Volume 25, Issue 1 , Pages 25-30, January 2011
Mutation H63D in the HFE gene confers risk for the development of type 2 diabetes mellitus but not for chronic complications☆☆☆
Article Outline
Abstract
Purpose
To evaluate the frequency of mutations in the HFE gene (C282Y and H63D) in type 2 diabetes mellitus (DM) patients and their possible association with diabetic chronic complications.
Methods
A case-control study with 723 subjects was performed. All diabetic subjects (n=519) underwent a clinical and laboratory evaluation. Diabetic retinopathy (DR) was evaluated by an ophthalmologist. Diabetic nephropathy (DN) was categorized by urinary albumin excretion (UAE) as normoalbuminuria (n=247), microalbuminuria (n=68), macroalbuminuria (n=70), or the presence of end-stage renal disease (dialysis; n=134). Data available for blood donors (n=204) were limited to age, sex, body mass index, and absence of previous diagnosis of diabetes and normal fasting plasma glucose. The mutations C282Y and H63D in the HFE gene were genotyped based on PCR protocols and digested with the restriction enzymes SnabI (C282Y) and MboI (H63D).
Results
There was an association of type 2 DM with H63D polymorphism (genotypes HD/DD: OR=1.7, 95% CI=1.2–2.6), but not with C282Y polymorphism (OR=0.7, 955 CI=0.4–1.4). In respect to the chronic complications, there was no difference in the prevalence of DR, DN, or ischemic heart disease among the different genotypes.
Conclusions
Mutation H63D in the HFE gene was associated with a higher risk of type 2 DM, but did not appear to confer risk for diabetic chronic complications. The mutation C282Y was not associated with diabetes or its chronic complications.
Keywords: Diabetes mellitus, Genetic predisposition, Hemochromatosis, HFE gene
1. Introduction
Iron is a transitional metal whose serum levels must be closely regulated to avoid diseases associated with both its lack (iron deficiency anemia) and its accumulation (hemochromatosis). Hereditary hemochromatosis (HH) is a classical example of iron overload and its complications. HH is an autosomal recessive iron metabolism disorder in which there is excessive absorption of this element compared with the amount needed by the body. Initially, this leads to the expansion of plasma iron (with increased transferrin saturation), with a later increase of body iron reserves (rise of ferritin) (Pietrangelo, 2006). Accumulation is progressive and, consequently, it compromises several organs (liver, pancreas, pituitary gland, heart, and joints). Mutations in the HFE gene are responsible for most cases of HH in European descendants (Feder et al., 1996). Protein HFE binds to β2-microglobulin favoring cell uptake of transferrin-bound iron, and it also modulates the expression of hepcidin, the “iron-regulating hormone” (Pietrangelo, 2006). Nevertheless, the precise mechanism by which HFE regulates iron metabolism has not yet been fully explained. Two mutations in this gene are mainly responsible for HH: C282Y (substitution of tyrosine by cysteine in Position 282) and H63D (substitution of histidine by aspartic acid in Position 63) (Feder et al., 1996). Most patients with HH are homozygotes for mutation C282Y and only 1–2% are compound heterozygotes (C282Y/H63D) (Rochette et al., 1999).
Diabetes mellitus (DM) develops in 10–40% of patients with HH. This depends mainly on how the diagnosis is made (genotypic, phenotypic, or laboratory) (McClain et al., 2006). Therefore a higher prevalence of mutations in the HFE gene could be expected among patients with type 2 DM. However, this issue is still controversial. Some authors report a higher prevalence of mutations in HFE among patients with type 2 DM (Conte et al., 1998), and others do not show this association (Halsall, McFarlane, Luan, Cox, & Wareham, 2003). Moreover, some studies have correlated changes in iron metabolism with the development of type 2 DM (Ford & Cogswell, 1999) and its complications (Loebstein et al., 1998). Mutations in the HH gene have been described as associated with diabetic nephropathy (DN) and proliferative diabetic retinopathy (DR) in Caucasian patients with type 2 DM (Moczulski et al., 2001, Peterlin et al., 2003).
The aim of this study was to evaluate the prevalence of HH mutations (C282Y and H63D) in white patients with type 2 DM from southern Brazil and to determine whether these mutations were associated with diabetic complications.
