Efficient Detection of Mediterranean β-Thalassemia Mutations by Multiplex Single-Nucleotide Primer Extension

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.

Presence of the IVS-I-6-Mutated Allele in Beta-Thalassemia Major Patients Correlates with Extramedullary Hematopoiesis Incidence

“…None of the rest of the mutations detected in the patients with EMH was related with its occurrence (IVS-I-110, p = 0.900; CD39, p = 0.134; IVS-I-1, p = 0.733; IVS-II-745, p = 0.474). The IVS-I-6 (T→C) mutation is one of the most common causes of β-thalassemia among individuals of Mediterranean and Middle Eastern ancestry [39,40]. Its high incidence has been reported among populations from Portugal [41]; Spain [42,43]; Albania [44]; the region of former Yugoslavia [45,46,47,48]; Greece [49,50,51,52]; Algeria [53]; Egypt [54]; Palestine [55,56]; Lebanon [57]; Syria [58,59]; Cyprus [60]; Turkey [61,62,63,64,65]; Saudi Arabia [66,67]; Iraq [68,69,70,71]; Iran [72,73,74]; and elsewhere.…”

Novel Βeta (β)-Thalassemia Mutation in Turkish Children

“…b-Thal clinically occurs in three main forms: major, intermedia and minor according to requirement of transfusion [3]. b-Thal major is characterized by completely inhibited synthesis of beta chains [4] so b-Thal major must be treated (generally transfusion therapy) otherwise 85 % of patients die in early childhood due to severe anemia [5].…”

“…Genomic sequence of b-globin gene was scanned for mutations working direct DNA sequence analysis. This assay enables more comprehensive detection of rare and novel mutations [3]. We reported the number and the frequencies of known mutations, and type of b-Thal mutations and then carried out mutation analysis of b-Thal in Gaziantep of Turkey.…”

SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

“…Moreover the assay tests for polyT variants (5T, 7T and 9T) in intron 8 (c.1210-12T[5_9]). The methodology for screening is based on single tube multiplex PCR reaction followed by multiplex fluorescent single-nucleotide primer extension coupled with automated capillary electrophoresis which has already been successfully implemented for several other applications in our laboratory [32] , [33] , [34] , [35] .…”