Identification of microsatellite markers <1 Mb from the FMR1 CGG repeat and development of a single-tube tetradecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of fragile X syndrome.

Abstract

To develop a single-tube polymerase chain reaction (PCR) panel of highly polymorphic markers for preimplantation genetic diagnosis (PGD) of fragile X syndrome (FXS). An in silico search was performed to identify all markers within 1 Mb flanking the FMR1 gene. Selected markers were optimized into a single-tube PCR panel and their polymorphism indices were determined from 272 female samples from three populations. The single-tube assay was also validated on 30 single cells to evaluate its applicability to FXS PGD. Thirteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube PCR panel together with AMELX/Y for gender determination. Analysis of 272 female samples confirmed the high polymorphism (PIC > 0.5) of most markers, with expected and observed heterozygosities ranging from 0.31 to 0.87. More than 99% of individuals were heterozygous for at least three markers, with 95.8% of individuals heterozygous for at least two markers on either side of the FMR1 CGG repeat. The tetradecaplex marker assay can be performed directly on single cells or after whole-genome amplification, thus supporting its use in FXS PGD either as a standalone linkage-based assay or as a complement to FMR1 mutation detection.Genet Med 18 9, 869-875.