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EASL clinical practice guidelines for HFE hemochromatosis

Published:April 19, 2010DOI:https://doi.org/10.1016/j.jhep.2010.03.001
      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 [
      • Feder J.N.
      • Gnirke A.
      • Thomas W.
      • Tsuchihashi Z.
      • Ruddy D.A.
      • Basava A.
      • et al.
      A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.
      ]. 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) [
      • Guyatt G.H.
      • Oxman A.D.
      • Vist G.E.
      • Kunz R.
      • Falck-Ytter Y.
      • Alonso-Coello P.
      • et al.
      GRADE: an emerging consensus on rating quality of evidence and strength of recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is ‘quality of evidence’ and why is it important to clinicians?.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Jaeschke R.
      • Helfand M.
      • Liberati A.
      • et al.
      Incorporating considerations of resources use into grading recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • et al.
      Going from evidence to recommendations.
      ,
      • Atkins D.
      • Best D.
      • Briss P.A.
      • Eccles M.
      • Falck-Ytter Y.
      • Flottorp S.
      • et al.
      Grading quality of evidence and strength of recommendations.
      ]. 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 [
      • Guyatt G.H.
      • Oxman A.D.
      • Vist G.E.
      • Kunz R.
      • Falck-Ytter Y.
      • Alonso-Coello P.
      • et al.
      GRADE: an emerging consensus on rating quality of evidence and strength of recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is ‘quality of evidence’ and why is it important to clinicians?.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Jaeschke R.
      • Helfand M.
      • Liberati A.
      • et al.
      Incorporating considerations of resources use into grading recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • et al.
      Going from evidence to recommendations.
      ,
      • Atkins D.
      • Best D.
      • Briss P.A.
      • Eccles M.
      • Falck-Ytter Y.
      • Flottorp S.
      • et al.
      Grading quality of evidence and strength of recommendations.
      ]. 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.
      ExampleNoteSymbol
      Quality of evidence
      HighRandomized trials that show consistent results, or observational studies with very large treatment effectsFurther research is very unlikely to change our confidence in the estimate of effectA
      ModerateRandomized trials with methodological limitations, or observational studies with large effectFurther research is likely to have an important impact on our confidence in the estimate of effect and may change the estimateB
      Low and very LowObservational 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 evidenceFurther 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 uncertainC
      Strength of recommendations
      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]).
      StrongDefined as being ‘confident that adherence to the recommendation will do more good than harm or that the net benefits are worth the costs’1
      WeakDefined 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 downsides2
      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.
      • Guyatt G.H.
      • Oxman A.D.
      • Vist G.E.
      • Kunz R.
      • Falck-Ytter Y.
      • Alonso-Coello P.
      • et al.
      GRADE: an emerging consensus on rating quality of evidence and strength of recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is ‘quality of evidence’ and why is it important to clinicians?.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Jaeschke R.
      • Helfand M.
      • Liberati A.
      • et al.
      Incorporating considerations of resources use into grading recommendations.
      ,
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Falck-Ytter Y.
      • Vist G.E.
      • Liberati A.
      • et al.
      Going from evidence to recommendations.
      ,
      • Atkins D.
      • Best D.
      • Briss P.A.
      • Eccles M.
      • Falck-Ytter Y.
      • Flottorp S.
      • et al.
      Grading quality of evidence and strength of recommendations.
      ).

      What is the prevalence of C282Y homozygosity?

