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
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-thalass...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2016-01-01
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| Series: | Advanced Biomedical Research |
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| Online Access: | http://www.advbiores.net/article.asp?issn=2277-9175;year=2016;volume=5;issue=1;spage=71;epage=71;aulast=Ramezanzadeh |
| _version_ | 1817997357936017408 |
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| author | Mahboubeh Ramezanzadeh Mansour Salehi Rasoul Salehi |
| author_facet | Mahboubeh Ramezanzadeh Mansour Salehi Rasoul Salehi |
| author_sort | Mahboubeh Ramezanzadeh |
| collection | DOAJ |
| description | 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.
Materials and 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. |
| first_indexed | 2024-04-14T02:35:54Z |
| format | Article |
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| issn | 2277-9175 |
| language | English |
| last_indexed | 2024-04-14T02:35:54Z |
| publishDate | 2016-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
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| series | Advanced Biomedical Research |
| spelling | doaj.art-b7f36989465b4dcca38cee453adaceb82022-12-22T02:17:26ZengWolters Kluwer Medknow PublicationsAdvanced Biomedical Research2277-91752016-01-0151717110.4103/2277-9175.180640Assessment 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 methodMahboubeh RamezanzadehMansour SalehiRasoul SalehiBackground: 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. Materials and 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.http://www.advbiores.net/article.asp?issn=2277-9175;year=2016;volume=5;issue=1;spage=71;epage=71;aulast=RamezanzadehAmplification refractory mutation systembeta-thalassemia mutationshigh resolution meltingsequencing |
| spellingShingle | Mahboubeh Ramezanzadeh Mansour Salehi Rasoul Salehi 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 Advanced Biomedical Research Amplification refractory mutation system beta-thalassemia mutations high resolution melting sequencing |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Amplification refractory mutation system beta-thalassemia mutations high resolution melting sequencing |
| url | http://www.advbiores.net/article.asp?issn=2277-9175;year=2016;volume=5;issue=1;spage=71;epage=71;aulast=Ramezanzadeh |
| work_keys_str_mv | AT mahboubehramezanzadeh assessmentofhighresolutionmeltanalysisfeasibilityforevaluationofbetaglobingenemutationsasareproduciblecostefficientandfastalternativetothepresentconventionalmethod AT mansoursalehi assessmentofhighresolutionmeltanalysisfeasibilityforevaluationofbetaglobingenemutationsasareproduciblecostefficientandfastalternativetothepresentconventionalmethod AT rasoulsalehi assessmentofhighresolutionmeltanalysisfeasibilityforevaluationofbetaglobingenemutationsasareproduciblecostefficientandfastalternativetothepresentconventionalmethod |