目的:血红蛋白遗传性疾病是世界上最常见的遗传性疾病,导致显著的发病率和死亡率。与血红蛋白β基因(HBB)相关的大量突变使得通过高分辨率熔解(HRM)PCR进行基因扫描成为一种有吸引力的诊断方法。然而,现有的HRM-PCR测定不能检测所有常见的点突变,并且仅具有非常有限的检测较大基因重排的能力。本研究的目的是开发一种HBB检测方法,可以用作高度异质人群的筛选测试,用于检测点突变和较大的基因重排。
方法:该测定基于常规HRM-PCR和新型基因比率分析拷贝数(GRACE)PCR方法的组合。HRM-PCR被广泛优化,其中包括使用未标记的探针和将通用碱基掺入引物中以防止常见非病理多态性的干扰。使用GRACE-PCR来确定HBB基因相对于参考基因的拷贝数,使用解链曲线分析来检测HBB基因中的重排。通过分析410个样品来评估测定的性能。
结果:共检测到44种不同的病理基因型。与参考方法相比,该试验的灵敏度为100%,特异性为98%.
结论:我们开发了一种检测HBB基因点突变和更大重排的方法。这个化验很快,敏感,特异性和成本效益使其适合作为初始筛选测试,可用于高度异质的队列。
OBJECTIVE: Inherited disorders of haemoglobin are the world\'s most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements.
METHODS: The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples.
RESULTS: A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %.
CONCLUSIONS: We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.