HFE Gene Detail
Hereditary hemochromatosis (HH), is a genetic, iron storage disorder where excess iron accumulates in the blood, leading to an iron overload. If left undiagnosed and untreated it can damage the body’s organs and joints and become fatal; increasing risk for developing certain diseases and conditions such as bone and joint disease (osteoarthritis and osteoporosis), liver disease (cirrhosis, cancer, liver failure), heart disease (irregular heart-beat, enlarged heart, congestive heart failure), endocrine disorders (diabetes, hypothyroidism, infertility, impotence, hormone imbalances) and skin discolouration. Hemochromatosis is only diagnosed when blood iron levels exceed threshold levels. Initial symptoms might manifest as joint pain, stiffness, fatigue, abdominal pain and weight loss.
The excess iron is deposited in multiple organs, causing oxidative tissue damage and this can lead to the above-mentioned conditions such as liver cirrhosis, cancer and cardiomyopathy. Fatigue and arthralgias are the most common symptoms seen in early disease with progressive liver injury with cirrhosis and hepatocellular carcinoma being possible long-term consequences of severe iron overload. Progressive iron deposition in parenchymal tissues may lead to liver and other organ toxicity and progressive iron overload can manifest clinically as fibrosis, cirrhosis and hepatocellular carcinoma.
Symptoms usually develop earlier in men than women, generally in the fourth or fifth decade of life, and in women, symptoms usually appear post menopause when they are no longer experiencing blood loss through menstruation and pregnancy.
HFE Cys282Tyr (C282Y) & HFE His63Asp (H63D)
The HFE gene is located on chromosome 6 and is best known for its association with hemochromatosis. The HFE protein regulates iron absorption by regulating the interaction of the transferrin receptor with transferrin.
Its main mode of action is through regulation of the iron storage hormone, hepcidin, which is considered to be the principle iron-regulatory hormone. A reduced expression of the iron regulatory protein, hepcidin, results in an inappropriate increase in intestinal iron absorption.
The body has an efficient mechanism by which iron is recycled by the body. Dietary iron absorption is normally dependent on the body’s current iron status; iron taken in from the diet is absorbed mainly to compensate for any iron losses in the body and to a limited extent for iron storage in the liver. When dietary iron is taken up by enterocytes in the lumen of the gastrointestinal tract, tissue ferritin binds and stores iron in the enterocyte, preventing it from directly entering the bloodstream, which is of particular importance when iron stores in the body are already high. Ferroportin allows iron to be transported out of the enterocytes and into the plasma, where it is bound to transferrin and delivered to various organs (bone marrow being the most notable) for haem synthesis and to the liver for storage.
A hepatic peptide, hepcidin, is the principle iron regulatory hormone. Hepcidin interacts with, and deactivates, the iron export protein, ferroprotein. This limits the amount of dietary iron entering the plasma from enterocytes, recycled from macrophages and stored iron from the hepatocytes. When iron storage in the liver increases, hepcidin synthesis increases in order to limit further absorption of iron, and similarly iron deficiency results in a downregulation of hepcidin in the liver allowing for an increase in iron absorption. In adult hereditary haemochromatosis, defective HFE or TFR2 gene results in a downstream deficiency of hepcidin, leading to excessive ferroportin expression at the cell surface and resultant increased plasma iron and increased transferrin saturation, which further leads to parenchymal iron excess. The exact mechanism of the HFE gene’s influence on hepcidin production is not yet clear.
There are several known mutations in the HFE gene but currently there are three that are available for testing i.e. C282Y, H63D and S65C.
SNP Description And Amino Acid Change
C282Y and H63D are the two polymorphisms which are most associated with the majority of the clinical cases of iron overload. Those most at risk for developing hemochromatosis are individuals who are homozygote for C282Y and compound heterozygote for both C282Y and H63D.
HFE C282Y (rs1800562), also known as Cys282Tyr, is the SNP that is most commonly responsible for hemochromatosis, accounting for 80-90% of all cases. This gene sees a mutation at amino acid 282 where there is a substitution of tyrosine for cysteine in the protein product. The homozygous YY (AA) genotype is the risk genotype.
