HFE C282Y homozygotes have reduced LDL cholesterol: the Atherosclerosis Risk in Communities (ARIC) Study

Abstract

Recent studies have raised questions about the long-term health risks for individuals with mutations in the HFE gene, although previous studies may have been plagued by selection bias or lack of population-based comparison groups. We examined cardiovascular disease risk factors, iron and liver biomarkers, and morbidity and mortality associated with the C282Y and H63D variants of HFE in the ARIC study, a population-based cohort of nearly 16,000 U.S. white and black men who were 45–64 years old at baseline. Subjects were followed for an average of 15 years for death, incident coronary heart disease, stroke, and heart failure, and 8 years for incident diabetes. The prevalence of C282Y homozygosity was 0.42% (45/10,800) in whites, similar to other North American population-based studies. C282Y homozygotes had statistically significantly lower mean LDL cholesterol and fibrinogen and higher mean levels of iron (ferritin, transferrin saturation) and liver biomarkers (alanine aminotransferase, Hepascore) compared to HFE wild-type subjects. Rates of all-cause mortality, cardiovascular disease, and diabetes were similar across HFE genotypes. These prospective, population-based data indicate higher serum iron indices and possible mild liver dysfunction or disease in some C282Y homozygotes, but provide little evidence that HFE C282Y or H63D mutations are related to all-cause mortality, cardiovascular disease, or diabetes. Reduced LDL in C282Y homozygotes may be due to effect of excess iron on cholesterol metabolism and lipoprotein formation in the liver.

Introduction

Hereditary hemochromatosis is a genetic disorder that may lead to excessive iron accumulation in various body tissues. Shortly after the discovery in 1996 that homozygosity for the 845 G->A [C282Y] mutation in the hemochromatosis gene (HFE) was the principal cause of hereditary hemochromatosis in individuals of northern European descent [1], it was assumed that most C282Y homozygotes would eventually develop excessive body iron deposition and clinically overt disease, most notably in the liver, heart, pancreas, joints, or pituitary [2]. However, large screening studies from Norway [3] and southern California [4] showed that the prevalence of clinical disease in target organs was similar in C282Y homozygotes compared to subjects without the mutation, and none of the 23 homozygotes in another population-based developed clinical signs or symptoms suggesting hereditary hemochromatosis during 25 years of follow-up [5]. Some of these studies have been criticized for a failure to ascertain subclinical disease, such as liver fibrosis [6]. Further concerns have been raised about the possibility of selection bias, as individuals who already had clinical manifestations were unlikely to be screened or may have died prematurely [7], although at least in some studies, evaluation of Hardy-Weinberg equilibrium found no deficit in the anticipated number of C282Y homozygotes [8].

Although it is unlikely that the vast majority of C282Y homozygotes eventually develop serious morbidity and early mortality, as was suggested less than a decade ago [2], the clinical significance of HFE mutations remains uncertain [9]. The U.S. Preventive Services Task Force recently concluded that there is “insufficient evidence to confidently project the impact of, or estimate the benefit from, widespread or high-risk genetic screening for hereditary hemochromatosis [9].” Because health benefits of screening for a disease depend largely on the natural history, determining the incidence of clinically significant morbidity or mortality related to HFE mutations is highly relevant. In the present report, we evaluated morbidity and mortality associated with the C282Y and H63D mutations of HFE in a U.S. cohort ascertained using carefully designed and executed population-based sampling techniques.

Methods

Study population

In 1987–89 the Atherosclerosis Risk in Communities (ARIC) Study enrolled 15,792 subjects ages 45–64 years in four U.S. locations: Forsyth County, NC; Jackson, MS; seven northwestern suburbs of Minneapolis, MN; and Washington County, MD [10]. Black residents were over-sampled as part of the area-probability sampling of Forsyth County, NC, while enrollment at the Jackson, MS, site was restricted to black residents. Eligible residents were invited to participate in a baseline clinical examination (visit 1) and three subsequent follow-up examinations approximately three years apart (visits 2–4). For the present analysis, we excluded 48 participants who were not white or black, 43 who did not provide consent for genetic studies, and 1,216 with missing data for either the C282Y or H63D variants, leaving 14,485 subjects (10,800 whites and 3685 blacks) available for analysis. The research was carried out according to the principles of the Declaration of Helsinki. Institutional review boards at each study institution provided approval and all participants gave informed consent.

