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Effect of hemochromatosis genotype and lifestyle factors on iron and red cell indices in a community population.

Hereditary hemochromatosis is a common iron overload disease with autosomal recessive inheritance occurring predominantly in individuals of northern European origin. A novel candidate gene, termed HFE for hereditary hemochromatosis, containing two missense mutations was identified in 1996 (1), and homozygosity for the C282Y mutation has been observed in 85-90% of patients of northern European origin with typical hereditary hemochromatosis (2-4). The H63D mutation is heterozygous in 15-20% of the population and may contribute to increased hepatic iron concentrations, especially when combined with the C282Y mutation (5,6). The proportion of heterozygotes carrying one C282Y mutated allele is high in Anglo-Celtic-based populations. Heterozygosity for the C282Y mutation can occur as either a C282Y wild-type heterozygous (C282Y/wt) [7] or compound heterozygous (C282Y/H63D) HFE genotype. Our recent population study of asymptomatic Australians indicated prevalences of 11.9% for C282Y/wt heterozygosity, 2.2% for C282Y/H63D heterozygosity, and 0.53% (1 in 188) for C282Y homozygosity (7,8).

Two recent prospective population-based studies have reported an association between heterozygosity for the C282Y mutation of the HFE gene for hereditary hemochromatosis and vascular events (9,10). These findings have led to speculation that C282Y/wt subjects had either increased serum ferritin compared with wild-type subjects or had reached the same ferritin concentrations at an earlier age (11). We therefore studied the effect of HFE genotype, age, gender, and lifestyle factors (obesity and consumption of alcohol and red meat) on iron indices.

Materials and Methods

PATIENTS

Busselton is a town in the southwest of Western Australia that has been prospectively studied since 1966 and is in many respects similar to the Framingham population (12). The population is essentially ethnically homogeneous, with 90% being of Anglo-Celtic descent. The most recent follow-up study of this population was in 1994. At this evaluation, clinical assessment was performed, and whole blood and serum samples were obtained from ~5000 Caucasian subjects. All blood tests were performed in the fasting state. From this group, we randomly selected 1488 female and 1522 male nonrelated subjects 20-79 years of age.

Permission was granted for this study by the Busselton Population Medical Research Foundation and The Committee for Human Rights at The University of Western Australia.

MEASUREMENT OF SERUM INDICES

Serum iron concentrations were measured using a standard colorimetric method, and the transferrin concentration was determined by rate immunoturbidimetry on a Hitachi 917 analyzer. Serum transferrin saturation was calculated from these results as follows: transferrin saturation (%) = serum iron ([micro]mol/L)/[2 x transferrin ([micro]mol/L)] x 100. Serum ferritin concentrations were measured by chemiluminescence immunoassay on a Chiron ACS-180 analyzer.

MEASUREMENT OF RED CELL INDICES

Hemoglobin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) measurements were performed on a Coulter STKS automated hematology analyzer.

DETERMINATION OF THE C282Y AND H63D MUTATIONS

Analysis was performed on DNA extracted from whole blood spotted onto neonatal screening cards as described by Walsh et al. (13). PCR amplification of the regions containing the missense mutations was performed using the published primer sequences of Feder et al. (1) (Gen-Bank Accession No. U60319) and cycling conditions described by Cullen et al. (14). Mutations were identified using restriction enzyme digestion followed by analysis on a 2% agarose gel. The C282Y missense mutation leads to the formation of a unique SnaBI restriction site, whereas the H63D mutation leads to the loss of a DpnII site. The status of all C282Y homozygous subjects was confirmed by separate testing with the primer sequence described by Jeffrey et al. (15) to avoid possible false-positive results attributable to the G5569A polymorphism. The H63D mutation was determined only in subjects who were heterozygous for the C282Y mutation to ascertain the prevalence of C282Y wild-type heterozygous (C282Y/ wt) and compound heterozygous (C282Y/H63D) genotypes. Wild-type refers to absence of the C282Y mutation.