       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.
      AuthorsRef.Country – PopulationIndividuals screenedAllele frequency for
      c.845 C > A (Y282)c.187 C > G (D63)
      Beckman et al. (1997)
      • Beckman L.E.
      • Saha N.
      • Spitsyn V.
      • Van Landeghem G.
      • Beckman L.
      Ethnic differences in the HFE codon 282 (Cys/Tyr) polymorphism.
      Mordvinia850.0176
      Finland1730.052
      Sweden – Saamis1510.0199
      Sweden – Saamis2060.0752
      Merryweather-Clarke et al. (1997)
      • Merryweather-Clarke A.T.
      • Pointon J.J.
      • Shearman J.D.
      • Robson K.J.
      Global prevalence of putative haemochromatosis mutations.
      UK3680.0600.12
      Ireland450.10.189
      Iceland900.0670.106
      Norway940.0740.112
      Former USSR1540.0100.104
      Finland3800.118
      Denmark370.0950.22
      Netherlands390.0260.295
      Germany1150.0390.148
      Ashkenazi3500.086
      Italy910.0050.126
      Greece1960.0130.135
      Turkey7000.136
      Spain780.0320.263
      Datz et al. (1998)
      • Datz C.
      • Lalloz M.R.
      • Vogel W.
      • Graziadei I.
      • Hackl F.
      • Vautier G.
      • et al.
      Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis.
      Austria2710.0410.258
      Burt et al. (1998)
      • Burt M.J.
      • George P.M.
      • Upton J.D.
      • Collett J.A.
      • Frampton C.M.
      • Chapman T.M.
      • et al.
      The significance of haemochromatosis gene mutations in the general population: implications for screening.
      New Zealand of European ancestry10640.0700.144
      Jouanolle et al. (1998)
      • Jouanolle A.M.
      • Fergelot P.
      • Raoul M.L.
      • Gandon G.
      • Roussey M.
      • Deugnier Y.
      • et al.
      Prevalence of the C282Y mutation in Brittany: penetrance of genetic hemochromatosis?.
      France – Brittany10000.065
      Merryweather-Clarke et al. (1999)
      • Merryweather-Clarke A.T.
      • Simonsen H.
      • Shearman J.D.
      • Pointon J.J.
      • Norgaard-Pedersen B.
      • Robson K.J.
      A retrospective anonymous pilot study in screening newborns for HFE mutations in Scandinavian populations.
      Scandinavia8370.0510.173
      Distante et al. (1999)
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
      Norway5050.0780.229
      Olynyk et al. (1999)
      • Olynyk J.K.
      • Cullen D.J.
      • Aquilia S.
      • Rossi E.
      • Summerville L.
      • Powell L.W.
      A population-based study of the clinical expression of the hemochromatosis gene.
      Australia30110.0757
      Marshall et al. (1999)
      • Marshall D.S.
      • Linfert D.R.
      • Tsongalis G.J.
      Prevalence of the C282Y and H63D polymorphisms in a multi-ethnic control population.
      USA – non-Hispanic whites1000.050.24
      Beutler et al. (2000)
      • Beutler E.
      • Felitti V.
      • Gelbart T.
      • Ho N.
      The effect of HFE genotypes on measurements of iron overload in patients attending a health appraisal clinic.
      USA – whites76200.0640.154002625
      Steinberg et al. (2001)
      • Steinberg K.K.
      • Cogswell M.E.
      • Chang J.C.
      • Caudill S.P.
      • McQuillan G.M.
      • Bowman B.A.
      • et al.
      Prevalence of C282Y and H63D mutations in the hemochromatosis (HFE) gene in the United States.
      USA – non-Hispanic whites20160.06370.153769841
      Andrikovics et al. (2001)
      • Andrikovics H.
      • Kalmar L.
      • Bors A.
      • Fandl B.
      • Petri I.
      • Kalasz L.
      • et al.
      Genotype screening for hereditary hemochromatosis among voluntary blood donors in Hungary.
      Hungarian blood donors9960.0340.014
      Pozzato et al. (2001)
      • Pozzato G.
      • Zorat F.
      • Nascimben F.
      • Gregorutti M.
      • Comar C.
      • Baracetti S.
      • et al.
      Haemochromatosis gene mutations in a clustered Italian population: evidence of high prevalence in people of Celtic ancestry.
      Italy – Celtic populations1490.036910.144295302
      Byrnes et al. (2001)
      • Byrnes V.
      • Ryan E.
      • Barrett S.
      • Kenny P.
      • Mayne P.
      • Crowe J.
      Genetic hemochromatosis, a Celtic disease: is it now time for population screening?.
      Ireland8000.12750.171875
      Beutler et al. (2002)
      • Beutler E.
      • Felitti V.J.
      • Koziol J.A.
      • Ho N.J.
      • Gelbart T.
      Penetrance of 845G → A (C282Y) HFE hereditary haemochromatosis mutation in the USA.
      USA – non-Hispanic whites30,6720.0622
      Guix et al. (2002)
      • Guix P.
      • Picornell A.
      • Parera M.
      • Galmes A.
      • Obrador A.
      • Ramon M.M.
      • et al.
      Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain).
      Spain – Balearic Islands6650.02030.201503759
      Deugnier et al. (2002)
      • Deugnier Y.
      • Jouanolle A.M.
      • Chaperon J.
      • Moirand R.
      • Pithois C.
      • Meyer J.F.
      • et al.
      Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.
      France93960.07636228
      Cimburova et al. (2002)
      • Cimburova M.
      • Putova I.
      • Provaznikova H.
      • Horak J.
      Hereditary hemochromatosis: detection of C282Y and H63D mutations in HFE gene by means of guthrie cards in population of Czech Republic.
      Czech Republic2540.039370080.142
      Van Aken et al. (2002)
      • Van Aken M.O.
      • De Craen A.J.
      • Gussekloo J.
      • Moghaddam P.H.
      • Vandenbroucke J.P.
      • Heijmans B.T.
      • et al.
      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.
      Netherlands12130.06141797
      Phatak et al. (2002)
      • Phatak P.D.
      • Ryan D.H.
      • Cappuccio J.
      • Oakes D.
      • Braggins C.
      • Provenzano K.
      • et al.
      Prevalence and penetrance of HFE mutations in 4865 unselected primary care patients.
      USA32270.05070.1512
      Jones et al. (2002)
      • Jones D.C.
      • Young N.T.
      • Pigott C.
      • Fuggle S.V.
      • Barnardo M.C.
      • Marshall S.E.
      • et al.
      Comprehensive hereditary hemochromatosis genotyping.
      UK1590.0850.173
      Candore et al. (2002)
      • Candore G.
      • Mantovani V.
      • Balistreri C.R.
      • Lio D.
      • Colonna-Romano G.
      • Cerreta V.
      • et al.
      Frequency of the HFE gene mutations in five Italian populations.
      Italy – five regions5780.0250.147
      Salvioni et al. (2003)
