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Biochemical expression of heterozygous hereditary hemochromatosis
European Journal of Internal Medicine 11 (2000) 317–321 www.elsevier.com / locate / ejim
Original article
Biochemical expression of heterozygous hereditary hemochromatosis a a b a, B. de Valk , R.S.G.M. Witlox , Y.T. van der Schouw , J.J.M. Marx * a
Department of Internal Medicine and Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands b Julius Center for Patient Oriented Research, University Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands Received 25 October 1999; received in revised form 25 April 2000; accepted 2 May 2000
Abstract Background: Hereditary hemochromatosis (HH) is a common autosomal recessive disease caused by an iron overload. Two mutations (C282Y and H63D) on the responsible HFE gene have been described. HH heterozygotes may have a slight iron overload that does not cause clinical disease. Compound heterozygosity may be associated with higher iron stores than C282Y heterozygosity. We studied biochemical iron parameters in HH C282Y and compound heterozygotes without a clinically significant iron overload. Methods: Data on hemoglobin, hematocrit, mean corpuscular volume, serum ferritin, serum iron, transferrin, and transferrin saturation were obtained from 40 C282 wild type controls (irrespective of H63D genotype), 61 C282Y heterozygotes, and 18 compound (C282Y/ H63D) heterozygotes without clinical iron overload disease. Results: Serum ferritin levels were significantly higher in female HH heterozygotes, particularly in compound heterozygotes, than in normal women. In male heterozygotes, no difference in serum ferritin was found. We found higher mean serum iron and transferrin saturation levels in male and female HH heterozygotes than in normal controls, the highest in the group of compound heterozygotes. Conclusions: Mean serum ferritin (only in women), serum iron, and transferrin saturation are highest in compound heterozygotes and lowest in controls. C282Y heterozygotes seem to be an intermediate group between compound heterozygotes and the normal population. 2000 Elsevier Science B.V. All rights reserved. Keywords: Iron; Ferritin; Transferrin saturation; HFE; Heterozygotes
1. Introduction Hereditary hemochromatosis (HH) is a common autosomal recessive disease caused by an iron overload. It is caused by inappropriately high iron absorption from the gut that leads to iron deposition in various organs, resulting in impaired function of these organs, e.g. liver cirrhosis, diabetes mellitus, or cardiac failure [1–3]. Diagnosis is made by laboratory investigations (a high serum ferritin and transferrin saturation) and by liver biopsy, which shows an elevated hepatic iron concentration, and hemosiderin deposits characteristically appearing first in the hepatocytes in the periportal area [1–3]. The gene responsible for HH–HFE – situated on the short arm of *Corresponding author. Tel.: 131-30-2509111; fax: 131-30-2518328. E-mail address: [email protected] (J.J.M. Marx).
chromosome 6, was recently discovered [4]. Most but not all patients with HH are homozygous for a mutation of this gene, resulting in a cysteine instead of a tyrosine at position 282 (C282Y) [4–6]. The significance of another mutation found in the HFE gene, the substitution of a histidine for aspartic acid at position 63 (H63D), is unclear [4,7]. After genotyping of HH patients became available, some patients with clinical symptoms of HH appeared to be heterozygous for the C282Y mutation; they were identified as compound heterozygotes (i.e. C282Y/ H63D heterozygotes) or were even missing both HFE mutations, indicating a different molecular defect [7–10]. Results from earlier studies in which HH heterozygotes were identified by HLA typing may be unreliable as iron overload could have been caused by non-HFE-linked disease modalities [11–14]. It has been suggested that the heterozygosity of the C282Y mutation protects young
0953-6205 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0953-6205( 00 )00111-4
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women from iron deficiency [15]. Compound heterozygosity is probably associated with higher iron stores than C282Y heterozygosity [7–10,16]. Recently, heterozygous HH has been identified as an independent risk factor for cardiovascular mortality and myocardial infarction [17,18]. Long-term exposure to small amounts of iron may play a role in atherogenesis and ischemia / reperfusion damage by catalyzing free radical formation [19], so even minimally increased iron stores in heterozygous HH could be detrimental and might explain the increased cardiovascular risk in heterozygous HH. We studied conventional biochemical iron parameters in Dutch HH C282Y and compound heterozygotes without a clinically significant iron overload who would need phlebotomy treatment and we compared them with the iron parameters of a control group containing subjects with wild type HFE (H63D heterozygotes and H63D homozygotes) also without a clinically significant iron overload.
