1. Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays
- Author
-
Angelique Delasaux, Philippe Joly, Caroline Garcia, Philippe Lacan, and Alain Francina
- Subjects
Time Factors ,Light ,Clinical Biochemistry ,Single-nucleotide polymorphism ,Anemia, Sickle Cell ,beta-Globins ,Biology ,HindIII ,Nucleic Acid Denaturation ,Polymorphism, Single Nucleotide ,Biochemistry ,High Resolution Melt ,Cohort Studies ,Gene cluster ,Fluorescence Resonance Energy Transfer ,Transition Temperature ,SNP ,Genotyping ,Genetics ,Base Sequence ,Biochemistry (medical) ,Haplotype ,Reproducibility of Results ,General Medicine ,Molecular biology ,Haplotypes ,Multigene Family ,biology.protein ,Restriction fragment length polymorphism - 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 βS 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 e, XmnI, HindIII Gγ, HindIII Aγ, HincII δ and a high resolution melting (HRM) assay for HincII ψβ. These six SNPs are sufficient in most cases to determine the βS haplotype. Results Our methodology allowed us to successfully determine the β-globin haplotypes of 139 patients suffering from sickle cell disease. For some βS / β0-patients, a supplementary SNP has been identified on the HindIII Gγ 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.
- Published
- 2011
- Full Text
- View/download PDF