Význam DNA vyšetření mutací C282Y, H63D a S65C v HFE genu

Authors: Monika Drastíková;  Martin Beránek;  Jaroslava Hegerová;  Daniela Putzová
Authors place of work: Univerzita Karlova v Praze, Lékařská fakulta Hradec Králové, Ústav klinické biochemie a diagnostiky FN
Published in the journal: Čas. Lék. čes. 2012; 151: 428-431
Category: Původní práce


Hereditary hemochromatosis is a relatively common genetic disease characterized by increased iron absorption and deposition in major organs of the body. The aim of this study was to determine the prevalence of C282Y, H63D and S65C mutations in the HFE gene in patients suspected of hereditary hemochromatosis and to compare it with healthy subjects (control group).

Material and methods:
The group of patients consisted of 95 males and 45 females (median age 55 years, range 20 to 83 years). The control group was represented by 167 volunteers of Caucasian origin (65 males and 102 females, median age 25 years, range 18 to 62 years). The PCR/RFLP genetic analysis was used to detect mutations in the HFE gene.

Allelic frequencies of C282Y, H63D, and S65C in the groups of patients were 18.2 %, 17.5 %, and 1.8 %, respectively. The frequencies of the alleles in the control group were 5.7 % (C282Y), 12.3 % (H63D), and 0.6 % (S65C).

Our results show significant differences in the frequency of C282Y mutation between the patients suspected of hereditary hemochromatosis and the control group (18.2 % vs. 5.7 %). The prevalence of H63D and S65C mutations in both groups was not statistically significant.

Key words:
hemochromatosis, HFE gene, C282Y, H63D, S65C, PCR/RFLP


Hereditary hemochromatosis is an inherited disease characterized by increased iron absorption from the intestine and deposition in the liver, heart, pancreas, pituitary gland, joints, and skin. The excessive iron accumulation in the liver influences hepatic stellate cell activation and transformation into myofibroblast–like cells producing higher amounts of collagen type I., II. and IV. (1, 2). The collagen hypersecretion contributes to hepatic fibrosis. HH is associated with overproduction of hydroxyl radicals by Fenton's reagent, which causes hepatocyte necrosis and hepatocellular carcinoma (3, 4, 5).

The prevalence of HH in the Caucasian population is approximately 1:200–500 (6, 7) and is among the most common inherited disorders. Hereditary hemochromatosis is divided into five subtypes: HH1 – HH5.  HH1 is the most frequent subtype and is caused by mutations in the HFE gene. Other subtypes are associated with genetic polymorphisms in genes for hemojuvelin, hepcidin, transferrin receptor 2, ferroportin, transferrin, ceruloplasmin and H-chains of ferritin. The prevalence and clinical symptoms of HH2–HH5 are not as significant as in subtype HH1 (8, 9). 

In 1996, the HFE gene was located on the short arm of chromosome 6 (6p21.3). It is a member of the major histocompatibility complex class I family with high homology to HLA I. class genes. The HFE protein with 343 amino acids is made up of three extracellular domains α1–α3. The HFE α3 domain is non-covalently bound to β2-microglobulin and this heterodimer is transported to the cell surface where it occupies the transferrin receptor (10). Diferric transferrin can crowd out the HFE protein from this bound and thus indicates a sufficient level of iron in the organism. Free HFE protein modulates hepcidin expression which, due to ferroportin degradation, decreases iron intake from enterocytes, hepatocytes and macrophages into the blood’s circulation (11).

A missense mutation C282Y in the fourth exon of the HFE gene is the most common cause of HH1 in Caucasians. Due to the substitution of tyrosine for cysteine at amino acid 282, the HFE protein loses one of four disulfide bonds. This conformation change influences the affinity of HFE to β2-microglobulin and its transport on the cell surface, thus resulting in increased iron absorption.

The second significant genetic change in the HFE gene is a substitution of aspartate for histidine at amino acid 63 (H63D). This mutation does not affect the production of HFE/β2-microglobulin heterodimers and the clinical symptoms may not manifest or can be milder. The mechanism of the higher iron uptake has not yet been accurately described. Also, the role of the third HFE mutation (S65C) in the pathogenesis of HH is not clear.

HH1 is an autosomal recessive disease whose phenotype is predisposed by the presence of mutations in both HFE alleles. Subjects with clinical symptoms of HH are either homozygotes for the same type of mutations (very often with genotype 282Y/282Y) or compound heterozygotes (2–6 % cases of HH) (12, 13).