Baseline data collection

At baseline, interviewers collected information on menopausal status, smoking status, and previous diagnoses, including cancer [10]. Fasting lipids, glucose, insulin, and fibrinogen were measured at a central laboratory using methods described previously [11–13], and hematology was performed at local laboratories. Prevalent coronary heart disease was defined as a self-reported history of a physician-diagnosed heart attack, evidence of an old myocardial infarction (MI) by electrocardiogram based on the Minnesota codes, or reported history of coronary revascularization. Prevalent stroke was defined as a reported stroke diagnosed by a physician.

Prevalent diabetes was defined as reported physician diagnosis, use of anti-diabetes medications, fasting (≥ 8hr post prandial) serum glucose ≥ 7 mmol/L (140 mg/dL), or non-fasting glucose of ≥ 11.1 mmol/L (200 mg/dL). Prevalent heart failure was defined as reported current intake of heart failure medication at visit 1 or evidence of manifest heart failure defined by the Gothenburg criteria stage 3 [14, 15]. Carotid intimal-medial thickness was measured by B-mode ultrasound [16, 17] and the mean intimal-medial thickness across three segments (internal carotid, bifurcation, common carotid) was used in analysis. History of fatigue was assessed at visit 2 by the question “Do you often feel tired?” History of arthritis was assessed at visit 4 by the question “Has a doctor ever told you that you have arthritis?” HFE variants were typed at the ARIC DNA Laboratory, University of Texas Health Sciences Center at Houston using a standard Taqman method. All C282Y homozgyotes (C282Y/C282Y) were verified at CLIA-certified Clinical Laboratories of the University of Minnesota Medical Center, Fairview using a restriction endonuclease method [1].

Iron and liver biomarkers

Because of limited resources, iron and liver biomarkers were measured only in C282Y homozygotes and a comparison group of 48 wild-type subjects randomly selected from white ARIC controls in the Longitudinal Investigation of Thromboembolism Etiology study [18], frequency matched to C282Y homozygotes by age group (45–54, 55–64, or 65–74 years) at the time of venipuncture. When available, serum from the most recent full cohort exam (visit 4, 1996–98) was used; serum from visit 2 (n=8) or visit 1 (n=3) was used for a few subjects, and was unavailable for all visits in one C282Y homozygote. Transferrin saturation was measured using automated spectrophotometric measurement of iron and unsaturated iron binding capacity on a Roche/Hitachi 911 analyzer using commercially available reagents (Roche Diagnostics/Boehringer Mannheim Corp., Indianapolis, IN). Ferritin, alanine aminotransferase, γ-glutamyltransferase, and total bilirubin were measured using the same equipment and commercially available assays from Roche Diagnostics. Alpha-2-macroglobulin was assayed using the Immage analyzer and commercially available reagents (Beckman-Coulter, Inc., Brea, CA), and hyaluronic acid was measured using an ELISA assay kit (Corgenix Inc., Westminster, CO). Coefficients of variation for all assays were less than 5.4% except ferritin and unsaturated iron binding capacity, which were 9.9% and 8.4%, respectively. To assess liver fibrosis, we used a predictive model (Hepascore) based on bilirubin, γ-glutamyltransferase, hyaluronic acid, α₂-macroglobulin, age, and sex previously validated in subjects with chronic hepatitis C infection [19].

