Assessment of high resolution melt analysis feasibility for evaluation of beta‑globin gene mutations as a reproducible, cost‑efficient and fast alternative to the present conventional method

Beta‑thalassemia is the most prevalent monogenic disease throughout the world. It was the first genetic disorder nominated for nation‑wide prevention programs involving population screening for heterozygotes and prenatal diagnosis (PND) in Iran. Due to the high prevalence of beta‑thalassemia, the shift from conventional mutation detection methods to more recently developed techniques based on novel innovative technologies are essential. We aimed to develop a real‑time polymerase chain reaction (PCR) based protocol using high resolution melting (HRM) analysis for diagnosis of common beta‑thalassemia mutations.

Forty DNA samples extracted from peripheral blood of suspected beta‑thalassemia carriers participated in this study were subjected to amplification refractory mutation system (ARMS). We then used 20 of these samples for HRM optimization. When 100% sensitivity and specificity was obtained with HRM procedure, we applied the technique for mutation detection on another remaining 20 samples as thalassemia cases with unknown mutations (detected mutations with ARMS‑PCR kept confidential). Finally, the HRM procedure applied on 2 chorionic villous sample (CVS) biopsied from 12 weeks gestational age pregnant women for routine PND analysis.

In the first step of study, Fr 8/9 (+G), IVSI‑1 (G > A), IVSI‑5 (G > C), IVSI‑110 (G > A), and CD44 (−C) mutations were diagnosed in samples under study using ARMS‑PCR technique. Finally, the HRM procedure applied on 20 unknown samples and 2 CVS The results of HRM were in complete concordance with ARMS and confirmed by sequencing.

The advantages of HRM analysis over conventional methods is high throughput, rapid, accurate, cost‑effective, and reproducible.