title: Validation of an unlabeled probe melting analysis assay combined with high-throughput extractions for genotyping of the most common variants in HFE-associated hereditary hemochromatosis, C282Y, H63D, and S65C.
abstract: Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to a toxic accumulation of iron in various organs and impaired organ function. Three variants in the HFE gene (p.C282Y, p.H63D, and p.S65C) are commonly associated with the development of the disease. Of these, p.C282Y homozygotes are at the highest risk. Compound heterozygotes of p.C282Y along with p.H63D or p.S65C have reduced penetrance. Furthermore, p.H63D homozygotes are not at an increased risk and little is known about the risk associated with homozygocity for p.S65C. Our current clinical assay for the three common HFE variants utilizes the LightCycler platform and paired probes employing fluorescent resonance energy transfer. To increase throughput and decrease costs, we developed a method whereby automated extraction was combined with unlabeled probes and differential melt profiles to detect these variants using the LightCycler 480 instrument. Using this approach, 43 samples extracted with three different extraction platforms were correctly genotyped. These data demonstrate that the newly developed assay to genotype the HFE mutations p.C282Y, p.H63D, and p.S65C, combined with high-throughput extraction platforms, is accurate and reproducible and represents an alternative to previously described tests.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology® , Salt Lake City, Utah 84108-1221, USA. kelli.sumner@aruplab.com
authors.forename: Kelli
authors.initials: K
authors.lastname: Sumner
authors.forename: Lindsey
authors.initials: L
authors.lastname: Hubley
authors.forename: Genevieve
authors.initials: G
authors.lastname: Pont-Kingdon
authors.forename: Sheri
authors.initials: S
authors.lastname: Mitchell
authors.forename: Tyler
authors.initials: T
authors.lastname: Wayman
authors.forename: Andrew
authors.initials: A
authors.lastname: Wilson
authors.forename: Cindy
authors.initials: C
authors.lastname: Meadows
authors.forename: Kerry
authors.initials: K
authors.lastname: Elenitoba-Johnson
authors.forename: David
authors.initials: D
authors.lastname: Pattison
authors.forename: Steven
authors.initials: S
authors.lastname: Dobrowolski
authors.forename: Hunter
authors.initials: H
authors.lastname: Best
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
mesh_terms: D019943:Amino Acid Substitution; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006432:Hemochromatosis / Q000175:diagnosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D006579:Heterozygote; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006720:Homozygote; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D020125:Mutation, Missense
chemical_list: D015342:DNA Probes; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins