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Heterozygosity for the C282Y mutation in the hemochromatosis gene is associated with increased serum iron, transferrin saturation, and hemoglobin in young women: a protective role against iron deficiency?

¹ First Department of Internal Medicine, St. Johanns Spital, Muellner-Haupstrasse 48, 5020 Salzburg, Austria.

² Institute of Biostatistics, University of Innsbruck, 6020 Innsbruck, Austria.

³ Department of Laboratory Medicine, St. Johanns Spital, 5020 Salzburg, Austria.
^a Author for correspondence. Fax 43-662-4482-881; e-mail b.paulweber{at}lkasbg.gv.at.

	Abstract

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Genetic hemochromatosis (GH) is the most common autosomal-recessive disorder (1 in 300 in populations of Celtic origin). Homozygosity for a C282Y mutation in the hemochromatosis (HFE) gene is the underlying defect in ~80% of patients with GH, and 3.2–13% of Caucasians are heterozygous for this gene alteration. Because the high frequency of this mutation may result from a selection advantage, the hypothesis was tested that the C282Y mutation confers protection against iron deficiency in young women. To address this question the genotype of codon 282 was determined in a cohort of 468 unrelated female healthcare workers, ages 18–40 years. In all study participants, a complete blood count was obtained, and erythrocyte distribution width, serum iron, transferrin, transferrin saturation, and ferritin were measured. Two individuals were homozygous for the C282Y mutation, 44 were heterozygous, and 416 were homozygous for the wild-type allele. Heterozygous women had significantly higher values for hemoglobin (P = 0.006), serum iron (P = 0.013), and transferrin saturation (P = 0.006) than women homozygous for the wild-type allele. Our data provide evidence for a protective role of the C282Y mutation in the HFE gene against iron deficiency in young women and suggest that a more efficient utilization of nutritional iron may have contributed to the high prevalence of the mutation in Caucasian populations.

	Introduction

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The high prevalence of iron deficiency and iron deficiency anemia (1)(2), especially in women of childbearing age, and its association with central nervous dysfunction (3)(4)(5), impaired work performance and response to exercise (6), impaired immune response (7), thermogenesis (8) and energy metabolism, and adverse outcome of pregnancy (9) represent a serious health problem. Worldwide, iron deficiency is the most common nutritional disorder. Women are more prone to suffer from the consequences of iron deficiency anemia because of menstrual blood loss and enhanced iron requirements during pregnancy and lactation, especially in developing countries (10).

Genetic hemochromatosis (GH), a disorder that causes iron overload, is the most common autosomal recessive disorder, affecting 1 in 300 individuals in Northern European populations (11). Recently, mutations in the hemochromatosis (HFE) gene that are responsible for GH have been identified (12). A G-to-A transition at cDNA position 845 produces a substitution of tyrosine for a highly conserved cysteine residue at position 282 of the HFE protein. Homozygosity for this mutation has been found in 67–100% of patients with GH (12)(13)(14)(15)(16)(17), and 3.2–13% of Caucasians have been found to be heterozygous for this gene alteration (12)(13)(14)(15)(16)(17). Such a high prevalence of a single mutation is unusual for a disorder with an autosomal- recessive mode of inheritance. Analysis of microsatellites at the HFE gene locus revealed that the 282 mutation occurs on chromosomes with the same haplotype in different populations (18)(19). This observation is a strong argument for a founder effect of the mutation arising on a single chromosome. The high frequency of this particular genetic defect may therefore be the result of a selection advantage for heterozygous carriers of this mutation. The metabolic consequences of the heterozygous state on iron metabolism may confer such a biological advantage. In the present study, the hypothesis was tested that the C282Y mutation in the HFE gene represents a protective factor against iron deficiency in young women.

	Materials and Methods

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patients and study design
Blood was taken after an overnight fast from 468 unrelated, nonpregnant female healthcare workers, ages 18–40 years. Information about demographic variables and factors that are known to potentially affect iron metabolism, such as age, family history of liver disease, surgery or major trauma, blood transfusions, blood donations (standard unit donation of 400 mL), iron supplementation, time from menarche, abnormal menstruation, metrorrhagia, number of pregnancies, and meat and alcohol consumption was obtained by questionnaires. In all subjects, various laboratory variables were obtained, including a complete blood count, red cell distribution width, C-reactive protein, alanine aminotransferase, serum creatinine, serum iron, transferrin, transferrin saturation, and ferritin. Individuals showing evidence of chronic inflammatory liver disease (n = 4) or signs of an acute inflammatory process as judged by increases in C-reactive protein (n = 2) were excluded from the analysis. Iron deficiency was defined as a ferritin concentration <12 µg/L and a serum transferrin saturation <15% (2). The hemoglobin cutoff for anemia was calculated as the mean of the study population minus 1.96 SD. Subjects were considered to be anemic if their hemoglobin was <11.5 g/L.

hematologic and biochemical assays
Complete blood counts were determined with a Sysmex NE-1500 automated hematology analyzer (Toa Medical Electronics, Ltd.). Serum iron was measured by the ferrozin method, and transferrin and C-reactive protein were determined by turbidimetric procedures using a Hitachi 917 analyzer (Boehringer Mannheim Diagnostics) and the respective kits (cat. nos. 1490869, 1552180, and 17300371; Boehringer Mannheim). Transferrin saturation was calculated by dividing the serum iron by the total iron-binding capacity and multiplying by 100. Serum ferritin was determined using the AxSYM microparticle enzyme immunoassay (Abbott Laboratories).

dna isolation and amplification
Genomic DNA was prepared from blood, using the Isolate 1 DNA extraction kit (Cruachem Ltd). DNA fragments were amplified by PCR, using the primers described (5'-ACATGGTTAAGGCCTGTTGC-3' and 5'-GCCACATCTGGCTTGAAATT-3' for the C187G mutation at codon 63; 5'-TGGCAAGGGTAAACAGATCC-3' and 5'-CTCAGGCACTCCTCTCAACC-3' for the G845A mutation at codon 282 of the HFE gene) (12). Amplification of both regions of the HFE gene was performed using the same PCR conditions. Each 100-µL reaction contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L each dNTP, 2.5 U of AmpliTaq DNA Polymerase (Perkin-Elmer), 200 ng of genomic DNA, and 200 ng of each oligonucleotide primer. A "hot start" PCR at 96 °C was followed by 35 cycles of denaturation at 96 °C for 30 s, annealing at 56 °C for 60 s, and extension at 72 °C for 60 s. PCR products were desalted using Ultrafree R-MC (30 000 NMWL) filter units (Millipore Corp.).

restriction fragment length analysis
Mutations were detected by restriction fragment length analysis. The G-to-A transition at nucleotide 845 creates a recognition site for the restriction enzyme SnaBI (New England Biolabs); the C-to-G transversion at nucleotide 187 abolishes the DNA sequence recognition site for endonuclease BclI (New England Biolabs). Restriction fragments were separated on 2% NuSieve^® GTG agarose and 1% agarose MP (Boehringer Mannheim) gels containing ethidium bromide and visualized by ultraviolet transillumination.

statistical analysis
Statistical analysis was carried out using the S-PLUS 3.3 or SPSS 7.5 software for Windows. Univariate comparisons of demographic and biochemical variables between the two HFE genotype groups were performed with the Student t-test and the test, respectively. Continuous variables were checked for gaussian distribution. Ferritin measures were log-transformed because their distribution was skewed. All statistical tests were two-sided. To assess the effect of HFE genotype on hemoglobin and other variables known to potentially affect hemoglobin, data for these variables were analyzed using multiple linear regression modeling.

Written informed consent was obtained from all study participants. The study was approved by the local ethics committee and was performed according to the guidelines of the Helsinki Declaration.

	Results

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The results of the demographic and genotype analyses are summarized in Table 1 . Of 462 subjects studied, two (0.4%) were found to be homozygous for the C282Y mutation. The first woman, a 19-year-old nurse, exhibited increased values for serum iron (17.4 µg/L) and transferrin saturation (55%). Her serum ferritin, hemoglobin, and red cell distribution width were within the health-related reference range. The second subject was a 40-year-old nurse with a history of hysterectomy 2 years before investigation and who showed moderate biochemical signs of iron overload as evidenced by a serum iron of 22.1 µg/L, a transferrin saturation of 90%, and a ferritin concentration of 403 µg/L. Her hemoglobin, red cell distribution width, and serum transaminases were within the health-related reference ranges.

