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High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

ABSTRACT: BACKGROUND:Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology. MATERIALS AND METHODS:In order to explore and validate HRM as a new screening method, we analysed 26 HB patients with previous CSGE-detected mutations, 22 patients with CSGE-undetectable mutations and 13 HB patients who had not been previously investigated. RESULTS:All 61 investigated samples, including the previously investigated and not previously investigated samples, proved to be HRM-positive, with the new screening method showing good efficiency and higher sensitivity than the previously used method. Mixing normal and unknown DNA to generate heterozygote conditions proved an excellent strategy to push the detection performance to its maximum. DISCUSSION:Mutation scanning by HRM analysis seems to be ideal in our context because it is rapid, cheap and capable of detecting the vast majority of mutations in HB patients. Nevertheless, to improve the detection ability of this scanning technology, it is recommended to start with a good strategy, based on good quality samples and optimised polymerase chain reaction amplification parameters, especially regarding primers and length of the amplicons.

SUBMITTER: Salviato R

PROVIDER: S-EPMC6343595 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

Salviato Roberta R Belvini Donata D Radossi Paolo P Tagariello Giuseppe G

Blood transfusion = Trasfusione del sangue 20180228 1

<h4>Background</h4>Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology.<h4>Materials and methods</h4>In order to explore and validate HRM as a new screening method, we analyse ...[more]

PMID: 29517974

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Project description:BACKGROUND: TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. RESULTS: We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. CONCLUSION: Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Project description:BackgroundHigh resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer.MethodsThe minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing.ResultsThe limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%.ConclusionThe comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

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High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

Publications

High resolution melting for F9 gene mutation analysis in patients with haemophilia B.

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