Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET
C B Moysés1, E S Moreira, P F Asprino
1Instituto de Pesquisa Fleury, São Paulo, SP, Brasil. cinthia.moyses@fleury.com.br
Insights
Hereditary hemochromatosis (HH) diagnosis is improved by a new real-time PCR method. This accurate and rapid assay simultaneously detects key HFE gene mutations, enabling faster presymptomatic diagnosis and treatment.
Area of Science:
- Genetics and Molecular Biology
- Clinical Diagnostics
- Human Metabolism
Background:
- Hereditary hemochromatosis (HH) is a common autosomal iron metabolism disorder.
- Key mutations in the hemochromatosis gene (HFE), including C282Y, H63D, and S65C, are associated with HH.
- Early diagnosis and treatment of HH are crucial to prevent severe clinical complications like liver cirrhosis and cardiac failure.
Purpose of the Study:
- To develop and validate a reliable method for simultaneous detection of C282Y, H63D, and S65C mutations in the HFE gene.
- To provide an accurate, rapid, and cost-effective diagnostic assay for presymptomatic diagnosis of HH.
- To compare the performance of the new method with conventional techniques like PCR-RFLP and sequencing.
Main Methods:
- Simultaneous detection of HFE gene mutations (C282Y, H63D, S65C) using real-time PCR with fluorescence resonance energy transfer (FRET) probes.
- Utilized a quencher instead of an acceptor fluorophore for increased multiplexing capabilities.
- Compared results with conventional PCR followed by restriction digestion and agarose gel electrophoresis (PCR-RFLP) and DNA sequencing for 80 individuals.
Main Results:
- Full agreement was observed between the quenched-FRET real-time PCR method and the conventional PCR-RFLP method for genotyping 80 individuals.
- DNA sequencing confirmed the accuracy of the new real-time PCR assay.
- The real-time PCR method demonstrated significant time savings in sample processing compared to PCR-RFLP.
Conclusions:
- Real-time PCR with quenched-FRET probes is an accurate, rapid, and cost-effective method for simultaneous HFE gene mutation detection.
- This assay facilitates efficient presymptomatic diagnosis of hereditary hemochromatosis.
- The method offers advantages over PCR-RFLP, including reduced processing time, elimination of toxic reagents, lower contamination risk, and automation potential.
Abstract:
Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.