STATISTICAL ANALYSIS

The Fisher exact test, the [chi square] test, and generalized linear models adjusted for multiple means testing using the least significant difference method were used. The normality test was carried out on all variables. Ferritin was highly skewed, and log transformation was used for all subsequent analyses. Statistical analyses were performed with SAS software (16).

Results

GENOTYPE PREVALENCE

The prevalences of wild types (wt/wt), heterozygotes (C282Y/wt), compound heterozygotes (C282Y/H63D), and homozygotes (C282Y/C282Y) for the C282Y mutation are shown in Table 1. The prevalences of the population with one mutated C282Y allele (C282Y/wt and C282Y/ H63D genotypes combined) were 14.1% in females and 14.5% in males. These prevalences were consistent with those predicted with the Hardy-Weinberg equation, based on an allelic frequency of 7.6% for the C282Y mutation. There were no significant between-gender differences in genotype prevalence ([chi square] test, P = 0.95). Homozygosity for the C282Y mutation occurred in 0.53% (1 in 188) of the subjects, nine females and seven males. Phenotypic presentation and clinical data for these subjects with hereditary hemochromatosis have been reported separately (7).

IRON AND HEMATOLOGY STUDIES

The data for serum iron, red cell indices, and body mass index (BMI) according to genotype in 1479 females are shown in Table 2. A threshold of 45% for transferrin saturation has been proposed for population screening for hereditary hemochromatosis (17), and the proportions exceeding this value are shown. Iron depletion was defined by a ferritin concentration <20 [micro]g/L (18) and iron deficiency by a ferritin concentration <12 [micro]g/L and a transferrin saturation <15% (19). The significance values are shown for comparison of either C282Y/wt or C282Y/ H63D genotypes to the wild type.

The iron indices in C282Y/wt females were not significantly different from those in the wild-type genotype; however, the mean MCV and MCH values were significantly increased. Compound heterozygous females had increased means for serum iron, transferrin saturation (21.9% exceeded a saturation of 45%), MCV, and MCH compared with the wild-type genotype. There were no significant differences between genotypes for the prevalence of iron depletion or deficiency.

Comparable data for serum iron, red cell indices, and BMI according to genotype in 1515 males are shown in Table 3. Compared with the wild-type genotype, C282Y/wt males had significantly increased means for serum iron, transferrin saturation, MCV, and MCH, and the C282Y/H63D males also had increased means for ferritin. The prevalences of both C282Y/wt and C282Y/ H63D males with transferrin saturation exceeding a threshold of 45% were significantly higher than the prevalence for wild-type males. There were no significant differences between genotypes for the prevalence of iron depletion or deficiency.

IRON INDICES AND AGE

Box and whisker plots for ferritin according to deciles of age in wild-type and C282Y/wt genotype males are shown in Fig. 1. Initial univariate regression analyses showed that there was no age-related increase in ferritin. A significant overall decrease of ferritin with age occurred in men with the wild-type genotype (coefficient [+ or -] SE, -0.0029 [+ or -] 0.0014; P = 0.05) but not the C282Y/wt genotype. Subsequent multiple comparison tests of age deciles showed that ferritin values in wild-type males significantly increased between the 20-29 years and 30-39 years deciles (geometric means, 126 and 181 [micro]g/L, respectively; P < 0.0001), and no subsequent changes occurred. Similar analysis by age deciles for C282Y/wt men confirmed the univariate regression analysis results and showed that there were no significant differences in ferritin values throughout the age range we examined (20-79 years). The results in Table 3 for the entire age range show that there were no significant differences between ferritin values in wild-type males and their C282Y/wt counterparts.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Box and whisker plots for ferritin according to deciles of age in wild-type and C282Y/wt females are shown in Fig. 2. For females 20-49 years of age, both univariate analysis and multiple comparison tests of age deciles showed no significant differences in mean ferritin values for either the wild-type or the C282Y/wt genotype. There was a significant increase in ferritin values for both wild-type and C282Y/wt genotypes occurring between the fourth and fifth decades, consistent with the average age of menopause for our population (51 years). There were no differences in the mean ferritin values between wild-type and C282Y/wt genotypes either before (geometric means for 20-49 years, 42 and 36 [micro]g/L, respectively) or after menopause (geometric means for 50-79 years, 84 and 87 [micro]g/L, respectively).

