Molecular Human Reproduction, Vol. 5, No. 11, 1089-1094,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Diagnosing genetic disease |
Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells
1 Department of Obstetrics and Gynaecology and 2 Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville 5011, South Australia, Australia, 3 Institute of Obstetrics and Gynaecology, The 2nd People's Hospital, Guangzhou, 510150, People's Republic of China and 4 South Australian Clinical Genetics Service, The Women's and Children's Hospital, North Adelaide, 5006, South Australia
Abstract
We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (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 male (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 out 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 four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.
DMDgene/Duchenne muscular dystrophy/preimplantation genetic diagnosis/single cell PCR
Notes
5 Present address: Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, East Melbourne, 3002, Victoria, Australia
6 To whom correspondance should be addressed
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