It is found in about 5-10% of the Caucasian population with approximately 1 in 200 people who are of European ancestry carrying this mutation. These carrier rates are between 10 & 15% amongst populations of Northern European ancestry, making it the most common mutation associated with a clinical disease. The frequency of a homozygous C282Y substitution has been reported to be 0.4% in European countries, with prevalence varying widely on a global distribution. Based on studies done in Europe, Aranda et al (2010) reported the prevalence of homozygous and heterozygous C282Y genotypes was <1.5% and <29% respectively, with the highest rates occurring in Northern Europe. Incidence of iron overload related disease in people carrying this homozygous mutation is less than 30% in men and 1% in women. This is partly due to environmental factors including physiological blood loss as well as the interaction between other genetic factors. These homozygous carriers are more likely to experience liver injury under the presence of certain co-factors, discussed later.
C282Y heterozygotes (genetic carriers for haemochromatosis) may have mildly elevated iron studies, but will not develop significant clinical disease unless cofactors are present, such as heavy alcohol consumption or steatohepatitis.
HFE H63D (rs1799945), also known as His63Asp, sees a substitution of aspartate for histidine at amino acid position 63 and is associated with a mild form of hemochromatosis. This risk is more likely to be seen when someone carries the H63D and C282Y variant, as described below. Radford Smith et al, reported that patients carrying this homozygous variation (DD) are no more likely to develop clinical disease compared to control population, but may still see an increase in serum ferritin levels.
Based on studies done in Europe, Aranda et al (2010) reported prevalence of the H63D mutations to be <8% and <38.8% for the homozygous and heterozygous prevalence respectively, with prevalence of these SNPS being most common in Southern Europe.
People who carry one risk allele of both the Y, for the SNP C282Y, and the D allele for the SNP H63D (compound heterozygotes), may be affected by a mild form of hemochromatosis.
Based on studies done in Europe, prevalence of C282Y/H63D compound heterozygote was <4%.
SNP Phenotypic Trait
People who are homozygous for HFE C282Y (YY) have the highest risk of developing hereditary haemochromatosis. Individuals who are homozygous for HFE H63D (DD) and people who are compound heterozygous for both H63D and C282Y have an increased risk of developing hereditary haemochromatosis, characterised by excess iron levels accumulating in the blood.
The characteristic biochemical abnormalities seen in people with haemochromatosis are raised serum transferrin and serum saturation levels. Iron status can be assessed by standard blood tests such as measuring the levels of serum iron, transferrin, transferrin saturation (TS), iron and serum ferritin (SF).
A SF concentration above 300ug/L in men and 200ug/L in women is an early indication of the disease, however levels fluctuate depending on patient’s gender and age. It should be noted that SF can be raised as an acute phase reactant in the presence of co-morbid disease and testing TS is also useful, as elevated TS is often present in early stages of the disease.
If left untreated, it can damage the body’s organs and joints, increasing risk for a number of diseases, as discussed above.
Figure 1, taken from Radford Smith (2018) (included below) illustrates the natural history of untreated hereditary haemochromatosis. With a prompt diagnosis and treatment (such as with phlebotomy), progression of the disease to organ damage in C282Y homozygotes is unlikely, unless other environmental factors are involved.
The biochemical phenotypic expression of the homozygous and heterozygous H63D genotype and of C282Y heterozygote genotype is much lower than the C282Y homozygote or in the compound C282Y / H63D.
Although these HFE mutations are used as an indicator to assess the risk of iron overload, it should be noted that there are people with iron overload and the associated risks mentioned below, who do not have the mutations and that not everyone with these HFE gene mutations necessarily develops iron overload.
A study was done to examine the relationship among HFE genotypes, elevated iron phenotypes and telomere length. Results found that elevated serum biochemical tests of iron status were associated with shorter telomere length, however this was independent of HFE gene mutations. The HFE gene mutations were not associated with shortened telomere length. Results supported the idea that health impacts from the HFE genotype are largely through the pathway of raised iron levels in the body and resultant oxidative stress, but it was unclear as to whether lifestyle variables increased the tendency for phenotypic expression in these individuals. Finding interventions to minimize gene expression and keep iron levels under control is a reasonable approach to health promotion in individuals with HFE genotypes.
Figure 1: The Natural history of (untreated) hereditary haemochromatosis. Prompt diagnosis and treatment with phlebotomy en ensure that progression of disease to organ damage in C282Y homozygotes is unlikely, unless environmental factors are involved.
Radford Smith et al. Haemochromatosis: a clinical update for the practicing physician. Internal medicine journal. 2018(509-516).
