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

Abstract

OBJECTIVE: To compare MutaGel HFE (commercial kit based on ARMSPCR)with in-house designed polymerase chain reaction-restrictionfragment length polymorphism (PCR-RFLP) techniques for the diagnosisof C282Y and H63D point mutations in HFE-gene, and to recommendthe feasibility of each method for individual laboratories according totheir requirements.METHODOLOGY: This quasi–experimental study was conducted atdepartment of forensic and biomedical sciences, university of LincolnUK from July 2006 to January 2007. Twenty-two samples were analyzedby two different molecular diagnostic techniques (ARMS & RFLP) forthe same genetic disorder (C282Y and H63D mutation in HFE-gene).Blood samples were procured from healthy volunteers from differentgeographical areas. Two controls (one positive and one negative) wererun with each batch. ARMS-PCR was carried out by using Mutagel HFEcommercial kit by immunodiagnostik whereas PCR-RFLP was performedas per UK Haemochromatosis consortium directives.RESULTS: Out of 22 samples for C282Y mutation; 20 showed normalgenotype, one was heterozygous, none was homozygous and 1 showedsample contamination. For H63D mutation, 8 showed normal genotype,one was homozygous, 12 were heterozygous while one sample showedcontamination.There was no statistically significant difference in diagnostic sensitivityand specificity of both groups. Each method had its unique performancecharacteristics that could be utilized by different laboratories dependingon their requirements.CONCLUSION: Both the techniques were equally good for diagnosis ofhaemochromatosis but RFLP semed more suitable for a laboratory withlow workload whereas MutaGel HFE seemed more appropriate for highworkload, less manpower and short turn-around time.KEYWORDS: RFLP (Restriction Fragment Length Polymorphism), PCR(Polymerase Chain Reaction), ARMS (Amplification Refractory MutationSystem), HFE gene mutation, Haemochromatosis.