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1 Medical Genetics, Athens University, and
2 Research Institute for the Study of Genetic and Malignant Disorders in Childhood, St. Sophia’s Children’s Hospital, Athens 11527, Greece.
aAddress correspondence to this author at: Medical Genetics, Athens University, St. Sophia’s Children’s Hospital, Choremio Research Laboratory, Thivon & Levadias Str., Athens 11527, Greece. Fax 301-779-5553; e-mail ekanavak{at}cc.uoa.gr.
Background: Hemoglobinopathies are priority genetic diseases for prevention programs. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis for carrier couples.
Methods: As a model, we designed a protocol based on the LightCyclerTM technology to screen for a spectrum of ß-globin gene mutations in the Greek population. Design was facilitated by dual fluorochrome detection and close proximity of many mutations. Three probe sets were capable of screening 95% of ß-globin gene mutations in the Greek population, including IVSII-745C
G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1GA, IVSI-5GA, IVSI-6TC, IVSI-110GA, and Cd39 CT.
Results: The protocol, standardized by analysis of 100 ß-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek ß-thalassemia heterozygotes with unknown mutations found 96 of 100 samples heterozygous for 1 of the 10 mutations, although melting curves were indistinguishable for mutations HbS/Cd6 and IVSI-5/IVSI-1, indicating a need of alternative methods for definitive diagnosis. One sample demonstrating a unique melting curve was characterized by sequencing as Cd8/9+G. Three samples carried mutations outside the gene region covered by the probes. The protocol was 100% accurate in 25 prenatal diagnosis samples, with 14 different genotype combinations diagnosed. The protocol was also flexible, detecting five ß-globin gene mutations from other population groups (IVSI-1GT, IVSI-5GC, IVSI-116TG, Cd37 TGGTGA, and Cd41/42 -TCTT).
Conclusions: The described LightCycler system protocol can rapidly screen for many ß-globin gene mutations. It is appropriate for use in many populations for directing definitive mutation diagnosis and is suited for rapid prenatal diagnosis with low cost per assay.
The following articles in journals at HighWire Press have cited this article:
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  G. Kokkali, J. Traeger-Synodinos, C. Vrettou, D. Stavrou, G.M. Jones, D.S. Cram, E. Makrakis, A.O. Trounson, E. Kanavakis, and K. Pantos Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study Hum. Reprod., May 1, 2007; 22(5): 1443 - 1449. [Abstract] [Full Text] [PDF]
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 K. Glynou, P. Kastanis, S. Boukouvala, V. Tsaoussis, P. C. Ioannou, T. K. Christopoulos, J. Traeger-Synodinos, and E. Kanavakis High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format Clin. Chem., March 1, 2007; 53(3): 384 - 391. [Abstract] [Full Text] [PDF]
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 L. Stromqvist Meuzelaar, K. Hopkins, E. Liebana, and A. J. Brookes DNA Diagnostics by Surface-Bound Melt-Curve Reactions J. Mol. Diagn., February 1, 2007; 9(1): 30 - 41. [Abstract] [Full Text] [PDF]
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G. Kokkali, C. Vrettou, J. Traeger-Synodinos, G.M. Jones, D.S. Cram, D. Stavrou, A.O. Trounson, E. Kanavakis, and K. Pantos Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of {beta}-thalassaemia major: Case report Hum. Reprod., July 1, 2005; 20(7): 1855 - 1859. [Abstract] [Full Text] [PDF]
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 J. Cheng, Y. Zhang, and Q. Li Real-time PCR genotyping using displacing probes Nucleic Acids Res., April 15, 2004; 32(7): e61 - e61. [Abstract] [Full Text] [PDF]
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