title: Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays.
abstract: BACKGROUND
  β-Globin haplotypes are important to predict the clinical development of patients suffering from sickle cell disease (SCD). Five main haplotypes (Benin, Bantu, Senegal, Cameroon and Arabic-Indian) are defined for β(S) chromosomes and their determination usually requires the genotyping by restriction fragment length polymorphism (RFLP) of six to eight single nucleotide polymorphisms (SNPs). However, RFLP is time-consuming and can lead to a misdiagnosis in case of a supplementary SNP on the restriction sequence. We propose a rapid β-globin haplotyping method using fluorescence resonance transfer (FRET) and high resolution melting (HRM) assays.
  
  
  METHODS
  We have settled a fluorescence resonance energy transfer (FRET) assay for HincII ε, XmnI, HindIII (G)γ, HindIII (A)γ, HincII δ and a high resolution melting (HRM) assay for HincII ψβ. These six SNPs are sufficient in most cases to determine the β(S) haplotype.
  
  
  RESULTS
  Our methodology allowed us to successfully determine the β-globin haplotypes of 139 patients suffering from sickle cell disease. For some β(S) / β(0)-patients, a supplementary SNP has been identified on the HindIII (G)γ restriction sequence leading to a false-negative RFLP result.
  
  
  CONCLUSIONS
  Combination of FRET and HRM assays is a rapid and reliable method for the β-globin gene cluster haplotyping.
authors: Unité de Pathologie Moléculaire du Globule Rouge, Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Edouard-Herriot, Hôpitaux de Lyon, Lyon, France.
authors: Philippe
authors: P
authors: Joly
authors: Philippe
authors: P
authors: Lacan
authors: Caroline
authors: C
authors: Garcia
authors: Angelique
authors: A
authors: Delasaux
authors: Alain
authors: A
authors: Francina
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics*; D001483:Base Sequence; D015331:Cohort Studies; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006239:Haplotypes / Q000235:genetics*; D008027:Light; D005810:Multigene Family / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D013997:Time Factors; D044366:Transition Temperature; D055544:beta-Globins / Q000235:genetics*
chemical_list: D055544:beta-Globins