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February 2000 • Volume 31 • Number 2


Hepatology Elsewhere
 Iron beware: A common HFE gene polymorphism may prevent the accurate molecular diagnosis of homozygous hemochromatosis in low-risk, but not high-risk groups

Richard D. Press, M.D., Ph.D. [MEDLINE LOOKUP]

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(1) Jeffrey GP, Chakrabarti S, Hegele RA, Adams PC. Polymorphism in intron 4 of HFE may cause overestimation of C282Y homozygote prevalence in haemochromatosis. Nat Genet 1999;22:325-326.

(2) DeVilliers JN, Kotze MJ. Significance of linkage disequilibrium between mutation C282Y and a MseI polymorphism in population screening and DNA diagnosis of hemochromatosis. Blood Cells Mol Dis 1999;25:250-252. Reprinted with permission.


   Abstract  TOP 

An increasing number of studies demonstrate a lack of phenotypic expression in subjects found to be homozygous for the common hereditary hemochromatosis (HH) mutation, C282Y. In this study the impact of possible overestimation of C282Y homozygosity, as a consequence of a MseI polymorphism identified in intron 4 of the HFE gene, was investigated in South African subjects. Utilization of a modified polymerase chain reaction (PCR)-based assay highlighted the potential implications with respect to genotype/phenotype correlation studies, particularly in the general population. Mistyping rather than lack of disease association provides a plausible explanation for the phenomenon of C282Y homozygosity without iron overload. Reassessment of C282Y mutation status in such cases may result in justified population screening in HH.


   Comments  TOP 

Hereditary hemochromatosis (HH), the most common genetic disorder in Caucasians, affects approximately 1 in 200 individuals with an adult-onset multi-system chronic disease resulting from an unregulated, increased intestinal iron absorption.1 Although hemochromatosis is common and can be definitively identified with blood, tissue, and now genotype-based laboratory tests, it is significantly under-diagnosed by the medical community,2most likely because of its heterogeneous and nonspecific clinical symptoms. Unlike most other genetic diseases, a delayed or missed diagnosis of HH is particularly unfortunate given the ability of therapeutic phlebotomy to restore normal life expectancy to those diagnosed before (but not after) the onset of cirrhosis.3-5

Since the 1996 discovery of the “hemochromatosis gene,” encoding an MHC-I–homologous protein known as HFE,6 laboratories throughout the world have shown that 80% to 90% of patients with HH have a single homozygous HFE point mutation (C282Y) that destroys its normal ability to bind the transferrin receptor and down-regulate the receptor's affinity for Fe-bound transferrin.7,8 Without functional HFE, intracellular uptake of iron proceeds without negative regulation and iron overload ensues. The fact that the HFE C282Y mutation is the true mediator of the pathological iron overload in HH patients comes from additional observations that the normal population frequency of the C282Y HFE mutation (0.3%-0.5% homozygotes; 12% heterozygotes) agrees with HH prevalence estimates from phenotypic iron-based screening tests,9 and that HFE-deficient “knockout” mice develop iron overload that mimics human HH.10

The consensus first-line laboratory assay for the diagnosis of HH is the transferrin saturation (TS) test (defined as serum iron divided by TIBC), a sensitive, but nonspecific marker for hemochromatosis. In those with initially elevated TS screens (>45%), confirmatory testing requires serum ferritin levels and, ultimately, hepatic histology and iron loads to confirm hepatic iron overload. However, as 80% to 90% of HH patients carry a disease-specific homozygous HFE C282Y mutation, the direct DNA-based determination of this pathological mutation has now become a sensitive, specific, noninvasive method to confirm the diagnosis of HH in those with clinical or laboratory evidence of iron overload—and perhaps more importantly, in their presymptomatic family members. Although most C282Y homozygotes will have documented iron overload and will benefit from curative, palliative, or preventative phlebotomy therapy, a small percentage of young homozygotes (approximately 20%) may show no clinical signs of disease (despite universal elevations in TS levels).9,11

