RS46(DXS548) GENOTYPING OF REPRODUCTIVE CELLS - APPROACHING PREIMPLANTATION TESTING OF THE FRAGILE-X SYNDROME

In order to approach preimplantation testing for the fragile-X syndrom e, we used genotyping of the polymorphic RS46(DXS548) locus closely li nked to the FMR-1 gene, in single reproductive cells of females. The R S46(DXS548) amplification was adjusted to the single cell level by a t wo-round polymerase chain reaction (PCR) procedure. Unfertilized oocyt es and extruded polar bodies were subjected to PCR. RS46(DXS548) genot yping at the single cell level was successful in 95% of the samples. I n two-third of the metaphase II oocytes and first polar bodies obtaine d from women who were heterozygous at the RS46(DXS548) locus, both mat ernal RS46(DXS548) alleles were observed because of crossing over duri ng the first meiotic division. This makes gamete selection by first po lar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR pr ocedure can be performed within 16 h after blastomere biopsy. Conseque ntly, the selection and transfer of the diagnosed embryos can be carri ed out within an acceptable time. Therefore, preimplantation testing f or the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an al ternative choice for prenatal testing for those carrier females who ar e heterozygous (informative) at the RS46(DXS548) locus.