Risk Factors For Developing Hemochromatosis
Diet, alcohol consumption, use of supplements, age and gender can all affect hemochromatosis risk, especially amongst people who are genetically at risk. This is discussed in more detail further on. Women are less likely to have hemochromatosis because of their blood losses during menstruation; however, they may be affected post menopause.
The older someone is, the more likely they are to have accumulated more iron in the blood and therefore age can affect risk, with complications more likely to develop between ages 40 and 60 in men and post-menopausal in women. Regular blood donors are less likely to be affected.
Regular phlebotomy remains the first line of treatment for hereditary haemochromatosis. Removal of blood prior to the development of cirrhosis allows patients to have a normal life expectancy, along with a reduction in some, if not all, of the symptoms, with notable improvements in overall wellbeing (especially reduced fatigue), liver function and skin pigmentation, however if cirrhosis is already well established it is often irreversible.
People who are unable to receive phlebotomy can consider erythrocytapheris to remove excess iron (5,9) or iron chelation, so that the individual can lead a normal life.
Proton Pump inhibitors have also been shown to reduce iron absorption in patients with HH, allowing for an increased time between phlebotomies.
Adams et al. (2018) developed guidelines with the aim to provide objective, simple, brief and practical recommendations about therapeutic aspects of HFE haemochromatosis for C282Y homozygous carriers (C282Y/C282Y) based on published scientific studies.
Interventions And Studies
Gallego et al. evaluated the diagnostic rate and estimated the penetrance of iron overload and associated organ damage for the two most common HH genotypes in a densely phenotyped cohort selected for reasons associated with HH. From a cohort of approximately 35,000 participants, 95 people homozygous for C282Y (Cys282Tyr) and 392 compound heterozygotes for C282Y and H63D (His63Asp), with information on HH diagnosis, were included for further analysis. Before exclusion of a few of the candidates, the mean age of participants was 66.4 years (+-15.8) and the average age at HH at diagnosis was 59.6 years (+_12.5).
The frequency of HH diagnosis was 24.4% in males homozygous for Cys282Tyr (282YY) and 3.5% in males compound heterozygote for Cys282Tyr and His63Asp, compared to a diagnostic rate of 14% and 2.3% in females homozygous for Cys282Tyr and compound heterozygote for both Cys282Tyr and His63Asp, respectively. This prevalence is higher than the diagnostic rate previously reported in the literature. For many of the signs of HH, penetrance was higher in Cys282Tyr homozygotes compared to Cys282Tyr; His63Asp, compound heterozygote individuals, although there were some signs that did not differ by phenotype. In males (but not in females), transferrin saturation levels above 50% was more common in homozygotes than compound heterozygotes (100% vs. 37.5% respectively) and serum ferritin levels above 300ng/ml was more frequent in homozygous compared to compound heterozygotes (77.8% vs. 33.3% respectively). This is similar to estimates previously reported.
Males had an overall prevalence of liver disease of 34.3% in homozygotes and 24.4% in compound heterozygotes respectively, while for females it was 29% for both genotypes and the rate of liver biopsies was more common between homozygotes and compound heterozygotes for males (10.9% vs. 1.8%) and females (9.1% vs. 2%). There was no significant difference in genotypes for other liver phenotypes looked at which included the presence of any liver disease, liver cirrhosis, other chronic liver diseases, hepatocellular carcinoma, elevated transaminases, hepatomegaly and ascites.
The proportion of individuals with a history in phlebotomy was higher in homozygotes vs. compound heterozygotes (19.6% vs. 2.9% respectively in males and 8% vs. 0.5% respectively in females). Rates of diabetes in males were 44.7% vs. 28% and family history of HH in males was 8.1% vs. 0% for homozygous vs. compound heterozygotes, whereas no differences were seen in females for these phenotypes. The proportion of hand X rays to evaluate arthritis was 24.5% vs. 11.5% in females, for homozygous carriers and compound heterozygous carriers respectively. There was no significant difference between genotypes for either gender for the rates of congestive heart failure, cardiomyopathy, osteoarthritis, hypogonadism, history of alcohol or tobacco use, use of over the counter medication for arthritis, diabetes medication, the proportion of individuals who used imaging studies such as abdominal ultrasound or echocardiogram, the presence of arthralgia, skin hyperpigmentation or pain on palpitation of proximal interphalangeal or metacarpophalangeal joints. Liver cirrhosis, hepatocellular carcinoma and cardiac phenotypes, specifically cardiomyopathies are considered extreme phenotypes of hemochromatosis, however the result of no difference across genotypes make sense as these days they are not as commonly seen as hey can be prevented by early detection of HH and treatment with phlebotomy. Diabetes, another late complication of HH, was found to be more frequent amongst homozygous compared to compound heterozygote males. The early signs such as fatigue, arthritis and skin hyperpigmentation might have not differed significantly for reasons such as not being captured correctly in the electronic health record, might already be occurring more commonly in the population or might be difficult to define.
