Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

Abstract

Background: Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population.

Methods: Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing.

Results: The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5-14.2%), 1.8% (95% CI 1.4-2.5%) and 3.6% (95% CI 3.0-4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions.

Conclusion: The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in the Slovenian population agree with those reported for the Central European populations although some deviations where observed in comparison with other populations of Slavic origin. Regional distribution of the mutations should be considered when planning population screening.

Figure 1

Validation of the real-time PCR assay for the 193A→T (S65C) mutation detection. (A) A multicomponent real-time amplification plots created by the SDS software. The genotypes are indicated with bold. The C_T values of individual probes for all genotypes are presented. Curves in the plots correspond to the indicated fluorophores or water (see the legend); ROX, internal reference dye; mse, mean squared error; NTC, no-template control. (B) End-point fluorescence detection is shown by the scattered diagram. Clustering of genotypes is based on the relative fluorescence from each well on 96-well plate. The expected area for 193 mutant homozygote samples clustering is indicated by red circle.

Figure 2

H63D, S65C and C282Y allele frequency distribution in 1282 blood donors among different Slovenian regions. A) Regional distribution of HFE polymorphisms. The regions, numbered from 1 to 11, are arranged from west to east, and marked on the map below. Average frequency for Slovenia is presented in the last column (total). In the region 9 H63D allele frequency is significant higher comparing to other regions (p = 0.005). (B) Geographic regions of Slovenia.