      • Salvioni A.
      • Mariani R.
      • Oberkanins C.
      • Moritz A.
      • Mauri V.
      • Pelucchi S.
      • et al.
      Prevalence of C282Y and E168X HFE mutations in an Italian population of Northern European ancestry.
      Italy – North6060.04702970.143564356
      Papazoglou et al. (2003)
      • Papazoglou D.
      • Exiara T.
      • Speletas M.
      • Panagopoulos I.
      • Maltezos E.
      Prevalence of hemochromatosis gene (HFE) mutations in Greece.
      Greece26400.089015152
      Sanchez et al. (2003)
      • Sanchez M.
      • Villa M.
      • Ingelmo M.
      • Sanz C.
      • Bruguera M.
      • Ascaso C.
      • et al.
      Population screening for hemochromatosis: a study in 5370 Spanish blood donors.
      Spain53700.031564250.208007449
      Mariani et al. (2003)
      • Mariani R.
      • Salvioni A.
      • Corengia C.
      • Erba N.
      • Lanzafame C.
      • De Micheli V.
      • et al.
      Prevalence of HFE mutations in upper Northern Italy: study of 1132 unrelated blood donors.
      Italy – North11320.0320.134
      Altes et al. (2004)
      • Altes A.
      • Ruiz A.
      • Barcelo M.J.
      • Remacha A.F.
      • Puig T.
      • Maya A.J.
      • et al.
      Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1146 newborns from a region of Northern Spain.
      Spain – Catalonia10430.02828380.19894535
      Adams et al. (2005)
      • Adams P.C.
      • Reboussin D.M.
      • Barton J.C.
      • McLaren C.E.
      • Eckfeldt J.H.
      • McLaren G.D.
      • et al.
      Hemochromatosis and iron-overload screening in a racially diverse population.
      USA – whites44,0820.068259150.153157751
      Barry et al. (2005)
      • Barry E.
      • Derhammer T.
      • Elsea S.H.
      Prevalence of three hereditary hemochromatosis mutant alleles in the Michigan Caucasian population.
      USA – non-Hispanic whites35320.0570.14
      Meier et al. (2005)
      • Meier P.
      • Schuff-Werner P.
      • Steiner M.
      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.
      Germany7090.044
      Matas et al. (2006)
      • Matas M.
      • Guix P.
      • Castro J.A.
      • Parera M.
      • Ramon M.M.
      • Obrador A.
      • et al.
      Prevalence of HFE C282Y and H63D in Jewish populations and clinical implications of H63D homozygosity.
      Jewish populations – Chuetas2550.007843140.123529412
      Hoppe et al. (2006)
      • Hoppe C.
      • Watson R.M.
      • Long C.M.
      • Lorey F.
      • Robles L.
      • Klitz W.
      • et al.
      Prevalence of HFE mutations in California newborns.
      USA – non-Hispanic whites9910.054994950.134207871
      Aranda et al. (2007)
      • Aranda N.
      • Viteri F.E.
      • Fernandez-Ballart J.
      • Murphy M.
      • Arija V.
      Frequency of the hemochromatosis gene (HFE) 282C → Y, 63H → D, and 65S → C mutations in a general Mediterranean population from Tarragona, Spain.
      Spain – Northeastern8120.031403940.219211823
      Terzic et al. (2006)
      • Terzic R.
      • Sehic A.
      • Teran N.
      • Terzic I.
      • Peterlin B.
      Frequency of HFE gene mutations C282Y and H63D in Bosnia and Herzegovina.
      Bosnia and Herzegovina2000.02250.115
      Floreani et al. (2007)
      • Floreani A.
      • Rosa Rizzotto E.
      • Basso D.
      • Navaglia F.
      • Zaninotto M.
      • Petridis I.
      • et al.
      An open population screening study for HFE gene major mutations proves the low prevalence of C282Y mutation in Central Italy.
      Italy – Central5020.01892430.148406375
      Raszeja-Wyszomirska et al. (2008)
      • Raszeja-Wyszomirska J.
      • Kurzawski G.
      • Suchy J.
      • Zawada I.
      • Lubinski J.
      • Milkiewicz P.
      Frequency of mutations related to hereditary haemochromatosis in northwestern Poland.
      Poland – Northwestern15170.044166120.154251813
      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).
      Figure thumbnail gr1
      Fig. 1Frequency of the C282Y allele in different European regions. (For detailed information see .)
      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.
      AuthorsRef.Study populationPrevalence of HLA/HFE among clinical hemochromatosis cases
      No. of casesC282Y homozygoteC282Y/H63D compound heterozygoteWild type both alleles
      Feder et al. (1996)
      • Feder J.N.
      • Gnirke A.
      • Thomas W.
      • Tsuchihashi Z.
      • Ruddy D.A.
      • Basava A.
      • et al.
      A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.
      USA – Multicenter18714821
      Jazwinska et al. (1996)
      • Jazwinska E.C.
      • Cullen L.M.
      • Busfield F.
      • Pyper W.R.
      • Webb S.I.
      • Powell L.W.
      • et al.
      Haemochromatosis and HLA-H.
      Australia1121120
      Jouanolle et al. (1996)
      • Jouanolle A.M.
      • Gandon G.
      • Jezequel P.
      • Blayau M.
      • Campion M.L.
      • Yaouanq J.
      • et al.
      Haemochromatosis and HLA-H.
      France656530
      Beutler et al. (1996)
      • Beutler E.
      • Gelbart T.
      • West C.
      • Lee P.
      • Adams M.
      • Blackstone R.
      • et al.
      Mutation analysis in hereditary hemochromatosis.
      USA – European origin147121
      Borot et al. (1997)
      • Borot N.
      • Roth M.
      • Malfroy L.
      • Demangel C.
      • Vinel J.P.
      • Pascal J.P.
      • et al.
      Mutations in the MHC class I-like candidate gene for hemochromatosis in French patients.
      France – Toulouse9468418
      Carella et al. (1997)
      • Carella M.
      • D’Ambrosio L.
      • Totaro A.
      • Grifa A.
      • Valentino M.A.
      • Piperno A.
      • et al.
      Mutation analysis of the HLA-H gene in Italian hemochromatosis patients.
      Italy – Northern75485
      Datz et al. (1998)
      • Datz C.
      • Lalloz M.R.
      • Vogel W.
      • Graziadei I.
      • Hackl F.
      • Vautier G.
      • et al.
      Predominance of the HLA-H Cys282Tyr mutation in Austrian patients with genetic haemochromatosis.
      Austria4031
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      A high prevalence of HLA-H 845A mutations in hemochromatosis patients and the normal population in eastern England.
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      The UK Haemochromatosis Consortium. A simple genetic test identifies 90% of UK patients with haemochromatosis. Gut 1997;41(6):841–4.

