Preimplantation genetic diagnosis of X-linked retinoschisis using multiple displacement amplification (MDA)

Abstract

Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis and allows selection of unaffected IVF embryos to establish pregnancies in couples at risk of transmitting a genetic disorder. X-linked juvenile retinoschisis (RS) is a recessively inherited vitreo-retinal degeneration characterized by macular pathology and intraretinal splitting of the retina. The underlying defect in X-linked juvenile retinoschisis is due to a defective XLRS1 gene on chromosome X. We investigate the use of MDA in the RS–PGD program. We investigate the use of MDA from single cells as a universal first step for PGD of single defects using MDA to amplify single blastomere DNA, which was then used in the RS–PGD program. Two couples which the wife carries a causative mutation 599 G > A in the exon 6 of the XLRS1 gene that causes X-linked RS. The affected wife from family 1 and 2 were heterozygous at TAAA2 and CAG locus. To identify the sex chromosomes X22 informativity test were performed. MDA was used to amplify the whole-genome directly from a single cell. The MDA product was used for polymerase chain reaction (PCR) analysis of informative markers, used in the RS–PGD program. Segregation studies of the both families were performed and showed that the markers were informative. Three cycles were carried out for the two couples. The development of a RS–PGD protocol allowed for the diagnosis of 12 embryos none of which remained without diagnosis. Five embryos were shown to be affected males and 2 were carried female, while 3 were unaffected males and 2 non-carrier females of which 5 were transferred. One singleton ongoing pregnancy ensued. 12 cells were analysed, none of which showed no amplification. Only one cell showed ADO. We describe the first MDA–PGD for X-linked RS. The MDA technique is useful for overcoming the problem of insufficient genomic DNA in PGD, since enough DNA from a single cell to allow for multiple PCR analyses. At the same time, high amplification efficiency and low ADO rates are seen compared with nested multiplex PCR. The use of MDA as an initial step in PGD allows for DNA amplification in quantities sufficient for the diagnosis of a wide spectrum of single-gene defects using standard methods.