Clinical Chemistry and Laboratory Medicine (CCLM)
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High resolution melting analysis to genotype the most common variants in the HFE gene
1Dipartimento di Medicina Chirurgia e Odontoiatria, Università degli Studi di Milano, Milan, Italy
2U.O. Diagnostica Molecolare Infettivologica, Azienda Ospedaliera San Paolo, Milano, Italy
Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 49, Issue 9, Pages 1453–1457, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2011.237, June 2011
- Published Online:
Background: High resolution melting (HRM) analysis of PCR amplicons was recently introduced as a closed-tube, rapid, and inexpensive method of genotyping. This study evaluated this system as an option for detecting the three most common mutations in the HFE gene (C282Y, H63D, S65C), accounting for the main form of hereditary haemochromatosis.
Methods: Ninety samples, previously screened by direct sequencing, and 27 controls were used. The analysis were performed on the Rotor Gene Q, using the commercial HRM mix containing the Eva Green dye (Qiagen). Specific primers allowed the amplification of the regions of interest in the HFE gene. Following amplification, a HRM analysis was conducted to detect DNA variants. The thermal denaturation profiles of the samples were compared with those of the controls.
Results: One hundred percent of heterozygous and homozygous samples were readily identified. Heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves, but significant differences were also present in the melting curves of the homozygous carries compared with those of the wild-type subjects.
Conclusions: HRM analysis is an appealing technology for HFE gene screening. It is a robust technique that can be widely adopted in diagnostic laboratories to facilitate gene mutation screening.
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