Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion

Abstract

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3-18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.

Preimplantation Diagnosis of Duchenne Muscular Dystrophy

“…Our results are in agreement with those of Holding et al, who described amplification success rates of 67-89% in a single cell deletion analysis of the dystrophin gene, and 97-100% in two cell analysis, and suggested two blastomeres would be necessary and sufficient for reliability PGD [35]. High amplification efficiency of exon 17 was reported by Liu et al to be almost 100% in 50 blastomeres [14].…”

“…In the first PGD for identifying the dystrophin gene mutation, six embryos were diagnosed at the cleavage stage after intracytoplasmic sperm injection (ICSI). Four of these embryos appeared to be unaffected, 3 of them were transferred to the uterus, and an unaffected non-carrier female was born [14]. Since this assay is only available for those families carrying exon 13-18 deletions, assays to detect the full range of mutations are needed.…”

“…The first approach is gender determination of embryos by either PCR or a fluorescence in situ hybridization (FISH)-based method [1,[3][4][5]. The second approach is diagnosis of specific gene mutation, which is attempted by a PCR-based method [14,35,36]. The third approach is linkage analysis by means of linked markers [35].…”

Well-devised quantification analysis for duplication mutation of Duchenne muscular dystrophy aimed at preimplantation genetic diagnosis

“…The main disadvantage of this approach is that half of the discarded male embryos are unaffected. Accordingly, allelic diagnosis by direct detection of a mutant gene has also been performed in the course of PGD [12].…”

“…The first specific PGD for DMD was performed for a family carrier of a deletion encompassing exon 17 of the dystrophin gene, through the study of this exon in single blastomeres [12]. There are several reports that describe a single cell multiplex PCR protocol allowing the analysis of dystrophin gene combined with gender determination [11,13,14].…”