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      Prevalence of the Cys282Tyr and His63Asp HFE gene mutations in Spanish patients with hereditary hemochromatosis and in controls.
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      Hemochromatosis in Ireland and HFE.
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      High incidence of the Cys 282 Tyr mutation in the HFE gene in the Irish population – implications for haemochromatosis.
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      HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.
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      A genotypic study of 217 unrelated probands diagnosed as ‘genetic hemochromatosis’ on ‘classical’ phenotypic criteria.
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      HFE genotype in patients with hemochromatosis and other liver diseases.
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      • et al.
      Mutation analysis in the HFE gene in patients with hereditary haemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, Canada).
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      • et al.
      Mutation analysis in the HFE gene in patients with hereditary haemochromatosis in Saguenay-Lac-Saint-Jean (Quebec, Canada).
      Canada – Quebec321438
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      • Papanikolaou G.
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      • Loukopoulos D.
      Hereditary hemochromatosis: HFE mutation analysis in Greeks reveals genetic heterogeneity.
      Greece1035
      Guix et al. (2000)
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      • Parera M.
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      • et al.
      Prevalence of the C282Y mutation for haemochromatosis on the Island of Majorca.
      Spain – Balearic Islands1413
      Brandhagen et al. (2000)
      • Brandhagen D.J.
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      • et al.
      Prevalence and clinical significance of HFE gene mutations in patients with iron overload.
      USA82702
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      • Cappuccio J.
      • Gallagher M.
      • Phatak P.D.
      Asymptomatic hemochromatosis subjects: genotypic and phenotypic profiles.
      USA – Minnesota12374156
      Van Vlierberghe et al. (2000)
      • Van Vlierberghe H.
      • Messiaen L.
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      • De Paepe A.
      • Elewaut A.
      Prevalence of the Cys282Tyr and His63Asp mutation in Flemish patients with hereditary hemochromatosis.
      Belgium – Flemish494621
      Bell et al. (2000)
      • Bell H.
      • Berg J.P.
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      • Distante S.
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      • et al.
      The clinical expression of hemochromatosis in Oslo, Norway. Excessive oral iron intake may lead to secondary hemochromatosis even in HFE C282Y mutation negative subjects.
      Norway120923
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      • Bosserhoff A.K.
      • Seegers S.
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      • et al.
      Mutation analysis of the HFE gene in German hemochromatosis patients and controls using automated SSCP-based capillary electrophoresis and a new PCR–ELISA technique.
      Germany – Southern362632
      de Juan et al. (2001)
      • de Juan D.
      • Reta A.
      • Castiella A.
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      HFE gene mutations analysis in Basque hereditary haemochromatosis patients and controls.
      Spain – Basque population352042
      Guix et al. (2002)
      • Guix P.
      • Picornell A.
      • Parera M.
      • Galmes A.
      • Obrador A.
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      • et al.
      Distribution of HFE C282Y and H63D mutations in the Balearic Islands (NE Spain).
      Spain – Balearic Islands302720
      De Marco et al. (2004)
      • De Marco F.
      • Liguori R.
      • Giardina M.G.
      • D’Armiento M.
      • Angelucci E.
      • Lucariello A.
      • et al.
      High prevalence of non-HFE gene-associated haemochromatosis in patients from southern Italy.
      Italy – Southern4691011
      Bauduer et al. (2005)
      • Bauduer F.
      • Scribans C.
      • Degioanni A.
      • Renoux M.
      • Dutour O.
      Distribution of the C282Y and H63D polymorphisms in hereditary hemochromatosis patients from the French Basque Country.
      France – Basque population1582
      Cukjati et al. (2007)
      • Cukjati M.
      • Vaupotic T.
      • Rupreht R.
      • Curin-Serbec V.
      Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay.
      Slovenia211022
      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 [
      • Walsh A.
      • Dixon J.L.
      • Ramm G.A.
      • Hewett D.G.
      • Lincoln D.J.
      • Anderson G.J.
      • et al.
      The clinical relevance of compound heterozygosity for the C282Y and H63D substitutions in hemochromatosis.
      ,
      • Rossi E.
      • Olynyk J.K.
      • Cullen D.J.
      • Papadopoulos G.
      • Bulsara M.
      • Summerville L.
      • et al.
      Compound heterozygous hemochromatosis genotype predicts increased iron and erythrocyte indices in women.
      ,
      • Lim E.M.
      • Rossi E.
      • De-Boer W.B.
      • Reed W.D.
      • Jeffrey G.P.
      Hepatic iron loading in patients with compound heterozygous HFE mutations.
      ].

