title: Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis.
abstract: Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic L-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, and Hospital, National Taiwan University, Taipei, Taiwan; Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Hsin-Fu
authors.initials: HF
authors.lastname: Chen
authors.affiliation: Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan; Department of Life Science, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Shun-Ping
authors.initials: SP
authors.lastname: Chang
authors.affiliation: Department of Life Science, National Chung-Hsing University, Taichung, Taiwan.
authors.forename: Sheng-Hai
authors.initials: SH
authors.lastname: Wu
authors.affiliation: Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan.
authors.forename: Wen-Hsiang
authors.initials: WH
authors.lastname: Lin
authors.affiliation: Department of Neurology, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Yi-Chung
authors.initials: YC
authors.lastname: Lee
authors.affiliation: Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Yen-Hsuan
authors.initials: YH
authors.lastname: Ni
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, and Hospital, National Taiwan University, Taipei, Taiwan.
authors.forename: Chi-An
authors.initials: CA
authors.lastname: Chen
authors.affiliation: Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan; Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Gwo-Chin
authors.initials: GC
authors.lastname: Ma
authors.affiliation: Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
authors.forename: Norman A
authors.initials: NA
authors.lastname: Ginsberg
authors.affiliation: Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan.
authors.forename: En-Min
authors.initials: EM
authors.lastname: You
authors.affiliation: Poyuan Women Clinic, Changhua, Taiwan.
authors.forename: Feng-Po
authors.initials: FP
authors.lastname: Tsai
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, and Hospital, National Taiwan University, Taipei, Taiwan; Department of Genomic Medicine, Changhua Christian Hospital, Changhua, Taiwan; Department of Life Science, National Chung-Hsing University, Taichung, Taiwan; Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan; Department of Life Science, Tunghai University, Taichung, Taiwan. Electronic address: mchen_cch@yahoo.com.
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
mesh_terms: D000328:Adult; D004624:Embryo Transfer; D004622:Embryo, Mammalian; D005260:Female; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D060005:Genotyping Techniques; D006801:Humans; D008297:Male; D009154:Mutation; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
keywords: ARMS-qPCR; Cleavage-stage embryo biopsy; Fresh embryo transfer (FET); Pre-implantation genetic diagnosis (PGD); Trophectoderm biopsy