2. Research design and methods
A case-control study was performed based on 723 white subjects. Patients with type 2 DM (n=519) were identified from a multicentric study that started recruiting patients in Southern Brazil in 2002 (Canani et al., 2005). That project aims at studying risk factors for chronic complications of DM in a state's representative sample of patients with type 2 DM. It includes five centers located at general hospitals in the state of Rio Grande do Sul, namely, Grupo Hospitalar Nossa Senhora da Conceição, Hospital São Vicente de Paulo, Hospital Universitário de Rio Grande, Fundação Universitária de Rio Grande, and Hospital de Clínicas de Porto Alegre. Type 2 DM was defined by diagnosis of diabetes after the age of 35 years with no use of insulin during the first 5 years after diagnosis. DNA samples from nondiabetic white blood donors were identified from a central blood bank and were used as controls (n=204). This blood bank concentrates donations from the entire state. The ethnicity was self-defined by the subject. The white individuals in Rio Grande do Sul are descended mainly from Portuguese, Spanish, Italian, and German immigrants (Crispim et al., 2005).
2.1. Subjects evaluation
The clinical evaluation of patients with type 2 DM included measuring weight and height on an anthropometric scale (without shoes and in light clothing); body mass index (BMI) was calculated (weight in kilograms divided by height in meters squared); and the mean of two measurements of systemic arterial blood pressure (BP), after a 5-min interval, in a seated position, was recorded with a mercury column sphygmomanometer (Phases I and V of the Korotkoff sounds). Hypertension was defined as BP ≥140/90 mmHg and/or use of antihypertensive medication.
DR was evaluated by an ophthalmologist who performed direct fundoscopy and was classified as absent, nonproliferative (microaneurysms, hard exudates, retinal hemorrhages, and intraretinal microvascular abnormalities), or proliferative (newly formed blood vessels and/or growth of fibrous tissue into the vitreous cavity) (Boelter et al., 2006). Patients with panphotocoagulation were classified as presenting proliferative DR.
DN was categorized according to urinary albumin excretion (UAE; three samples, without ACE inhibitors or angiotensin type I receptor blockers for at least 1 week) as normoalbuminuria (n=247), microalbuminuria (n=68), macroalbuminuria (n=70), or the presence of end-stage renal disease defined as patients performing hemodialysis for at least 3 months (n=134). Two different types of UAE collections were used to define DN: 24-h timed urine collections or random spot urine samples. The cutoff values used to define the stages of renal involvement followed American Diabetes Association recommendations for timed urine collections (µg/min). For spot samples, urine concentration (mg/l) cutoffs were used as previously validated in our central laboratory (Zelmanovitz et al., 1997) as normoalbuminuria (UAE <20 µg/min or <17 mg/l), microalbuminuria (UAE 20–199 µg/min or 17–174 mg/l), and macroalbuminuria (UAE >200 µg/min or >174 mg/l).
Ischemic heart disease was evaluated as previously described (Costa, Canani, Lisboa, Tres, & Gross, 2004) in 381 patients. Briefly, ischemic heart disease was established as the presence of angina or possible infarct according to the World Health Organization Cardiovascular Questionnaire, and/or the presence of resting ECG abnormalities [Minnesota Code: Q and QS patterns (1.1–2, 1.3); S-T junction (J) and segment depression (4.1–4); T-wave items (5.1–3) and complete left bundle block (7.1)], and/or the presence of perfusion abnormalities (fixed or variable) upon myocardial scintigraphy at rest and after dipyridamole administration.
Data available on the blood donors were limited to age, sex, BMI, and absence of previous diagnosis of diabetes and/or fasting plasma glucose <100 mg/dl.
2.2. Laboratory evaluation
The laboratory evaluation included measuring UAE by immunoturbidimetry (Sera-Pak immunomicroalbuminuria; Bayer, Tarrytown, NY, USA; intra- and interassay coefficients of variation: 4.5% and 11.0%, respectively); A1c test by an ion-exchange high-performance liquid chromatography procedure (Merck-Hitachi L-9100 glycated hemoglobin analyzer, reference range 2.7–4.3%; Merck, Darmstadt, Germany); glucose using the glucose oxidase method; total cholesterol, HDL, and triglycerides by enzymatic methods; creatinine by the Jaffé reaction; and the estimated glomerular filtration rate (eGFR) was calculated by the abbreviated Modification of Diet in Renal Disease Study formula (Levey et al., 1999).
2.3. Molecular analysis
Total DNA was extracted from peripheral blood leucocytes. All participants were genotyped by polymerase chain reaction with specific primers and conditions for mutation C282Y (Jeffrey, Chakrabarti, Hegele, & Adams, 1999) and H63D (Feder et al., 1996). The material of this PCR was then digested with the restriction enzymes SnabI (C282Y) and MboI (H63D). The products resulting from cleavage with the restriction enzymes were separated into agarose gels at 2.5%, stained with ethidium bromide, according to the size of each fragment to be studied.
