Rapid, sensitive and discriminatory HbS and HbC mutation detection using High Resolution Melting Analysis.

Background: Hemoglobinopathies are common monogenic diseases forming a major public health problem due to their severity and disabling nature. Genetic identification of the two most frequently observed missense mutations in the beta-globin gene, HbS and HbC, are important epidemiologically and aid in prevention of the sickle cell trait and other serious hemoglobin disorders.

Aim: Our increasing patient population of Mediterranean, African and Middle Eastern origin urge for the need of a rapid, inexpensive and high-throughput genetic testing for HbS and HbC variants.

Results: Classical high resolution melting analysis with an optimized PCR amplicon of 110 bp allowed a sensitive and reliable identification of the two neighbouring HbS (c.20A>T) and HbC (c.19G>A) mutations. Discriminatory melting profiles were observed for all possible combinations of mutations: HbAA, HbAS, HbAC, HbSS, HbCC and HbSC, and confirmed the results obtained by Hb-chromatography and PCR followed by restriction digestion. Within the wild type control population tested, mainly consisting of Belgian and North African individuals, two other aberrant melting patterns were observed. Sequencing of all samples with aberrant melting patterns revealed one polymorphism at position c.9 either in heterozygous or homozygous state. Further testing including samples from over 35 other nationalities did not disclose other melting profiles.

Conclusion: This HbS and HbC HRM seems a promising, inexpensive and high-throughput alternative to PCR and restriction digestion analysis, although further validation is needed prior to implementation in a post- and prenatal diagnostic setting.