto*
RESULTADOS
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RESULTADOS
ControlHermano PapáMamáTío
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RESULTADOS
ControlMamáPapá
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5. DISCUSSION
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AUTOR QUOTE AGREE DISAGREE
Wirth B. [7] Because 94% of clinically typical SMA patients have the homozygous deletion in SMN1. .
Miskovic M et al [13] Among the second group of families with at least 1 affected child without molecular genetic confirmation of the disease, prenatal diagnosis was offered on the basis of data from a previous study showing that 81% of Serbian individuals affected with SMA had the homozygous SMN1 deletion that causes the disease.
.
DISCUSSION
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AUTOR QUOTE AGREE DISAGREE
Somerville MJ et al [16] False negative results might be expected in cases of individuals who are compound heterozygous or (very rarely) homozygous for small mutations in the SMN1 gene.
.
Wirth B et al [17] Another concern in the prenatal prediction of SMA is that the SMN1 locus has a high rate of the novo deletions and rearrangements, which are found in 2% of affected individuals.
.
DISCUSSION
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6. CONCLUSSIONS
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• Genetic mapping was really useful for this study because it can locate the gene which is responsible of a hereditary disease, even without knowing the identity of the gene or the molecular basis of the disease. This technique can be used in a lot of inherited disorders such as SMA, with the purpose of decreasing the morbidity and mortality rate of the disease by doing prenatal diagnosis on the fetuses.
CONCLUSSIONS
Daniela Hincapié Tabares
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• PCR, RFLP and genetic mapping like MLPA, are laboratory techniques with a huge utility in the field of medicine, especially in the discovery of genetic diseases because these methods give an amplification of the DNA which allows a more detailed observation.
CONCLUSSIONS
Daniela Hincapié Tabares
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• By understanding the mechanisms affected by the absence of the SMN1 gene, we will be able to design effective therapeutic strategies to prevent neuronal degeneration and death in patients with SMA.
CONCLUSSIONS
Maria Alejandra Gómez Otálvaro
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• Early diagnosis of any congenital defect in the fetus is of vital importance as it would help to avoid unnecessary risks to the mother and child and try to improve the quality of life of the newborn.
CONCLUSSIONS
Maria Alejandra Gómez Otálvaro
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7. MAPAS CONCEPTUALES
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Daniela Hincapié Tabares
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Maria Alejandra Gómez Otálvaro
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8. BIBLIOGRAFÍA
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BIBLIOGRAFÍA• Martínez L.M., Vargas N., Toro A.E., Pamplona A.P., Quevedo
E. Biología molecular. 7°edición. Medellín, Colombia: UPB; 2012.
• Gómez J.E., González A., Castaño J.C., Patarroyo M.A. Biología molecular: principios y aplicaciones. Medellín, Colombia: CIB; 2011.