MOLECULAR DIAGNOSTICS AT YNHH: HEREDITARY HEMOCHROMATOSIS

Patients with hemochromatosis are often identified through follow-up of abnormally high serum iron levels and by investigation of asymptomatic relatives of affected individuals. Transferrin saturation (100 x serum iron / TIBC, normal range 16-50%) is a good screening test for HHC since transferrin saturation is >60% in 90% of homozygous HHC patients. Serum ferritin is similarly somewhat useful, with 71% of homozygous HHC patients having increased levels. Although these laboratory values are elevated in a variety of other diseases, including alcoholic or viral hepatitis or hepatic malignancy, the combination of transferrin saturation and serum ferritin has a sensitivity of 94% and a specificity of 97% for diagnosing HHC. If these values are confirmed to be elevated, HHC should be strongly considered. Secondary causes of iron overload, including generalized liver disease, excess ingestion of oral iron supplements, and multiple red cell transfusions associated with thalassemia or sickle cell anemia should also be considered. Traditionally, the diagnosis of HHC has been confirmed by liver biopsy and the demonstration of parenchymal iron deposits, as well as the quantitation of increased hepatic iron content.

Recent linkage studies of HHC patients have identified a candidate disease gene termed HLA-H. The HLA-H gene product is related to the major histocompatibility complex (MHC) class I family and is believed to play a role in iron metabolism. Among 178 HHC patients, 83% were found to be homozygous for a G to A nucleotide change which results in a cysteine to tyrosine substitution at amino acid position 282 in the HLA-H protein (C282Y); all individuals with homozygosity for C282Y had clinical evidence for iron overload. The C282Y mutation is predicted to inactivate the HLA-H protein by elimination of a critical disulfide bridge present in MHC class I molecules. A second HLA-H mutation at nucleotide 187 (C to G switch), resulting in a histidine to aspartic acid substitution at amino acid 63 (H63D), was also described in these studies, although the association of H63D with HHC is much less significant than C282Y. Homozygous H63D was found at a three-fold higher frequency in HHC, while individuals doubly heterozygous for both H63D and C282Y had a five-fold higher risk.

The YNHH molecular diagnostics laboratory now tests for the presence of both the C282Y and H63D mutations as an aid in the diagnosis of hereditary hemochromatosis. The test uses a polymerase chain reaction (PCR) to amplify the DNA regions encoding for these amino acids. For the former, the G to A nucleotide change results in the creation of a new Rsa I restriction enzyme site; thus, the mutation can be identified by the appearance of a unique DNA fragment after Rsa I digestion of the PCR product and agarose gel electrophoresis. For the latter, the C to G nucleotide change abolishes an Mbo I restriction enzyme site, and the mutation is detected by the absence of digestion fragments. Patient samples are first tested for homozygosity for the C282Y mutation; if the patient is homozygous for C282Y, no further testing is required. Samples which do not have or are heterozygous for the C282Y mutation, are then tested for the H63D mutation, which appears to have an increased presence in hereditary hemochromatosis patients.

The genetic test for hereditary hemochromatosis requires one tube (lavender top) of EDTA anticoagulated blood which should be kept at room temperature and sent to the Immunology Laboratory at YNHH between Monday and Friday. Questions can be referred to the Immunology Laboratory at 785-2440.

References

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2. Little, D.R. Hemochromatosis: diagnosis and management. Am Fam Physician 53(8):2623-2632, 1996.
3. Conrad, M.E., Umbreit, J.N., Moore, E.G., and Parmley, R.T. Hereditary hemochromatosis: a prevalent disorder of iron metabolism with an elusive etiology. Am J Hematol 47(3):218-224, 1994.
4. Edwards, C.Q. and Kushner, J.P. Screening for hemochromatosis. N Engl J Med 328(22):1616-1620, 1993.
5. Barton, J.C. and Bertoli, L.F. Hemochromatosis: the genetic disorder of the twenty-first century. Nature Medicine 2(4):394-395, 1996.
6. Feder, J.N., Gnirke, A., Thomas, W. et. al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis Nat Genet 13(4):399-408, 1996.

Michael E. Ripps M.D., Ph.D.
John Greg Howe Ph.D.