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European Journal of Heart Failure 2005 7(1):103-108; doi:10.1016/j.ejheart.2004.03.007
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© 2004 European Society of Cardiology

Hereditary hemochromatosis gene (HFE) mutations C282Y, H63D and S65C in patients with idiopathic dilated cardiomyopathy

Jokke Hannukselaa,b,*, Mari Leppilampia, Keijo Peuhkurinenc, Satu Kärkkäinenc, Eija Saastamoinenc, Tiina Heliöd, Maija Kaartinend, Markku S. Nieminend, Pentti Nieminene and Seppo Parkkilaa,b

a Department of Clinical Chemistry, University of Oulu P.O. Box 5000, Oulu, Finland
b Institute of Medical Technology, University of Tampere and Tampere University Hospital Finland
c Department of Internal Medicine, University of Kuopio Finland
d Department of Internal Medicine, University of Helsinki Finland
e Medical Informatics Group, University of Oulu Oulu, Finland

* Corresponding author. Tel.: +358-50-351-0270; fax: +358-3-215-8597. E-mail address: jokkeha{at}paju.oulu.fi


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: Hereditary hemochromatosis (HH), a common autosomal recessive disease, leads to excessive iron accumulation in some organs, including the heart. It is therefore not surprising that cardiomyopathy is one of the most severe complications of HH. The HFE gene defects have been thought to contribute to idiopathic dilated cardiomyopathy (IDCM) in some patients, even though the results of genotype analyses have so far been contradictory. Hence we set out here to evaluate the prevalence and potential role of HFE mutations in patients with IDCM.

Methods: A total of 91 IDCM patients and 102 controls were subjected to HFE mutation analyses, in which C282Y, H63D and S65C mutations were determined for each patient. We also analyzed the impact of the C282Y and H63D mutations on the left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and New York Heart Association (NYHA) functional classes.

Results: The prevalences of heterozygosity for the C282Y, H63D and S65C mutations in the IDCM patients were 13.2%, 22.0% and 2.2%, respectively. LVEDD was significantly higher (P=0.037) in those with the C282Y mutation at the end of the follow-up period than in those with no mutation.

Conclusions: Our data showed no significant deviations in C282Y, H63D and S65C mutation frequencies between the IDCM patients and controls, suggesting that these mutations do not increase the risk of IDCM. Heterozygosity for the C282Y mutation may nevertheless be a modifying factor contributing to LV dilatation and remodeling.

Key Words: Hereditary hemochromatosis • Hereditary hemochromatosis gene • C282Y • H63D • S65C • Idiopathic dilated cardiomyopathy

Received December 8, 2003; Revised January 6, 2004; Accepted March 20, 2004


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism leading to iron accumulation in parenchymal cells [1]. More than 90% of HH patients carry the C282Y or H63D mutation in the HFE gene, most of them being homozygous for the same missense mutation C282Y [2,3]. The frequencies of C282Y heterozygotes in the North American and European populations are 9.0% and 9.2%, respectively [3]. Corresponding values for H63D heterozygotes are 23% in North America and 22% in Europe. In addition to these mutations, the S65C mutation in the HFE gene has recently been shown to be clinically significant, although it seems to be associated with a mild form of HH [4,5]. The reported carrier frequencies of the S65C mutation in Caucasians are 1-4% [4–9]. Untreated HH can lead to severe complications due to an iron overload in some organs, including liver cirrhosis, diabetes, hypogonadotrophic hypogonadism, arthritis and cardiomyopathy [10].

Idiopathic dilated cardiomyopathy (IDCM), a primary myocardial disease, is characterized by ventricular dilation and impaired systolic function. It has been demonstrated that several cases previously thought to be idiopathic are indeed familial, and future molecular genetic studies may improve our knowledge of the etiology of IDCM [11,12]. Cardiomyopathy is one of the most severe complications of HH, and physicians have been aware of iron overload cardiomyopathies for a long time. Interestingly, hemochromatosis protein (HFE) bears structural similarities to major histocompatibility complex class I molecules, and these molecules are probably involved in the pathogenesis of a subgroup of dilated cardiomyopathy and influence the severity of the disease [13,14]. Since the discovery of the HFE gene, three studies have been published in which HFE mutations are analyzed in patients with cardiomyopathy. The hypothesis that HFE gene mutations account for a subgroup of IDCM was supported by the findings of Mahon et al. [15]. Even though they concluded that body iron status does not correlate with the severity of the disease or reduced survival in IDCM patients, they observed that the frequency of the H63D mutation in the HFE gene is significantly increased in patients with IDCM. Two later studies have not found any such association between H63D mutation and IDCM, however [16,17].

