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

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. •We evaluate ARMS-qPCR for preimplantation genetic diagnosis of monogenic disorders.•Day-3 embryos are genotyped without the co-amplification of polymorphic markers.•Testing results are available within one day, enabling fresh embryo transfer (FET).•Diagnostic rates are favorable but allele-dropout should be cautioned.•The assay may complement current methods, especially for timely diagnosis for FET.