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Iron overload in humans is associated with a variety of genetic and acquired conditions. Of these, HFE hemochromatosis (HFE-HC) is by far the most frequent and most well-defined inherited cause when considering epidemiological aspects and risks for iron-related morbidity and mortality. The majority of patients with HFE-HC are homozygotes for the C282Y polymorphism [
]. Without therapeutic intervention, there is a risk that iron overload will occur, with the potential for tissue damage and disease. While a specific genetic test now allows for the diagnosis of HFE-HC, the uncertainty in defining cases and disease burden, as well as the low phenotypic penetrance of C282Y homozygosity poses a number of clinical problems in the management of patients with HC. This Clinical Practice Guideline will therefore, focus on HFE-HC, while rarer forms of genetic iron overload recently attributed to pathogenic mutations of transferrin receptor 2, (TFR2), hepcidin (HAMP), hemojuvelin (HJV), or to a sub-type of ferroportin (FPN) mutations, on which limited and sparse clinical and epidemiologic data are available, will not be discussed. We have developed recommendations for the screening, diagnosis, and management of HFE-HC.
Introduction
This Clinical Practice Guideline (CPG) has been developed to assist physicians and other healthcare providers as well as patients and interested individuals in the clinical decision making process for HFE-HC. The goal is to describe a number of generally accepted approaches for the diagnosis, prevention, and treatment of HFE-HC. To do so, four clinically relevant questions were developed and addressed:
(1)
What is the prevalence of C282Y homozygosity?
(2)
What is the penetrance of C282Y homozygosity?
(3)
How should HFE-HC be diagnosed?
(4)
How should HFE-HC be managed?
Each question has guided a systematic literature review in the Medline (PubMed version), Embase (Dialog version), and the Cochrane Library databases from 1966 to March 2009. The study selection was based on specific inclusion and exclusion criteria (Table 1). The quality of reported evidence has been graded according to the Grades of Recommendation, Assessment, Development, and Evaluation system (GRADE) [
]. The GRADE system classifies recommendations as strong or weak, according to the balance of the benefits and downsides (harms, burden, and cost) after considering the quality of evidence (Table 2). The quality of evidence reflects the confidence in estimates of the true effects of an intervention, and the system classifies quality of evidence as high, moderate, low, or very low according to factors that include the study methodology, the consistency and precision of the results, and the directness of the evidence [
]. Every recommendation in this CPG is followed by its GRADE classification in parentheses.
Table 1Inclusion and exclusion criteria for the literature search.
Inclusion and exclusion criteria for searching references
Inclusion criteria
1. Populations: adults age >18 years, population applicable to Europe, North America, Australia, New Zealand, screening population with elevated iron measures, asymptomatic iron overload, or HFE C282Y homozygosity (all ages were included for questions on C282Y prevalence)
2. Disease: symptomatic (liver fibrosis, cirrhosis, hepatic failure, hepatocellular carcinoma, diabetes mellitus, cardiomyopathy, or arthropathy hypogonadism, attributable to iron overload) or asymptomatic with or without C282Y homozygosity
3. Design:
a. Questions on prevalence: cohort or cross-sectional studies (also studies in newborns)
b. Questions on burden, natural history, penetrance: cross-sectional and longitudinal cohort studies
c. Questions on therapeutics: RCTs and large case series
4. Outcomes: incidence, severity, or progression of clinical hemochromatosis or iron measures, nonspecific symptoms (for questions on therapy)
Exclusion criteria
1. Non-human study
2. Non-English-language
3. Age: <18 years unless adult data are analyzed separately
4. Design: case-series with <15 patients, editorial, review, letter, congress abstract (except research letters)
5. For questions on epidemiology and diagnosis: does not include HFE genotyping
6. Does not report relevant prevalence or risk factors (for questions on prevalence–penetrance), does not report relevant outcomes (for questions on therapy)
7. Not phlebotomy treatment (for questions on therapy)
Table 2Quality of evidence and strength of recommendations according to GRADE.
Example
Note
Symbol
Quality of evidence
High
Randomized trials that show consistent results, or observational studies with very large treatment effects
Further research is very unlikely to change our confidence in the estimate of effect
A
Moderate
Randomized trials with methodological limitations, or observational studies with large effect
Further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate
B
Low and very Low
Observational studies without exceptional strengths, or randomized trials with very serious limitations; unsystematic clinical observations (e.g. case reports and case series; expert opinions) as evidence of very-low-quality evidence
Further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate. Any estimate of effect is very uncertain
Factors that affect the strength of a recommendation are: (a) quality of evidence; (b) uncertainty about the balance between desirable and undesirable effect; (c) uncertainty or variability in values and preferences; (d) uncertainty about whether the intervention represents a wise use of resources (see Refs. [2–6]).