       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 [
      • Cadet E.
      • Capron D.
      • Perez A.S.
      • Crepin S.N.
      • Arlot S.
      • Ducroix J.P.
      • et al.
      A targeted approach significantly increases the identification rate of patients with undiagnosed haemochromatosis.
      ,
      • Swinkels D.W.
      • Aalbers N.
      • Elving L.D.
      • Bleijenberg G.
      • Swanink C.M.
      • van der Meer J.W.
      Primary haemochromatosis: a missed cause of chronic fatigue syndrome?.
      ,
      • Vital Durand D.
      • Francois S.
      • Nove-Josserand R.
      • Durupt S.
      • Durieu I.
      • Morel Y.
      • et al.
      [Haemochromatosis screening in 120 patients complaining with persistent fatigue].
      ]. None of the three studies found the prevalence of C282Y homozygosity to be increased.

       Arthralgia

      Most available studies investigated the prevalence of C282Y mutations in patients with inflammatory arthritis [
      • Willis G.
      • Scott D.G.
      • Jennings B.A.
      • Smith K.
      • Bukhari M.
      • Wimperis J.Z.
      HFE mutations in an inflammatory arthritis population.
      ,
      • Li J.
      • Zhu Y.
      • Singal D.P.
      HFE gene mutations in patients with rheumatoid arthritis.
      ,
      • Rovetta G.
      • Grignolo M.C.
      • Buffrini L.
      • Monteforte P.
      Prevalence of C282Y mutation in patients with rheumatoid arthritis and spondylarthritis.
      ]; there are few studies in patients with non-inflammatory arthralgia or chondrocalcinosis [
      • Cadet E.
      • Capron D.
      • Perez A.S.
      • Crepin S.N.
      • Arlot S.
      • Ducroix J.P.
      • et al.
      A targeted approach significantly increases the identification rate of patients with undiagnosed haemochromatosis.
      ,
      • Timms A.E.
      • Sathananthan R.
      • Bradbury L.
      • Athanasou N.A.
      • Wordsworth B.P.
      • Brown M.A.
      Genetic testing for haemochromatosis in patients with chondrocalcinosis.
      ]. In the majority of studies of patients with undifferentiated osteoarthritis the prevalence of C282Y homozygosity did not exceed that of the control population [
      • Guyatt G.H.
      • Oxman A.D.
      • Kunz R.
      • Vist G.E.
      • Falck-Ytter Y.
      • Schunemann H.J.
      What is ‘quality of evidence’ and why is it important to clinicians?.
      ,
      • Rovetta G.
      • Grignolo M.C.
      • Buffrini L.
      • Monteforte P.
      Prevalence of C282Y mutation in patients with rheumatoid arthritis and spondylarthritis.
      ]. 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 [
      • Carroll G.J.
      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.
      ,
      • Cauza E.
      • Hanusch-Enserer U.
      • Bischof M.
      • Spak M.
      • Kostner K.
      • Tammaa A.
      • et al.
      Increased C282Y heterozygosity in gestational diabetes.
      ]. A higher prevalence of C282Y homozygosity was only found in patients with well-characterized chondrocalcinosis [
      • Timms A.E.
      • Sathananthan R.
      • Bradbury L.
      • Athanasou N.A.
      • Wordsworth B.P.
      • Brown M.A.
      Genetic testing for haemochromatosis in patients with chondrocalcinosis.
      ].

       Diabetes

      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 [
      • Acton R.T.
      • Barton J.C.
      • Passmore L.V.
      • Adams P.C.
      • Speechley M.R.
      • Dawkins F.W.
      • et al.
      Relationships of serum ferritin, transferrin saturation, and HFE mutations and self-reported diabetes in the Hemochromatosis and Iron Overload Screening (HEIRS) study.
      ,
      • Hahn J.U.
      • Steiner M.
      • Bochnig S.
      • Schmidt H.
      • Schuff-Werner P.
      • Kerner W.
      Evaluation of a diagnostic algorithm for hereditary hemochromatosis in 3500 patients with diabetes.
      ,
      • Frayling T.
      • Ellard S.
      • Grove J.
      • Walker M.
      • Hattersley A.T.
      C282Y mutation in HFE (haemochromatosis) gene and type 2 diabetes.
      ,
      • Davis T.M.
      • Beilby J.
      • Davis W.A.
      • Olynyk J.K.
      • Jeffrey G.P.
      • Rossi E.
      • et al.
      Prevalence, characteristics and prognostic significance of HFE gene mutations in type 2 diabetes: The Fremantle Diabetes Study.
      ,
      • Habeos I.G.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Psilopanagiotou A.
      • Papavassiliou A.G.
      • Vagenakis A.G.
      The role of Hemochromatosis C282Y and H63D mutations in the development of type 2 diabetes mellitus in Greece.
      ,
      • Qi L.
      • Meigs J.
      • Manson J.E.
      • Ma J.
      • Hunter D.
      • Rifai N.
      • et al.
      HFE genetic variability, body iron stores, and the risk of type 2 diabetes in U.S. women.
      ,
      • Halsall D.J.
      • McFarlane I.
      • Luan J.
      • Cox T.M.
      • Wareham N.J.
      Typical type 2 diabetes mellitus and HFE gene mutations: a population-based case-control study.
      ,
      • Fernandez-Real J.M.
      • Vendrell J.
      • Baiget M.
      • Gimferrer E.
      • Ricart W.
      C282Y and H63D mutations of the hemochromatosis candidate gene in type 2 diabetes.
      ,
      • Braun J.
      • Donner H.
      • Plock K.
      • Rau H.
      • Usadel K.H.
      • Badenhoop K.
      Hereditary haemochromatosis mutations (HFE) in patients with Type II diabetes mellitus.
      ,
      • Malecki M.T.
      • Klupa T.
      • Walus M.
      • Czogala W.
      • Greenlaw P.
      • Sieradzki J.
      A search for association between hereditary hemochromatosis HFE gene mutations and type 2 diabetes mellitus in a Polish population.
      ,
      • Sampson M.J.
      • Williams T.
      • Heyburn P.J.
      • Greenwood R.H.
      • Temple R.C.
      • Wimperis J.Z.
      • et al.
      Prevalence of HFE (hemochromatosis gene) mutations in unselected male patients with type 2 diabetes.
      ,
      • Njajou O.T.
      • Alizadeh B.Z.
      • Vaessen N.
      • Vergeer J.
      • Houwing-Duistermaat J.
      • Hofman A.
      • et al.
      The role of hemochromatosis C282Y and H63D gene mutations in type 2 diabetes: findings from the Rotterdam Study and meta-analysis.
      ]. Apart from one exception, no association between type 2 diabetes and C282Y homozygosity was found [
      • Cadet E.
      • Capron D.
      • Perez A.S.
      • Crepin S.N.
      • Arlot S.
      • Ducroix J.P.
      • et al.
      A targeted approach significantly increases the identification rate of patients with undiagnosed haemochromatosis.
      ]. A higher prevalence of the C282Y allele was found in proliferative diabetic retinopathy and nephropathy complicating type 2 diabetes [
      • Peterlin B.
      • Globocnik Petrovic M.
      • Makuc J.
      • Hawlina M.
      • Petrovic D.
      A hemochromatosis-causing mutation C282Y is a risk factor for proliferative diabetic retinopathy in Caucasians with type 2 diabetes.
      ], 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%) [
      • Ellervik C.
      • Mandrup-Poulsen T.
      • Nordestgaard B.G.
      • Larsen L.E.
      • Appleyard M.
      • Frandsen M.
      • et al.
      Prevalence of hereditary haemochromatosis in late-onset type 1 diabetes mellitus: a retrospective study.
      ].