The first symptoms of HH usually appear between the ages of 40 and 50 but they are relatively nonspecific. Initial complaints may include fatigue, hepatomegaly, and joint and muscle pain; in advanced stages of HH irregular heartbeat, hypogonadism, diabetes mellitus, cirrhosis and hepatocellular carcinoma may also appear. The signs of HH are frequently complicated by other disease like liver steatosis, alcoholism, hematopoiesis disorders, metabolic syndrome, etc.

The diagnosis of HH is based on a clinical and physical examination and biochemical tests, including transferrin saturation, serum ferritin, serum iron and hepatic iron concentration. Biochemical analysis should be followed by molecular genetic testing, which is important for finding the cause of the disease and for screening relatives of patients with an increased susceptibility of developing the phenotype. Without appropriate laboratory diagnostics the nonspecific signs of HH could be overlooked and the disease could be left untreated in the long term.

The aim of this study was to determine the prevalence of C282Y, H63D and S65C mutations in the HFE gene in patients suspected of hereditary hemochromatosis and to compare it with healthy subjects (control group).

Material and Methods

The experimental group contained 140 patients (95 men, 45 women at a median age of 55 years, range 20−83 years) collected from twelve clinical centers specialized in diseases of liver and iron metabolism. The control group of 167 healthy subjects of Caucasian origin (65 men, 102 women, median age 25 years, range 18−62 years) was analyzed with the written informed consent of all subjects. The study was approved by the local Ethical Committee of the Faculty of Medicine and Faculty Hospital Hradec Kralove.

DNA extraction (QIAamp Blood Mini Kit, Qiagen, Germany) was performed from 200 µl of K3EDTA blood (experimental group) or from buccal cells (control group) collected using FlogSwabs (Copan Flock Technologies, Italy). The polymerase chain reaction (PCR) mixture for HFE exon 2 (25 μl) contained 100 ng DNA, 10x concentrated PCR buffer with 15mM magnesium chloride (TaKaRa, Japan), 200 µM dNTPs, 0.4 µM primers – forward primer 5´‑ACA TGG TTG AGG CCT GTT GC‑3´ and reverse primer 5´‑GCC ACA TCT GGC TTG AAA TT‑3´ (Generi Biotech, Czech Republic) and 1.5 U HS Taq polymerase (TaKaRa). The PCR mixture for HFE exon 4 differs from the above described mixture by sequences of primers; forward exon 4 primer: 5´‑ CTG GAT AAC CTT GGC TGT ACC CCC ‑3´ and reverse primer: 5´‑ CAG ATC CTC ATC TCA CTG ‑3´. The temperature profile consisted of heating to 95 °C for 5 min followed by 30 PCR cycles (denaturation 94 °C for 30 sec, annealing 50 °C for 30 sec, and elongation 72 °C for 30 sec) in the thermocycler Veriti™ 96-Well Thermal Cycler, Applied Biosystems, USA.

Restriction mixtures composed of 10 µl of PCR products, 1 µl of restriction buffer, and 1 µl of the appropriate restriction enzyme (all components New England Biolabs, USA): RsaI enzyme for C282Y, BclI for H63D and HinfI for S65C mutation analysis. The incubation time was 16 hours at 37 °C (RsaI and Hinf enzymes) or 50 °C (BclI enzyme). The analysis of restriction fragments (RFLP) was performed on 3% agarose gel with ethidium bromide. The presence of C282 wild-type allele was indicated by restriction fragments of 171 and 18 bp, while mutant 282Y fragments had lengths 142 bp, 29 bp, and 18 bp. In the case of 63D mutation, the BclI recognition site was changed and 208 bp PCR products were visible in the gel. In contrast, shorter fragments of 138 bp and 70 bp identified H63 wild-type alleles. In S65C analysis, 208 bp bands characterized 65C mutant alleles, and 147 bp and 61 bp restriction fragments identified S65 wild-type alleles of the HFE gene.  


In the experimental group we found 18 homozygotes (12.9 %) and 10 heterozygotes (7.1 %) for C282Y; 7 homozygotes (5.0 %) and 32 heterozygotes (22.9 %) for H63D; and three heterozygotes (2.1 %) for S65C. Compound heterozygosity was observed in 5 cases (3.5 %): three cases of 282Y/63D (2.1 %) and two cases (1.4 %) of 282Y/65C (Tab.1).

