1 INSERM EMI 01-15, Laboratoire de Génétique Moléculaire, CHU Morvan, Brest, France.
2 Etablissement Français du Sang-Bretagne, Site de Brest, Brest, France.
3 Etablissement Français du Sang-Bretagne, Site de Quimper, Quimper, France.
4 Service dHépato-Gastroentérologie, Centre Hospitalier Universitaire La Cavale Blanche, Brest, France.
Received for publication August 29, 2002; accepted for publication January 22, 2003.
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ABSTRACT |
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alcohol drinking; disease susceptibility; hemochromatosis; hereditary diseases; iron overload
Abbreviations: Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; OR, odds ratio.
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INTRODUCTION |
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Hereditary hemochromatosis is one of the sole genetic diseases benefiting from simple and efficient treatment when implemented early. Treatment relies on regular therapeutic venesection, generally weekly, until iron depletion occurs (i.e., normalization of iron parameters) and is followed by a maintenance treatment. Without early implementation of such a treatment, the disease has a poor prognosis and therefore can evolve toward irreversible damage, such as hepatocellular carcinoma or heart failure (13).
In 1996, a candidate gene for hereditary hemochromatosis was cloned on chromosome 6, at position 6p21.3 (4). This gene, HFE, encodes the HFE protein, which is a transmembrane glycoprotein implied in modulation of iron uptake (4, 5). The gene contains a main mutation, C282Y, corresponding to substitution of a tyrosine for a cysteine at amino acid 282 and preventing formation of a disulfide bond (4, 6). This mutation, whose allelic frequency varies between 0.5 and 10 percent in Caucasian populations (7, 8), is present in a homozygous state in 8095 percent of patients from northern Europe (4, 7, 911). In some US populations, only 60 percent of hemochromatosis patients are homozygous for this mutation (12). Besides this C282Y mutation, two other susceptibility factors associated with milder forms of hereditary hemochromatosis (H63D, S65C) and about 10 rare mutations have been identified in the HFE gene (9, 1316).
Discovery of the HFE gene has enabled a better understanding of the physiopathologic mechanisms implied in hereditary hemochromatosis. However, this pathology remains complex and presents a large phenotypic heterogeneity (1719). The different mutations identified in the HFE gene do not have the same penetrance, and rapidity of the evolution of iron overload can be modified by factors that may reduce the iron stores (blood donation) or increase them (intake of iron). Moreover, the severity of the disease can vary in patients with similar degrees of iron overload. For example, the risk of cirrhosis is increased by excessive alcohol consumption or the presence of viral hepatitis (20), while the risk of cardiomyopathy is increased by high intake of vitamin C, which potentiates iron uptake. Phenotypic expression of hereditary hemochromatosis can therefore be influenced by environmental factors and is the result of interactions between the gene and modifying factors.
The aim of the present study (19772002) was to identify the influence of excessive alcohol consumption on expression of the disease in patients homozygous for the main mutation (C282Y). To do so, we analyzed a cohort of 378 hemochromatosis subjects treated in a blood center of western Brittany, France, where the frequency of the disease is particularly high (21). This study showed that excessive alcohol consumption significantly increases the phenotypic expression of hereditary hemochromatosis.
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MATERIALS AND METHODS |
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Clinical questionnaire
At a patients first visit to the blood center, a physician specialist completed a clinical questionnaire. It contained information on sociodemographic characteristics of the patient (e.g., gender, age, age at disease onset, circumstances of disease diagnosis, lifestyle factors, height, weight), clinical signs (fatigue, skin pigmentation, hepatomegaly, arthritis, cardiomyopathy, metabolic disorders (i.e., diabetes, hypercholesterolemia, hypertriglyceridemia)), and biochemical parameters (serum ferritin, serum iron, transferrin saturation, alanine aminotransferase (ALT), aspartate aminotransferase (AST)). Also available were data on the treatment of patients, such as number and quantity of venesections and quantity of iron removed. This questionnaire also included a detailed item on daily consumption of alcohol, measured by the number of glasses of alcohol drunk each day (including wine, beer, and liquors). These data enabled the quantity of ethanol (in grams) consumed each day, by each patient in the cohort, to be determined. Excessive alcohol consumption was defined as daily consumption of more than or equal to 60 g. Very moderate alcohol consumption was defined as less than 20 g of ethanol per day.
