Dataset Information

Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

ABSTRACT: Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for ? globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, ? globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from ?-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for ?-thalassaemia gene scanning.

SUBMITTER: Chassanidis C

PROVIDER: S-EPMC4924799 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

Chassanidis Christos C Boutou Effrossyni E Voskaridou Ersi E Balassopoulou Angeliki A

PloS one 20160628 6

Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a ...[more]

PMID: 27351925

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Project description:BackgroundMutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is the most commonly used method for mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%-10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application.MethodsWe used serial dilution of cell lines containing the TP53 exon 8 mutation to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step for cross-hybridization, followed by preferential denaturation and amplification at optimized critical temperature (full-COLD-PCR), to further enrich low-level mutations before HRM with or without DMSO, and we used droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional PCR-HRM and full-COLD-PCR-HRM with and without DMSO were used for mutation scanning of TP53 exon 8 in cancer samples containing known mutations and myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined.ResultsThe detection sensitivity of PCR-HRM scanning increases 2- to 5-fold in the presence of DMSO, depending on mutation type and sequence context, and can typically detect mutation abundance of approximately 1%. When mutation enrichment is applied during amplification with full-COLD-PCR followed by HRM in the presence of DMSO, mutations with 0.2%-0.3% abundance in TP53 exon 8 can be detected.ConclusionsDMSO improves HRM mutation scanning sensitivity with saturating dyes. When full-COLD-PCR is used, followed by DMSO-HRM, the overall improvement is about 20-fold compared with conventional PCR-HRM.

Project description:BACKGROUND:Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92?+?1 - c.92?+?130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS:Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G?>?A, c.92?+?5G?>?C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G?>?A, c.92G?>?C, c.92?+?130G?>?C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS:Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.

Dataset Information

Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

Publications

Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

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