Serum iron and transferrin saturation values were not correlated with age in either gender.

IRON INDICES AND OBESITY

A BMI between 20 and 25 kg/[m.sup.2] is usually considered normal, and obesity is defined as a value >30. Obesity as assessed by BMI had highly significant effects on iron indices. Table 4 shows coefficients [+ or -] SE for univariate regression analyses of the relationship between age-adjusted BMI (dependent variable) and serum iron, transferrin saturation, and log ferritin (independent variables) according to HFE genotype. In wild-type subjects of both genders, the age-adjusted BMI was negatively correlated with serum iron and transferrin saturation and positively correlated with ferritin. Obesity tends to increase ferritin, whereas it decreases serum iron and transferrin saturation. In male C282Y/wt subjects, the age-adjusted BMI was not correlated with serum iron, transferrin saturation, or ferritin, but in female C282Y/wt subjects, it was correlated with serum iron. No correlations were detected between BMI and serum iron, transferrin saturation, or log ferritin in C282Y/H63D subjects.

ALCOHOL CONSUMPTION AND FERRITIN

Males admitted to a higher alcohol consumption than females, and 63% of males compared with 28% of females estimated their intake to be >10 g/day. Fig. 3 shows box and whisker plots for ferritin according to alcohol consumption and gives ferritin concentrations and levels of significance compared with a baseline intake of 1-10 g/day. Ferritin concentrations (median, interquartile range) for both males and females showed a significant increase with increasing alcohol consumption. Ferritin values for males consuming either 11-50 g of alcohol/day or >50 g/day were both significantly higher than for those who consumed 1-10 g/day (P = 0.0007 and <0.0001, respectively). Similarly, females consuming either 11-50 g of alcohol/day or >50 g/day had significantly higher ferritin values than those who consumed 1-10 g/day (P = 0.0002 and 0.006, respectively).

[FIGURE 3 OMITTED]

MEAT CONSUMPTION AND FERRITIN

The frequency of red meat (beef) consumption was high, with 89% of men and 81% of women reporting eating red meat three or more times per week. Fig. 4 shows box and whisker plots for ferritin according to the frequency of red meat consumption and gives ferritin concentrations and levels of significance compared with a baseline intake of one to two times per week. Ferritin concentrations (methan, interquartile range) for both males and females were significantly increased with increasing frequency of consumption. Ferritin values for males consuming red meat either three to six times per week or every day were significantly higher than for those who consumed one to two times per week (P = 0.0002 and 0.0001, respectively). Similarly, ferritin values for females consuming red meat either three to six times per week or every day were significantly higher than for those who consumed one to two times per week (P = 0.007 and 0.005, respectively). We compared the log-ferritin values for C282Y/wt and wild-type subjects who consumed meat every day by two-sample t-test: although male C282Y/wt subjects had higher median ferritin values than wild-type subjects, the difference did not achieve significance (medians, 221 vs 190 [micro]g/L; P = 0.098), and there was no difference in the females (medians, 75 vs 70 [micro]g/L; P = 0.691).

[FIGURE 4 OMITTED]

Discussion

The prevalences of the C282Y/wt and C282Y/H63D genotypes were 12.0% and 2.1% in females and 12.3% and 2.2% in males, respectively (Table 1);14.1% of the females and 14.5% of the males therefore had one mutated C282Y allele. These relatively high prevalence rates for the C282Y mutation make this population ideal for studying the effect of HFE genotype.