Strong
Defined as being ‘confident that adherence to the recommendation will do more good than harm or that the net benefits are worth the costs’
1
Weak
Defined as being ‘uncertain that adherence to the recommendation will do more good than harm OR that the net benefits are worth the costs’
The uncertainty associated with weak recommendations follows either from poor-quality evidence, or from closely balanced benefits versus downsides
2
∗ Factors that affect the strength of a recommendation are: (a) quality of evidence; (b) uncertainty about the balance between desirable and undesirable effect; (c) uncertainty or variability in values and preferences; (d) uncertainty about whether the intervention represents a wise use of resources (see Refs.
The prevalence of HFE gene polymorphisms in the general population
The frequency of HC-associated HFE gene polymorphisms in the general population was determined in 36 screening studies, which fulfilled the inclusion criteria (Table 3). The allelic frequency of C282Y was 6.2% in a pooled cohort of 127,613 individuals included in the individual patient meta-analysis from these 36 studies (Table 3).
Table 3Prevalence of the common HFE polymophisms C282Y and H63D in the general population.
High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
No increase in mortality and morbidity among carriers of the C282Y mutation of the hereditary haemochromatosis gene in the oldest old: the Leiden 85-plus study.
Hemochromatosis gene HFE Cys282Tyr mutation analysis in a cohort of Northeast German hospitalized patients supports assumption of a North to South allele frequency gradient throughout Germany.
From this allelic frequency for C282Y, a genotype frequency of 0.38% or 1 in 260 for C282Y homozygosity can be calculated from the Hardy–Weinberg equation. The reported frequency of C282Y homozygosity is 0.41%, which is significantly higher than the expected frequency. This probably reflects a publication or ascertainment bias.
Significant variations in frequencies of the C282Y allele between different geographic regions across Europe have been reported with frequencies ranging from 12.5% in Ireland to 0% in Southern Europe (Fig. 1).
Fig. 1Frequency of the C282Y allele in different European regions. (For detailed information see Table 3.)
In addition to C282Y, which is also known as the ‘major’ HFE-associated polymorphism, H63D, considered to be the ‘minor’ HFE polymorphism, has been found more frequently in HC patients than in the control population. The frequency of the H63D polymorphism shows less geographic variation, with an average allelic frequency of 14.0% from pooled data (23,733 of 170,066 alleles). An additional HFE polymorphism is S65C, which can be associated with excess iron when inherited in trans with C282Y on the other parental allele. The allelic frequency of this polymorphism is ∼0.5% and appears to be higher in Brittany, France.
The prevalence of homozygosity for C282Y in the HFE gene in clinically recognized hemochromatosis
The prevalence of C282Y homozygosity in clinically recognized individuals with iron overload was assessed in a meta-analysis including 32 studies with a total of 2802 hemochromatosis patients of European ancestry (Table 4). This analysis of pooled data shows that 80.6% (2260 of 2802) of HC patients are homozygous for the C282Y polymorphism in the HFE gene. Compound heterozygosity for C282Y and H63D was found in 5.3% of HC patients (114 of 2117, Table 4). In the control groups, which were reported in 21 of the 32 studies, the frequency of C282Y homozygosity was 0.6% (30 of 4913 control individuals) and compound heterozygosity was present in 1.3% (43 of 3190 of the control population).
Table 4Prevalence of C282Y homozygosity and C282Y/H63D compound heterozygosity in clinically recognized hemochromatosis.
Authors
Ref.
Study population
Prevalence of HLA/HFE among clinical hemochromatosis cases
The clinical expression of hemochromatosis in Oslo, Norway. Excessive oral iron intake may lead to secondary hemochromatosis even in HFE C282Y mutation negative subjects.
Mutation analysis of the HFE gene in German hemochromatosis patients and controls using automated SSCP-based capillary electrophoresis and a new PCR–ELISA technique.