Morbidity and mortality follow-up

Liver disease was assessed at visit 3 by the question “Has a doctor ever said you had cirrhosis or another chronic liver disease?” and searches of underlying and contributing causes of death (ICD-9 570–573 or 155; ICD-10 K7 or C22) and hospital discharge diagnoses (ICD-9 CM 570–573 or 155). Clinical diagnosis of hemochromatosis was ascertained as a discharge diagnosis of hemochromatosis (ICD-9 CM 275.0). All-cause mortality was ascertained by reviews of death certificates, annual follow-up interviews, hospital charts, and other means. Details on quality assurance for ascertainment and classification of coronary heart disease (CHD) and stroke events have been published elsewhere [20–22]. Incident CHD events were defined as a validated definite or probable hospitalized MI, definite CHD death, unrecognized myocardial infarction defined by ARIC electrocardiogram readings, or coronary revascularization. Incident ischemic stroke was defined as validated definite or probable hospitalized embolic or thrombotic brain infarctions. Incident heart failure was defined as a hospital discharge diagnosis coded with heart failure at any position (ICD-9 CM 428) or death with heart failure coded as the underlying cause of death (ICD-9 428; ICD-10 I50) [23]. Subjects were defined as having incident diabetes if they met any of the criteria listed above for prevalent diabetes at any of the follow-up visits with date of diagnosis interpolated between visits [24].

Statistical analysis

We tested the C282Y and H63D variants separately for Hardy Weinberg equilibrium using χ² goodness of fit tests. Pairwise differences in baseline characteristics between each combined C282Y and H63D genotype group and a common reference group of subjects wild-type for both variants were tested by analysis of variance (continuous measures) or logistic regression (dichotomous measures) as implemented in SAS (version 9.1, SAS Institute Inc., Cary, NC). For iron and liver biomarkers, differences between C282Y homozygotes and wild-type subjects were tested by analysis of covariance, adjusting for age (matching variable) and sex. Triglycerides, insulin, ferritin, bilirubin, γ-glutamyltransferase, hyaluronic acid, and alanine aminotransferase were log-transformed before analysis. For longitudinal outcomes, we calculated person-years at risk for each participant as the time between the baseline examination date and the last date of follow-up (e.g., December 31, 2004 for all outcomes except diabetes), date of loss-to-follow-up, date of death, or date of disease diagnosis. We used the Epitab procedure in STATA (release 8, StataCorp, College Station, TX) to estimate rate ratios and exact confidence intervals.

Results

Genotype frequencies for C282Y and H63D did not deviate statistically from those expected under Hardy-Weinberg equilibrium in whites or blacks (p>0.05). Among whites, there were slightly more C282Y homozygotes (n=45) than expected under Hardy-Weinberg equilibrium conditions (n=43.3). Only four black subjects were either C282Y homozygotes or C282Y/H63D compound heterozygotes (Table 1), thereby precluding detailed analyses of those genotypes. All further analyses were restricted to whites.

Table 1.

HFE C282Y and H63D Genotype Frequencies

	C282Y/C282Y	C282Y/H63D	H63D/H63D	C282Y/+	H63D/+	+/+
Whites, N (%)	45 (0.4)	193 (1.8)	257 (2.4)	1084 (10.0)	2453 (22.7)	6768 (62.7)
Blacks, N (%)	1 (0.03)	3 (0.1)	3 (0.1)	100 (2.7)	189 (5.1)	3389 (92.0)

Mean LDL cholesterol and fibrinogen levels at baseline were substantially lower among C282Y homozygotes compared to wild-type subjects (Table 2). Similar patterns were observed for LDL during follow-up: mean levels were 0.31 mmol/L (12 mg/dL; p=0.06), 0.44 mmol/L (17 mg/dL; p=0.007), and 0.47 mmol/L (18 mg/dL; p=0.003) lower in C282Y homozygotes at visits 2, 3, and 4, respectively. No adjustment was made for lipid-lowering medications in these analyses. At visit 4, self-reported use of lipid-lowering medications was nearly twice as common among wild-type subjects than C282Y homozygotes (16% vs. 9%), although this difference was not statistically significant. At baseline, mean hematocrit and hemoglobin were higher in carriers of the C282Y or H63D mutations compared to wild-type subjects, although for C282Y homozygotes differences were not statistically significant.

