title: 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.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  The advantages of HRM analysis over conventional methods is high throughput, rapid, accurate, cost-effective, and reproducible.
authors.affiliation: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mahboubeh
authors.initials: M
authors.lastname: Ramezanzadeh
authors.affiliation: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mansour
authors.initials: M
authors.lastname: Salehi
authors.affiliation: Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Rasoul
authors.initials: R
authors.lastname: Salehi
keywords: Amplification refractory mutation system; beta-thalassemia mutations; high resolution melting; sequencing