Hence, 19.4% of clinically characterized HC patients have the disease in the absence of C282Y homozygosity. Although compound heterozygosity (H63D/C282Y) appears to be disease associated, in such individuals with suspected iron overload, cofactors should be considered as a cause [
The prevalence of HFE genotypes in selected patient groups
Fatigue
To date, there are only cross-sectional or case-control studies investigating the prevalence of C282Y homozygosity in patients with fatigue or chronic fatigue syndrome [
]. In the majority of studies of patients with undifferentiated osteoarthritis the prevalence of C282Y homozygosity did not exceed that of the control population [
]. In patients with osteoarthritis in the 2nd and 3rd metacarpophalangeal joints, higher allele frequencies of the HFE-polymorphisms (C282Y and H63D) were found, although this was not accompanied by an increased frequency of C282Y homozygotes [
Primary osteoarthritis in the ankle joint is associated with finger metacarpophalangeal osteoarthritis and the H63D mutation in the HFE gene: evidence for a hemochromatosis-like polyarticular osteoarthritis phenotype.
Association of the C282Y polymorphism with diabetes mellitus has been mainly evaluated in patients with type 2 diabetes mellitus in cross-sectional and case-control studies [
Relationships of serum ferritin, transferrin saturation, and HFE mutations and self-reported diabetes in the Hemochromatosis and Iron Overload Screening (HEIRS) study.
], although the frequency of C282Y homozygosity was not increased. The prevalence of C282Y homozygotes in patients with type 1 diabetes mellitus has been addressed in only one study where a significantly higher rate of C282Y homozygotes was detected (odds ratio 4.6; prevalence 1.26%) [
]. Three to 5.3% of patients were C282Y-homozygous, which is about 10-fold higher than the reported prevalence in the general population. The prevalence of C282Y homozygosity increased to 7.7% if patients were selected on the basis of a transferrin saturation of >45% [
Hepatocellular carcinoma (HCC) is a recognized complication of HFE-HC. Nevertheless few studies have analyzed the frequency of C282Y homozygosity in patients with HCC and these are limited with respect to their size [
]. Subgroup analysis for gender specific prevalence and different etiologies were statistically underpowered. However, three studies in HCC reported a frequency of C282Y-homozygotes of 5.5–10% [
] did not find an association between HCC and the C282Y-polymorphism.
Hair loss, hyperpigmentation, amenorrhea, loss of libido
There were no hits according to the search criteria.
Porphyria cutanea tarda
The prevalence of C282Y homozygosity among patients with porphyria cutanea tarda (PCT) was found to be increased significantly compared with control populations, ranging from 9% to 17% in several studies [
The C282Y mutation in the haemochromatosis gene (HFE) and hepatitis C virus infection are independent cofactors for porphyria cutanea tarda in Australian patients.
]. The association between PCT and the common HFE gene polymorphisms C282Y and H63D is illustrated by a recent meta-analysis, where the odds ratios for PCT were 48 (24–95) in C282Y homozygotes, and 8.1 (3.9–17) in C282Y/H63D compound heterozygotes [
High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
Relationships of serum ferritin, transferrin saturation, and HFE mutations and self-reported diabetes in the Hemochromatosis and Iron Overload Screening (HEIRS) study.
]. The positive predictive value of an elevated ferritin for detection of C282Y-homozygotes was 1.6–17.6% (Table 5). The frequency of a ferritin concentration above 1000 μg/L was 0.2–1.3% in non-selected populations [
High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri.
Population screening for hemochromatosis: a comparison of unbound iron-binding capacity, transferrin saturation, and C282Y genotyping in 5211 voluntary blood donors.
Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in Native Americans and whites in the Hemochromatosis and Iron Overload Screening Study.
High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
Population screening for hemochromatosis: a comparison of unbound iron-binding capacity, transferrin saturation, and C282Y genotyping in 5211 voluntary blood donors.
Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in Native Americans and whites in the Hemochromatosis and Iron Overload Screening Study.
] (Table 5). The positive predictive value of elevated transferrin saturation for the detection of C282Y-homozygotes was 4.3–21.7% (Table 5).
What is the penetrance of C282Y homozygosity?
Differences in inclusion criteria and in the definition of biochemical and disease penetrance have produced a range of estimates for the penetrance of C282Y homozygosity. The disease penetrance of C282Y homozygosity was 13.5% (95% confidence interval 13.4–13.6%) when 19 studies were included in the meta-analysis and the results of individual studies weighted on the inverse variance of the results of the individual study (Fig. 2) [
Fig. 2Forest plot of studies on the penetrance of hemochromatosis. Studies are weighted on the inverse of the confidence interval. (For detailed information see Table 6).