Table 2.

Baseline Characteristics (Means (Standard Errors) or Percentages) by HFE Genotype, ARIC Whites

Characteristic	C282Y/C282Y (N=45)	C282Y/H63D (N=193)	H63D/H63D (N=257)	C282Y/+ (N=1084)	H63D/+ (N=2453)	+/+ (N=6768)
Age (years)	53.4 (0.9)	54.1 (0.4)	54.5 (0.4)	54.3 (0.2)	54.4 (0.1)	54.4 (0.1)
Gender (% men)	47	40	49	45	48	48
Post-menopausal, women (%)	55	66	72	63	65	66
Current smoking (%)	18	30	25	25	25	25
Body mass index (kg/m2)	26.5 (0.7)	27.5 (0.3)	27.3 (0.3)	27.1 (0.1)	27.1 (0.1)	26.9 (0.1)
LDL cholesterol (mmol/L)	3.18 (0.15)	3.58 (0.07)	3.59 (0.06)	3.52 (0.03)	3.56 (0.02)	3.56 (0.01)
HDL cholesterol (mmol/L)	1.41 (0.06)	1.30 (0.03)	1.28 (0.03)	1.31 (0.01)	1.30 (0.01)	1.31 (0.01)
Triglycerides (mmol/L)†	1.18 (0.80–1.94)	1.37 (0.98–1.91)	1.39 (1.05–1.94)	1.33 (0.93–1.88)	1.31 (0.93–1.88)	1.28 (0.93–1.83)
Fasting glucose (mmol/L)‡	5.46 (0.08)	5.46 (0.04)	5.50 (0.03)	5.45 (0.02)	5.48 (0.01)	5.47 (0.01)
Fasting insulin (pmol/L)‡	65.2 (8.4)	75.9 (4.1)	69.2 (3.7)	72.6 (1.7)	73.1 (1.2)	72.4 (0.7)
Fibrinogen (g/L)	2.79 (0.09)	3.05 (0.04)	3.00 (0.04)	2.97 (0.02)	2.98 (0.01)	2.97 (0.01)
Hematocrit (volume fraction)	0.425 (0.006)	0.431 (0.003)	0.426 (0.002)	0.424 (0.001)	0.423 (0.001)	0.421 (0.001)
Hemoglobin (mmol/L)	2.218 (0.030)	2.250 (0.015)	2.220 (0.013)	2.205 (0.006)	2.197 (0.004)	2.182 (0.002)
White cell count (109 cells/L)	6.02 (0.30)	6.52 (0.14)	6.48 (0.13)	6.27 (0.06)	6.38 (0.04)	6.27 (0.02)
Platelet count (109 cells/L)	243 (11)	252 (6)	256 (5)	257 (2)	256 (2)	260 (1)
Hypertension (%)	22	33	34	28	29	26
Carotid IMT (mm)	0.724 (0.031)	0.716 (0.015)	0.719 (0.013)	0.722 (0.006)	0.725 (0.004)	0.727 (0.002)
History of cancer (%)	9.1	5.7	7.4	5.1	6.5	7.2
History of fatigue (%)§	40	49	47	45	45	43
History of arthritis (%)‖	29	32	42	41	40	38

IMT: intimal-medial thickness

estimates listed in bold are significantly different (p<0.05) compared to +/+ subjects

^†medians and interquartile ranges are shown

^‡among subjects without diabetes

^§measured at visit 2

^‖measured at visit 4

Among C282Y homozygotes, 11 (25%) had ferritin levels greater than 1000 µg/L, while none of the 48 age-matched wild-type subjects had levels exceeding this cutpoint. Mean ferritin, transferrin saturation, and total iron were significantly higher while unsaturated and total iron binding capacities were significantly lower in C282Y homozygotes (Table 3). The Hepascore index was significantly higher in C282Y homozygotes (p=0.003), although there were only small differences for individual components of the index, including bilirubin, γ-glutamyltransferase, hyaluronic acid, and α₂-macroglobulin.

