Abstract
BackgroundHereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted byiron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. p.C282Y and p.H63D diagnosis is usually made byrestriction enzyme analysis. However, the use of this technique is largelylimited to research laboratories because they are relativelyexpensive, time-consuming, and difficult to transform into a high throughput format.MethodsSingle nucleotide variations in target DNA sequences can be readily identified using molecular beacon fluorescent probes. These are quenched probes with loop and hairpin structure, and they become fluorescent upon specific target recognition. We developed high throughput homogeneous real-time PCR assays using molecular beacon technology, to genotype p.C282Y and p.H63D variants. Representative samples of different genotypes for these variants were assayed by restriction enzyme analysis and direct sequencing as bench mark methods for comparison with the newly developed molecular beacon-based real-time PCR assay.ResultsComplete concordance was achieved by all three assay formats. Homozygotes (mutant and wildtype) and heterozygotes were readily differentiated by the allele specific molecular beacons as reported by the associated fluorophore in the real-time assay developed in this study. Additionally, these assays were used in a high throughput format to establish the allele frequency of C282Y and H63D in Saudis for the first time.ConclusionThese assays may be reliably applied as a diagnostic test or large scale method for population screening.
Highlights
Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted byiron-overload
These assays may be reliably applied as a diagnostic test or large scale method for population screening
C282Y and H63D genotyping of HFE by PCR/restriction enzyme analysis Both of p.C282Y and p.H63D mutations alter restrictionenzyme sites, providing a method for genotyping. c.845G>A creates a recognition site for RsaI; c.187C>G abolishes the DNA sequence recognition site for MboI
Summary
Hereditary Hemochromatosis (HH) is an autosomal recessive disorder highlighted byiron-overload. Two popular mutations in HFE, p.C282Y and p.H63D, have been discovered and found to associate with HH in different ethnic backgrounds. A cysteine-to-tyrosine substitution at position282 in the HFE protein (p.C282Y), has (page number not for citation purposes). Large populationbased studies are required to definitively establish the prevalence of these mutations on an ethnic and or geographic basis. A recently described third mutation, which substitutes cysteine for serine at position (p.S65C) of the HFE protein, is enriched in HH individuals who have a mild form of the disease and lack the p.C282Y or p.H63D mutations [8]. Whilst many other mutations of HFE associated with the hemochromatosis phenotype have been described, they appear to be rare or "private" mutations [9]
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