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 Franco
Paula E Montoya
Medicine students
Molecular Biology

2. 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 .

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

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. Types of stem cells
1. Embryonic stem cells, which are isolated from the
inner cell mass of blastocysts
2. Adult stem cells, which are found in various tissues.

6. Lysosomes

7. lysosomes
Classification
Over 60 lysosomal enzymes are • Primary: those that contain
known. 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 – hydrolyse
phosphates
•Glucosidases – hydrolyse glycogen
•Carboxylases - hydrolyse carboxyl
groups
•Sulphatases – hydrolyse sulphate

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.

10. PGD
Preimplantation 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.

12. 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.

13. Materiales y Métodos
• Tay Sachs
4 (PGD)-5(prenatal diagnosis)
Double carrier

14. • Gaucher
Familia 2 : heterocigoto /mutación paterna
Familia 3:mujer GS/marido mutación 84GG -50%

15. • 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

16. 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

17. 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 .

20. 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

21. Tabla 3

22. Tabla 3

23. 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.

24. Figura 1

25. 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

26. 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.

27. 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.

28. 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].

29. 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 was
A. 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].

30. Conclusions
ause it
very int eresting bec
The article was prevention m
ethod
tics of a
show s us the statis diseases, suc
h as
serious
for 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.

31. 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 genes
Despite being a tech
nique in which is riske
embryos, is a proced d the lives of many
ure whose purpose is
quality for babies born to provide better
.

32. Concept map

33. MAPA
PAULA

34. GRACIAS