COMPARISON OF MUTAGEL HFE AND POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP) FOR THE INVESTIGATION OF C282Y AND H63D POINT MUTATION IN HFE GENE

OBJECTIVE: To compare MutaGel HFE (commercial kit based on ARMS-PCR) with in-house designed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques for the diagnosis of C282Y and H63D point mutations in HFE-gene, and to recommend the feasibility of each method for individual laboratories according to their requirements.METHODOLOGY: This quasi–experimental study was conducted at department of forensic and biomedical sciences, university of Lincoln UK from July 2006 to January 2007. Twenty-two samples were analyzed by two different molecular diagnostic techniques (ARMS RFLP) for the same genetic disorder (C282Y and H63D mutation in HFE-gene).Blood samples were procured from healthy volunteers from different geographical areas. Two controls (one positive and one negative) were run with each batch. ARMS-PCR was carried out by using Mutagel HFE commercial kit by immunodiagnostik whereas PCR-RFLP was performed as per UK Haemochromatosis consortium directives.RESULTS: Out of 22 samples for C282Y mutation; 20 showed normal genotype, one was heterozygous, none was homozygous and 1 showed sample contamination. For H63D mutation, 8 showed normal genotype, one was homozygous, 12 were heterozygous while one sample showed contamination.There was no statistically significant difference in diagnostic sensitivity and specificity of both groups. Each method had its unique performance characteristics that could be utilized by different laboratories depending on their requirements.CONCLUSION: Both the techniques were equally good for diagnosis of haemochromatosis but RFLP semed more suitable for a laboratory with low workload whereas MutaGel HFE seemed more appropriate for high workload, less manpower and short turn-around time.