Denaturing HPLC-based assay for molecular screening of nondeletional mutations causing alpha-thalassemias

Alpha-thalassemias are recessively inherited hemoglobin disorders caused by loss of function of either one of the two duplicated alpha-globin genes (alpha1 and alpha2). Although less frequent than large genomic deletions, at least 48 different nondeletional mutations have also been reported as causative mutations of alpha+-thalassemia. In this study, we have evaluated the performance of a relatively simple and semiautomated technique, denaturing HPLC (DHPLC), for the molecular identification of alpha-globin gene nondeletional mutations in 50 heterozygous carriers of Italian origin and in 50 wild-type individuals, as controls. Using our protocol we detected 12 different nondeletional mutations, including 10 point mutations, 1 five-nucleotide deletion and 1 point mutation associated with the –alpha3.7 rearranged alpha2/alpha1 gene.In conclusion, this study shows that DHPLC is a reliable, sensitive and specific screening method to rapidly detect the most common alpha-globin nucleotide mutations described in the Italian population.[...