[Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR].
OBJECTIVE
To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.


METHODS
Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.


RESULTS
In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.


CONCLUSIONS
The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.
Wu
Yue-Li
YL
National Laboratory of Medical Genetics, Xiangya Hospital, Central South University, Changsha, China.
Wu
Ling-Qian
LQ
Li
Yan-Ping
YP
Liu
Dong-E
DE
Zeng
Qiao
Q
Zhu
Hai-Yan
HY
Pan
Qian
Q
Liang
De-Sheng
DS
Hu
Hao
H
Long
Zhi-Gao
ZG
Li
Juan
J
Dai
He-Ping
HP
Xia
Kun
K
Xia
Jia-Hui
JH
D053523:Amelogenin / Q000235:genetics*; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D041321:Chromosomes, Human, X / Q000235:genetics; D041322:Chromosomes, Human, Y / Q000235:genetics; D020732:Cytogenetic Analysis / Q000379:methods; D005091:Exons / Q000235:genetics; D005260:Female; D017353:Gene Deletion; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*
D053523:Amelogenin