Denaturing HPLC-Based Assay for Molecular Screening of Nondeletional Mutations Causing α-Thalassemias

α-Thalassemias (OMIM 141850 and 141800; GenBank accession no. NT037887) are recessively inherited hemoglobin disorders caused by loss of function of either one of the two duplicated α-globin genes (α1 and α2), both located on chromosome 16p13.3 (1)(2). More than 95% of α-thalassemia phenotypes result from meiotic unequal recombinational events between the highly homologous α1- and α2-globin loci, which lead mainly to large genomic deletions (3–100 kb), which remove one to four α-globin genes, and rarely to α-gene triplication or quadruplication. Although less frequent, at least 48 different nondeletional mutations (including point mutations and deletions/insertions of a few nucleotides), mostly located in the α2-globin gene, have also been reported as causative mutations of α+-thalassemia (3)(4). At present, molecular identification of this type of nucleotide mutation is carried out by specific PCR amplification of the α2 or α1 gene, followed by methods that have only a limited rate of detection (60–80%) and are technically demanding, such as single-strand conformation polymorphism (SSCP) analysis and denaturing gradient gel electrophoresis (5)(6), or are costly, cumbersome, and time-consuming, such as direct sequencing and reverse dot-blot analysis (7)(8). We have evaluated the performance of a relatively simple and semiautomated technique, denaturing HPLC (DHPLC), that separates heteroduplex and homoduplex molecules on a stationary phase under partially denaturing conditions (9)(10). We tested a Transgenomic™ Wave DHPLC-based protocol for the molecular identification of α-globin gene nondeletional mutations in 50 wild-type individuals and 50 heterozygous carriers of Italian origin whose genes had previously been molecularly defined by restriction endonuclease digestion of PCR fragments and/or reverse dot-blot analysis. Blood samples were collected from heterozygous individuals and healthy controls at the Centro Studi Microcitemie (Rome), the Galliera Hospital Genova, and the Oasi ONLUS Troina after informed consent was given. Genomic …