These HFE mutations were selected because they have been previously related to significant alterations in the iron metabolism (Pietrangelo, 2006).
2.4. Statistical analysis
Considering that the prevalence of C282Y mutations in the American population is 9.5% (Hanson, Imperatore, & Burke, 2001), the study was powered to detect an odds ratio (OR) of 2.2 at 5% significance and 80% power with a cases–controls ratio of 2:1.
The χ2 test with Yates's correction was used to compare the frequency of alleles and genotypes among the groups. The continuous data were compared using the Student's t test and categorical data with the χ2 test. Variables without normal distribution (UAE, serum creatinine, and triglycerides) were log transformed for analysis. Continuous data were presented as mean±S.D. or median with range. The Hardy–Weinberg equilibrium was examined using the χ2 test. Because of the small prevalence of the H63D and C282Y polymorphisms the major genotypes were compared to the other two groups (HH vs. HD/DD and CC vs. CY/YY). The population attributive risk was calculated using the following equation: 100×[prevalence (OR−1)/prevalence (OR−1)+1]. The magnitude of effect was assessed through the OR (Rockhill, Newman, & Weinberg, 1998). A two-sided P value <.05 was considered statistically significant. All statistical analyses were performed using the software SPSS version 13.0 (SPSS, Chicago, IL, USA).
3. Results
The proportions of female patients in the type 2 DM group and in the blood donors were 50.3% (n=261) and 48.5% (n=99), respectively, with a mean age of 60.3±9.9 and 52.6±8.8 years (P<.001). The genotype frequency did not deviate from the Hardy–Weinberg equilibrium in the control group for both mutations. However, in the type 2 DM group, the frequency of homozygotes for mutation C282Y (two patients) was higher than expected (CC/CY/YY—472/27/02, P=.023), while the mutation H63D remained at the Hardy–Weinberg equilibrium (HH/HD/DD—355/151/13, P=.51). The distribution of HFE genotypes did not differ statistically among sexes in either group.
The HD/DD genotypes (H63D) were more prevalent in the group of patients with type 2 DM (OR=1.7, 95% CI=1.2–2.6] (Table 1). The frequency of D allele was 0.17 in patients with type 2 DM compared to 0.10 in the control group (P=.003). Considering a 21.1% frequency of risk with the genotypes combined (HD/DD) in the general population, the attribute risk for this mutation to predispose to DM in the white Brazilian population is 12.9%. Regarding the C282Y polymorphism and compound heterozygotes (i.e., H63D plus C282Y heterozygosis), no differences were found between diabetic and nondiabetic subjects (four patients in both groups).
Table 1. Frequency of mutations H63D and C282Y in the HFE gene among patients with and without type 2 DM
| HFE mutation | Blood donors, n (%) | Type 2 DM, n (%) | OR (95% CI) | P |
|---|---|---|---|---|
| H63D | ||||
 Genotype | ||||
| HH | 161 (78.9) | 355 (68.4) | 1 | |
| HD/DD | 43/0 (21.1/0.0) | 151/13 (29.1/2.5) | 1.7 (1.2–2.6) | .006 |
| Alleles | ||||
| H | 365 (89.5) | 861 (83.0) | ||
| D | 43 (10.5) | 177 (17.0) | .003 | |
| C282Y | ||||
| Genotype | ||||
| CC | 185 (92.0) | 472 (94.2) | 1 | |
| CY/YY | 16/0 (8.0/0.0) | 27/2 (5.4/0.4) | 0.7 (0.4–1.4) | .373 |
| Alleles | ||||
| C | 386 (96.0) | 971 (96.9) | ||
| Y | 16 (4.0) | 31 (3.1) | .503 | |
Table 2 describes the clinical and laboratory characteristics of the group of patients with type 2 DM according to the H63D mutation, since in the present study only this mutation was associated with DM. Patients with D allele (HD/DD) had higher fasting plasma glucose (166.9±67.6 vs. 181.5±75.8 mg/dl, P=.04); however, there was no difference between groups in the A1c test (6.9±2.1% vs. 7.0±2.2%, P=.59). Males with D allele (HD/DD) have lower waist circumference (99.7±10.3 vs. 95.8±10.8 cm, P=.05). Regarding the other variables, the two groups were similar.
