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A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis.
Prenat Diagn. 1999 Dec; 19(13):1209-16.PD

Abstract

Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.

Authors+Show Affiliations

First Department of Pediatrics, Athens University, St Sophia's Children's Hospital, Greece.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

10660957

Citation

Vrettou, C, et al. "A Widely Applicable Strategy for Single Cell Genotyping of Beta-thalassaemia Mutations Using DGGE Analysis: Application to Preimplantation Genetic Diagnosis." Prenatal Diagnosis, vol. 19, no. 13, 1999, pp. 1209-16.
Vrettou C, Palmer G, Kanavakis E, et al. A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenat Diagn. 1999;19(13):1209-16.
Vrettou, C., Palmer, G., Kanavakis, E., Tzetis, M., Antoniadi, T., Mastrominas, M., & Traeger-Synodinos, J. (1999). A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenatal Diagnosis, 19(13), 1209-16.
Vrettou C, et al. A Widely Applicable Strategy for Single Cell Genotyping of Beta-thalassaemia Mutations Using DGGE Analysis: Application to Preimplantation Genetic Diagnosis. Prenat Diagn. 1999;19(13):1209-16. PubMed PMID: 10660957.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. AU - Vrettou,C, AU - Palmer,G, AU - Kanavakis,E, AU - Tzetis,M, AU - Antoniadi,T, AU - Mastrominas,M, AU - Traeger-Synodinos,J, PY - 2000/2/8/pubmed PY - 2000/3/4/medline PY - 2000/2/8/entrez SP - 1209 EP - 16 JF - Prenatal diagnosis JO - Prenat. Diagn. VL - 19 IS - 13 N2 - Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common. SN - 0197-3851 UR - https://www.unboundmedicine.com/medline/citation/10660957/A_widely_applicable_strategy_for_single_cell_genotyping_of_beta_thalassaemia_mutations_using_DGGE_analysis:_application_to_preimplantation_genetic_diagnosis_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0197-3851&date=1999&volume=19&issue=13&spage=1209 DB - PRIME DP - Unbound Medicine ER -