Heteroduplex analysis for the three common HFE variants: methodology, reliability and analysis of over 5000 requests for testing.

Objective: To describe the analysis of over 5300 patient samples for the HFE genotype.

Methods: Blood samples received from hospitals in England, Wales and Ireland were analysed with a single, multiplex PCR using heteroduplex generators for the C282Y, H63D and S65C variants of the HFE gene. PCR products labelled with fluorescent dyes were analysed by capillary electrophoresis. Genotype frequencies were analysed according to the reasons given for testing.

Results: Analysis of 400 samples sent in duplicate revealed one error that was associated with reporting rather than the methodology. Of 5327 samples received, 1122 were for family testing, 2470 for diagnostic testing and in 1735 cases no reason was given. Overall, homozygosity for C282Y was found in 14% of samples received for family testing and in 16% of the remaining samples. Clinical indications such as "liver disease" were of little predictive value for homozygosity for C282Y, but this increased if a raised serum ferritin concentration or transferrin saturation was indicated. When the diagnosis was iron overload, 39% of subjects tested were homozygous for C282Y. Compound heterozygosity (C282Y/H63D) was more frequent than in the general population but the frequency was not further increased in subjects for whom there was a diagnosis of iron overload. The frequencies of heterozygosity for H63D or S65C and homozygosity for H63D were not significantly increased in any group compared with the general population frequency.

Conclusion: These results demonstrate the reliability of the methodology and confirm the difficulty of identifying genetic haemochromatosis purely on the basis of clinical suspicion that haemochromatosis may be responsible for liver disease, diabetes or arthritis.