2. Methods
2.1. Study subjects Data on genotypically HH heterozygotes, identified by family screening for hemochromatosis (which we perform when a homozygous patient is identified) in the period from 1996 to 1998, were obtained from the Utrecht Medical Center Hereditary Hemochromatosis database. Seventy-nine HH heterozygotes were eligible for this study; 61 of them were C282Y heterozygotes and H63 wild type and 18 were compound heterozygotes. As a control group, we took the 40 family members of HH homozygotes, identified as C282 wild type, irrespective of their H63D genotype. Subjects with chronic inflammatory diseases, infectious diseases, increased ESR, or liver diseases were excluded because those conditions influence the estimations of serum ferritin, serum iron, and transferrin saturation [20]. Subjects who were referred to our hospital for iron overload and who were diagnosed as homozygous hemochromatosis patients but who later turned out to be HFE C282Y or compound heterozygotes were also excluded from this analysis since they represent
a group of subjects who are clinically homozygous (probably because another, still unknown, genetic factor is involved) and would incorrectly influence the mean values of iron parameters of asymptomatic HH heterozygotes.
2.2. Laboratory measurements Hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), serum ferritin, serum iron, serum transferrin, and transferrin saturation were measured using routine laboratory methods. HFE genotyping for the C282Y mutation as well as for the H63D mutation was performed as previously described [21].
2.3. Statistics Serum ferritin was adjusted for age because of its known age dependency [22]. Differences between genotypes were tested with a one-way ANOVA. All statistical analyses were performed using SPSS 8.0 for Windows. Although the size of our study was not particularly large, the power to detect differences of the magnitudes reported in our study varied between 81% and 93% for serum iron levels and transferrin saturation, and was 68% for female serum ferritin levels.
3. Results Data were obtained from 40 controls (21 women and 19 men), 61 C282Y heterozygotes (41 women and 20 men), and 18 compound heterozygotes (11 women and 7 men). All tests were performed on all subjects. The results are shown in Tables 1 and 2.
3.1. Hemoglobin, hematocrit, and MCV There was no difference in the mean values for Hb, Ht, or MCV between controls, C282Y heterozygotes, and compound heterozygotes, either in men or in women.
Table 1 Mean (6S.D.) of laboratory values in female C282Y heterozygotes, compound (C282Y/ H63D) heterozygotes and controls
n Hb (mmol / l) Ht MCV (fl) Serum ferritin (m-g / l)a Serum iron (mmol / l) Transferrin (g / l) Transferrin saturation a
Means adjusted for age.
Controls
C282Y
C282Y/ H63D
P-value
21 8.660.6 0.4160.03 8963 70650 15.964.5 2.9860.43 0.2360.08
41 8.560.5 0.4060.02 9064 76652 18.566.9 2.8660.53 0.2960.12
11 8.460.7 0.3960.04 9164 1396123 21.666.5 2.7260.56 0.3560.12
0.339 0.166 0.732 0.015 0.052 0.379 0.016
B. de Valk et al. / European Journal of Internal Medicine 11 (2000) 317 – 321
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Table 2 Mean (6S.D.) of laboratory values in male C282Y heterozygotes, compound (C282Y/ H63D) heterozygotes and controls
n Hb (mmol / l) Ht MCV (fl) Serum ferritin (mg / l)a Serum iron (mmol / l) Transferrin (g / l) Transferrin saturation a
Controls
C282Y
C282Y/ H63D
P-value
19 9.660.5 0.4560.04 8963 171673 19.065.6 2.8660.39 0.3060.09
20 9.560.6 0.4460.03 9065 1626118 19.166.7 2.7060.37 0.3260.15
7 9.660.4 0.4460.02 8865 185690 25.765.7 2.7160.43 0.4160.09
0.800 0.416 0.473 0.858 0.038 0.407 0.090
Means adjusted for age.
