HFE H63D PCR Assay
The Viennalab HFE H63D RealFast Assay detects the H63D variant in the HFE gene by real-time PCR and discriminates the three possible genotypes in a human genomic DNA extract: HH (normal), HD (heterozygous) and DD (homozygous mutant). Variants in HFE are the cause of the most common form of hereditary haemochromatosis, an iron overload disorder in which iron accumulates in the liver, heart and pancreas. Homozygous carriers of H63D show only a slightly raised iron absorption and rarely develop the disorder; the variant becomes clinically relevant in combination with C282Y or with other risk factors.
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This kit in detail
- Detects HFE H63D (g.8671C>G, rs1799945)
- Discriminates the HH, HD and DD genotypes
- Real-time PCR with allele-specific hydrolysis probes, 120 bp amplicon
- WT-Control and MUT-Control supplied
- Whole peripheral blood, dried blood spots, buccal swabs or saliva
- 32 or 100 reactions per kit, REF 7-143 and REF 7-140
The Viennalab HFE H63D RealFast Assay determines the genotype of the H63D variant in the HFE gene, one of the two variants that account for most cases of hereditary haemochromatosis.
Clinical background
Hereditary haemochromatosis is a group of inherited iron overload disorders in which iron is absorbed in excess and deposited in the liver, heart and pancreas, with liver cirrhosis, diabetes, cardiomyopathy and joint complaints as consequences. HFE encodes an atypical MHC class I molecule involved in the regulation of iron absorption. Most patients are homozygous for C282Y; a smaller group is compound heterozygous for C282Y and H63D.
Interpretation
Homozygous carriers of H63D show only a slightly raised iron absorption and rarely develop haemochromatosis. The variant carries weight in combination with C282Y, or alongside other factors such as obesity, hypertension, type 2 diabetes, smoking or oral contraceptive use. The assay reports HH, HD or DD.
Method
The assay is a real-time PCR based on the 5′ nuclease principle. A 120 bp fragment of the HFE gene is amplified and read with two allele-specific hydrolysis probes: HEX for the wild-type allele and FAM for the H63D allele. The kit supplies the RealFast 2x Genotyping Mix, the HFE H63D Assay Mix, a WT-Control and a MUT-Control; a heterozygous control is prepared by mixing the two 1:1. The recommended DNA input is 10 to 100 ng per reaction, at a concentration of 2 to 20 ng/µl, and the limit of detection is 0.2 ng of genomic DNA per reaction. DNA isolated from whole peripheral blood, dried blood spots, buccal swabs or saliva can be used, and DNA extraction reagents are not supplied with the kit.
- RealFast™ 2x Genotyping Mix: Includes HotStart Taq DNA polymerase and dNTPs in an optimized buffer system for PCR amplification.
- HFE H63D Assay Mix: Contains gene-specific primers and two allele-specific, dual-labeled hydrolysis probes for genotype discrimination.
- HFE H63D WT-Control: This control represents the wild-type (normal) genotype for validation purposes.
- HFE H63D MUT-Control: Included control for the homozygous mutant genotype to ensure accurate test results.
Datasheets and regulatory documentation
Everything your quality team needs for evaluation and procurement.
Instructions for Use
Frequently paired with this kit
Other assays and instruments lab teams add alongside the HFE H63D PCR Assay.
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