HFE C282Y RealFast™ Assay: Detecting Hereditary Hemochromatosis Risk

Introduction: Hereditary hemochromatosis (HH) is a group of genetic iron overload disorders characterized by excessive iron absorption in the body. The HFE C282Y RealFast™ Assay, developed by ViennaLab Diagnostics GmbH, is a cutting-edge real-time PCR-based test designed to accurately detect the C282Y mutation in the HFE gene, which is associated with the most common form of HH. This assay allows for the identification of individuals at risk for hereditary hemochromatosis by distinguishing between normal, heterozygous, and homozygous mutant genotypes.

Understanding Hereditary Hemochromatosis: HH is a complex disorder caused by mutations in genes involved in iron regulation, particularly the HFE gene. The HFE protein plays a crucial role in regulating iron absorption in the small intestine. Mutations, such as the C282Y mutation, disrupt this regulation, leading to excess iron accumulation in various organs, including the liver, heart, and pancreas.

Prevalence and Genetic Basis: The C282Y mutation in the HFE gene is the most prevalent genetic risk factor for HH, particularly among individuals of Caucasian descent. Approximately 80% of HH patients are homozygous for this mutation. Compound heterozygotes, who carry both C282Y and H63D mutations, are less common and typically have a milder iron absorption phenotype.

Clinical Implications: Homozygous carriers of the C282Y mutation are at the highest risk of developing iron overload-related complications, including liver cirrhosis, diabetes, and cardiomyopathy. Even heterozygous carriers may be at an increased risk of iron-related disorders when other genetic or environmental factors are present. Early detection of these mutations is essential for effective management and prevention of HH-related complications.

Principle of the Test: The HFE C282Y RealFast™ Assay employs a fluorogenic 5’ nuclease assay, known as the TaqMan® assay. Each reaction includes gene-specific primer pairs that amplify a 144 bp fragment of the HFE gene, along with two dual-labeled, allele-specific hydrolysis probes. These probes hybridize to the target sequence during PCR, preventing the fluorescent reporter from fluorescing. As PCR progresses, the Taq DNA polymerase cleaves the reporter, generating a fluorescent signal proportional to the accumulated PCR product.

In normal samples, the HEX-labeled C282Y wild type probe binds to the complementary strand, emitting a strong signal in the HEX channel and minimal signal in the FAM channel. Vice versa, in homozygous mutant samples, the FAM-labeled C282Y mutant probe binds, resulting in a strong signal in the FAM channel and minimal signal in the HEX channel. Heterozygous samples exhibit intermediate signals in both channels.

Conclusion: The HFE C282Y RealFast™ Assay is a powerful diagnostic tool for identifying individuals at risk of hereditary hemochromatosis. With its precision and speed, this assay allows for early detection and intervention, ultimately improving patient outcomes by preventing iron overload-related complications. Early screening and management are essential for individuals with genetic predispositions to HH, and this assay plays a pivotal role in achieving that goal.