Significantly increased serum iron and transferrin saturation values were observed in females with the C282Y/ H63D but not the C282Y/wt genotype (Table 2). Thus, 21.9% of female C282Y/H63D subjects exceeded a transferrin saturation value of 45%, which has been proposed as a threshold for the investigation of subjects for hereditary hemochromatosis (17). Previous studies have reported increased transferrin saturation values in female heterozygotes (20, 21), but these studies were conducted before the availability of genotyping for HFE mutations and putative heterozygotes were identified on the basis of HLA typing in family studies of hereditary hemochromatosis probands. Subjects with the C282Y/H63D genotype would have been included as heterozygotes, and we have shown that this group has significantly increased transferrin saturation values. No significant between-genotype differences were observed in females for ferritin or hemoglobin, although both C282Y/wt and C282Y/H63D genotypes had increased MCV and MCH indices.

Both C282Y/wt and C282Y/H63D males had significantly increased serum iron, transferrin saturation, MCV, and MCH values compared with wild-type subjects (Table 3). These results agree with previous studies of heterozygous males (20, 21).

The mean ferritin values for both female and male C282Y/wt subjects were not significantly different from wild-type subjects (Tables 2 and 3, respectively). The assertion that heterozygotes have significantly increased serum ferritin compared with wild-type subjects is based on studies conducted before the availability of genotyping for HFE mutations of heterozygotes identified in family studies of hereditary hemochromatosis patients (20, 21). Both studies identified putative heterozygotes on the basis of HLA typing in family studies of hereditary hemochromatosis probands. A US study reported significantly higher mean ferritin concentrations for 209 male and 260 female heterozygotes 31-60 years of age compared with healthy controls in the same age range (20). Another study of 255 heterozygotes in Canada (21) found that the mean [+ or -] SE for serum ferritin for heterozygotes was significantly higher than in control subjects (140 [+ or -] 10.2 vs 87 [+ or -] 8.5 [micro]g/L; P <0.05). There may have been a selection bias resulting from studying hereditary hemochromatosis families rather than a community population. In the case of males, the inadvertent inclusion of the C282Y/H63D genotype as heterozygotes would have increased the mean ferritin values obtained in both studies (Table 3).

Our finding that ferritin did not differ significantly between C282Y/wt and wild-type subjects has been corroborated in a large community sample of 1233 complete pairs of Australian twins (22) in whom both HFE mutations were assessed. The latter study concluded that the effects of the HFE gene on serum ferritin are minor compared with the effects on serum iron and transferrin saturation. We found that the C282Y/wt genotype caused a significant increase in transferrin saturation in males, whereas no such effect was observed for ferritin in either gender. The mechanism for the effects of the C282Y/wt genotype on iron absorption and iron stores have not been fully studied. However, the main effect of C282Y heterozygosity seems to be to change the interactions between transferrin, its receptor, and the HFE protein (23), which may account for the increased transferrin saturation and implies that the effects on iron stores and hence ferritin may be secondary consequences.

Two recent prospective population-based studies have reported an association between heterozygosity for the C282Y mutation of the HFE gene for hereditary hemochromatosis and vascular events. A study of 12 239 Dutch women showed that C282Y/wt subjects were at significantly increased risk of death from either myocardial infarction or cerebrovascular disease compared with wild-type subjects (9). Similar findings were reported in a prospective study of 1150 Finnish men where C282Y/wt subjects were at a 2.3-fold increased risk of acute myocardial infarction (10). In an editorial accompanying these studies, it was stated that heterozygous subjects had higher mean serum ferritin concentrations than wild-type subjects and that it was likely that even heterozygotes with statistically normal ferritin concentrations achieved them at an earlier age (11). We have demonstrated that C282Y/wt subjects do not have higher ferritin concentrations than their wild-type equivalents. However, we present evidence that C282Y/wt males have achieved steady-state ferritin concentrations by their 20-29 years age decile, whereas wild-type males do not do so until the 30-39 years decile. Thus, there is a longer period of steady-state ferritin concentrations in C282Y/wt males than in wild-type subjects. This effect was not observed in females, and there were no significant increases of ferritin when the population was segregated into premenopausal (ages 20-49 years) and postmenopausal (ages 50-79 years) age groups.