       Liver disease

      There are a limited number of studies reporting C282Y-homozygosity in unselected patients with liver disease [
      • Poullis A.
      • Moodie S.J.
      • Ang L.
      • Finlayson C.J.
      • Levin G.E.
      • Maxwell J.D.
      Routine transferrin saturation measurement in liver clinic patients increases detection of hereditary haemochromatosis.
      ,
      • Poullis A.
      • Moodie S.J.
      • Maxwell J.D.
      Clinical haemochromatosis in HFE mutation carriers.
      ,
      • Nichols L.
      • Dickson G.
      • Phan P.G.
      • Kant J.A.
      Iron binding saturation and genotypic testing for hereditary hemochromatosis in patients with liver disease.
      ]. 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% [
      • Poullis A.
      • Moodie S.J.
      • Ang L.
      • Finlayson C.J.
      • Levin G.E.
      • Maxwell J.D.
      Routine transferrin saturation measurement in liver clinic patients increases detection of hereditary haemochromatosis.
      ].

       Hepatocellular carcinoma

      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 [
      • Willis G.
      • Bardsley V.
      • Fellows I.W.
      • Lonsdale R.
      • Wimperis J.Z.
      • Jennings B.A.
      Hepatocellular carcinoma and the penetrance of HFE C282Y mutations: a cross sectional study.
      ,
      • Cauza E.
      • Peck-Radosavljevic M.
      • Ulrich-Pur H.
      • Datz C.
      • Gschwantler M.
      • Schoniger-Hekele M.
      • et al.
      Mutations of the HFE gene in patients with hepatocellular carcinoma.
      ,
      • Hellerbrand C.
      • Poppl A.
      • Hartmann A.
      • Scholmerich J.
      • Lock G.
      HFE C282Y heterozygosity in hepatocellular carcinoma: evidence for an increased prevalence.
      ,
      • Boige V.
      • Castera L.
      • de Roux N.
      • Ganne-Carrie N.
      • Ducot B.
      • Pelletier G.
      • et al.
      Lack of association between HFE gene mutations and hepatocellular carcinoma in patients with cirrhosis.
      ,
      • Lauret E.
      • Rodriguez M.
      • Gonzalez S.
      • Linares A.
      • Lopez-Vazquez A.
      • Martinez-Borra J.
      • et al.
      HFE gene mutations in alcoholic and virus-related cirrhotic patients with hepatocellular carcinoma.
      ,
      • Pirisi M.
      • Toniutto P.
      • Uzzau A.
      • Fabris C.
      • Avellini C.
      • Scott C.
      • et al.
      Carriage of HFE mutations and outcome of surgical resection for hepatocellular carcinoma in cirrhotic patients.
      ]. The etiology of HCC differed significantly between the studies. Patients with clinical HC were specifically excluded in one study [
      • Hellerbrand C.
      • Poppl A.
      • Hartmann A.
      • Scholmerich J.
      • Lock G.
      HFE C282Y heterozygosity in hepatocellular carcinoma: evidence for an increased prevalence.
      ]. 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% [
      • Willis G.
      • Bardsley V.
      • Fellows I.W.
      • Lonsdale R.
      • Wimperis J.Z.
      • Jennings B.A.
      Hepatocellular carcinoma and the penetrance of HFE C282Y mutations: a cross sectional study.
      ,
      • Cauza E.
      • Peck-Radosavljevic M.
      • Ulrich-Pur H.
      • Datz C.
      • Gschwantler M.
      • Schoniger-Hekele M.
      • et al.
      Mutations of the HFE gene in patients with hepatocellular carcinoma.
      ,
      • Pirisi M.
      • Toniutto P.
      • Uzzau A.
      • Fabris C.
      • Avellini C.
      • Scott C.
      • et al.
      Carriage of HFE mutations and outcome of surgical resection for hepatocellular carcinoma in cirrhotic patients.
      ] and three further studies found an increased prevalence of C282Y heterozygosity [
      • Hellerbrand C.
      • Poppl A.
      • Hartmann A.
      • Scholmerich J.
      • Lock G.
      HFE C282Y heterozygosity in hepatocellular carcinoma: evidence for an increased prevalence.
      ,
      • Lauret E.
      • Rodriguez M.
      • Gonzalez S.
      • Linares A.
      • Lopez-Vazquez A.
      • Martinez-Borra J.
      • et al.
      HFE gene mutations in alcoholic and virus-related cirrhotic patients with hepatocellular carcinoma.
      ,
      • Fargion S.
      • Stazi M.A.
      • Fracanzani A.L.
      • Mattioli M.
      • Sampietro M.
      • Tavazzi D.
      • et al.
      Mutations in the HFE gene and their interaction with exogenous risk factors in hepatocellular carcinoma.
      ]. Only one study [
      • Boige V.
      • Castera L.
      • de Roux N.
      • Ganne-Carrie N.
      • Ducot B.
      • Pelletier G.
      • et al.
      Lack of association between HFE gene mutations and hepatocellular carcinoma in patients with cirrhosis.
      ] 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 [
      • Bonkovsky H.L.
      • Poh Fitzpatrick M.
