Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays
Rapid and reliable β-globin gene cluster haplotyping of sickle cell disease patients by FRET Light Cycler and HRM assays
复制标题
DOI：
10.1016/j.cca.2011.03.025
复制
发表时间：
2011-06-11
影响因子：
5
通讯作者：
Francina, Alain
中科院分区：
文献类型：
--
作者：
Joly, Philippe;Lacan, Philippe;Francina, Alain
背景：β -珠蛋白单倍型对预测镰状细胞病（SCD）患者的临床发展具有重要意义。5种主要的单倍型（贝宁、班图、塞内加尔、喀麦隆和阿拉伯-印度）被定义为β (s)染色体，它们的确定通常需要6到8个单核苷酸多态性（SNPs）的限制性片段长度多态性（RFLP）基因分型。然而，RFLP是耗时的，并且在限制性内切序列上存在补充SNP的情况下可能导致误诊。我们提出了一种使用荧光共振转移（FRET）和高分辨率熔化（HRM）测定的快速β -珠蛋白单倍型方法。方法：建立了HincII epsilon、XmnI、HindIII (G)gamma、HindIII (a)gamma、HincII delta的荧光共振能量转移（FRET）法和HincII psi beta的高分辨率熔化（HRM）法。在大多数情况下，这6个snp足以确定β (s)单倍型。结果：我们的方法使我们能够成功地确定139例镰状细胞病患者的β -珠蛋白单倍型。对于一些β (s)/ β(0)-患者，在HindIII (G) γ限制性序列上发现了一个补充SNP，导致RFLP结果为假阴性。结论：FRET和HRM联合检测是一种快速、可靠的β -珠蛋白基因簇单倍型分析方法。(C) 2011 Elsevier B.V.版权所有
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.