We observed a striking correlation of age-adjusted BMI with iron indices for wild-type but not heterozygous subjects (Table 4). Obesity tended to increase ferritin, whereas it decreased serum iron and transferrin saturation in wild-type subjects of either gender. These results agree with findings in the study of Australian twins (22). The physiological mechanism responsible for these effects is unknown at this time.

We confirm a previous report showing that the frequency of meat intake and quantity of alcohol consumed are important lifestyle factors affecting serum ferritin concentrations for both genders (24). The heme content of red meat provides a dietary iron content of very high bioavailablity. Intestinal absorption of heme iron is relatively unaffected by the status of body iron stores, whereas absorption of non-heme iron is regulated according to the demands of iron stores (25). We observed significant increases in median ferritin concentrations with increasing frequency of red meat consumption above a baseline of one to two times per week, consistent with lack of regulation of iron uptake from dietary heme.

Median ferritin concentrations for both males and females showed a significant increase with increasing alcohol consumption above a baseline of 1-10 g/day. The mechanism for the effect of alcohol consumption on serum ferritin is poorly understood. Alcohol appears to have many modes of action that can affect serum ferritin concentrations. These include induction of an inflammatory response in the liver with resulting de novo ferritin synthesis, causing ferritin release from liver cells and changing gut permeability, thereby altering iron absorption (26).

In conclusion, we report iron and red cell indices on a large community population in whom the prevalence of heterozygosity for the C282Y mutation is relatively high. Previous studies found increased transferrin saturation and ferritin concentrations in putative heterozygotes; however, we confirmed significantly increased transferrin saturation only in male C282Y/wt subjects. Ferritin values for our C282Y/wt subjects were not significantly different from the wild-type genotype, although male C282Y/wt subjects achieved maximal ferritin concentrations in their second rather than their third decade. Compound heterozygous (C282Y/H63D) subjects formed a separate category of C282Y heterozygotes in whom both iron and red cell indices were significantly increased compared with the wild-type genotype.

We are indebted to the Busselton Population Medical Research Foundation for their invaluable cooperation.

Received September 5, 2000; accepted December 4, 2000.

References

(1.) Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class 1-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399-408.

(2.) Rossi E, Henderson S, Chin C, Olynyk J, Beilby JP, Reed WD, Jeffrey GP. Genotyping as a diagnostic aid in genetic haemochromatosis. J Gastroenterol Hepatol 1999;14:423-6.

(3.) Jouanolle A-M, Gandon G, Jezequal P, Blayau M, Campion ML, Yaouanq J, et al. Haemochromatosis and HLA-H. Nat Genet 1996;14:251-2.

(4.) The UK Haemochromatosis Consortium. A simple genetic test identifies 90% of UK patients with haemochromatosis. Gut 1997; 41:841-4.

(5.) Bacon BR, Olynyk JK, Brunt EM, Britton RS, Wolff RK. HFE genotype in patients with hemochromatosis and in patients with liver disease. Ann Intern Med 1999:130;953-62.

(6.) Moirand R, Jouanolle AM, Brissot P, Le Gall JY, David V, Deugnier Y. Phenotypic expression of HFE mutations: a French study of 1110 unrelated iron-overloaded patients and relatives. Gastroenterology 1999;116:372-7.

(7.) 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;341:718-24.

(8.) Rossi E, Olynyk JK, Cullen DJ, Papadopoulos G, Bulsara M, Summerville L, Powell LW. Compound heterozygous hemochromatosis genotype predicts increased iron and erythrocyte indices in women. Clin Chem 2000;46:162-6.

(9.) Roest M, van der Schouw YT, de Valk B, Marx JJ, Tempelman MJ, de Groot PG, et al. Heterozygosity for a hereditary hemochromatosis gene is associated with cardiovascular death in women. Circulation 1999;100:1268-73.

(10.) Tuomainen TP, Kontula K, Nyyssonen K, Lakka TA, Helio T, Salonen JT. Increased risk of acute myocardial infarction in carriers of the hemochromatosis gene Cys282Tyr mutation: a prospective cohort study in men in eastern Finland. Circulation 1999;100:1274-9.

(11.) Sullivan JL. Iron and the genetics of cardiovascular disease. Circulation 1999;100:1260-3.

(12.) Knuiman MW, Vu HT. Prediction of coronary heart disease mortality in Busselton, Western Australia: an evaluation of the Framingham, national health epidemiologic follow up study, and WHO ERICA risk scores. J Epidemiol Community Health 1997;51:515-9.

(13.) Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 1991;10:506-13.

(14.) Cullen LM, Summerville L, Glassick TV, Crawford DH, Powell LW, Jazwinska EC. Neonatal screening for the hemochromatosis defect. Blood 1997;90:4236-7.

(15.) Jeffrey GP, Chakrabarti S, Hegele RA, Adams PC. Polymorphism in intron 4 of HFE may cause overestimation of C282Y homozygote prevalence in hemochromatosis. Nat Genet 1999;22:325-6.

(16.) SAS Institute. SAS/STAT users guide, Ver. 6, 4th ed., Vol. 1. Cary, NC: SAS Institute Inc, 1989:943pp.

(17.) McLaren CE, McLachlan GJ, Halliday JW, Webb SI, Leggett BA, Jazwinska EC, et al. Distribution of transferrin saturation in an Australian population: relevance to the early diagnosis of hemochromatosis. Gastroenterology 1998;114:543-9.

(18.) Hastka J, Lasserre JJ, Schwarzbeck A, Hehlmann R. Central role of zinc protoporphyrin in staging iron deficiency. Clin Chem 1994; 40:768-73.

(19.) Looker AC, Dallman PR, Carroll MD, Gunter EW, Johnson CL. Prevalence of iron deficiency in the United States. JAMA 1997; 277:973-6.

(20.) Bulaj ZJ, Griffen LM, Jorde LB, Edwards CQ, Kushner JP. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med 1996;335:1799-805.

(21.) Adams PC. Prevalence of abnormal iron studies in heterozygotes for hereditary hemochromatosis: an analysis of 255 heterozygotes. Am J Hematol 1994;45:146-9.

(22.) Whitfield JB, Cullen LM, Jazwinska EC, Powell LW, Heath AC, Zhu G, et al. Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins. Am J Hum Genet 2000 66:1246-58.

(23.) Salter-Cid L, Brunmark A, Li Y, Leturcq D, Peterson PA, Jackson MR, Yang Y. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci U S A 1999;96:5434-9.

(24.) Leggett BA, Brown NN, Bryant SJ, Duplock L, Powell LW, Halliday JW. Factors affecting the concentrations of ferritin in serum in a healthy Australian population. Clin Chem 1990;36:1350-5.

(25.) Hunt JR, Roughead ZK. Adaptation of iron absorption in men consuming diets with high or low iron bioavailability. Am J Clin Nutr 2000; 71:94-102.

(26.) Moirand R, Lescoat G, Delamaire D, Lauvin L, Campion JP, Deugnier Y, Brissot P. Increase in glycosylated and nonglycosylated serum ferritin in chronic alcoholism and their evolution during alcohol withdrawal. Alcohol Clin Exp Res 1991;15:963-9.

[7] Nonstandard abbreviations: wt, wild type; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; and BMI, body mass index.

ENRICO ROSSI, [1] * MAX K. BULSARA, [2] JOHN K. OLYNYK, [3] DIGBY J. CULLEN, [4,5] LESA SUMMERVILLE, [6] and LAWRIE W. POWELL [6]

[1] Biochemistry Section, Pathcentre, QE II Medical Centre, Nedlands, Western Australia 6009, Australia

[2] Department of Public Health and

[3] University Department of Medicine, University of Western Australia, Fremantle, Western Australia 6160, Australia

[4] Department of Gastroenterology, Fremantle Hospital, Fremantle, Western Australia 6160, Australia

[5] Busselton Population Medical Research Foundation, Perth, Western Australia 6907, Australia

[6] Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia

*Author for correspondence. Fax 61-5-9346-3882; e-mail ric.rossi@health.wa.gov.au
Table 1. Genotype prevalence.

 Females Males

Genotype (a) n Prevalence, % n Prevalence, %

wt/wt 1268 85.2 1295 85.1
C282Y/wt 179 12.0 187 12.3
C282Y/H63D 32 2.1 33 2.2
C282Y/C282Y 9 0.60 7 0.46
 1488 1522

(a) wt, wild-type (refers to absence of the C282Y mutation);
C282Y/wt, hetero- zygote; C282Y/H63D, compound heterorygote;
C282Y/C282Y, C282Yhomorygote.

Table 2. Iron metabolism and hematology variablesa in
1479 women according to HFE genotype.

 wt/wt C282Y/wt

n 1268 179
Age, years 53.4 (15.3) 52.8 (16.1)
Iron, [micro]mol/L 17.1 (5.6) 17.9 (6.1)
Transferrin sat, (d) % 26.0 (15.2) 28.1 (11.5)
Ferritin, median 65 (35-124) 64 (33-120)
 (interquartile
 range), [micro]g/L
Hb, g/L 134 (10.0) 135 (10.0)
MCV, fL 88.4 (4.5) 89.2 (4.7) (c)
MCH, pg 30.2 (1.7) 30.5 (1.8) (c)
BMI, kg/[m.sup.2] 25.7 (4.6) 26.3 (4.6)
Transferrin sat >45% 2.5% 4.5%
 (n = 32) (n = 8)
Ferritin <20 [micro]g/L 11.9% 15.1%
 (n = 151) (n = 27)
Ferritin <12 [micro]g/L; 3.2% 3.4%
transferrin sat <15% (n = 41) (n = 6)

(a) Data are means (SD), except where indicated.

(b,c) P for comparison to the wild-type (wt/wt)
genotype: (b) P <0.001; (c) P <0.05.

(d) sat, saturation; Hb, hemoglobin.

 C282Y/H63D

n 32
Age, years 52.7 (13.8)
Iron, [micro]mol/L 21.2 (8.8) (b)
Transferrin sat, (d) % 36.3 (11.7) (b)
Ferritin, median 63 (45-164)
 (interquartile
 range), [micro]g/L
Hb, g/L 137 (10.3)
MCV, fL 91.3 (3.0) (b)
MCH, pg 31.3 (1.1) (b)
BMI, kg/[m.sup.2] 25.0 (4.1)
Transferrin sat >45% 21.9% (b)
 (n = 7)
Ferritin <20 [micro]g/L 6.3%
 (n = 2)
Ferritin <12 [micro]g/L; 0%
transferrin sat <15% (n = 0)

(a) Data are means (SD), except where indicated.

(b,c) P for comparison to the wild-type (wt/wt)
genotype: (b) P <0.001; (c) P <0.05.

(d) sat, saturation; Hb, hemoglobin.

Table 3. Iron metabolism, BMI, and hematology variablesa
in 1515 men according to HFE genotype.

 wt/wt C282Y/wt

n 1295 187
Age, years 51.5 (15.5) 51.8 (16.0)
Iron, [micro]mol/L 18.8 (5.5) 19.7 (6.4) (b)
Transferrin sat, (e) 29.5 (9.8) 32.5 (11.6) (c)
Ferritin, median 177 (108-277) 162 (96-291)
 (interquartile
 range), [micro]g/L
Hb, g/L 151 (10.0) 151.00
MCV, fL 88.8 (3.9) 89.7 (4.2) (d)
MCH, pg 30.5 (1.4) 30.9 (1.5) (c)
BMI, kg/[m.sup.2] 26.7 (3.4) 26.4 (3.0)
Transferrin 6.9% 10.6% (d)
 sat >45% (n = 90) (n = 30)
Ferritin <20 [micro]g/L 1.7% 3.7%
 (n = 22) (n = 7)
Ferritin <12 [micro]g/L; 0.3% 1.1%
 transferrin (n = 4) (n = 2)
 sat <15%

