Abstract
β-Thalassemias and abnormal hemoglobin variants are among the most common hereditary abnormalities in humans. Molecular characterization of the causative genetic variants is an essential part of the diagnostic process. In geographic areas with high hemoglobinopathy prevalence, such as the Mediterranean region, a limited number of genetic variants are responsible for the majority of hemoglobinopathy cases. Developing reliable, rapid and cost-effective mutation-specific molecular diagnostic assays targeting particular populations greatly facilitates routine hemoglobinopathy investigations. We developed a one-tube single-nucleotide primer extension assay for the detection of eight common Mediterranean β-thalassemia mutations: Codon 5 (-CT); CCT(Pro)->C–, Codon 6 (-A); GAG(Glu)->G-G, Codon 8 (-AA); AAG(Lys)->–G, IVS-I-1 (G->A), IVS-I-6 (T->C), IVS-I-110 (G->A), Codon 39 (C->T), and IVS-II-745 (C->G), as well as the hemoglobin S variant beta 6(A3) Glu>Val. We validated the new assay using previously genotyped samples obtaining 100% agreement between independent genotyping methods. Our approach, applicable in a range of Mediterranean countries, offers a combination of high accuracy and rapidity exploiting standard techniques and widely available equipment. It can be further adapted to particular populations by including/excluding assayed mutations. We facilitate future modifications by providing detailed information on assay design.
Highlights
Hemoglobinopathies are the most abundant group of monogenic abnormalities in humans representing a health burden comparative to that of other major diseases [1,2]
We took advantage of the extensive genetic information collected through hemoglobinopathy diagnostics in our laboratory in order to design a mutation-specific assay custom-tailored for our purposes
We selected the top eight most common b-thalassemia mutations to include in the minisequencing assay (Table 1 and Figure S1)
Summary
Hemoglobinopathies are the most abundant group of monogenic abnormalities in humans representing a health burden comparative to that of other major diseases [1,2]. They are caused by genetic defects affecting the globin genes encoding for the hemoglobin a and b chains. Some geographic areas including the Mediterranean region have high prevalence of b-thalassemias [5]. Another subclass of HBB mutations, rather than decreasing the amount of b chain as in b-thalassemia, lead to the production of abnormal hemoglobin variants. Homozygous patients suffer from sickle cell disease, a serious condition with numerous complications including acute hemolytic and vaso-occlusive crises [1]
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