Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects

A great majority of patients seeking preimplantation genetic diagnosis (PGD ) are women >35 years of age. In addition to being carriers for single gene defects, these women also have a higher risk of having children with Down' s syndrome (trisomy 21). For these patients, it would be advantageous if a diagnostic test for trisomy 21 was developed, which could be used in conjun ction with tests for single gene defects. Here, we assessed the feasibility of developing an accurate genetic test for diagnosing trisomy 21 and the m utation causing spinal muscular atrophy (SMA) in single cells using multipl ex fluorescence polymerase chain reaction (PCR). Single- and two-round PCR were developed using a combination of primers for the survival motor neuron (SMN) gene exons 7 and 8 and two chromosome 21 short tandem repeats (STRs) , D21S226 and D21S11. After only 36 cycles, 88 and 68% of normal single cel ls were screened for SMA mutations and trisomy 21 respectively. In multiple x PCR using only two primers (SMN exon 7 and D21S11) instead of four, the e fficiency of SMA diagnosis was increased to 93%, In the same reactions, the D21S11 alleles were detected in 83% of the normal single cells. Clinical a pplications of this assay should enable detection of those embryos that hav e inherited three heterozygous alleles and, therefore, benefit many PGD pat ients who are at an increased risk of Down's syndrome.