Prevention of Lysosomal Storage Diseases and Derivation of Mutant StemCell Lines by Preimplantation

Genetic Diagnosis.

Gheona Altarescu, Rachel Beeri, Rachel Eiges, Silvina Epsztejn-Litman, Talia Eldar-Geva, Deborah Elstein, Ari Zimran, Ehud J.Margalioth, Ephrat Levy-Lahad, and Paul Renbaum.

Tatiana Gil Franco Paula Montoya

INTRUDUCTION Preimplantation genetic diagnosis( PGD) is a great tool for avoiding the transmission for genetic diseases to descendants. This is a challenging method because have analyze de disorder in a single cell , and have to build protocols for each specific mutation.Is necessarily that give the results in a short time, that can used in testing two or more indications at once and accuracy rates approaching 100%; to get this, use information of genomic DNA from family and polar bodies , how give maternal autosomal dominant.

Stem cells •Undifferentiated cell capable of self-renewal and differentiate into more specialized cells.

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.

Types of stem cells

1. Ambryonic stem cells, which are isolated from the inner cell mass of blastocysts

2. Adult stem cells, which are found in various tissues.

Lysosomes

Are membrane cytoplasmic organelles

Hydrolytic enzymes

Intracellular digestion of

macrumolecules,old cell parts,

and microorganisms

lysosomesClassification

Over 60 lysosomal enzymes are known. There is a hydrolyse for each type of biological molecule: • Peptidases – hydrolyse proteins• DNAases – hydrolyse DNA• RNAases – hydrolyse RNA• Lipases – hydrolyse lipids• Phosphatases – hydrolyse

phosphates• Glucosidases – hydrolyse

glycogen• Carboxylases - hydrolyse

carboxyl groups• Sulphatases – hydrolyse

sulphate

• Primary: those that contain enzymes solon.

•  Secondary: besides containing enzymes, also digestion materials.

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.

PGDPreimplantation genetic testing is a technique used to identify genetic defects in embryos created through in vitro fertilization (IVF) before pregnancy. Preimplantation genetic diagnosis (PGD) refers specifically to when one or both genetic parents has a known genetic abnormality and testing is performed on an embryo to determine if it also carries a genetic abnormality.

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.

Materiales y Métodos •Tay Sachs

Double carrier

4 (PGD)-5(prenatal diagnosis)

•Gaucher

Familia 2 : heterocigoto /mutación paternaFamilia 3:mujer GS/marido mutacion 84GG -50%

•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 –CVS3: mujer –análisis prenatal –interrupción –doble mutación

IVF- estimulación ovárica, recuperación del ovocito , 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

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.

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

Tabla 3

20 Familias con mutaciones

56 ciclos de PGD

Análisis de 329 ovocitos

Tasa de embarazo: 38%

Tabla 3

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.

Figura 1

Figura 1 (A) Electroforesis: método de laboratorio en el que se utiliza una corriente eléctrica controlada con la finalidad de separar biomoleculas según su tamaño y carga eléctrica a través de una matriz 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

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 Gaucher.

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 producen in vitro después de ser cultivadas en un medio carente del FACTOR INHIBIDOR LEUCÉMICO.

Discussion

AUTOR OPINION AGREE OR DISAGREE

K. Toyooka

In the second couple there was no known infertility but since more than half of female carriers of Fabry disease develop symptoms during life [22], they preferred medical sex

election for males.

G. L. Harton, M. De Rycke, F. Fiorentino.

Since PGD was first performed in 1991, by using different strategies, the technique has become very accurate with a misdiagnosis rate of less than 1% [26].

DiscussionAUTOR OPINION AGREE OR

DISAGREE

P. Kozlowski, A. Knippel, and R.

Stressig.

Both of these invasive methods are accompanied by a small risk of abortion due to the procedure [28].

A. Ribner, G. Altarescu, A. Zimran, and

D. Elstein.

One Gaucher stem cell line was derived from mutant embryos caring 84GG and N370S mutations is of particular of interest due to recent evidence of correlations between Parkinson disease and Gaucher [29].

Conclusions

The article was very interesting becouse

it shows us the statistics of a prevention

method for orphan and serious diseases,

such as lysosomal storage diseases.

It is also interesting to note the good prognosis that has this therapy in the prevention of the children’s birth with mutations.

ConclusionsIt is satisfying to know that PGD is a

procedure in which is cared the embryo’s

stability and also is forecasted the future

mutations in genes of the embryo.

Despite being a technique in which is risked the lives of many embryos, is a procedure whose purpose is to provide better quality for babies born.

Concept map

MAPA PAULA

GRACIAS