3.2. Serum ferritin
4. Discussion
In women, mean serum ferritin was twice as high in the group of compound heterozygotes (1396123 mg / l) as it was in the other two groups (controls 70650 mg / l, C282Y heterozygotes 76652 mg / l, P ANOVA50.015). Ferritin in the C282Y group was slightly, but not significantly, higher than in the control group. In men, there was no difference in serum ferritin between the three groups (P ANOVA5 0.858).
The prevalence of heterozygous hereditary hemochromatosis is estimated to be about 6–14% [23–26]. While some HH heterozygotes develop a clinically significant iron overload, suggesting that another genetic factor may be present in these cases [7–10,16], most heterozygotes have no clinical symptoms of the disease [11–14,27]. In a large study among 1058 HH heterozygotes identified by HLA-typing, mean serum ferritin and iron concentrations and mean transferrin saturation were higher in HH heterozygotes than in 321 normal controls, but liver biopsy abnormalities were only seen in association with other conditions, such as alcohol abuse, hepatitis, or porphyria cutanea tarda [11]. In another study of 255 HH heterozygotes also identified by HLA-typing, 11% of the heterozygotes had an elevated serum ferritin and 8.6% had elevated transferrin saturation values [14]. In both studies [11,14], the mean levels of serum ferritin, serum iron, and transferrin saturation were in the normal range. In a study by Valberg and coworkers [13], a higher serum iron and transferrin saturation and a lower serum transferrin were found in male and female HH heterozygotes than in normal controls. Mean serum ferritin was increased in females but not in males. Cartwright and associates have also reported increased mean serum iron and transferrin saturation, as well as a limited increase in the amount of liver iron, in HH heterozygotes without clinical manifestations [12]. In contrast to our study, these previously mentioned investigations were performed in HH heterozygotes identified by HLA-typing [11–14] and obviously cannot distinguish between C282Y heterozygotes and compound heterozygotes. In a recent French study [16], C282Y heterozygotes without an iron overload were not significantly different from controls with regard to serum ferritin, liver iron concentration, or hepatic iron index. Compound heterozygotes had a slight increase in transferrin saturation compared with C282Y heterozygotes and controls. In our study, we investigated the data of genotyped HH heterozygotes and controls who were examined at the outpatient clinic of the Utrecht University Hospital from 1996 to 1998 because a family member had homozygous symptomatic HH. Despite the absence of a
3.3. Serum iron In women, a trend was observed for increasing serum iron in C282Y heterozygotes and compound heterozygotes, with compound heterozygotes having higher serum iron values than C282Y heterozygotes, and C282Y heterozygotes having higher serum iron values than control subjects (21.666.5 mmol / l, 18.566.9 mmol / l and 15.964.5 mmol / l, respectively, P ANOVA50.052). In men, compound heterozygotes had higher serum iron values than C282Y heterozygotes and controls (25.765.7 mmol / l, 19.166.7 mmol / l, and 19.065.6 mmol / l, respectively, P ANOVA50.038). No difference was observed in the serum iron levels of C282Y heterozygotes and controls.
3.4. Transferrin and transferrin saturation Transferrin levels were decreased in compound heterozygotes and in C282Y heterozygotes compared with controls, but this was far from significant (for women 2.7260.56 g / l, 2.8660.53 g / l, and 2.9860.43 g / l, respectively, P ANOVA50.379 and for men 2.7160.43 g / l, 2.7060.37 g / l, and 2.8660.39 g / l, respectively, P ANOVA50.407). Transferrin saturation, however, was the highest in both female and male compound heterozygotes compared to the other two groups. Again, C282Y heterozygotes seem to be intermediate between compound heterozygotes and controls in women but not in men (for women 0.3560.12, 0.2960.12, and 0.2360.08 respectively, P ANOVA50.016; for men 0.4160.09, 0.3260.15, and 0.3060.09 respectively, P ANOVA50.09).
