Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays

Background: beta-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 beta(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 beta-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 epsilon, XmnI, HindIII (G)gamma, HindIII (A)gamma, HincII delta and a high resolution melting (HRM) assay for HincII psi beta. These six SNPs are sufficient in most cases to determine the beta(s) haplotype. Results: Our methodology allowed us to successfully determine the beta-globin haplotypes of 139 patients suffering from sickle cell disease. For some beta(s)/beta(0)-patients, a supplementary SNP has been identified on the HindIII (G)gamma restriction sequence leading to a false-negative RFLP result. Conclusion: Combination of FRET and HRM assays is a rapid and reliable method for the beta-globin gene cluster haplotyping. (C) 2011 Elsevier B.V. All rights reserved.