title: Rapid screening of multiple beta-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes.
abstract: 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 LightCycler technology to screen for a spectrum of beta-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 beta-globin gene mutations in the Greek population, including IVSII-745C-->G, HbS, Cd5-CT, Cd6-A, Cd8-AA, IVSI-1G-->A, IVSI-5G-->A, IVSI-6T-->C, IVSI-110G-->A, and Cd39 C-->T.
  
  
  RESULTS
  The protocol, standardized by analysis of 100 beta-thalassemia heterozygotes with known mutations, was 100% reliable in distinguishing wild-type from mutant alleles. Subsequent screening of 100 Greek beta-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 beta-globin gene mutations from other population groups (IVSI-1G-->T, IVSI-5G-->C, IVSI-116T-->G, Cd37 TGG-->TGA, and Cd41/42 -TCTT).
  
  
  CONCLUSIONS
  The described LightCycler system protocol can rapidly screen for many beta-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.
authors.affiliation: Medical Genetics, Athens University, St Sophia's Children's Hospital, Athens 11527, Greece.
authors.forename: Christina
authors.initials: C
authors.lastname: Vrettou
authors.forename: Joanne
authors.initials: J
authors.lastname: Traeger-Synodinos
authors.forename: Maria
authors.initials: M
authors.lastname: Tzetis
authors.forename: George
authors.initials: G
authors.lastname: Malamis
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011296:Prenatal Diagnosis / Q000379:methods*; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D005914:Globins