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B. de Valk et al. / European Journal of Internal Medicine 11 (2000) 317 – 321
significant iron overload (which was an exclusion criterion since we wanted to study the iron parameters in asymptomatic HH heterozygotes identified by family screening), serum ferritin levels were significantly higher in female HH compound heterozygotes than in HH C282Y heterozygotes and normal controls. In male HH heterozygotes, no difference in serum ferritin was found. We found a higher mean serum iron level and a trend towards higher transferrin saturation levels in male compound HH heterozygotes than in normal controls, and a borderline higher mean serum iron level and higher transferrin saturation levels in female compound HH heterozygotes than in normal controls. In women, a trend towards increasing serum iron and transferrin saturation was observed in C282Y heterozygotes and compound heterozygotes, with compound heterozygotes having higher serum iron and transferrin saturation levels than C282Y heterozygotes, and C282Y heterozygotes having higher serum iron and transferrin saturation values than control subjects. In men, no such trend was observed. However, the number of male heterozygotes was less than the number of female heterozygotes. While serum ferritin and transferrin saturation in women, and serum iron in men, were significantly different between the three genotypes, differences of serum iron in women and transferrin saturation in men lack statistical significance. However, there is a statistical trend for increased serum iron in women (P ANOVA50.052) and for transferrin saturation in men (P ANOVA50.090), both the highest in compound heterozygotes, intermediate in C282Y heterozygotes and the lowest in normal controls. A larger study is necessary to confirm our results. However, our findings are in concordance with the previously mentioned studies, in which larger numbers of subjects are described [11–14,16]. Thus, as shown in Tables 1 and 2, our results suggest that iron stores, assessed by serum ferritin (only in women), serum iron and transferrin saturation, are higher in HH heterozygotes than in control subjects. The iron stores probably are the highest in compound heterozygotes and the lowest in the control group, C282Y heterozygous hemochromatosis being an intermediate group between compound heterozygosity and the normal population. Despite being in the normal range (see Tables 1 and 2), these higher iron stores could probably explain the excess cardiovascular risk in HH heterozygotes [17,18], because catalyzing lipid peroxidation and increasing the magnitude of reperfusion damage only needs little amounts of iron [19].
Acknowledgements Financial support was received by a grant from the University Hospital Utrecht.
References [1] Nichols GM, Bacon BR. Hereditary hemochromatosis: pathogenesis and clinical features of a common disease. Am J Gastroenterol 1989;84:851–62. [2] Bothwell TH, MacPhail AP. Hereditary hemochromatosis: etiologic, pathologic, and clinical aspects. Semin Hematol 1998;35:55–71. [3] Andrews NC, Levy JE. Iron is hot: an update on the pathophysiology of hemochromatosis. Blood 1998;92:1845–51. [4] 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;13:399–408. [5] Jazwinska EC, Cullen LM, Busfield F et al. Haemochromatosis and HLAH. Nat Genet 1996;14:249–51. [6] Jouanolle AM, Gandon G, Jezequel P et al. Haemochromatosis and HLAH. Nat Genet 1996;14:251–2. [7] Burke W, Press N, McDonnell SM. Hemochromatosis: genetics helps to define a multifactorial disease. Clin Genet 1998;54:19. [8] Sham RL, Ou CY, Cappuccio J, Braggins C, Dunnigan K, Phatak PD. Correlation between genotype and phenotype in hereditary hemochromatosis: analysis of 61 cases. Blood Cells Mol Dis 1997;23:314–20. [9] Aguilar Martinez P, Biron C, Blanc F et al. Compound heterozygotes for hemochromatosis gene mutations: may they help to understand the pathophysiology of the disease? Blood Cells Mol Dis 1997;23:269–76. [10] 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– 8. [11] Bulaj ZJ, Griffen LM, Jorde LB, Edwards CQ, Kushner JP. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. New Engl J Med 1996;335:1799–805. [12] Cartwright GE, Edwards CQ, Kravitz K et al. Hereditary hemochromatosis. Phenotypic expression of the disease. New Engl J Med 1979;301:175–9. [13] Valberg LS, Lloyd DA, Ghent CN et al. Clinical and biochemical expression of the genetic abnormality in idiopathic hemochromatosis. Gastroenterology 1980;79:884–92. [14] Adams PC. Prevalence of abnormal iron studies in heterozygotes for hereditary hemochromatosis: an analysis of 255 heterozygotes. Am J Hematol 1994;45:146–9. [15] Datz C, Haas T, Rinner H, Sandhofer F, Patsch W, Paulweber B. 