      • Pimstone N.
      • Obando J.
      • Di Bisceglie A.
      • Tattrie C.
      • et al.
      Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in North America.
      ,
      • Chiaverini C.
      • Halimi G.
      • Ouzan D.
      • Halfon P.
      • Ortonne J.P.
      • Lacour J.P.
      Porphyria cutanea tarda, C282Y, H63D and S65C HFE gene mutations and hepatitis C infection: a study from southern France.
      ,
      • Cribier B.
      • Chiaverini C.
      • Dali Youcef N.
      • Schmitt M.
      • Grima M.
      • Hirth C.
      • et al.
      Porphyria cutanea tarda, hepatitis C, uroporphyrinogen decarboxylase and mutations of HFE gene. A case-control study.
      ,
      • Egger N.G.
      • Goeger D.E.
      • Payne D.A.
      • Miskovsky E.P.
      • Weinman S.A.
      • Anderson K.E.
      Porphyria cutanea tarda: multiplicity of risk factors including HFE mutations, hepatitis C, and inherited uroporphyrinogen decarboxylase deficiency.
      ,
      • Frank J.
      • Poblete Gutierrez P.
      • Weiskirchen R.
      • Gressner O.
      • Merk H.F.
      • Lammert F.
      Hemochromatosis gene sequence deviations in German patients with porphyria cutanea tarda.
      ,
      • Gonzalez Hevilla M.
      • de Salamanca R.E.
      • Morales P.
      • Martinez Laso J.
      • Fontanellas A.
      • Castro M.J.
      • et al.
      Human leukocyte antigen haplotypes and HFE mutations in Spanish hereditary hemochromatosis and sporadic porphyria cutanea tarda.
      ,
      • Hift R.J.
      • Corrigall A.V.
      • Hancock V.
      • Kannemeyer J.
      • Kirsch R.E.
      • Meissner P.N.
      Porphyria cutanea tarda: the etiological importance of mutations in the HFE gene and viral infection is population-dependent.
      ,
      • Kratka K.
      • Dostalikova Cimburova M.
      • Michalikova H.
      • Stransky J.
      • Vranova J.
      • Horak J.
      High prevalence of HFE gene mutations in patients with porphyria cutanea tarda in the Czech Republic.
      ,
      • Lamoril J.
      • Andant C.
      • Gouya L.
      • Malonova E.
      • Grandchamp B.
      • Martasek P.
      • et al.
      Hemochromatosis (HFE) and transferrin receptor-1 (TFRC1) genes in sporadic porphyria cutanea tarda (sPCT).
      ,
      • Martinelli A.L.
      • Zago M.A.
      • Roselino A.M.
      • Filho A.B.
      • Villanova M.G.
      • Secaf M.
      • et al.
      Porphyria cutanea tarda in Brazilian patients: association with hemochromatosis C282Y mutation and hepatitis C virus infection.
      ,
      • Mehrany K.
      • Drage L.A.
      • Brandhagen D.J.
      • Pittelkow M.R.
      Association of porphyria cutanea tarda with hereditary hemochromatosis.
      ,
      • Nagy Z.
      • Koszo F.
      • Par A.
      • Emri G.
      • Horkay I.
      • Horanyi M.
      • et al.
      Hemochromatosis (HFE) gene mutations and hepatitis C virus infection as risk factors for porphyria cutanea tarda in Hungarian patients.
      ,
      • Roberts A.G.
      • Whatley S.D.
      • Morgan R.R.
      • Worwood M.
      • Elder G.H.
      Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda.
      ,
      • Stolzel U.
      • Kostler E.
      • Schuppan D.
      • Richter M.
      • Wollina U.
      • Doss M.O.
      • et al.
      Hemochromatosis (HFE) gene mutations and response to chloroquine in porphyria cutanea tarda.
      ,
      • Stuart K.A.
      • Busfield F.
      • Jazwinska E.C.
      • Gibson P.
      • Butterworth L.A.
      • Cooksley W.G.
      • et al.
      The C282Y mutation in the haemochromatosis gene (HFE) and hepatitis C virus infection are independent cofactors for porphyria cutanea tarda in Australian patients.
      ,
      • Tannapfel A.
      • Stolzel U.
      • Kostler E.
      • Melz S.
      • Richter M.
      • Keim V.
      • et al.
      C282Y and H63D mutation of the hemochromatosis gene in German porphyria cutanea tarda patients.
      ,
      • Toll A.
      • Celis R.
      • Ozalla M.D.
      • Bruguera M.
      • Herrero C.
      • Ercilla M.G.
      The prevalence of HFE C282Y gene mutation is increased in Spanish patients with porphyria cutanea tarda without hepatitis C virus infection.
      ]. No association between PCT and the C282Y polymorphism was found in Italian patients [
      • Sampietro M.
      • Piperno A.
      • Lupica L.
      • Arosio C.
      • Vergani A.
      • Corbetta N.
      • et al.
      High prevalence of the His63Asp HFE mutation in Italian patients with porphyria cutanea tarda.
      ]. 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 [
      • Ellervik C.
      • Birgens H.
      • Tybjaerg Hansen A.
      • Nordestgaard B.G.
      Hemochromatosis genotypes and risk of 31 disease endpoints: meta-analyses including 66,000 cases and 226,000 controls.
      ].

