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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.
Hum Mutat. 2004 May; 23(5):513-21.HM

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.

Authors+Show Affiliations

Medical Genetics, Athens University, St. Sophia's Children's Hospital, Athens, Greece.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15108284

Citation

Vrettou, Christina, et al. "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." Human Mutation, vol. 23, no. 5, 2004, pp. 513-21.
Vrettou C, Traeger-Synodinos J, Tzetis M, et al. 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. Hum Mutat. 2004;23(5):513-21.
Vrettou, C., Traeger-Synodinos, J., Tzetis, M., Palmer, G., Sofocleous, C., & Kanavakis, E. (2004). 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. Human Mutation, 23(5), 513-21.
Vrettou C, et al. 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. Hum Mutat. 2004;23(5):513-21. PubMed PMID: 15108284.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Vrettou,Christina, AU - Traeger-Synodinos,Joanne, AU - Tzetis,Maria, AU - Palmer,Giles, AU - Sofocleous,Christalena, AU - Kanavakis,Emmanuel, PY - 2004/4/27/pubmed PY - 2004/9/21/medline PY - 2004/4/27/entrez SP - 513 EP - 21 JF - Human mutation JO - Hum. Mutat. VL - 23 IS - 5 N2 - 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. SN - 1098-1004 UR - https://www.unboundmedicine.com/medline/citation/15108284/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_ L2 - https://doi.org/10.1002/humu.20022 DB - PRIME DP - Unbound Medicine ER -