Improved single-cell protocol for preimplantation genetic diagnosis of spinal muscular atrophy.
OBJECTIVE
To develop and validate a simple and reliable single-cell analysis protocol for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA).


METHODS
Molecular tests based on specific enzymatic digestion have already been described for SMA diagnosis. We modified the amplified DNA fragments so as to introduce a novel restriction site that provides an internal control for the completeness of the digestion.


METHODS
The genetics and reproduction departments of two teaching hospitals.


METHODS
Six informed couples at risk of transmitting SMA.


METHODS
All patients underwent standard procedures associated with intracytoplasmic sperm injection.


METHODS
Improvement of SMA diagnostic efficiency and accuracy on single cell.


RESULTS
One hundred fifty lymphocytes were analyzed with our protocol. One hundred percent diagnostic accuracy was achieved from both homozygous normal and SMN1-deleted leukocytes. Successful molecular analysis was achieved for 36 of 42 biopsied embryos (86%). Twenty-five normal embryos were transferred, but no pregnancy was achieved.


CONCLUSIONS
We developed an improved protocol for PGD of SMA that is simple, robust, and accurate; unfortunately, no pregnancies were achieved for any of the six patients who have undergone PGD in the program thus far.
Burlet
Philippe
P
Département de Génétique, U393, Hôpital Necker Enfants Malades, Paris, France.
Frydman
Nelly
N
Gigarel
Nadine
N
Bonnefont
Jean Paul
JP
Kerbrat
Violaine
V
Tachdjian
Gérard
G
Frydman
René
R
Munnich
Arnold
A
Steffann
Julie
J
Ray
Pierre F
PF
D001483:Base Sequence; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D005260:Female; D005820:Genetic Testing / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods* / Q000706:statistics & numerical data