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? Clin Chem 1998;44:2429–32. [16] Moirand R, Jouanolle AM, Brissot P, Le Gall JY, David V, Deugnier Y. Phenotypic expression of HFE mutations: A french study of 1110 unrelated ironoverloaded patients and relatives. Gastroenterology 1999;116:372–7. [17] Roest M, van der Schouw YT, de Valk B et al. Heterozygosity for a hereditary hemochromatosis gene is associated with cardiovascular death in women. Circulation 1999;100:1268–73. [18] Tuomainen TP, Kontula K, Nyyssonen K et al. Hemochromatosis gene HFE polymorphism is associated with twofold increased risk at acute myocardial infarction in men. Circulation 1999;100:1274–9. [19] de Valk B, Marx JJM. Iron, atherosclerosis and ischemic heart disease. Arch Intern Med 1999;159:1542–8. [20] Worwood M. Laboratory determination of iron status. In: Brock JH, Halliday JW, Pippard MJ, Powell LW, editors, Iron metabolism in health and disease, London: W.B. Saunders, 1994, pp. 449–76. [21] Santos M, Clevers H, Marx JJM. Mutations of the hereditary hemochromatosis candidate gene HLAH in porphyria cutanea tarda. New Engl J Med 1997;336:1327–8.
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[25] Jouanolle AM, Fergelot P, Raoul ML et al. Prevalence of the C282Y mutation in Brittany: penetrance of genetic hemochromatosis? Ann Genet 1998;41:195–8. [26] Ryan E, O’Keane C, Crowe J. Hemochromatosis in Ireland and HFE. Blood Cells Mol Dis 1998;24:428–32. [27] Powell LW, Jazwinska EC. Hemochromatosis in heterozygotes. New Engl J Med 1996;335:1837–9.
Original article
Biochemical expression of heterozygous hereditary hemochromatosis a a b a, B. de Valk , R.S.G.M. Witlox , Y.T. van der Schouw , J.J.M. Marx * a
Department of Internal Medicine and Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands b Julius Center for Patient Oriented Research, University Medical Center, P.O. Box 85500, 3508 GA Utrecht, The Netherlands Received 25 October 1999; received in revised form 25 April 2000; accepted 2 May 2000
Abstract Background: Hereditary hemochromatosis (HH) is a common autosomal recessive disease caused by an iron overload. Two mutations (C282Y and H63D) on the responsible HFE gene have been described. HH heterozygotes may have a slight iron overload that does not cause clinical disease. Compound heterozygosity may be associated with higher iron stores than C282Y heterozygosity. We studied biochemical iron parameters in HH C282Y and compound heterozygotes without a clinically significant iron overload. Methods: Data on hemoglobin, hematocrit, mean corpuscular volume, serum ferritin, serum iron, transferrin, and transferrin saturation were obtained from 40 C282 wild type controls (irrespective of H63D genotype), 61 C282Y heterozygotes, and 18 compound (C282Y/ H63D) heterozygotes without clinical iron overload disease. Results: Serum ferritin levels were significantly higher in female HH heterozygotes, particularly in compound heterozygotes, than in normal women. In male heterozygotes, no difference in serum ferritin was found. We found higher mean serum iron and transferrin saturation levels in male and female HH heterozygotes than in normal controls, the highest in the group of compound heterozygotes. Conclusions: Mean serum ferritin (only in women), serum iron, and transferrin saturation are highest in compound heterozygotes and lowest in controls. C282Y heterozygotes seem to be an intermediate group between compound heterozygotes and the normal population. 2000 Elsevier Science B.V. All rights reserved. Keywords: Iron; Ferritin; Transferrin saturation; HFE; Heterozygotes
1. Introduction Hereditary hemochromatosis (HH) is a common autosomal recessive disease caused by an iron overload. It is caused by inappropriately high iron absorption from the gut that leads to iron deposition in various organs, resulting in impaired function of these organs, e.g. liver cirrhosis, diabetes mellitus, or cardiac failure [1–3]. Diagnosis is made by laboratory investigations (a high serum ferritin and transferrin saturation) and by liver biopsy, which shows an elevated hepatic iron concentration, and hemosiderin deposits characteristically appearing first in the hepatocytes in the periportal area [1–3]. The gene responsible for HH–HFE – situated on the short arm of *Corresponding author. Tel.: 131-30-2509111; fax: 131-30-2518328. E-mail address: [email protected] (J.J.M. Marx).