Table 2. Characteristics of patients with type 2 DM
| All (N=519) | Mutation H63D | P⁎ | ||
|---|---|---|---|---|
| HH (n=355) | HD/HD (n=164) | |||
| Age (years) | 60.3±9.9 | 60.5±10.1 | 60.1±9.6 | .73 |
| Male sex, n (%) | 258 (49.7) | 175 (49.3) | 83 (50.6) | .85 |
| Diabetes duration (years) | 14.3±8.1 | 14.4±8.3 | 14.0±7.8 | .61 |
| Age at diagnosis (years) | 46.4±10.2 | 46.3±10.3 | 46.6±10.0 | .82 |
| Waist (cm) | ||||
| Male | 98.6±10.6 | 99.7±10.3 | 95.8±10.8 | .05 |
| Female | 97.4±12.0 | 97.9±12.2 | 96.3±11.6 | .47 |
| BMI (kg/m2) | 28.5±4.8 | 28.6±4.8 | 28.1±4.8 | .29 |
| Systolic blood pressure (mmHg) | 144.9±24.2 | 145.1±24.2 | 144.5±24.3 | .80 |
| Diastolic blood pressure (mmHg) | 86.1±13.1 | 86.0±12.6 | 86.5±14.3 | .69 |
| Hypertension (%) | 72.3 | 72.3 | 72.3 | 1.00 |
| Glucose (mg/dl) | 171.4±70.4 | 166.9±67.6 | 181.5±75.8 | .04 |
| A1c test (%) | 7.0±2.1 | 6.9±2.1 | 7.0±2.2 | .59 |
| Total cholesterol (mg/dl) | 206.3±46.0 | 204.3±46.7 | 211.2±43.9 | .21 |
| LDL-C (mg/dl) | 127.9±45.6 | 130.0±47.1 | 122.4±41.3 | .20 |
| HDL-C (mg/dl) | 44.1±11.8 | 43.9±11.3 | 44.6±13.1 | .57 |
| Triglycerides (mg/dl), median (range) | 185.0 (42–1265) | 199.3 (42–892) | 179.5 (47–1265) | .20 |
| UAE (µg/min), median (range) | 11.3 (0.1–10,100) | 7.4 (0.1–10,100) | 10.1 (1.0–7680) | .23 |
| Creatinine (mg/dl) | 1.7±2.1 | 1.7±2.1 | 1.7±1.9 | .99 |
| eGFR (ml/min per 1.73 m2) | 67.2±31.1 | 67.1±31.1 | 67.4±31.3 | .94 |
| Diabetic nephropathy (n=515) | ||||
| Microalbuminuric, n (%) | 67 (13.0) | 40 (11.3) | 27 (16.8) | .12 |
| Macroalbuminuric/ESRD, n (%) | 202 (39.2) | 136 (38.4) | 66 (41.0) | |
| Diabetic retinopathy (n=390) | ||||
| Nonproliferative, n (%) | 124 (31.8) | 89 (33.2) | 35 (28.7) | .66 |
| Proliferative, n (%) | 96 (24.6) | 64 (23.9) | 32 (26.2) | |
| Ischemic heart disease (n=381) (%) | 42.5 | 40.8 | 46.5 | .31 |
⁎P for HH vs. HD+DD. |
The prevalence of DN, DR, and ischemic heart disease was not different between those carrying the D allele and those without it (Table 2). Likewise, there were no differences concerning the frequency of diabetic chronic complications and the C282Y mutation (data not shown). Because of the different clinical manifestations of HH among men and women, a stratified analysis was performed by gender. There were no differences in the frequency of chronic complications regarding genotypes (data not shown).
4. Discussion
In the present study, the H63D polymorphism was associated with type 2 DM in the Brazilian population of European descent. This is the first study to address the role of HH genes in DM in this population. However, both mutations evaluated (C282Y and H63D) were not related with micro- and macrovascular complications of DM.
The H63D mutation was associated with presenting type 2 DM (OR=1.7). On the other hand, no increased risk was observed for the C282Y mutation. These results are in accordance with those found in a recent meta-analysis of 17 studies (4245 patients with type 2 DM and 5982 controls) that did not find an association between mutation C282Y and the risk of type 2 DM (OR=1.0, 95% CI=0.9–1.2) but showed a moderately increased risk in the carriers of mutation H63D (OR=1.1, 95% CI=1.0–1.2) (Qi et al., 2005). Furthermore, H63D mutation has been described in linkage disequilibrium with other genetic variants (Cardoso et al., 2002). This association of H63D and other polymorphisms could explain the discordant results between different HFE gene mutations (C282Y and H63D) and risk of DM, because they may interact in the pathological process.