Forty-four (9.5%) women were heterozygous for the C282Y mutation, and 416 (90.1%) were homozygous for the wild-type allele. Compared with the women heterozygous for the C282Y mutation, wild-type subjects did not display any differences in age, body mass index, and history of surgery or major trauma. No difference was observed between the heterozygous and wild-type groups in the proportion of subjects who had received blood transfusions, had a history of blood donation, or had used iron supplementation. Their histories of abnormal menstruation, number of pregnancies, and meat and alcohol consumption were similar, and no difference in time from menarche was observed. Significantly more subjects heterozygous for the C282Y mutation had a positive family history of liver disease (P <0.001; Table 1 ). As shown in Table 2 , analysis of the biochemical variables revealed significantly higher values for hemoglobin (P = 0.002), serum iron (P = 0.013), and transferrin saturation (P = 0.006) in individuals heterozygous for GH. The association between the HFE genotype and hemoglobin remained highly significant (P <0.01) in multivariate analysis, adjusting for variables such as family history of liver disease, history of surgery or major trauma, number of subjects receiving transfusions, medical iron supplementation, number of pregnancies, and alcohol consumption. We could not detect a difference in ferritin concentrations (P = 0.35) and red cell distribution width (P = 0.38) between heterozygous and wild-type homozygous individuals. Twenty-eight subjects (6.7%) homozygous for the wild-type allele presented with iron deficiency; nine of them fulfilled the criteria for iron deficiency anemia. Only two of the heterozygous individuals (4.5%) were iron deficient, and neither of them was anemic. These differences were not statistically significant. In the subgroup of women with two or more pregnancies, the difference in hemoglobin concentrations between subjects with different HFE genotypes was more pronounced than among nulliparous women. Again, this difference was not statistically significant (data not shown).

	Discussion

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The data presented support the hypothesis of a biological advantage for young female carriers of the HFE mutation. Several biochemical measures of iron status showed significant differences between women heterozygous for C282Y and those homozygous for the wild-type allele. These included serum iron and transferrin saturation, both of which were increased. Although serum ferritin values were also higher in heterozygotes, the range of ferritin concentrations was large, and differences were not significant. In a recently reported analysis of GH families, similar findings were obtained by Bulaj et al. (19) and Powell and Jazwinska (20) for a group of heterozygous females of comparable age, identified by HLA typing, whose serum iron and transferrin saturation were increased but whose ferritin concentrations were similar to those of controls. In contrast to this study, we investigated women, unrelated in reproductive age, who were most prone to develop iron deficiency. Furthermore, in our study a direct mutation detection assay was used.

Although it has been demonstrated recently that the HFE gene product complexes with the transferrin receptor and decreases the affinity of transferrin binding (21), full understanding of the observed changes in standard laboratory indices will not be possible until the function of the HFE gene product in iron metabolism is known. Nevertheless, we speculate that the C282Y mutation in the HFE gene is conducive to increased intestinal iron absorption, thereby conferring protection against iron deficiency and iron deficiency anemia.

The C282Y mutation is believed to have originated in a Celtic population. We suggest that the importance of its biological advantage decreased over time because iron deficiency is now less common because of reduced birth rates, the use of oral contraceptives, oxytocic drugs, medical iron supplementation, and improved nutrition. Notwithstanding a putative importance of the C282Y mutation in the past, its protective effect may still be relevant in present times for conditions of enhanced iron demand that may result from recurring pregnancies. Influences of the mutation on iron metabolism are supported by our study, which showed a difference in iron supplementation between the heterozygous and wild-type subjects. In addition, a history of metrorrhagia was reported by a greater percentage of heterozygous individuals, and this group also contained a higher proportion of multiparous women. These facts could have attenuated genotypic effects on some indicators reflecting iron metabolism. Interestingly, the prevalence of iron deficiency and iron deficiency anemia was considerably lower in our study population, which consisted exclusively of healthcare workers, than in other study cohorts of women with comparable age (2). Conceivably, such a sample bias could have attenuated the relevance of our results.

In conclusion, our data strongly suggest a role of the C282Y mutation in the HFE gene in preventing iron deficiency in young females. Protection against iron deficiency and its morbid consequences may have conferred a selection advantage for heterozygous carriers of the mutation in the past and may explain the high prevalence of this particular mutation, which accounts for the most frequent genetic disorder with an autosomal mode of inheritance.

	Acknowledgments

 
This work was supported by grants from the Medizinische Forschungsgesellschaft Salzburg and by the Jubilaeums-fonds Projekt No. 6041/2 of the Oesterreichische National Bank. We thank C. Talman, E. Meisl, and G. Sander for technical assistance.

	References

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