Preimplantation genetic diagnosis for fragile X syndrome using multiplex nested PCR

Abstract Fragile X syndrome is caused by a dynamic mutation in the FMR1 gene. Normal individuals have 200 repeats. Female carriers are at risk of having affected offspring. A multiplex nested polymerase chain reaction protocol is described for preimplantation genetic diagnosis (PGD) of fragile X syndrome with simultaneous amplification of the CGG-repeat region, the Sry gene and several flanking polymorphic markers. The amplification efficiency was ≥96% for all loci. The allele dropout rate in heterozygotic females was 9% for the FMR1 CGG-repeat region and 5–10% for the polymorphic markers. Amplification failure for Sry occurred in 5% of single leukocytes isolated from males. PGD was performed in six patients who underwent 15 cycles. Results were confirmed in all cases by amniocentesis or chorionic villous sampling. Five clinical pregnancies were obtained (31% per cycle), four of which resulted in a normal delivery and one miscarried. This technique is associated with high efficiency and accuracy and may be used in carriers of full mutations and unstable high-order premutations.