The clinical utility of direct HFE mutation tests to confirm an HH diagnosis has led to the growing availability of polymerase chain reaction (PCR)-based assays to directly detect the causative C282Y mutation—typically using the originally described Feder et al. PCR primer pair.6 The above report by Jeffrey et al.,12 which stated that 48% of the putative C282Y “homozygotes” diagnosed with a widely-used PCR-based assay did not, on closer inspection, really carry 2 mutant C282Y alleles was thus an alarming revelation to those of us making clinical decisions using diagnostic HFE genotyping tests. As shown by these investigators,12 this 48% false-positive genotype frequency was due to the presence of a very common intron 4 HFE gene polymorphism near the 3´ end of the ubiquitously-utilized reverse PCR primer.6 In those with one mutant C282Y HFE allele, the presence of this functionally silent HFE G5569A primer binding site polymorphism (on the other HFE allele) resulted in inefficient PCR amplification of the normal C282 allele, preferential amplification of the disease-associated Y282 allele, and an ultimate false-positive “C282Y homozygous” interpretive pattern. As this G5569A polymorphism is extremely common (heterozygotes representing 21% of the normal population),12 a large number of patients previously identified as C282Y “homozygotes” could, in fact, really be 5569A/282Y compound heterozygotes without substantial disease risk—a worrisome scenario for patients, clinicians, and public health officials.

Fortunately, as shown by the second above report by deVilliers et al.,13 and later confirmed by other investigators,14-17 the risks of generating a false positive “homozygous” genotype in a true clinical setting are, for a number of reasons, considerably smaller than the original Jeffrey et al. report would have initially suggested. First, in comparison with the iron overloaded patients that would be tested for HFE mutations in a clinical scenario (with a high HH disease prevalence), the group being screened by Jeffrey et al. was composed of healthy volunteer blood donors with a low pretest risk of hemochromatosis. As the C282Y and G5569A polymorphisms are never found on the same chromosome,12 the prevalence of confounding 5569A alleles (and the corresponding risk of false genotyping) would then, by definition, be much higher in a healthy population than in a 282Y-enriched clinical referral group. Second, the allele frequencies of the G5569A and C282Y polymorphisms (approximately 11% and 6%, respectively) predict a 1.3% population frequency of compound heterozygotes,12 yet Jeffrey et al. detected only 0.29% (15 of 5,211) of their normal blood donor screenees as being mistyped for C282Y (P < .0001). Most of the predicted compound heterozygotes therefore carried a 5569A allele that PCR amplified efficiently enough to avoid a false positive “homozygote” diagnosis. Third, as originally shown by Jeffrey et al.,12and later confirmed by other groups,13,15 the amplification efficiency of the polymorphic 5569A allele is highly dependent on experimental PCR primer annealing temperatures, lower temperatures favoring enhanced amplification. The relatively high 62°C annealing temperature used in the screening study by Jeffrey et al. did, in fact, result in a higher frequency of false positive genotypes than occurred in a similar genotyping assay at a lower annealing temperature.12

The report by deVilliers et al.,13 in comparison, showed that in a group of iron-overloaded subjects being clinically referred for hemochromatosis diagnostic testing, all of the 65 patients originally defined as being homozygous for the C282Y mutation (using the PCR primers noted by Feder et al.6) were true hemochromatosis homozygotes (as confirmed with nonpolymorphic HFE PCR primers). However, similar to the experience of Jeffrey et al., when the same polymorphic PCR primers from Feder et al. were used for population-based genotype screens in normal individuals, a “C282Y homozygous” diagnosis was falsely assigned to 2/283 (0.7%) of these non–iron-overloaded subjects, both of whom were C282Y/G5569A compound heterozygotes.13 This relatively low proportion of false-positive genotypes might have been even higher if not for the moderate 60°C PCR annealing temperature. Not surprisingly, when the polymorphic PCR primers from Feder et al. were annealed at 55°C, both the 5569A allele and the 282Y allele amplified with equal efficiency.13

The report by deVilliers et al. thus suggests that the common G5569A polymorphism may sometimes confound the accuracy of HFE genotype results in low-prevalence screening studies, but not in clinical evaluations of high-risk iron-overloaded subjects. Similar conclusions have been reached in 3 additional recent reports.14-16 A European consortium of 11 clinical diagnostic laboratories, for example, on re-typing the DNA samples from their hemochromatosis referral populations, failed to find any falsely-assigned HFE genotypes among the 575 patients initially defined as “C282Y homozygous” using the polymorphic PCR primers from Feder et al.14 The confounding effect of the G5569A polymorphism appears to be similarly benign in the United States, where none of the 67 clinical diagnostic laboratories blindly evaluating the same C282Y/G5569A compound heterozygous proficiency sample reported a false genotype result.15 Finally, my own laboratory has also reevaluated each of the 221 putative C282Y homozygotes referred to us for HH diagnostic testing. Of this cohort with documented iron overload (transferrin saturation = 58 ± 1.8%; serum ferritin = 940 ± 150 ng/mL), only 2 of 221 subjects (the only ones with the G5569A polymorphism) had been falsely-assigned a “C282Y homozygous” genotype, neither of whom had evidence of iron overload.16In comparison, the common G5569A polymorphism was present in 21% of our normal control individuals.