chromosome 6, was recently discovered [4]. Most but not all patients with HH are homozygous for a mutation of this gene, resulting in a cysteine instead of a tyrosine at position 282 (C282Y) [4–6]. The significance of another mutation found in the HFE gene, the substitution of a histidine for aspartic acid at position 63 (H63D), is unclear [4,7]. After genotyping of HH patients became available, some patients with clinical symptoms of HH appeared to be heterozygous for the C282Y mutation; they were identified as compound heterozygotes (i.e. C282Y/ H63D heterozygotes) or were even missing both HFE mutations, indicating a different molecular defect [7–10]. Results from earlier studies in which HH heterozygotes were identified by HLA typing may be unreliable as iron overload could have been caused by non-HFE-linked disease modalities [11–14]. It has been suggested that the heterozygosity of the C282Y mutation protects young
0953-6205 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0953-6205( 00 )00111-4
B. de Valk et al. / European Journal of Internal Medicine 11 (2000) 317 – 321
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women from iron deficiency [15]. Compound heterozygosity is probably associated with higher iron stores than C282Y heterozygosity [7–10,16]. Recently, heterozygous HH has been identified as an independent risk factor for cardiovascular mortality and myocardial infarction [17,18]. Long-term exposure to small amounts of iron may play a role in atherogenesis and ischemia / reperfusion damage by catalyzing free radical formation [19], so even minimally increased iron stores in heterozygous HH could be detrimental and might explain the increased cardiovascular risk in heterozygous HH. We studied conventional biochemical iron parameters in Dutch HH C282Y and compound heterozygotes without a clinically significant iron overload who would need phlebotomy treatment and we compared them with the iron parameters of a control group containing subjects with wild type HFE (H63D heterozygotes and H63D homozygotes) also without a clinically significant iron overload.
2. Methods
2.1. Study subjects Data on genotypically HH heterozygotes, identified by family screening for hemochromatosis (which we perform when a homozygous patient is identified) in the period from 1996 to 1998, were obtained from the Utrecht Medical Center Hereditary Hemochromatosis database. Seventy-nine HH heterozygotes were eligible for this study; 61 of them were C282Y heterozygotes and H63 wild type and 18 were compound heterozygotes. As a control group, we took the 40 family members of HH homozygotes, identified as C282 wild type, irrespective of their H63D genotype. Subjects with chronic inflammatory diseases, infectious diseases, increased ESR, or liver diseases were excluded because those conditions influence the estimations of serum ferritin, serum iron, and transferrin saturation [20]. Subjects who were referred to our hospital for iron overload and who were diagnosed as homozygous hemochromatosis patients but who later turned out to be HFE C282Y or compound heterozygotes were also excluded from this analysis since they represent
a group of subjects who are clinically homozygous (probably because another, still unknown, genetic factor is involved) and would incorrectly influence the mean values of iron parameters of asymptomatic HH heterozygotes.
2.2. Laboratory measurements Hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), serum ferritin, serum iron, serum transferrin, and transferrin saturation were measured using routine laboratory methods. HFE genotyping for the C282Y mutation as well as for the H63D mutation was performed as previously described [21].
2.3. Statistics Serum ferritin was adjusted for age because of its known age dependency [22]. Differences between genotypes were tested with a one-way ANOVA. All statistical analyses were performed using SPSS 8.0 for Windows. Although the size of our study was not particularly large, the power to detect differences of the magnitudes reported in our study varied between 81% and 93% for serum iron levels and transferrin saturation, and was 68% for female serum ferritin levels.