Measurement of iron parameters and determination of HFE genotype
Serum iron concentrations were measured by using standard biochemical methods. The normal range of serum ferritin for women was 15200 µg/liter and for men was 30300 µg/liter. The normal range of transferrin saturation was 2045 percent. DNA samples harboring the C282Y mutation were identified by polymerase chain reaction and restriction enzyme assays, as described previously (13, 21), and, more recently, by denaturing high-performance liquid chromatography (22).
Statistical analysis
In this paper, quantitative variables are expressed as mean (standard deviation). Differences in means between groups were tested by using Students t test or the Mann-Whitney U test. Qualitative values are presented as percentages and were compared by using the chi-square test or Fishers exact test in case of sample size samples. Because the distribution of the serum ferritin variable was highly skewed, logarithmic transformation was performed to normalize the measures for all statistical analyses. A p value of less than 5 percent was considered significant. Statistical analyses were conducted by using Epi-Info software (version 6.04; Centers for Disease Control and Prevention, Atlanta, Georgia).
The biochemical and clinical characteristics of the whole cohort were described. Then, the biologic characteristics of patients according to their alcohol consumption (≥60 vs. <60 g/day) were compared. The influence of this factor on disease expression, measured by levels of serum ferritin, serum iron, and transferrin saturation, was assessed by using a linear regression analysis. In a first step, we conducted a univariate analysis and then a multivariate analysis with adjustment for potential confounding factors such as gender and age. Moreover, the frequency of clinical signs observed in patients reporting excessive alcohol consumption was compared with that observed in patients reporting lower alcohol consumption. Odds ratios with 95 percent confidence intervals were calculated.
This study fulfilled the bioethical rules in place in France. Informed consent was obtained from patients before blood samples were taken.
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RESULTS |
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Biochemical parameters according to alcohol consumption
Results of the comparison of biochemical data between hereditary hemochromatosis patients who did and those who did not consume excessive quantities of alcohol are presented in table 2. Iron parameters were significantly increased in patients who drank at least 60 g of alcohol per day (serum ferritin: 1,745.2 vs. 968.7 µg/liter, p < 0.0001; serum iron: 39.9 vs. 36.0 µmol/liter, p = 0.0040; transferrin saturation: 87.1 vs. 80.1 percent, p = 0.0071). Table 2 also reports the results of the linear regression analyzing the relation between alcohol consumption and iron overload, measured by the logarithm of serum ferritin, serum iron, and transferrin saturation. These results remained unchanged after adjustment for gender and age. Moreover, ALT and AST levels were also significantly higher in subjects reporting heavy alcohol consumption (table 2). These data were available for only 265 and 262 patients, respectively. Mean of ALT was 66.3 IU/liter (standard deviation, 48.1) in patients declaring heavy alcohol consumption versus 41.1 IU/liter (standard deviation, 28.3) in those whose level of alcohol consumption was lower (p = 0.0003). Similarly, mean of AST was 56.2 IU/liter (standard deviation, 47.8) in the group of patients reporting excessive alcohol consumption versus 34.9 IU/liter (standard deviation, 18.4) in the other group (p = 0.0002). Patients who did not undergo ALT and AST tests corresponded to patients who had begun their treatment more than 10 years ago, before these examinations began to be conducted. The patients who had had ALT and AST tests were not significantly different from those who did not have these tests.
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DISCUSSION |
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In our study, the prevalence of excessive alcohol consumption among hereditary hemochromatosis patients (8.7 percent) was lower than that reported in previous studies, which were performed before the HFE gene was discovered and which should therefore have included, as hereditary hemochromatosis patients, subjects who had alcoholic siderosis (23, 24). For example, in a study performed in 1992, Loreal et al. (23) showed that in their cohort of 127 patients, 29 percent were alcoholic. Before the genetic test existed, the distinction between patients affected with hereditary hemochromatosis and those affected with alcoholic siderosis was difficult to establish. Therefore, most studies should have considered some patients with alcoholic siderosis as having hereditary hemochromatosis. Discovery of the HFE gene in 1996 has provided a complementary element for helping to set the diagnosis of hereditary hemochromatosis. Our study measured the effect of alcohol consumption in hereditary hemochromatosis patients whose diagnosis was not ambiguous and who were homozygous for the main mutation of the HFE gene. In 1995, Adams and Agnew (25) showed similar results. They focused their study on diagnosis criteria based on the presence of a human-lymphocyte-antigen identical sibling with iron overload. Heavy alcohol consumption (>80 g/day) was observed in 15 percent of their hemochromatosis patients (25).
