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Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis

Chen, Hsin-Fu, Chang, Shun-Ping, Wu, Sheng-Hai, Lin, Wen-Hsiang, Lee, Yi-Chung, Ni, Yen-Hsuan, Chen, Chi-An, Ma, Gwo-Chin, Ginsberg, Norman A., You, En-Min, Tsai, Feng-Po, Chen, Ming
Gene 2014 v.548 pp. 299-305
allelic dropouts, amino acids, biopsy, diagnostic techniques, embryo transfer, genetic markers, mutation, quantitative polymerase chain reaction, risk
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.