In the present study we determined the prevalence of the C282Y and H63D mutations in order to clarify their association with IDCM. Furthermore, we studied whether HFE mutations modify the phenotype of the disease. For the first time, we also studied the prevalence of the S65C mutation in patients with IDCM.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Subjects
The evaluation of the 91 IDCM patients from Eastern and Southern Finland (mean age 46.2, standard deviation (S.D.) 12.4, 64 male and 27 female) included HFE mutation analysis and phenotypic characterization. The patients were consecutively recruited and evaluated, and the diagnosis of IDCM was based on the commonly approved diagnostic criteria for DCM (left ventricular ejection fraction (LVEF)<45% and left ventricular end diastolic diameter (LVEDD)>27 mm/m2) and exclusion of secondary causes of DCM [18]. The patients were evaluated in terms of a personal and family history, physical examination, 12-lead electrocardiograph (ECG), chest X-ray and transthoracic echocardiography (M-mode, two-dimensional and Doppler). Altogether 88% of the patients had undergone diagnostic coronary angiography. C282Y, H63D and S65C mutations in the HFE gene were determined for each patient. The control group was a cohort of 102 healthy subjects (medical students and laboratory personnel) whose blood was also subjected to C282Y, H63D and S65C mutation analyses. The mean follow-up period of IDCM patients from the diagnosis was 6.0 years (S.D. 4.7). The investigation conforms to the principles outlined in the Declaration of Helsinki.

2.2. HFE genotyping
DNA was isolated from peripheral blood WBCs using a blood kit (Nucleospin, Macherey-Nagel, Düren, Germany) or by the proteinase-K phenol-chloroform or salt-precipitation method. Parts of the HFE gene were amplified by PCR using the sets of primers described by Feder et al. [2] and Jeffrey et al. [19]. The PCR protocol was the same as described earlier by Datz et al. [20], and the amplification products were then digested with the restriction enzymes SnaB I (New England BioLabs, Beverly, MA, USA), Bcl I (New England BioLabs) and Hinf I (New England BioLabs), since the C282Y mutation creates a restriction site and the H63D and S65C mutations abolish one. Digestion products were resolved on a 1.2% agarose gel containing nucleic acid gel stain (GelStar, BioWhittaker Molecular applications, Rockeland, ME, USA) and visualized by UV light stimulation.

2.3. Statistical analysis
The statistical differences between the prevalences of the various HFE genotypes in the IDCM patients and the control group were evaluated with the {chi}2-test and Fisher's exact probability test. The LVEDD and LVEF measurements are presented in the form of means and S.D. To evaluate the potential confounding effects of age, gender and medication, we analyzed the association of these variables with LVEDD and LVEF using correlation coefficients and Student's two sample t test. Differences in medication between the various categories of HFE genotypes before the diagnosis of IDCM and during the follow-up period were evaluated with {chi}2-test and Fisher's exact probability test. On this basis, statistical significances of the impact of various HFE genotypes and confounding factors on LVEDD and LVEF were assessed with a two-way analysis of variance. Multinomial logistic regression analysis [21] was used to evaluate the effect of various HFE genotypes on the New York Heart Association (NYHA) classes and analysis of variance with repeated measurements on alterations in LVEDD and LVEF within the follow-up period. The effect of duration of follow-up period was adjusted using the two-way analysis of variance and analysis of repeated measurements. The statistical analyses were performed using SPSS for Windows software (version 11.5).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
The frequencies of the C282Y, H63D and S65C mutations in the patients with IDCM were 13.2%, 22.0% and 2.2%, respectively, and these frequencies did not differ significantly from those observed in the control group (Table 1). The C282Y and H63D mutation frequencies observed in the control group were almost the same as those reported earlier by Parkkila et al. [22] in the same district. Hence our results confirm the relatively high frequencies of C282Y and H63D mutations in healthy population in Finland, equal to the frequencies observed in patients with IDCM.


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  		Table 1 Genotypic (a) and allelic frequencies (b) of HFE mutations among patients with idiopathic dilated cardiomyopathy (IDCM) and in the control group

 
We also evaluated the correlation of the C282Y and H63D mutations with the severity of symptoms and echocardiographic changes typical of IDCM. Parameters describing the severity of the clinical manifestations in C282Y and H63D heterozygous vs. wild type IDCM patients are presented in Table 2. Although the difference in LVEDD between the C282Y heterozygous and wild type patients did not reach statistical significance (P=0.106) at the time of diagnosis, a statistically significant LVEDD effect (P=0.037) was found between these patients at the end of the follow-up period. There was no statistically significant association between the HFE genotypes and LVEF or NYHA functional class. The changes in LVEDD or LVEF between the various categories of HFE genotypes during the follow-up were relatively small (Table 2). The effects of S65C heterozygosity on the variables were not calculated due to the low number of cases.