Note that declaration of alcohol consumption is subjective (26, 27) and that consumption must certainly be, at least in some cases, underestimated. However, this issue is addressed in all studies dealing with alcohol consumption and should not have affected our study more than others.
In this study, we showed that excessive alcohol consumption increased the severity of the disease, characterized by more frequent clinical signs, notably skin pigmentation, diabetes, and hepatomegaly. It is known that alcohol worsens the hepatic damage produced by iron in hereditary hemochromatosis. Several studies reported that excessive alcohol consumption greatly increased the prevalence of hepatic fibrosis and cirrhosis in hereditary hemochromatosis patients (23, 28). For example, in a recent study, Fletcher et al. (28) quantified the contribution of excessive alcohol consumption to the development of cirrhosis in a cohort of 224 C282Y-homozygous hemochromatosis subjects. These authors clearly observed that patients who reported excessive alcohol consumption (>60 g per day) seemed nine times more likely to develop cirrhosis compared with subjects who drank less than this quantity. The frequency of cirrhosis reached 61.1 percent in the group of heavy drinkers, whereas it was uncommon in the other group of patients (7.1 percent). Moreover, the authors showed that patients reporting heavy alcohol consumption were more likely to develop cirrhosis at an earlier age. Indeed, the mean age of patients who consumed excessive quantities of alcohol was 46.5 years (standard deviation, 10.5) compared with 53.7 years (standard deviation, 11.9) in patients who drank less than 60 g of alcohol per day. Fletcher et al. also reported that the findings were not significantly modified by considering a threshold, for alcohol consumption, of 40 g per day.
In our study, the influence of alcohol consumption on the risk of extensive liver fibrosis or cirrhosis could not be estimated. A liver biopsy was performed for the majority of the patients, but the population sample was not sufficient to enable us to draw conclusions.
Iron overload itself can result in hepatic fibrosis and cirrhosis. However, in hemochromatosis patients, this overload should be influenced by cofactors such as hepatitis or alcohol (20, 2325). According to Adams and Agnew, "patients with heavy alcohol consumption had a higher prevalence of cirrhosis at the time of diagnosis without a significant increase in iron overload, suggesting an additive hepatotoxic effect of the alcohol rather than any secondary effects on iron absorption or metabolism" (25, p. 726). Chronic alcohol consumption therefore has an additive hepatotoxic effect. In a recent editorial, Britton and Bacon (29) explained the possible mechanisms by which the combination of alcohol and iron overload lead to hepatic fibrogenesis. This occurrence may be the result of two phenomena: first, both iron overload and alcohol induce oxidative stress in the liver, causing oxidative injury and fibrogenesis; second, these two factors may also produce hepatocellular damage by nonoxidative mechanisms (29).
Our findings have implications for the public health field in terms of the adoption of preventive strategies. Alcohol consumption associated with genetic factors increases the severity of hereditary hemochromatosis and therefore the risk of cirrhosis and cancer. Consequently, patients who have the disease should be discouraged from consuming excessive quantities of alcohol because of the added hepatotoxicity it induces. In any case, treatment by regular venesection is recommended to reduce iron stores to normal values and to decrease the risk that the disease will progress, causing irreversible damage (28, 29).
In conclusion, phenotypic expression of hereditary hemochromatosis is influenced by exogenous risk factors such as alcohol consumption, as illustrated in this study. The severity of the disease is the result of interactions between genetic and environmental factors. Therefore, the pathology of hereditary hemochromatosis is multifactorial and, with this disease, it is interesting to analyze the complexity of gene-gene and gene-environment interactions (19).
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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