Previous studies reported an association between HFE mutations and the microvascular complications of DM such as DN (Moczulski et al., 2001) and DR (Peterlin et al., 2003). Moczulski et al. (2001) found a higher prevalence of DN among those with the H63D mutation (OR=1.8, 95% CI=1.2–2.8). Peterlin et al. (2003) reported an association of proliferative DR and the C282Y mutation (OR=3.0, 95% CI=1.2–8.0). In the present study, no association was found with either complication. One explanation for these discrepancies could be due to the characteristics of patients included. For instance, the prevalence of arterial hypertension and metabolic control were distinct in the Moczulski et al. study and ours. Regarding DR, the subjects included in the Peterlin et al. study had a longer duration of DM compared to those included in the present study (18.2 vs. 14.3 years, P<.001). DM duration is a well-known risk factor for developing DR. An alternative explanation could be due to other genetic environmental interactions. Qi et al. (2005) showed that the risk for developing DM associated with HFE gene mutations is modulated by the amount of heme iron in the diet. Therefore, local nutritional habits could explain some of the discordant results.
The relationship between HFE mutations and cardiovascular disease is a controversial issue. Some prospective studies suggested a greater risk of cardiovascular events among heterozygotes for C282Y mutation (Tuomainen et al., 1999). However, others (Campbell et al., 2003) failed to show the same relationship. A recent meta-analysis did not confirm the association of the mutations, C282Y or H63D, and cardiovascular disease (van der et al., 2006). The present study suggests that these mutations do not have a major effect on cardiovascular disease in patients with type 2 DM.
Recently, another study also evaluated HFE mutations and the prevalence of chronic diabetic complications. In the same way, no correlation was observed among ND, RD, ischemic heart disease, and C282Y mutation or compound heterozygosity (C282Y/H63D) (Davis et al., 2008). However, the role of H63D mutation alone was not evaluated.
Dubois-Laforgue, Caillat-Zucman, Boitard, and Timsit (2000) described a lower prevalence of obesity in subjects with type 2 DM and H63D mutation. Likewise, in the present study the H63D mutation was associated with a lower waist circumference in males.
The mechanism by which the iron metabolism could be associated with higher DM susceptibility has not yet been fully explained. None of the subjects in the present study has clinically overt HH. The expected effect on the predisposition to DM would be due to a selective toxic effect of iron on the beta cell. This occurs due to the high number of divalent metal transporter 1 (DMT1) in the beta cells which are necessary to transport the zinc used in processing the insulin granules (Andrews, 1999). Iron influx through this pathway and particular susceptibility of beta cells to oxidative stress might lead to apoptosis and reduction in the beta cell mass (Cooksey et al., 2004). Furthermore, the iron overload also reduces the hepatic sensitivity to insulin (Dmochowski, Finegood, Francombe, Tyler, & Zinman, 1993). Finally, the combination of reduced beta cell mass and insulin resistance results in hyperglycemia and development of DM (Hramiak, Finegood, & Adams, 1997).
The surplus number of homozygotes for mutation C282Y found in the present study, which disturbed the Hardy–Weinberg equilibrium, was also shown in other populations (Ellervik et al., 2001, Beutler et al., 2002). This could be explained by the higher tendency of individuals in the same demographic area to be related. In the present study, however, this is not likely to have occurred due to its multicentric design. Genotyping errors are unlikely, since samples with the mutations were confirmed in duplicate by independent experiments. Most probably, this is a statistical finding by chance since the significance comes from the excess of only one subject.
Case-control studies are more likely to have sampling biases; therefore to minimize this, we included cases from several centers all over the state of Rio Grande do Sul. In the same way, the controls were selected from a blood bank that received donations from the entire state. In this way, a putative representative and similar sample of cases and controls was obtained. We cannot exclude, however, that further studies increasing the number of patients could better clarify the findings obtained in our study. These studies could corroborate our results or detect small differences in the prevalence of chronic diabetic complications not found here.
In conclusion, there is an association between the H63D mutation of the HFE gene and type 2 DM. However, this mutation did not increase the risk of chronic complications of DM. Future studies should evaluate whether other genetic and environmental factors that modify iron metabolism are involved in the increase of the risk of type 2 DM.
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☆ Conflict of interest: the authors do not have any conflict of interest.
☆☆ This study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundo de Incentivo à Pesquisa (FIPE) do Hospital de Clínicas de Porto Alegre. LHC was a recipient of a grant from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). MLC is a recipient of a scholarship from the Brazilian Coordination for the Improvement of Higher Education Personnel.
PII: S1056-8727(09)00133-0
doi:10.1016/j.jdiacomp.2009.12.002
© 2011 Elsevier Inc. All rights reserved.
Volume 25, Issue 1 , Pages 25-30, January 2011