 C282Y/H63D

n 33
Age, years 52.0 (16.6)
Iron, [micro]mol/L 22.5 (7.3) (c)
Transferrin sat, (e) 39.9 (13.3) (c)
Ferritin, median 323 (193-457) (d)
 (interquartile
 range), [micro]g/L
Hb, g/L 154 (10.6)
MCV, fL 91.4 (4.0) (c)
MCH, pg 31.4 (1.4) (c)
BMI, kg/[m.sup.2] 26.2 (3.2)
Transferrin 21.2% (d)
 sat >45% (n = 7)
Ferritin <20 [micro]g/L 6.1%
 (n = 2)
Ferritin <12 [micro]g/L; 0%
 transferrin (n = 0)
 sat <15%

(a) Data are means (SD), except where indicated.

(b-d) P for comparison to the wild-type (wt/wt) genotype:
(b) P <0.05; (c) P<0.001; (d) P<0.01.

(e) sat, saturation; Hb, hemoglobin.

Table 4. Linear regression analyses for the relationship between
BMI and serum iron, transferrin saturation, and log ferritin
adjusted for age according to HFE genotype. (a)

 wt/wt

 Coefficient [+ or -] SE P

Females
 Iron, [micro]mol/L -0.1753 [+ or -] 0.0342 0.0001
 Transferrin sat, (b) % -0.3012 [+ or -] 0.0948 0.001
 Ferritin, [micro]g/L 0.0211 [+ or -] 0.0057 0.0002
Males
 Iron, [micro]mol/L -0.0965 [+ or -] 0.0457 0.03
 Transferrin sat, % -0.3365 [+ or -] 0.0813 0.0001
 Ferritin, [micro]g/L 0.0397 [+ or -] 0.0066 0.0001

 C282Y/wt

 Coefficient [+ or -] SE P

Females
 Iron, [micro]mol/L -0.2267 [+ or -] 0.1012 0.03
 Transferrin sat, (b) % -0.2871 [+ or -] 0.1892 NS
 Ferritin, [micro]g/L 0.0040 [+ or -] 0.0159 NS
Males
 Iron, [micro]mol/L -0.0970 [+ or -] 0.1597 NS
 Transferrin sat, % -0.1248 [+ or -] 0.2890 NS
 Ferritin, [micro]g/L -0.0005 [+ or -] 0.0217 NS

(a) No correlations were detected between BMI and serum iron,
transferrin saturation, or log ferritin in C282Y/H63D subjects.

(b) sat, saturation; NS, not significant.

Fig. 3. Effect of alcohol intake (g/day) on serum ferritin
concentrations ([micro]g/L).

Box and whisker plots showing median and interquartile range in
the box (50th, 25th, and 75th percentiles) and 5th and 95th
percentiles (whiskers). The table gives ferritin data as median
(interquartile range) and Pvalues for comparison to
subjects consuming 1-10 g/day. females; males.

 Ferritin, [micro]g/L, median
 (interquartile range)

Alcohol intake, g/day Females Males

1-10 60 (31-113) 160 (94-249)
11-50 71 (39-145) (a) 192 (122-295) (a)
>50 162 (67-197) (b) 234 (127-367) (a)

(a) P <0.001.

(b) P <0.01.

Fig. 4. Effect of frequency of red meat intake on serum ferritin
concentrations ([micro]g/L).

Box and whisker plots showing median and interquartile range in
the box (50th, 25th, and 75th percentiles) and 5th and 95th
percentiles (whiskers). The table gives ferritin data as median
(interquartile range) and Pvalues for comparison to
subjects consuming red meat one to two times/week. females; males.

 Ferritin, [micro]g/L, median
 (interquartile range)

Frequency of red meat Females Males

1-2/week 53(35-95) 138(81-204)
3-6/week 68 (36-129) (a) 181 (106-288) (b)
Every day 72 (35-150) (a) 196 (127-298) (b)

(a) P <0.01.

(b) P <0.001.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Rossi, Enrico; Bulsara, Max K.; Olynyk, John K.; Cullen, Digby J.; Summerville, Lesa; Powell, Lawrie
Publication:Clinical Chemistry
Date:Feb 1, 2001
Words:5094
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