Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning.β地中海贫血（β-thalassaemia）是全球最常见的常染色体隐性遗传病之一。该疾病在整个地中海区域高发，这一现状推动了分子诊断检测方法的建立，以阻断重症患儿的出生。因此，开发一种可靠、经济高效且快速的β珠蛋白基因点突变筛查方法，使其能够适配常规实验室的工作流程，已成为至关重要的需求。本研究首次报道了一种高分辨率熔解曲线分析（High-Resolution Melting Analysis, HRMA）方法的开发，该方法适用于筛查希腊人群中存在的高度异质性β珠蛋白基因突变，因此可推广至地中海区域及其他已发现此类突变的地区。在此研究框架下，我们将希腊人群中常见突变所在的β珠蛋白基因区域划分为10个重叠扩增子。我们的反应体系可实现多引物组的同时扩增与部分多重化，从而显著缩短实验时长。我们对携带明确基因型的β地中海贫血患者/携带者的DNA样本进行了检测。不同基因型可呈现出特征性的熔解曲线，从而实现突变的精准检测。本研究所述的HRMA方法可适配高通量检测场景，是一种快速、简便、经济高效、可靠、可行性强且灵敏度高的β地中海贫血基因筛查手段。