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

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.

Conclusion: Combination of FRET and HRM assays is a rapid and reliable method for the β-globin gene cluster haplotyping.