Prevention of lysosomal storage diseases and derivation of2

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  • familias portadoras de mutaciones en cuatro genes que causan las enfermedades lisosomales
  • un resumen de los ciclos de PGD se presentan en siguiente tabla. IMPORTANTES: 1,3, 7 Y 11
  • Embriones mutantes de estas 4 enfermedades lisosomales fueron donados para la derivación de líneas de células madre. (después de la aprobación del IRB y el consentimiento de la familia)
  • Fue empleado por primeras vez en el año 1937
  • Prevention of lysosomal storage diseases and derivation of2

    1. 1. Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Lines by Preimplantation Genetic Diagnosis. Gheona A ltar escu, Rachel Beer i, Rachel Eiges, Silvina Epsztejn-Litman, Talia Eldar -Geva, Debor ah Elstein, A r i Zimran, Ehud J.Margalioth, Ephr at Levy-Lahad, and Paul Renbaum.Tatiana Gil FrancoPaula E MontoyaMedicine studentsMolecular Biology
    2. 2. INTRUDUCTIONPreimplantation genetic diagnosis( PGD) is a great tool for avoiding thetransmission for genetic diseases to descendants. This is a challengingmethod because have analyze de disorder in a single cell , and haveto build protocols for each specific mutation.Is necessarily that give the results in a short time, that can used in testingtwo or more indications at once and accuracy rates approaching100%; to get this, use information of genomic DNA from family and polarbodies , how give maternal autosomal dominant .
    3. 3. Stem cells• Undifferentiated cell capable of self-renewal and differentiate into more specialized cells.
    4. 4. Stem cells • Self-renewal: the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. • Potency: the capacity to differentiate into specialized cell types.
    5. 5. Types of stem cells1. Embryonic stem cells, which are isolated from the inner cell mass of blastocysts2. Adult stem cells, which are found in various tissues.
    6. 6. Lysosomes
    7. 7. lysosomes ClassificationOver 60 lysosomal enzymes are • Primary: those that containknown. There is a hydrolyse for enzymes solon.each type of biological molecule:  •  Secondary: besides containing enzymes, also digestion materials.•Peptidases – hydrolyse proteins•DNAases – hydrolyse DNA•RNAases – hydrolyse RNA•Lipases – hydrolyse lipids•Phosphatases – hydrolysephosphates•Glucosidases – hydrolyse glycogen•Carboxylases - hydrolyse carboxylgroups•Sulphatases – hydrolyse sulphate
    8. 8. LSD• Lysosomal storage diseases are genetic disorders in which a genetic mutation affects the activity of one or more of the acid hydrolases. In such diseases, the normal metabolism of specific macromolecules is blocked and the macromolecules accumulate inside the lysosomes, causing severe physiological damage or deformity.
    9. 9. PGDPreimplantation genetic testing is atechnique used to identify geneticdefects in embryos createdthrough in vitro fertilization (IVF)before pregnancy. Preimplantationgenetic diagnosis (PGD) refersspecifically to when one or bothgenetic parents has a knowngenetic abnormality and testing isperformed on an embryo todetermine if it also carries a geneticabnormality.
    10. 10. Objective• Present the strategy and outcome of PGD for four lysosomal storage disorders ( TSD,GD,FD,HS) with the purpose of avoiding the transmission for genetic diseases and termination of pregnancy in couples at risk of transmitting of these disorders.
    11. 11. Materiales y Métodos• Tay Sachs 4 (PGD)-5(prenatal diagnosis) Double carrier
    12. 12. • Gaucher Familia 2 : heterocigoto /mutación paterna Familia 3:mujer GS/marido mutación 84GG -50%
    13. 13. • Fabry : 2 parejas : hombre enfermo de fabry azoospermia no obstructiva en una pareja hombres serán sanos –mujeres portadoras (ligado al X) no implantar portadores• Síndrome de Hunter 1 y 2 : hermanas –CVS 3: mujer –análisis prenatal –interrupción –doble mutación
