Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis

Abstract

Sickle-cell and beta-thalassemia syndromes are priority genetic diseases for prevention programs involving population screening with the option of prenatal diagnosis for carrier couples. Preimplantation genetic diagnosis (PGD) represents a specialized alternative to prenatal diagnosis and is most appropriately used for couples with an unsuccessful reproductive history and/or undergoing assisted reproduction. However, clinical application of PGD has been hindered by difficulties in reliably transferring molecular diagnostic protocols to the single-cell level. We standardized and validated a protocol involving first-round multiplex PCR, amplifying the region of the beta-globin gene containing most of the common disease mutations world-wide and two unlinked microsatellite markers (GABRB3 and D13S314), followed by: 1) analysis of beta-globin genotypes with real-time PCR and 2) microsatellite sizing to exclude chance contamination. The protocol was standardized on 100 single lymphocytes from a beta-thalassemia heterozygote, including 15 artificially contaminated samples, the latter demonstrated through microsatellite analysis. PCR failure and allele drop-out (ADO) were observed in one (uncontaminated) sample each (1.2%). A pilot study in six clinical PGD cycles with five different beta-globin genotype interactions achieved results (in 5-6 hr) in 46 out of 50 single blastomeres (92%), all concordant with results from an established PGD method applied simultaneously; microsatellite analysis detected only parental alleles, excluding contamination. Beta-globin genotypes were also confirmed in two blastomeres through prenatal diagnosis (twin pregnancy), and in 11 out of 12 spare embryos, revealing one incident of ADO. Overall, the protocol proved to be sensitive, accurate, reliable, rapid, and applicable for many genotype interactions, with internal monitoring of contamination, thus fulfilling all requirements for clinical PGD application.

Rapid and Reliable Genotyping Technique for GM1 Gangliosidosis in Shiba Dogs by Real-Time Polymerase Chain Reaction with TaqMan Minor Groove Binder Probes

“…2,5,6 Real-time PCR is generally considered more sensitive, rapid, and less time consuming than conventional PCR for the analysis of genetic variability. In the present study, therefore, the application of real-time PCR with TaqMan minor groove binder (MGB) probes a was investigated to establish a rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs.…”

Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing

“…All STRs were analyzed directly from the first round PCR product, on an automatic sequencer, apart from the MIB HLA STR and the 24TG STR, for which a nested PCR preceded fragment analysis on the automatic sequencer. Most primer sequences for direct and indirect mutation detection and HLA typing have been previously published and any re-designed or new primer sets are presented in Table 1 [ Kakourou et al 2014;Vrettou et al 2004;Zachaki et al 2011].…”

“…For beta-thalassaemia, mutation detection was performed on diluted first round PCR products by real-time nested PCR using LightCycler hybridization probes as previously described [Vrettou et al 2004]. For detection of the sideroblastic anaemia mutation, a second round PCR was performed on diluted (1:1000) first round PCR products.…”

Conception and pregnancy outcome in a patient with 11-bp deletion of the steroidogenic acute regulatory protein gene

“…Cleavagestage biopsy, using a noncontact laser (ZILOS-tk; Hamilton Thorne Biosciences, Beverly, MA), was performed on seven embryos that had developed beyond the six-cell stage (12). Manipulation of cells, cell lysis, and precautions against contamination were as previously described (12,13). Exon 6 of the StAR gene and another eight loci, including four throughout the Y-chromosome (SRY, Amel Y, and SY127, SY149 from the AZF region), three in the X-chromosome (Amel X, AR exon 6, and STR 45 microsatellite marker), and one autosomal microsatellite marker, D13S314, were amplified.…”