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).

DESIGN

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.

SETTING

The genetics and reproduction departments of two teaching hospitals.

PATIENT(S): Six informed couples at risk of transmitting SMA.

INTERVENTION(S): All patients underwent standard procedures associated with intracytoplasmic sperm injection.

MAIN OUTCOME MEASURE(S): Improvement of SMA diagnostic efficiency and accuracy on single cell.

RESULT(S): 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.

CONCLUSION(S): 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.