Table 3.

Serum iron and liver biomarkers by HFE C282Y Genotype, ARIC Whites

	C282Y/C282Y (N=44)		+/+ (N=48)
Biomarker	Median	Interquartile Range	Median	Interquartile Range	p-value*
Ferritin (µg/L)†	741	377–1143	157	82–238	<0.001
Transferrin saturation (fraction saturation)	0.77	0.62–0.95	0.30	0.24–0.35	<0.001
Total iron (µmol/L)	33.8	27.4–38.3	16.1	12.9–19.9	<0.001
Unsaturated iron binding capacity (µmol/L)	10.9	2.0–18.1	40.1	33.5–45.5	<0.001
Total iron binding capacity (µmol/L)	43.7	38.8–50.8	55.8	51.7–61.4	<0.001
Hepascore†	0.10	0.06–0.26	0.08	0.06–0.12	0.003
Bilirubin (µmol/L)†	6.8	5.1–10.3	6.8	5.1–7.7	0.15
Gamma glutamyltransferase (U/L)†	20	15–30	20	12–31	0.71
Hyaluronic acid (µg/L)†	24	15–34	19	13–27	0.05
α2-macroglobulin (g/L)	1.9	1.6–2.3	1.8	1.6–2.2	0.09
Alanine aminotransferase (U/L)†	20	14–29	11	8–14	<0.001

^*obtained from linear regression models adjusted for age and sex

^†log-transformed for linear regression analysis

Mean length of follow-up was 15 years for all-cause mortality, CHD, stroke, and heart failure, and 8 years for diabetes. Rates of all-cause mortality, incident CHD, stroke, heart failure and diabetes were similar across HFE genotypes (Table 4). Five C282Y homozygotes (11%) had clinical evidence of liver disease during follow-up, including two subjects who died with an underlying cause of death coded as hepatocellular carcinoma and cholangiocarcinoma, respectively, two subjects with hospital discharge diagnoses indicating liver disease, and one subject who self-reported liver disease at visit 3. Among wild-type subjects, 3% had clinical evidence of liver disease. Prior to any genetic testing as part of this study, four C282Y homozygotes (9%) had a hospital discharge diagnosis of hemochromatosis during follow-up, while only one wild-type subject (0.01%) had such a diagnosis.

Table 4.

All-Cause Mortality and Incident Disease by HFE Genotype, ARIC Whites

Outcome		C282Y/C282Y	C282Y/H63D	H63D/H63D	C282Y/+	H63D/+	+/+
All-Cause Mortality	Deaths	7	30	45	174	399	1066
	Person-years	704	2,954	3,900	16,717	37,599	103,755
	Rate per 1000 person-years	9.9	10.2	11.5	10.4	10.6	10.3
	Rate ratio (Exact 95% CI)	0.97 (0.39–2.00)	0.99 (0.66–1.42)	1.12 (0.81–1.51)	1.01 (0.86–1.19)	1.03 (0.92–1.16)	1.00 (ref)
Incident CHD	Cases	6	28	31	145	308	860
	Person-years	670	2,638	3,463	14,820	33,191	92,218
	Rate per 1000 person-years	9.0	10.6	9.0	9.8	9.3	9.3
	Rate ratio (Exact 95% CI)	0.96 (0.35–2.10)	1.14 (0.75–1.66)	0.96 (0.65–1.37)	1.05 (0.87–1.25)	1.00 (0.87–1.13)	1.00 (ref)
Incident Stroke	Cases	1	11	11	50	93	245
	Person-years	692	2,887	3,799	16,215	36,680	101,316
	Rate per 1000 person-years	1.4	3.8	2.9	3.1	2.5	2.4
	Rate ratio (Exact 95% CI)	0.60 (0.01–3.36)	1.58 (0.78–2.89)	1.20 (0.59–2.18)	1.27 (0.92–1.73)	1.05 (0.82–1.34)	1.00 (ref)
Incident Heart Failure	Cases	2	16	23	100	224	564
	Person-years	704	2,697	3,658	15,384	34,934	96,001
	Rate per 1000 person-years	2.8	5.9	6.3	6.5	6.4	5.9
	Rate ratio (Exact 95% CI)	0.48 (0.06–1.75)	1.01 (0.57–1.65)	1.07 (0.67–1.62)	1.11 (0.89–1.37)	1.09 (0.93–1.28)	1.00 (ref)
Incident Diabetes	Cases	3	19	20	87	241	566
	Person-years	299	1,312	1,583	7,400	16,397	45,805
	Rate per 1000 person-years	10.0	14.5	12.6	11.8	14.7	12.4
	Rate ratio (Exact 95% CI)	0.81 (0.17–2.39)	1.17 (0.70–1.85)	1.02 (0.62–1.59)	0.95 (0.75–1.19)	1.19 (1.02–1.39)	1.00 (ref)