    14. 14. IVF- estimulación ovárica, recuperación delovocito , fertilización y biopsia congelados- descongelados: Valerato de estradiol oral (Estrofem 4-8mg al día) y vaginal Utrogestan (progesterona micronizada 900 mg / día). Cuerpo polar y blastómero
    15. 15. Análisis Molecular• Se extrajo el ADN de las células de sangre periférica ( parejas, niños afectados y familiares de primer grado ).• Para cada enfermedad: Marcadores polimórficos de microsatélites que rodean el gen enfermo se identificaron y los marcadores informativos usados, se crearon haplotipos para cada familia. Estos marcadores y las mutaciones familiares se usaron para el desarrollo de ensayos múltiples de una sola célula. Una reacción de PCR múltiple se utiliza . Sólo las muestras que fueron informativas para un mínimo de tres marcadores polimórficos se consideraron para el diagnóstico. Precauciones estrictas para evitar cualquier foco de contaminación .
    16. 16. Derivación de líneas y mantenimiento• Derivación y mantenimiento de indiferenciados Shaare Zedek (SZ) Hunter y células de Gaucher se llevaron a cabo de acuerdo con protocolos aplican rutinariamente en blastocitos diagnosticados como mutante
    17. 17. Tabla 3
    18. 18. Tabla 3
    19. 19. Tabla 4• De los 28 embriones, se obtuvieron dos líneas de células madre embrionarias humanas (HESC).• Una de un embrión mutante Hunter hembra.• Otra de compuestos heterocigotos 84GG/N370S para la enfermedad de Gaucher.
    20. 20. Figura 1
    21. 21. Figura 1 (A )Electroforesis: método delaboratorio en el que se utiliza unacorriente eléctrica controlada conla finalidad de separarbiomoleculas según su tamaño ycarga eléctrica a través de unamatriz gelatinosa. (1937) Estas nuevas líneas presentan características típicas de células madre embrionarias humanas, que expresan un panel de marcadores no diferenciados, como NANOG, Oct4, Sox2, y REX
    22. 22. Figura 1 (b)• Análisis cromosómico por tinción de Giemsa, llevado a cabo en metafase.• Mostró un cariotipo normal, 46XX humano para la línea celular Hunter y 46XY para la línea de
    23. 23. Figura 1 (c) • Se observaron colonias de células madre embrionarias humanas (HESC) y cuerpos embrioides. • HESC: células que poseen la capacidad de dividirse por largos periodos, se pueden diferenciar en las células de distintos linajes. (Pluripotentenciales). • Cuerpos embrioides: son agregaciones espontáneas de HESC que se
    24. 24. Discussion AGREE OR AUTHOR OPINION DISAGREE In the second couple there was no known infertility but since more than half of female carriers of Fabry disease K. Toyooka develop symptoms during life [22], they preferred medical sex election for males. Since PGD was first performed in 1991,G. L. Harton, M. De Rycke, by using different strategies, the technique has become very accurate F. Fiorentino. with a misdiagnosis rate of less than 1% [26].
    25. 25. Discussion AGREE OR AUTOR OPINION DISAGREE P. Kozlowski, A. Knippel, Both of these invasive methods are accompanied by a small risk of and R. Stressig. abortion due to the procedure [28]. One Gaucher stem cell line wasA. Ribner, G. Altarescu, A. derived from mutant embryos caring Zimran, and 84GG and N370S mutations is of D. Elstein. particular of interest due to recent evidence of correlations between Parkinson disease and Gaucher [29].
    26. 26. Conclusions ause it very int eresting becThe article was prevention m ethod tics of ashow s us the statis diseases, suc h as seriousfor o rphan and ses. lysosomal storage disea It is also interestin g to note the go has this therapy od prognosis that in the prevention birth with mutation of the children’s s.
    27. 27. Conclusions in which is th at PGD is a procedure It is satisfying to know ed the stabilit y and also is forecast cared the embryo’s of the embryo. futu re mutations in genesDespite being a tech nique in which is riskeembryos, is a proced d the lives of many ure whose purpose isquality for babies born to provide better .
    28. 28. Concept map
    29. 29. MAPAPAULA
    30. 30. GRACIAS

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