Label Name: HFE MUTN HEM
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB833 
Last Review:  3/16/2017 2:52:41 PM
Specimen Type
  Whole Blood (EDTA)
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 5  ML
Label Reminders
  Be sure to include the patient's name, history #, date and time of collection, and collector's initials
Collection Notes
  • Peripheral Blood: One lavender-top EDTA tube (minimum of 3 mls) is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Buccal swab (using the MDL specimen collection kit): Scrape the inside of the mouth using 10 strokes with a sterile nylon bristle cytology brush. Dip the brush up and down 10 times in the provided solution (Cell Lysis Solution) contained in the 1.5mL microfuge tube. Detailed instructions and collection kits are available on request from the Clinical Molecular Diagnostics Laboratory.
  Deliver peripheral blood and buccal swab specimens to laboratory at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.
Turn Around Time -  Routine: 10 DAYS   Stat: N/A
Reference Values


No Reference Values
  This assay uses the amplification refractory mutation system (ARMS) technique to detect p.C282Y (c.845G>A) and p.H63D (c.187C>G) mutations in the HFE gene. Genomic DNA is extracted from peripheral blood or a buccal swab and used in a polymerase chain reaction (PCR) mediated tetra-primer ARMS assay. In this assay, four oligonucleotide primers specifically amplify the wild type HFE allele, the mutant HFE allele, and a control segment of DNA in a single PCR reaction. Separate amplification reactions are performed for the p.C282Y mutation and the p.H63D mutations. The resulting PCR products are resolved by EtBr-stained capillary gel electrophoresis. The presence of an HFE mutation can be determined by the size of the PCR amplification products.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Special Information
  • Additional Patient Information Required: Additional Patient Information Required: Due to the unique nature of genetic testing, patients offered this test should receive pre-test and post-test genetic counseling. Counseling should help the patient understand the strengths and limitations of DNA testing and the medical implications for the patient as well as for other family members. Patients are also required to give consent for testing.
Clinical Significance and Interpretive Data

Hereditary hemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism. Most cases of HHC are caused by mutations in the HFE gene, located on the short arm of chromosome 6 (6p21.3). Mutations in this gene cause defects in the HFE protein, a transmembrane protein located in duodenal crypt cells. Although the exact physiologic role of HFE is still debated, it is generally recognized that HFE complexes with transferrin receptor to inhibit enterocyte iron absorption. Mutations in the HFE gene lead to dysfunctional HFE protein, causing abnormally increased iron absorption. Excess iron is stored in the liver, skin, pancreas, heart, joints, testes, and pituitary gland. If left untreated, excess iron storage can lead to serious complications such as diabetes, cirrhosis, and cardiomyopathy (reviewed in 1).

Although numerous allelic variants of HFE have been described, only two mutations have been associated with hereditary hemochromatosis; C282Y and H63D. These mutations have a high frequency in the general US population (C282Y/C282Y = 0.4%, C282Y/H63D = 2%, H63D/H63D = 2%, C282Y/wt = up to 9.2%, H63D/wt = 23%), and most patients (>90%) with hereditary hemochromatosis are homozygous for C282Y, H63D or compound heterozygotes (C282Y/H63D) (2,3).

Hereditary hemochromatosis demonstrates incomplete penetrance, meaning that some individuals fail to develop hemochromatosis even though they inherit two mutant HFE alleles. In one large study, 28.4% of C282Y homozygous men and 1.2% of C282Y homozygous women developed documented iron-overload-related disease, while only one C282Y/H63D individual developed disease. No H63D homozygotes developed disease (4). Other studies designed to estimate disease penetrance suggest variable rates, from 1% to 50% in C282Y homozygotes and from 0.3 to 1.4% in C282Y/H63D compound heterozygotes and H63D homozygotes (reviewed in 1).


HFE genotyping can be used to confirm a clinical suspicion of hereditary hemochromatosis. Furthermore, identifying a patient with hemochromatosis can facilitate appropriate monitoring of iron status in family members. This may help identify iron overload, which can be treated by simple phlebotomy, before associated symptoms develop. Due to variable penetrance of C282Y and H63D mutations, diagnosis of hemochromatosis should not be made on the basis of HFE genotyping alone. Clinical, histological, and laboratory data, must be evaluated to render an appropriate diagnosis.


1. Bellissimo D. Hematologic Disorders: Hemochromatosis, Hemoglobinopathies, and Rh Incompatibility. In: Leonard D, ed. Molecular Pathology in Clinical Practice. New York, NY: Springer Science+Business Media; 2007:151-153.

2. Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399408.

3. Hanson EH, Imperatore G, Burke W. HFE gene and hereditary hemochromatosis: a HuGE review. Human Genome Epidemiology. Am J Epidemiol. 2001;154:193-206.

4. Allen KJ, Gurrin LC, Constantine CC, et al. Iron-Overload-Related Disease in HFE Hereditary Hemochromatosis. N Engl J Med 2008;358:221-230
    Testing for hereditary hemochromatosis is indicated in patients with clinical symptoms suggestive of this disease, including; cirrhosis, arthritis, cardiac arrhythmias, hypogonadism, diabetes mellitus and progressive skin pigmentation.
Test Synonyms
  Synonym(s):  C282Y
Synonym(s):  Ferritin
Synonym(s):  H63D
Synonym(s):  Hemochromatosis
Synonym(s): HFE
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

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