3. Results Data were obtained from 40 controls (21 women and 19 men), 61 C282Y heterozygotes (41 women and 20 men), and 18 compound heterozygotes (11 women and 7 men). All tests were performed on all subjects. The results are shown in Tables 1 and 2.
3.1. Hemoglobin, hematocrit, and MCV There was no difference in the mean values for Hb, Ht, or MCV between controls, C282Y heterozygotes, and compound heterozygotes, either in men or in women.
Table 1 Mean (6S.D.) of laboratory values in female C282Y heterozygotes, compound (C282Y/ H63D) heterozygotes and controls
n Hb (mmol / l) Ht MCV (fl) Serum ferritin (m-g / l)a Serum iron (mmol / l) Transferrin (g / l) Transferrin saturation a
Means adjusted for age.
Controls
C282Y
C282Y/ H63D
P-value
21 8.660.6 0.4160.03 8963 70650 15.964.5 2.9860.43 0.2360.08
41 8.560.5 0.4060.02 9064 76652 18.566.9 2.8660.53 0.2960.12
11 8.460.7 0.3960.04 9164 1396123 21.666.5 2.7260.56 0.3560.12
0.339 0.166 0.732 0.015 0.052 0.379 0.016
B. de Valk et al. / European Journal of Internal Medicine 11 (2000) 317 – 321
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Table 2 Mean (6S.D.) of laboratory values in male C282Y heterozygotes, compound (C282Y/ H63D) heterozygotes and controls
n Hb (mmol / l) Ht MCV (fl) Serum ferritin (mg / l)a Serum iron (mmol / l) Transferrin (g / l) Transferrin saturation a
Controls
C282Y
C282Y/ H63D
P-value
19 9.660.5 0.4560.04 8963 171673 19.065.6 2.8660.39 0.3060.09
20 9.560.6 0.4460.03 9065 1626118 19.166.7 2.7060.37 0.3260.15
7 9.660.4 0.4460.02 8865 185690 25.765.7 2.7160.43 0.4160.09
0.800 0.416 0.473 0.858 0.038 0.407 0.090
Means adjusted for age.
3.2. Serum ferritin
4. Discussion
In women, mean serum ferritin was twice as high in the group of compound heterozygotes (1396123 mg / l) as it was in the other two groups (controls 70650 mg / l, C282Y heterozygotes 76652 mg / l, P ANOVA50.015). Ferritin in the C282Y group was slightly, but not significantly, higher than in the control group. In men, there was no difference in serum ferritin between the three groups (P ANOVA5 0.858).
The prevalence of heterozygous hereditary hemochromatosis is estimated to be about 6–14% [23–26]. While some HH heterozygotes develop a clinically significant iron overload, suggesting that another genetic factor may be present in these cases [7–10,16], most heterozygotes have no clinical symptoms of the disease [11–14,27]. In a large study among 1058 HH heterozygotes identified by HLA-typing, mean serum ferritin and iron concentrations and mean transferrin saturation were higher in HH heterozygotes than in 321 normal controls, but liver biopsy abnormalities were only seen in association with other conditions, such as alcohol abuse, hepatitis, or porphyria cutanea tarda [11]. In another study of 255 HH heterozygotes also identified by HLA-typing, 11% of the heterozygotes had an elevated serum ferritin and 8.6% had elevated transferrin saturation values [14]. In both studies [11,14], the mean levels of serum ferritin, serum iron, and transferrin saturation were in the normal range. In a study by Valberg and coworkers [13], a higher serum iron and transferrin saturation and a lower serum transferrin were found in male and female HH heterozygotes than in normal controls. Mean serum ferritin was increased in females but not in males. Cartwright and associates have also reported increased mean serum iron and transferrin saturation, as well as a limited increase in the amount of liver iron, in HH heterozygotes without clinical manifestations [12]. In contrast to our study, these previously mentioned investigations were performed in HH heterozygotes identified by HLA-typing [11–14] and obviously cannot distinguish between C282Y heterozygotes and compound heterozygotes. In a recent French study [16], C282Y heterozygotes without an iron overload were not significantly different from controls with regard to serum ferritin, liver iron concentration, or hepatic iron index. Compound heterozygotes had a slight increase in transferrin saturation compared with C282Y heterozygotes and controls. In our study, we investigated the data of genotyped HH heterozygotes and controls who were examined at the outpatient clinic of the Utrecht University Hospital from 1996 to 1998 because a family member had homozygous symptomatic HH. Despite the absence of a
3.3. Serum iron In women, a trend was observed for increasing serum iron in C282Y heterozygotes and compound heterozygotes, with compound heterozygotes having higher serum iron values than C282Y heterozygotes, and C282Y heterozygotes having higher serum iron values than control subjects (21.666.5 mmol / l, 18.566.9 mmol / l and 15.964.5 mmol / l, respectively, P ANOVA50.052). In men, compound heterozygotes had higher serum iron values than C282Y heterozygotes and controls (25.765.7 mmol / l, 19.166.7 mmol / l, and 19.065.6 mmol / l, respectively, P ANOVA50.038). No difference was observed in the serum iron levels of C282Y heterozygotes and controls.
