Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells

We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to a nalyse five exons commonly deleted in deletion-type Duchenne muscular dystr ophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal ma le (220 cells), a normal female (24 cells) and a male DMD patient (40 cells ) carrying a deletion of exons 46-49 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 0-4.5% of normal male cells. One negative control ou t of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with f our of the embryos found to be male and one female. This method is therefor e suitable for preimplantation genetic diagnosis and will allow the transfe r of healthy embryos (both male and female) in families carrying DMD gene d eletions involving at least one of the five exons 17, 19, 44, 45 and 48.