Although the presence of the very common G5569A primer binding site polymorphism thus appears to only minimally impact the accuracy of HFE genotyping results, the most prudent immediate corrective action would be the use of nonpolymorphic PCR primers for all future HFE-based hemochromatosis genotyping studies. Perhaps more importantly, however, this initially alarming (but subsequently benign) incident should serve as a critical reminder that despite the power of molecular genetic testing to define previously undiagnosable disease states, DNA-based laboratory data, like all other clinical laboratory data, must always be interpreted in the context of the complete clinical and laboratory scenario.


   References  TOP 
    1.  Press RD. Hereditary hemochromatosis: impact of molecular and iron-based testing on the diagnosis, treatment, and prevention of a common, chronic disease. Arch Pathol Lab Med 1999;123:1053-1059.
    MEDLINE

    2.  Yang Q, McDonnell SM, Khoury MJ, Cono J, Parrish RG. Hemochromatosis-associated mortality in the United States from 1979 to 1992: an analysis of Multiple-Cause Mortality Data. Ann Intern Med 1998;129:946-953.
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    3.  Niederau C, Fischer R, Sonnenberg A, Stremmel W, Trampisch HJ. Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis. N Engl J Med 1985;313:1256-1262.
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    4.  Adams PC, Speechley M, Kertesz AE. Long-term survival analysis in hereditary hemochromatosis. Gastroenterology 1991;101:368-372.
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    5.  Niederau C, Fischer R, Purschel A, Stremmel W, Haussinger D. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110:1107-1119.
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    6.  Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Domingo R, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399-408.
    MEDLINE

    7.  Lebron JA, Bennett MJ, Vaughn DE, Chirino AJ, Snow PM, Mintier GA, Bjorkman PJ. Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor. Cell 1998;93:111-123.
    MEDLINE

    8.  Feder JN, Penny DM, Irrinki A, Lee VK, Lebron JA, Watson N, Sigal E, et al. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci U S A 1998;95:1472-1477.
    CROSSREF
    MEDLINE

    9.  Olynyk JK, Cullen DJ, Aquilia S, Rossi E, Summerville L, Powell LW. A population-based study of the clinical expression of the hemochromatosis gene. N Engl J Med 1999;341:718-724.
    MEDLINE

    10.  Zhou XY, Tomatsu S, Fleming RE, Parkkila S, Waheed A, Jiang J, Fei Y, et al. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A 1998;95:2492-2497.
    CROSSREF
    MEDLINE

    11.  Crawford DH, Jazwinska EC, Cullen LM, Powell LW. Expression of HLA-linked hemochromatosis in subjects homozygous or heterozygous for the C282Y mutation. Gastroenterology 1998;114:1003-1008.
    MEDLINE

    12.  Jeffrey GP, Chakrabarti S, Hegele RA, Adams PC. Polymorphism in intron 4 of HFE may cause overestimation of C282Y homozygote prevalence in haemochromatosis. Nat Genet 1999;22:325-326.
    MEDLINE

    13.  deVilliers JNP, Kotze M. Significance of linkage disequilibrium between mutation C282Y and an MseI polymorphism in population screening and DNA diagnosis of hemochromatosis. Blood Cells Mol Dis 1999;25:250-252.
    CROSSREF
    MEDLINE

    14.  Merryweather-Clarke AT, Pointon JJ, Shearman JD, Robson KJ, Jouanolle AM, Mosser A, David V, et al. Polymorphism in intron 4 of HFE does not compromise haemochromatosis mutation results. Nat Genet 1999;23:271.
    MEDLINE

    15.  Noll WW, Belloni DR, Stenzel TT, Grody WW. Polymorphism in intron 4 of HFE does not compromise haemochromatosis mutation results. Nat Genet 1999;23:271-272.

    16.  Gomez PS, Parks S, Ries R, Tran TC, Gomez PF, Press RD. Polymorphism in intron 4 of HFE does not compromise haemochromatosis mutation results. Nat Genet 1999;23:272.
    MEDLINE

    17.  Somerville MJ, Sprysak KA, Hicks M, Elyas BG, Vicen-Wyhony L. An HFE intronic variant promotes misdiagnosis of hereditary hemochromatosis. Am J Hum Genet 1999;65:924-926.
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