Preimplantation genetic diagnosis was used to avoid the transmission of four lysosomal storage disorders (TSD, GD, FD, HS) in couples at risk. 329 eggs from 56 cycles underwent PGD and analysis, resulting in 20 families avoiding transmission. Two human embryonic stem cell lines were derived from embryos diagnosed with Hunter syndrome and Gaucher disease. The cell lines expressed stem cell markers and had a normal karyotype. PGD is an effective technique for avoiding genetic transmission of lysosomal storage disorders and generating disease-specific stem cell lines.

1. 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 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

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
Intracellular
digestion of
Are membrane
Hydrolytic macromolecules,
cytoplasmic
enzymes old cell
organelles
parts, and
microorganisms

7. lysosomes
Classification
Over 60 lysosomal enzymes • Primary: those that
are known. There is a contain enzymes solon.
hydrolyse for each type of • Secondary: besides
biological molecule:
containing enzymes, also
• Peptidases – hydrolyse digestion materials.
proteins
• DNAases – hydrolyse DNA
• RNAases – hydrolyse RNA
• Lipases – hydrolyse lipids
• Phosphatases – hydrolyse
phosphates
• Glucosidases – hydrolyse
glycogen

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

11. Families
Transmission
or not Diseases of
lysosomes
LSD
Stem cells
(TSD,GD,FDHS)
PGD

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)
• Síndrome de no implantar portadores
Hunter
1 y 2 : hermanas –CVS
3: mujer –análisis prenatal –interrupción –

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.

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
Tasa de
embarazo:
Análisis de 38%
329
56 ciclos ovocitos
de PGD
20
Familias
con
mutacione
s

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

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

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

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
K. Toyooka disease develop symptoms
during life [22], they
preferred medical sex
election for males.
Since PGD was first
G. L. Harton, M. performed in 1991, by using
different strategies, the
De Rycke, F. technique has become very

29. Discussion
AGREE OR
AUTOR OPINION
DISAGREE
P. Kozlowski, A. Both of these invasive
methods are accompanied
Knippel, and R. by a small risk of abortion
Stressig. due to the procedure [28].
One Gaucher stem cell line
was derived from mutant
A. Ribner, G. embryos caring 84GG and
Altarescu, A. N370S mutations is of
Zimran, and particular of interest due to
recent evidence of
D. Elstein. correlations between
Parkinson disease and

30. Conclusions

31. Conclusions

32. Concept map

33. MAPA
PAULA

34. GRACIAS