Overall the study found that the rate of HH is higher than was previously reported and it confirmed that prevalence of iron overload is higher in HFE Cys282Tyr homozygotes than compound heterozygotes. It was recommended that the use of opportunistic screening for HFE Cys282Tyr homozygotes conferring risk of HH in those with existing genomic data, should be reconsidered and further genetic testing in the general population should be evaluated with studies of outcome research and cost effectiveness. There were some limitations of this study, which can be read up in more detail in the article, but included some incomplete data from pre-existing clinical records; the lack of a true control group who did not have genotypes at risk for HH and the heterogeneity in terms of clinical sites and subject selection, which was not random compared to the general population.
The clinical expression of haemochromatosis may be influenced by confounding factors that increase hepatocyte injury, including, but not limited to, excessive alcohol consumption and obesity.
Fletcher et al. compared liver biopsy data for 224 C282Y homozygous patients and found that those consuming ≥60g alcohol per day were almost 9 times more likely to develop severe fibroids or cirrhosis than those with a lower daily alcohol consumption. It was thought that this may be due to the cumulative hepatic oxidative effects of alcohol in addition to excess hepatocellular iron stores. Maintenance of a healthy body weight and levels of physical activity is important to reduce risk of developing comorbid liver disease such as non-alcoholic fatty liver disease (NAFLD). It was recommended that alcohol intake should be limited to guidelines by the National Health and Medical Research Council of ≤2standard drinks per day and alcohol should be avoided in the presence of cirrhosis and fibrosis.
Non-alcoholic fatty liver disease may also be a key cofactor in HH in patients with advanced liver damage. In a study of 214 patients with haemochromatosis and homozygote for C282Y who had undergone liver biopsy prior to phlebotomy, 41% presented with steatosis, due to both higher body mass index and alcohol consumption. The presence of steatosis was independently associated with liver fibrosis, male gender, excess alcohol and hepatic iron content.
Implementation of a healthy, well balanced diet is recommended as part of the disease management.
Aranda et al. carried out a study to investigate the effects of diet, alcohol intake and other lifestyle factors on the risk of iron overload, especially in people who are genetically at risk. A random sample was drawn from three communities in the Mediterranean Region of Tarragona in the N.E of Spain, who were of Caucasian origin and stratified by age and gender. A total of 815 participants (425 females and 390 males) aged 18-75 years were studied. Food intake was evaluated using the dietary recall method over three non-consecutive days, including a weekend/holiday.
Total iron intake was found to increase the values of TS and SF showing an increased iron absorption and total iron levels in the body, while calcium intake reduced TS and SF values. It appeared that subjects who were heterozygous for C282Y saw a decrease in the control for iron absorption, allowing for absorption from diets that were rich in bioavailable iron. Alcohol consumption resulted in increased TS and SF values. This has also been reported in other studies. Lower TS and SF levels were related to an increased intake of dietary calcium and fibre, amongst men with HFE mutations. It was therefore recommended that males with high risk HFE mutations should be encouraged to include more dietary fibre and calcium and abstain from alcohol.
Rossi et al. performed a cross sectional study on 1488 females and 1522 males, ages 20-79 years old from Busselton, Australia to assess the effects of HFE genotype, age, gender and lifestyle on serum iron and haematology indices. With regard to dietary/lifestyle interventions, ferritin concentrations increased significantly with increased alcohol consumption, in both males and females.