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  		Table 2 Comparison of clinical characteristics between the categories of HFE genotypes in the patients with idiopathic dilated cardiomyopathy

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The present series of mutation analyses performed on the HFE gene showed no significant changes in C282Y, H63D or S65C mutation frequencies in the IDCM patients as compared with the controls. Even though it is known that cardiomyopathy is one of the most severe complications of HH, the major conclusion to be drawn here is that these mutations per se do not seem to increase the risk of IDCM. Nevertheless, we cannot exclude the possibility that these mutations could contribute to the progression of the disease or the severity of its manifestations, at least in some patients.

The clinical impact of the HFE mutations on the severity of IDCM manifestations has largely remained unresolved. Mahon et al. [15] reported previously that C282Y heterozygous IDCM patients have less ventricular dilation and better fractional shortening. We, on the contrary, found that the left ventricles of the C282Y heterozygous IDCM patients were more dilated than those of the wild types, and that the difference became evident at the end of the follow-up period (Fig. 1a). There were no significant differences in the ejection fractions between the C282Y heterozygous and wild type patients, even though the LVEF values tended to improve only a little in the C282Y heterozygous patients during the follow-up period (Fig. 1b). The contradictions between these two studies may represent a sporadic bias or a genetic heterogeneity between the two distant populations studied, but we cannot rule out the possibility that C282Y mutation may play a role in modifying the pathophysiology of IDCM. It is also noteworthy that Mahon et al. [15] neither provided information about the medical treatment of the IDCM patients nor reported whether the difference in duration of the follow-up period between the C282Y heterozygous and wild type patients had any implications for their results.


Figure 1
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  		Fig. 1 Differences in (a) left ventricular end-diastolic diameter (LVEDD) and (b) ejection fraction (LVEF) between the C282Y heterozygotes (HET) and wild type (WT) patients with idiopathic dilated cardiomyopathy within the follow-up period.

 
Potential confounding effects of different medications and duration of the follow-up period between the C282Y heterozygous and wild type IDCM patients were considered in the statistical analyses of this study. The IDCM patients were followed for an average of six years (95% confidence interval, 4.9 to 7.0), and no statistically significant difference was observed in the length of the follow-up period between the C282Y heterozygous and wild type patients. Medications slightly differed between the various HFE categories before the IDCM diagnosis. This could possibly contribute to the difference in LVEDD between the C282Y heterozygous and wild type patients at the time of diagnosis, and explain why this difference did not reach statistical significance. Medications cannot, however, account for the difference observed in LVEDD at the end of the follow-up period and LVEF tended to improve only to a minor extent in the C282Y heterozygotes during the follow-up period. The therapeutic treatment that was administered for IDCM in C282Y heterozygous and wild type patients is shown in Table 3. The medical therapy that may influence on the long-term progression of LV remodeling, i.e. beta-blockers and blockers of the renin angiotensin aldosterone system, was very similar in carriers of the C282Y mutation to that given to the wild type patients [23–27]. Our findings point to a possibility that patients in the other HFE categories may have a better response to medical treatment and that C282Y heterozygotes may eventually generate more severe clinical features of IDCM. It has been suggested that left ventricular size is greater in patients having DCM with more severe symptoms [27] and that end diastolic left ventricular diameter could be an independent predictor of mortality in these patients [15,28], although opposite findings have also been reported [29,30]. Thus, it is difficult to assess the true clinical significance of our findings at the moment.


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  		Table 3 Medications in C282Y heterozygous (HET) and wild type (WT) patients with idiopathic dilated cardiomyopathy during the follow-up period

 
We did not find any differences between the H63D heterozygous and wild type patients in parameters describing the severity of the disease, and the effects of the C282Y and H63D mutations on alterations in LVEDD or LVEF were small during the follow-up. Further studies are required to show whether heterozygosity for the C282Y mutation has a substantive effect on the progression or severity of IDCM. A large number of participants would anyway be required in order to study the potential effects of various combinations of C282Y, H63D and S65C mutations on the phenotype of IDCM.

The frequency of the S65C mutation was determined for the first time in IDCM patients showing no significant difference relative to the control group. Furthermore, the low number of S65C mutations within the IDCM patients suggests that it is unlikely that this mutation alone is of any great importance for IDCM. The frequency of the S65C mutation has not been determined earlier for a healthy population in Finland, and the result (3.9%) was quite consistent with those reported in North America and Europe [4–9]. The high frequencies of the HFE mutations observed among the present control individuals are in agreement with other studies of Caucasian populations [3], thus suggesting that HH is a seriously underdiagnosed disease [31]. There has recently been intensive debate on the penetrance of the HFE mutations in general, and it would be fascinating to determine in future studies the definite penetrance of these mutations for cardiomyopathy.


   Acknowledgements
 
The work was supported by grants from Maud Kuistila Foundation to J.H, from Sigrid Juselius Foundation and Academy of Finland to S.P and from Finnish Foundation for Cardiovascular Research and Aarne and Aili Turunen Foundation to K.P. We greatfully acknowledge professor Aimo Ruokonen for helping with sample collection. We thank Anja Heikkinen, Sini Puustinen and Marjut Huovinen for technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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