Sixteen heterozygotes for C282Y (9.6 %), three homozygotes (1.8 %) and 32 heterozygotes for H63D (19.2 %), and two heterozygotes (1.2 %) for S65C were identified in the control group. Three subjects (1.8 %) were compound heterozygotes with 282Y/63D allelic combination. No homozygotes either for C282Y or S65C were found in controls.

The frequency of all three risk alleles for HH appearing in the experimental group was 37.5 % (18.2 % for C282Y, 17.5 % for H63D, and 1.8 % for S65C), twofold than in the controls: 18.6 % (5.7 % for C282Y, 12.3 % for H63D, and 0.6 % for S65C), see Tab.2. 

Among the patients, thirty subjects had one of the genotypes associated with possible clinical symptoms of HH (282Y/282Y, 63D/63D, 282Y/63D, and 282Y/65C). In the control group these genotypes were observed in six healthy subjects. The risk of developing HH was sevenfold higher in patients with liver or iron disorders than in the healthy volunteers (odds ratio 7.32; p<0.001; 95% confidence interval: 3.27−16.39 %).

Tab. 1. Frequency of HFE genotypes in experimental and control groups (wt = wild type)
Frequency of <em>HFE</em> genotypes in experimental and control groups (wt = wild type)

Tab. 2. Frequency of risk alleles for hereditary hemochromatosis in the HFE genes
Frequency of risk alleles for hereditary hemochromatosis in the <em>HFE</em> genes


Since 1996, when the HFE gene was first described, a lot of papers dealing with mutations in this gene have been published. C282Y is the major type of mutations associated with hereditary hemochromatosis (80–90 %), especially if it is present in the organism in the homozygous form (14, 15). On the other hand, the clinical penetrance of C282Y is not complete and HH manifests only in part of the C282Y homozygotes (in 50 % in men and 25 % in women) (16). 

Population studies revealed that the highest frequency of 282Y allele is in northwestern Europe, mainly in Ireland (14 %) and Great Britain (8 %). In northern Europe (Norway, Sweden, Denmark) the frequencies of 282Y range between 5.7–7.5 %. It is not known which population spread the C282Y mutation to other parts of Europe (Viking or Celtic expansions) (17, 18). In central Europe the 282Y mutation frequency is 3.4–4.0 %. In southeastern Europe (Bosna and Hercegovina, Romania, Serbia, Macedonia) the 282Y prevalence is 1.0–2.2 % (19). The occurrence of 282Y outside Europe is very low.

In our control group the frequency of 282Y allele was higher than was published previously by Čimburová et al. (5.7 % vs 3.4 %) (20). This discrepancy we explain by a low number of probands in our control group (n=167). Genetic analysis of the patients of clinical centers (n=140) revealed a threefold higher incidence of the 282Y allele (18.2 %) in comparison with the controls. Eighteen 282Y/282Y homozygotes were found.  

As mentioned above, C282Y is thought to be the most important factor for HH development. C282 homozygosity is present in 96 % of HH patients (21, 22). Clinical manifestation of HH symptoms in 282Y heterozygotes is rare. However, the heterozygosity can accelerate organ failure on the background of other diseases (chronic hepatitis C, liver steatosis, chronic alcoholism, etc.). 

The frequency of H63D mutation in Europe ranges between 10–20 %. Its highest prevalence was reported in the Basque population (30 %), Bulgaria, Spain, and Portugal (> 20 %). Čimburová et al. have estimated the H63D frequency in the Czech Republic to be 15 % (16).

In Caucasians, S65C mutation frequency ranges from 0.5 % (southeastern Italy) to 3 % (northern Scandinavia, Saami population) (23, 24). In our country, S65C frequency is about 1 % (16). A low frequency of S65C was also found in our control group (0.6 %).

As noted, HH symptoms in H63D homozygotes and compound heterozygotes (282Y/63D and 282Y/65C) are milder than in C282Y homozygotes. The occurrence of H63D (17.5 %) and S65C (1.8 %) mutations in our experimental group was not significantly different from the above mentioned control group and population data. Among S65C heterozygotes in the experimental group, we found a thirty-year woman with physiological values of biochemical analytes (transferrin saturation, ferritin, serum iron, serum aminotransferases, total bilirubin, glucose, and alkaline phosphatase) and three men with increased serum iron. However, theses findings may not be associated with S65C heterozygosity.  