Discussion

We found that genotype frequencies for C282Y and H63D in the population-based ARIC cohort were similar to those reported for whites and blacks in the HEIRS study [25], the U.S.-representative NHANES III investigation [26], and a health maintenance organization in southern California [8]. There was no evidence that C282Y homozygotes had been selectively lost from the population by the time subjects were enrolled in the ARIC cohort, similar to the findings of Beutler et al. [8]. Furthermore, our results indicated similar rates of all-cause mortality across all HFE genotypes over an average of 15 years of follow-up. Consistent with our results, cross-sectional studies in Finland [27], the Netherlands [28], and France [29] did not find lower frequencies of the C282Y mutation in older age groups, as might be expected if the mutation is an important cause of death. Similarly, the Melbourne Collaborative Cohort Study found that HFE genotypes were unrelated to all-cause mortality over 11.4 years of follow-up in 29,676 subjects of northern European ancestry [6].

C282Y homozygotes had a more favorable lipid profile, with mean LDL-cholesterol concentrations lower than wild-type subjects. To our knowledge, no previous studies have reported an association between C282Y homozygosity and LDL cholesterol. Interestingly, one study found that self-reported cholesterol problems were 3 to 5 times more frequent in wild-type subjects compared to C282Y/C282Y subjects [30]. Although this finding was considered likely due to chance [30], our results are consistent with less frequent use of cholesterol-lowering drugs among C282Y homozygotes. The effect of the HFE C282Y mutation on mean LDL levels in the ARIC study (0.38 mmol/L or 15 mg/dL) is similar in magnitude to the range of effects on LDL (0.05 to 0.23 mmol/L or 2 to 9 mg/dL per allele) for the eleven most significant single nucleotide polymorphisms, including those near the APOE/C1/C4, LDLR, APOB, and PCSK9 genes, in a combined analysis of three recent genome-wide association studies from Europe [31]. However, the frequency of the C282Y/C282Y genotype is only 0.4–0.5% in populations of European ancestry, making it unlikely that these genome-wide association studies of LDL were sufficiently powered to detect an effect of a rare variant such as HFE.

Experimental iron overload in rats has been found to lower LDL and raise HDL [32]. Excess iron could affect cholesterol metabolism due to increased intracellular oxidative stress, membrane peroxidation, and altered activity of liver enzymes involved in cholesterol metabolism and lipoprotein formation [32]. Alternatively, differences in cholesterol levels could reflect long-term liver damage due to iron overload in C282Y homozygotes. Among patients with chronic hepatitis C, total cholesterol levels have been found to be 0.31–0.41 mmol/L (12–16 mg/dL) lower, on average, among those with significant liver fibrosis compared to those with no significant fibrosis, possibly due to reduced cholesterol synthesis in more advanced forms of liver disease [33, 34].

