Genotyping of the hemochromatosis HFE p.H63D and p.C282Y mutations by high-resolution melting with the Rotor-Gene 6000® instrument

Abstract

The genotyping of HFE p.C282Y and p.H63D mutations is one of the most requested molecular analyses in the laboratorial routine. In this scenario, the main aim was to develop a genotyping assay that has advantages compared to other methods. Genotypes for the HFE p.C282Y (c.G845A; rs1800562) and p.H63D (c.C187G, rs1799945) mutations were assessed by polymerase chain reaction (PCR) followed by high resolution melting (HRM) analysis with the Rotor-Gene 6000(®) instrument. Validation studies were conducted in samples bi-directionally sequenced. The melting assay was developed in a unique procedure and to ensure the result in approximately 112 min (31 min for sample preparation and 81 min for the PCR-HRM step). Genotypes for the HFE p.C282Y mutation were easily distinguished in the region of 80-86°C. For the HFE p.H63D, genotypes were also easily distinguished in the region of 76-82°C, but using the addition of known wild-type genotype DNA in all unknown samples plus a reaction without addition. In validation, genotypes were 100% concordant between methods. Our genotyping assay with the Rotor-Gene 6000(®) instrument applies to the laboratorial routine with several advantages, especially in large-scale demand. The main advantages were the non-dependence on gel electrophoresis and on mutagenic reagents for visualization of fragments, reduction of the chances for contamination due to sample preparation, the lack of use of probe-based methods and cost-effectiveness.