Preimplantation genetic diagnosis of beta-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes.
Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells ( approximately 5cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38weeks' gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.
Hung
Chia-Cheng
CC
Graduate Institute of Clinical Genomics, National Taiwan University College of Medicine, Taipei, Taiwan.
Chen
Shee-Uan
SU
Lin
Shin-Yu
SY
Fang
Mei-Ya
MY
Chang
Li-Jung
LJ
Tsai
Yi-Yi
YY
Lin
Li-Ting
LT
Yang
Yu-Shih
YS
Lee
Chien-Nan
CN
Su
Yi-Ning
YN
D000328:Adult; D000595:Amino Acid Sequence; D001757:Blastomeres / Q000166:cytology; D015342:DNA Probes / Q000737:chemistry*; D047108:Embryonic Development; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D005817:Genetic Counseling; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D019836:Preimplantation Diagnosis; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000175:diagnosis / Q000235:genetics*
D015342:DNA Probes; D055544:beta-Globins