title: Preimplantation genetic diagnosis of beta-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes.
abstract: 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.
authors.affiliation: Graduate Institute of Clinical Genomics, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Chia-Cheng
authors.initials: CC
authors.lastname: Hung
authors.forename: Shee-Uan
authors.initials: SU
authors.lastname: Chen
authors.forename: Shin-Yu
authors.initials: SY
authors.lastname: Lin
authors.forename: Mei-Ya
authors.initials: MY
authors.lastname: Fang
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Chang
authors.forename: Yi-Yi
authors.initials: YY
authors.lastname: Tsai
authors.forename: Li-Ting
authors.initials: LT
authors.lastname: Lin
authors.forename: Yu-Shih
authors.initials: YS
authors.lastname: Yang
authors.forename: Chien-Nan
authors.initials: CN
authors.lastname: Lee
authors.forename: Yi-Ning
authors.initials: YN
authors.lastname: Su
mesh_terms: 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*
chemical_list: D015342:DNA Probes; D055544:beta-Globins