In 65 patients from the experimental group (46.5 %) we did not detect either C282Y, H63D, or S65C mutations in the HFE gene. In these patients, increased concentrations of serum iron might be caused by non-HFE mutations influencing higher iron absorption, transport or depositing. The symptoms of hemochromatosis can be also evoked by unsuitable eating habits, toxic or viral insults (HCV), obesity, or excessive long-term alcohol consumption (>60 g/day in men and >40 g/day in women). Alcohol inhibits hepcidin transcription and the oxidative stress increases the secretion of transferrin receptor 1 (5, 25).

Due to the high number of non-genetic factors occurring in HH etiopathogenesis, it is not possible to implement large-scale population screening for C282Y, H63D, and S65C mutations in the HFE gene. It is preferred to focus on targeted screening for HH in high-risk populations (16). For the right diagnostic strategy of hemochromatosis, combining molecular genetic analysis with biochemical tests involving transferrin saturation, unbound iron-binding capacity, serum ferritin, serum iron and hepatic iron concentration is currently recommended (26). Despite low specificity and the existence of many factors (age, diet, eating habits, menstruation and other blood losses) influencing laboratory results, biochemical markers can significantly contribute to monitoring treatment efficacy and dietary regimens,


Hereditary hemochromatosis is a considerably widespread genetic disease. The biochemical and molecular genetic analyses are used for diagnosing HH. Although mass screening programs are not yet common, it is necessary to diagnose high-risk individuals for hereditary hemochromatosis.

Our study points out that patients with elevated iron blood levels from clinical centers for diseases of liver and iron metabolism had significant differences in the frequency of C282Y mutation in comparison with the healthy population. Thus, molecular genetic tests contribute to a more reliable HH confirmation in patients and their family members.     

This study was supported by the student research project SVV no. 264902 (2012).


  • BclI – Bacillus caldolyticus
  • DNA – deoxyribonucleic acid
  • dNTPs – deoxynucleotide triphosphates
  • HCV – hepatitis C virus
  • HH – hereditary hemochromatosis
  • HinfI – Haemophilus influenzae
  • HLA – Human Leukocyte Antigen 
  • HS Taq – hot start Thermus aquaticus
  • K3EDTA – Ethylene diamine tetraacetic acid trisodium salt
  • PCR – polymerase chain reaction
  • RFLP – restriction fragment length polymorphism
  • RsaI – Rhodopseudomonas sphaeroides

Address for correspondence:

Mgr. Monika Drastíková

Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine and University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic

e-mail: monika.drastikova@fnhk.cz


1. Brůha R, Hůlek P, Petrtýl J. Jaterní cirhóza. In: Ehrmann J, Hůlek P, et al. Hepatologie. Praha: Grada Publishing 2010; 399–410.

2. Mormone E, George J, Nieto N. Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches. Chem Biol Interact. 2011; 193: 225–231.

3. Horák J. Genetická hemochromatóza. In: Ehrmann J, Hůlek P, et al. Hepatologie. Praha: Grada Publishing 2010; 339–445.

4. Kovář J. Biologický význam a fyziologické funkce železa. In: Horák J. Hemochromatóza. Praha: Grada Publishing 2010; 15–22.

5. Fargion S, Valenti L, Fracanzani A. L. Beyond hereditary hemochromatosis: new insights into the relationship between iron overload and chronic liver diseases. Dig Liver Dis 2011; 43: 89–95.

6. Phatak PD, Sham RL, Raubertas RF, et al. Prevalence of hereditary hemochromatosis in 16031 primary care patients. Ann Intern Med 1998; 129: 954–961.

7. Lyon E, Frank EL. Hereditary hemochromatosis since discovery of the HFE gene. Clin Chem 2001; 47: 1147–1156.

8. Whittington CA, Kowdley KV. Review article: haemochromatosis. Aliment Pharmacol Ther 2002; 16: 1963–1975.

9. Novotný J. Poruchy metabolismu železa II. Vnitř. Lék. 2005; 51: 995–1006.

10. Feder JN, Penny DM, Irrinki A, et al. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci USA 1998; 95: 1472–1477.

11. Deicher R, Hörl WH. New insights into the regulation of iron homeostasis. Eur J Clin Invest 2006; 36: 301–309.

12. Mura C, Raguenes O, Férec C. HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis. Blood 1999; 93: 2502–2505.

13. Gurrin LC, Bertalli NA, Dalton GW, et al. HFE C282Y/H63D compound heterozygotes are at low risk of hemochromatosis-related morbidity. Hepatology 2009; 50: 94–101.