According to previously published results from the ARIC Study [35], CHD risk was elevated by about 40% for every 1 mmol/L increment in LDL cholesterol. Therefore, the observed difference in LDL concentration between wild-type subjects and C282Y homozygotes (i.e., approximately 0.4 mmol/L) would predict a 16% lower rate of incident CHD among C282Y homozygotes. However, we found did not find a statistically significant association between either HFE C282Y or H63D mutations and risk of coronary heart disease, a finding consistent with other prospective studies [36–39]. Even large cohort studies such as our own are unlikely to detect small reductions in CHD risk possibly mediated by lower LDL concentrations because of the relatively small number of C282Y homozygotes available for follow-up. In contrast to our results, an earlier case-cohort analysis from the ARIC study [40] found that C282Y heterozygotes had a significantly increased risk of CHD after adjustment for traditional risk factors (HR=2.7; 95% CI: 1.2–6.1), but our results do not corroborate that previous analysis. In fully adjusted models identical to those used in the earlier report, hazard ratios for incident CHD in the present analysis were 0.97 (95% CI: 0.63–1.5) for events ascertained through calendar year 1991, the last date of follow-up in the earlier report, and 1.03 (95% CI: 0.88–1.2) when follow-up was extended through 2004. The discrepancy between the earlier report and present analysis is likely due to the fact that C282Y carriers were underrepresented due to random error introduced when only a sample of the cohort was selected for the earlier study. In the earlier report, the frequency of the HFE C282Y mutation in the ARIC cohort was estimated to be 6.1% based on a cohort random sample of only 535 subjects [40]; in the present analysis, the frequency of the C282Y mutation was found to be substantially higher (9.7%) in 10,800 white subjects from the full cohort.

Of the six genotype groups, wild-type subjects had the lowest mean hematocrit and hemoglobin values and highest mean platelet count. Similar patterns have been reported previously [41, 42]. As expected, C282Y homozygotes had higher levels of biomarkers of liver dysfunction and disease, including alanine aminotransferase and the Hepascore index. Hepascore provides an area under the curve of 0.76–0.82 for significant biopsy-determined liver fibrosis among patients with chronic hepatitis C infection [19, 43, 44]. Although only a few subjects had Hepascore values consistent with significant fibrosis, differences between C282Y homozygotes and wild-type subjects may reflect more subtle or early indications of liver damage. Beutler et al. similarly found that plasma collagen IV concentrations, another marker of mild hepatic fibrosis, were significantly higher in homozygotes compared to wild-type subjects [4].

Strengths of the present study include its large sample size, extensive follow-up, and population-based sampling that provides inference to the general population. A systematic review by the U.S. Preventive Services Task Force noted that the many previous studies of HFE may have been plagued by selection bias or lack of population-based comparison groups [9]. The ARIC cohort was enrolled nearly ten years before the discovery of HFE, making it unlikely subjects had been previously screened for mutations or that their participation was related to knowledge of their genotype. Limitations include the limited number of C282Y homozygotes and small number of controls tested for iron and liver biomarkers, incomplete information on liver disease, limited to self-report, hospital and death records, and an index of liver fibrosis (Hepascore) of uncertain validity in the context of hemochromatosis. Recent studies found that 5–20% of C282Y homozygotes had subclinical disease detected by biopsy, including cirrhosis and hepatic fibrosis [6, 45]. In addition, limited information was available regarding clinical diagnosis of hemochromatosis and markers of iron metabolism were only measured once, thereby providing an incomplete history of iron status. Studies of untreated C282Y homozygotes have demonstrated substantial short- and long-term intra-individual variability in serum ferritin and transferrin saturation levels [46, 47].

Speculations

In the ARIC study, C282Y homozygotes had significantly higher levels of serum iron measures and biomarkers of liver function, but there was little evidence that HFE genotypes are related to all-cause mortality, cardiovascular disease, or diabetes. Reduced LDL in C282Y homozygotes may be due to effect of excess iron on cholesterol metabolism and lipoprotein formation in the liver.

Abbreviations

LDL

low density lipoprotein

HDL

high density lipoprotein

CHD

coronary heart disease