3.4. Transferrin and transferrin saturation Transferrin levels were decreased in compound heterozygotes and in C282Y heterozygotes compared with controls, but this was far from significant (for women 2.7260.56 g / l, 2.8660.53 g / l, and 2.9860.43 g / l, respectively, P ANOVA50.379 and for men 2.7160.43 g / l, 2.7060.37 g / l, and 2.8660.39 g / l, respectively, P ANOVA50.407). Transferrin saturation, however, was the highest in both female and male compound heterozygotes compared to the other two groups. Again, C282Y heterozygotes seem to be intermediate between compound heterozygotes and controls in women but not in men (for women 0.3560.12, 0.2960.12, and 0.2360.08 respectively, P ANOVA50.016; for men 0.4160.09, 0.3260.15, and 0.3060.09 respectively, P ANOVA50.09).
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significant iron overload (which was an exclusion criterion since we wanted to study the iron parameters in asymptomatic HH heterozygotes identified by family screening), serum ferritin levels were significantly higher in female HH compound heterozygotes than in HH C282Y heterozygotes and normal controls. In male HH heterozygotes, no difference in serum ferritin was found. We found a higher mean serum iron level and a trend towards higher transferrin saturation levels in male compound HH heterozygotes than in normal controls, and a borderline higher mean serum iron level and higher transferrin saturation levels in female compound HH heterozygotes than in normal controls. In women, a trend towards increasing serum iron and transferrin saturation was observed in C282Y heterozygotes and compound heterozygotes, with compound heterozygotes having higher serum iron and transferrin saturation levels than C282Y heterozygotes, and C282Y heterozygotes having higher serum iron and transferrin saturation values than control subjects. In men, no such trend was observed. However, the number of male heterozygotes was less than the number of female heterozygotes. While serum ferritin and transferrin saturation in women, and serum iron in men, were significantly different between the three genotypes, differences of serum iron in women and transferrin saturation in men lack statistical significance. However, there is a statistical trend for increased serum iron in women (P ANOVA50.052) and for transferrin saturation in men (P ANOVA50.090), both the highest in compound heterozygotes, intermediate in C282Y heterozygotes and the lowest in normal controls. A larger study is necessary to confirm our results. However, our findings are in concordance with the previously mentioned studies, in which larger numbers of subjects are described [11–14,16]. Thus, as shown in Tables 1 and 2, our results suggest that iron stores, assessed by serum ferritin (only in women), serum iron and transferrin saturation, are higher in HH heterozygotes than in control subjects. The iron stores probably are the highest in compound heterozygotes and the lowest in the control group, C282Y heterozygous hemochromatosis being an intermediate group between compound heterozygosity and the normal population. Despite being in the normal range (see Tables 1 and 2), these higher iron stores could probably explain the excess cardiovascular risk in HH heterozygotes [17,18], because catalyzing lipid peroxidation and increasing the magnitude of reperfusion damage only needs little amounts of iron [19].
Acknowledgements Financial support was received by a grant from the University Hospital Utrecht.
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