Males consuming 11-50g alcohol/day or >50g/ day had significantly higher ferritin values than those consuming 1-10g/day. Red meat (beef) consumption was high with 89% men and 81% women reporting eating red meat 3 or more times per week. Ferritin levels increased with an increased meat consumption, with ferritin values significantly higher in those consuming red meat 3-6x/week compared to those consuming red meat 1-2x/week, in both males and females.From a cohort of UK women, 2531 women were genotyped for C282Y and H63D mutations in the hemochromatosis gene. Greenwood et al. found a statistically significant gene diet interaction between C282Y homozygotes and heme iron intake. Heme iron intake (from meat, fish and poultry) was found to be 2x greater for C282Y homozygotes than other groups. C282Y homozygotes had serum ferritin concentrations 2.4 times higher than wild types, while C282Y heterozygotes had similar concentrations to wild types. Compound heterozygotes had serum ferritin concentrations 1.2 times higher than subjects who were wild type for both mutations. Heme iron intake was strongly associated to iron status. Women consuming more heme iron had higher serum ferritin concentrations. This relationship was exacerbated in people who were homozygous for C282Y mutation, with the influence of heme iron intake being 2x as strong as in wild types, resulting in substantially raised serum ferritin concentrations. The presence of H63D mutation was not associated with higher serum ferritin concentrations except in those who were compound heterozygotes with the C282Y mutation. These findings confirm results of some smaller studies investigating heme and non heme iron absorption in controlled conditions. Lynch et al found that in healthy volunteers, the absorption of both heme and non heme iron was lower among those with higher serum ferritin concentrations. In patients with haemochromatosis, absorption of both types of iron was higher, but only heme iron absorption was free of any inverse association with serum ferritin. Their study also found that the absorption of iron from normal diets in heterozygotes was not much different from healthy volunteers. Greenwood et al concluded that women who are homozygous for C282Y should reduce their meat (heme iron) intake to reduce the rate of iron accumulation, as long as they are not anaemic. The larger group of women who are heterozygous for C282Y mutation do not have substantially higher levels of serum ferritin concentrations than the wild type and based on this study they do not need to reduce their red meat intake, other than as part of a healthy diet, nor do H63D homozygotes need to reduce theirs.
Adams et al. (2018) discussed dietary recommendations in his article on therapeutic recommendations in HFE haemochromatosis. Dietary considerations discussed were that Vitamin C (found in citrus fruit) has been found to increase iron absorption and polyphenols in tea and coffee (and other polyphenolic containing beverages) can reduce non-haem iron absorption when consumed together. A regular meat intake may also contribute to raised SF. He recommended including a healthy varied diet, avoiding foods that have been fortified with iron such as certain breakfast cereals, avoiding supplementation with iron, and vitamin C and avoiding a high alcohol intake. He emphasised that dietary restriction should not replace phlebotomy.
Summary Of Recommendations
In individuals with gene variations that put them at risk for haemochromatosis i.e. C282Y homozygote and C282Y / H63D compound heterozygotes, early diagnosis, blood test screenings and prompt treatment with phlebotomy is recommended. Regular monitoring of iron levels, particularly serum ferritin and transferrin saturation, is recommended.
Maintenance of a healthy body weight and increased physical activity is important to reduce risk of complications such as developing comorbid liver disease such as NAFLD.
Alcohol intake should be limited with some guidelines recommending limiting alcohol to ≤2standard drinks per day with avoidance in the presence of cirrhosis and fibrosis, and other guidelines recommending complete avoidance and some recommending limiting alcohol intake but without specifying actual amounts. This can be assessed based on which genotype an individual is carrying i.e. homozygous for HFE C282Y or compound heterozygous for C282Y and H63D, their gender (males appear to have an increased risk) and the presence of associated conditions, especially liver problems.
At risk individuals should reduce their intake of foods rich in heme iron, particularly by reducing their red meat intake. They should aim to avoid consuming foods that are very high in Vitamin C together with a meal containing iron, and avoid use of supplements containing iron. At risk individuals, particularly males, should also be encouraged to include more dietary fibre and calcium in their diet.
Dietary management is important but should not replace phlebotomy.
Radford Smith et al (2018) developed a simple algorithm for the investigation and management of elevated serum ferritin (SF) levels, which may be a useful tool in assessing and managing at risk individuals. (See figure 2 below).
Figure 2: Simplified algorithm for investigation and management of elevated serum ferritin levels.
Radford Smith et al. Haemochromatosis: a clinical update for the practicing physician. Internal medicine journal. 2018(509-516).
Haemochromatosis: a clinical update for the practicing physician
Radford-Smith et al, 2018.