14. Allen KJ, Gurrin LC, Constantine CC, et al. Iron-overload-related disease in HFE hereditary hemochromatosis. N Engl J Med 2008; 358: 221–230.

15. Gómez-Llorente C, Miranda-León MT, Blanco S, et al. Frequency and clinical expression of HFE gene mutations in a Spanish population of subjects with abnormal iron metabolism. Ann Hematol 2005; 84: 650–655.

16. Zlocha J, Kovács L, Požgayová S, et al. Molekulovo-genetická diagnostika a skríning hereditárnej hemochromatózy. Vnitř. Lék. 2006; 52: 602–608.

17. Pedersen P, Melsen GV, Milman N. Frequencies of the haemochromatosis gene (HFE) variants C282Y, H63D and S65C in 6020 ethnic Danish men. Ann Hematol 2008; 87: 735–740.

18. Olsson KS, Konar J, Dufva IH, et al. Was the C282Y mutation an Irish Gaelic mutation that the Vikings helped disseminate– Eur J Haematol 2011; 86: 75–82.

19. Adler G, Clark JS, Łoniewska B, et al. Prevalence of 845G>A HFE mutation in Slavic populations: an east-west linear gradient in South Slavs. Croat Med J 2011; 52: 351–357.

20. Čimburová M, Půtová I, Provazníková H, et al. S65C and other mutations in the haemochromatosis gene in the Czech Population. Folia Biol 2005; 51: 172–176.

21. Hanson EH, Imperatore G, Burke, W. HFE gene and hereditary hemochromatosis: a HuGE review. Am J Epidemiol 2001; 154: 193–206.

22. Cukjati M, Vaupotic T, Rupreht R, et al. Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay. BMC Med Genet 2007; 69: 1–9.

23. Pietrapertosa A, Vitucci A, Campanale D, et al. HFE gene mutations in Apulian population: allele frequencies. Eur J Epidemiol 2003; 18: 685–589.

24. Beckman LE, Sjoberg K, Eriksson S, et al. Haemochromatosis gene mutations in Finns, Swedes, and Swedish Saamis. Hum Hered 2001; 52: 110–112.

25. Pietrangelo A. Hemochromatosis: an endocrine liver disease. Hepatology 2007; 46: 1291–1301.

26. Husová L, Dastych M, Votava M, et al. Hereditární hemochromatóza – opomíjená diagnóza. Čes a Slov Gastroent a Hepatol 2005; 59: 188–194.

Adiktológia Alergológia a imunológia Anestéziológia a resuscitácia Angiológia Audiológia a foniatria Biochémia Dermatológia Detská dermatológia Detská gastroenterológia Detská gynekológia Detská chirurgia Detská kardiológia Detská nefrológia Detská neurológia Detská onkológia Detská otorinolaryngológia Detská pneumológia Detská psychiatria Detská rádiológia Detská reumatológia Detská urológia Diabetológia Endokrinológia Farmácia Farmakológia Fyzioterapia Gastroenterológia a hepatológia Genetika Geriatria a gerontológia Gynekológia a pôrodníctvo Hematológia Hygiena a epidemiológia Hyperbarická medicína Chirurgia cievna Chirurgia hrudná Chirurgia plastická Chirurgia všeobecná Infekčné lekárstvo Intenzívna medicína Kardiochirurgia Kardiológia Logopédia Mikrobiológia Nefrológia Neonatológia Neurochirurgia Neurológia Nukleárna medicína Nutričný terapeut Obezitológia Oftalmológia Onkológia Ortodoncia Ortopédia Otorinolaryngológia Patológia Pediatria Pneumológia a ftizeológia Popáleninová medicína Posudkové lekárstvo Praktické lekárstvo pre deti a dorast Protetika Psychológia Rádiodiagnostika Rádioterapia Rehabilitácia Reprodukčná medicína Reumatológia Sestra Sexuológia Súdne lekárstvo Stomatológia Telovýchovné lekárstvo Toxikológia Traumatológia Urgentná medicína Urológia Laboratórium Domáca starostlivosť Foniatria Algeziológia Zdravotníctvo Dentální hygienistka Student medicíny

Článok vyšiel v časopise

Časopis lékařů českých

Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.


Nemáte účet?  Registrujte sa