Reliability of DHPLC in mutational screening of β‐globin (HBB) alleles

The inherited disorders of hemoglobin represent the most common Mendelian disease worldwide, with a higher prevalence among Mediterraneans, Asians, Africans, and Indians. Altered β-globin sequences, causing either hemoglobinopathies or β-thalassemia syndromes, are due to more than 200 different mutations in the β-globin gene. Prevention programs based on postnatal and prenatal molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. We have developed a rapid and highly specific mutation screening test based on the denaturing high-performance liquid chromatography (DHPLC) system. The sensitivity and specificity of the method were tested on the full genomic region of the β-globin gene in 30 normal Italian subjects and 40 heterozygous carriers in which 25 different β-globin mutations had been previously characterized by multiplex-ARMS technique. The results showed DHPLC to be 100% sensitive and specific. All the 25 sequence alterations and two previously undetected polymorphisms were precisely identified with neither false positive nor false negative results. In addition, 12 compound heterozygous and four homozygous patients were successfully subjected to DHPLC. Overall, the method was able to rapidly identify the most common β-globin mutations, accounting for more than 97% of β-globin alleles in the Italian population. Compared to classical approaches of mutation screening, this method allows a rapid, highly sensitive, cost-effective, and semi-automated simultaneous mutational scanning of a large number of samples.