Although the majority of C282Y homozygotes may have a raised serum ferritin and transferrin saturation, this cannot be relied upon as secure evidence of iron overload. An individual patient data meta-analysis including 1382 C282Y homozygous individuals reported in 16 studies showed that 26% of females and 32% of males have increased serum ferritin concentrations (>200 μg/L for females and >300 μg/L in males) (Table 6). The prevalence of excess tissue iron (>25 μmoles/g liver tissue or increased siderosis score) in 626 C282Y homozygotes who underwent liver biopsy was 52% in females and 75% in males as reported in 13 studies. The higher penetrance of tissue iron overload is due to the selection of patients for liver biopsy, which is more likely to be carried out in patients with clinical or biochemical evidence of iron overload.
Table 6Data from studies addressing the penetrance of C282Y homozygotes.
High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri.
Two additional patients had serum ferritin of 1200 μg/L and 805 μg/L respectively, but did not undergo liver biopsy. Cirrhosis was found in 1 patient, fibrosis in 3 patients, and arthritis in 6 patients
In persons homozygous for the C282Y mutation, iron overload-related disease developed in a substantial proportion of men but in a small proportion of women
When all 1382 patients with reported iron parameters were included in the meta-analysis, the penetrance of excess liver iron was then 19% for females and 42% for males.
Clinical penetrance and progression
Disease penetrance based on symptoms (e.g. fatigue, arthralgia) is difficult to assess due to the non-specific nature and high frequency of such symptoms in control populations [
Disease penetrance based on hepatic histology has been studied but is biased by the fact that liver biopsy is usually reserved for patients with a high pre-test likelihood for liver damage. However, these studies give an estimate of disease expression in C282Y homozygotes. Elevated liver enzymes were found in 30% of males in one study [
The clinical expression of hemochromatosis in Oslo, Norway. Excessive oral iron intake may lead to secondary hemochromatosis even in HFE C282Y mutation negative subjects.
Penetrance is generally higher in male than in female C282Y homozygotes. C282Y homozygotes identified during family screening have a higher risk of expressing the disease (32–35%) when compared with C282Y homozygotes identified during population based studies (27–29%).
Three longitudinal (population screening) studies are available and show disease progression in only a minority of C282Y homozygotes [
]. Available data suggest that up to 38–50% of C282Y homozygotes may develop iron overload, with (as already stated) 10–33% eventually developing hemochromatosis-associated morbidity [
The prevalence and predictive value of abnormal serum iron indices for C282Y homozygosity in an unselected population
Serum iron studies are usually used as the first screening test when hemochromatosis is suspected. The predictive value of screening for serum iron parameters in the general population is highlighted by two studies [
The prevalence of persistently increased serum transferrin saturation upon repeated testing was 1% (622 of over 60,000). Of these individuals ∼50% also had hyperferritinemia (342 of 622). Homozygosity for C282Y could be detected in ∼90% of men and ∼75% of women with a persistently elevated transferrin saturation and increased serum ferritin. From a cross-sectional point of view, the disease penetrance of the C282Y/C282Y genotype in this study cohort, defined as the prevalence of liver cirrhosis, was ∼5.0% in men and <0.5% in women [
Genetic screening for HFE-HC is not recommended, because disease penetrance is low and only in few C282Y homozygotes will iron overload progress (1B).
Patient populations:
•
HFE testing should be considered in patients with unexplained chronic liver disease pre-selected for increased transferrin saturation (1C).
•
HFE testing could be considered in patients with:
–
Porphyria cutanea tarda (1B).
–
Well-defined chondrocalcinosis (2C).
–
Hepatocellular carcinoma (2C).
–
Type 1 diabetes (2C).
•
HFE testing is not recommended in patients with
–
Unexplained arthritis or arthralgia (1C).
–
Type 2 diabetes (1B).
How should HFE-HC be diagnosed?
The EASL CPG panel agreed on the following case definition for diagnosis of HFE-HC:
C282Y homozygosity and increased body iron stores with or without clinical symptoms.
The following section will address the genetic tests and tools for assessing body iron stores.
Genetic testing – Methodology
C282Y homozygosity is required for the diagnosis of HFE-HC, when iron stores are increased (see diagnostic algorithms). Any other HFE genotype must be interpreted with caution. The available methods are reported in Table 7. The intronic variant c.892+48 G>A may complicate amplification refractory mutation system (ARMS) – PCR for genetic testing [
Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload.
Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.
A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).
Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis.
Clinically overt hereditary hemochromatosis in Denmark 1948–1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients.
Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload.
Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.
A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).
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