PREIMPLANTATION PREVENTION OF X-LINKED DISEASE - RELIABLE AND RAPID SEX DETERMINATION OF SINGLE HUMAN-CELLS BY RESTRICTION ANALYSIS OF SIMULTANEOUSLY AMPLIFIED ZFX AND ZFY SEQUENCES

In vitro fertilization (IVF), blastomere biopsy of the 6 - 8 cell embr yo, and single cell DNA diagnosis allows couples at risk of transmitti ng an X-linked or autosomal disease to start a pregnancy knowing their child will not be affected. We present a quick and reliable nested PC R strategy for sex determination at the single cell level by simultane ous amplification and subsequent restriction fragment analysis of the homologous but non-allelic ZFX and ZFY genes present on the X and Y ch romosomes respectively. Amplified ZFX and ZFY sequences are of equal s ize and produce distinguishable HaeIII digestion products. In a random ized, blinded study of 194 individually isolated lymphoblasts, amniocy tes, chorion villus cells, and blastomeres, 191 amplified successfully (98.4% sensitivity). None of the sample blanks showed any PCR product , all 90 of the karyotypically XY cells were correctly genotyped as ZF X/ZFY, all 83 of the 84 XX cells that amplified were correctly genotyp ed as ZFX only, and analyses of all same-embryo blastomeres were compl etely concordant (100% specificity). This strategy avoids a source of misdiagnosis observed in methods which detect only Y-specific sequence s, where amplification failure in an XY cell results in an erroneous X X diagnosis. This rapid (6 hr) and simple method of analysis, when app lied to preimplantation embryo diagnosis, allows the avoidance of offs pring affected with an X-linked recessive disorder by transferring onl y female embryos for implantation and ensuing pregnancy.