       The prevalence of C282Y homozygosity in individuals with biochemical iron abnormalities

      There is considerable variation in the cut-off of ferritin and transferrin saturation used for genetic screening of hereditary hemochromatosis (HH).

       Serum ferritin

      The prevalence of elevated ferritin varies between 4% and 41% in healthy populations depending on the cut-off and the screening setting (Table 5) [
      • Burt M.J.
      • George P.M.
      • Upton J.D.
      • Collett J.A.
      • Frampton C.M.
      • Chapman T.M.
      • et al.
      The significance of haemochromatosis gene mutations in the general population: implications for screening.
      ,
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
      ,
      • Olynyk J.K.
      • Cullen D.J.
      • Aquilia S.
      • Rossi E.
      • Summerville L.
      • Powell L.W.
      A population-based study of the clinical expression of the hemochromatosis gene.
      ,
      • Deugnier Y.
      • Jouanolle A.M.
      • Chaperon J.
      • Moirand R.
      • Pithois C.
      • Meyer J.F.
      • et al.
      Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.
      ,
      • Acton R.T.
      • Barton J.C.
      • Passmore L.V.
      • Adams P.C.
      • Speechley M.R.
      • Dawkins F.W.
      • et al.
      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 [
      • Adams P.C.
      • Reboussin D.M.
      • Barton J.C.
      • McLaren C.E.
      • Eckfeldt J.H.
      • McLaren G.D.
      • et al.
      Hemochromatosis and iron-overload screening in a racially diverse population.
      ,
      • Waalen J.
      • Felitti V.J.
      • Gelbart T.
      • Beutler E.
      Screening for hemochromatosis by measuring ferritin levels: a more effective approach.
      ].
      Table 5Prevalence of C282Y homozygosity in patients with elevated serum ferritin and transferrin saturation.
      AuthorsRef.Study populationPrevalence of C282Y homozygotes among patients with elevated serum ferritinPrevalence of C282Y homozygotes among patients with elevated transferrin saturation (TS)Comments
      Prevalence of elevated serum ferritinPrevalence of C282YPrevalence of TS elevationPrevalence of C282Y
      Deugnier et al.
      • Deugnier Y.
      • Jouanolle A.M.
      • Chaperon J.
      • Moirand R.
      • Pithois C.
      • Meyer J.F.
      • et al.
      Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.
      Cross-sectional, n = 9396em76 of 981 (7.5%)21 of 76 (17.6%)70 of 993 (7%)26 of 70 (18%)Health care, young patients; ferritin available for a subgroup only
      Olynyk et al.
      • Olynyk J.K.
      • Cullen D.J.
      • Aquilia S.
      • Rossi E.
      • Summerville L.
      • Powell L.W.
      A population-based study of the clinical expression of the hemochromatosis gene.
      Cross-sectional, n = 3011fn405 of 3011 (13.5%)8 of 405 (2%)202 of 3011 (6.7%)15 of 202 (7.4%)Patient selection included persistently elevated TS (45% or higher) or homozygosity for the C282Y mutation
      Burt et al.
      • Burt M.J.
      • George P.M.
      • Upton J.D.
      • Collett J.A.
      • Frampton C.M.
      • Chapman T.M.
      • et al.
      The significance of haemochromatosis gene mutations in the general population: implications for screening.
      Cross-sectional, n = 1064gl42 of 1040 (4.0%)2 of 42 (4.8%)46 of 1040 (4.4%)5 of 46 (10.9%)Voters
      Distante et al.
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
      Cross-sectional, n = 505hl23 of 505 (4.6%)2 of 23 (8.7%)25 of 505 pts (5%)2 of 25 (8%)Health care
      McDonnell et al.
      • McDonnell S.M.
      • Hover A.
      • Gloe D.
      • Ou C.Y.
      • Cogswell M.E.
      • Grummer-Strawn L.
      Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri.
      Cross-sectional, n = 1450ioNo dataNo data60 of 1640 (3.7%)13 of 60 (21.7%)HMO employees; only data for TS
      Delatycki et al.
      • Delatycki M.B.
      • Allen K.J.
      • Nisselle A.E.
      • Collins V.
      • Metcalfe S.
      • du Sart D.
      • et al.
      Use of community genetic screening to prevent HFE-associated hereditary haemochromatosis.
      Cross-sectional, n = 11,307No dataNo dataNo dataNo data2 of 47 pts (biopsy in 6 pts) had precirrhotic fibrosis
      Adams et al.
      • Adams P.C.
      • Kertesz A.E.
      • McLaren C.E.
      • Barr R.
      • Bamford A.
      • Chakrabarti S.
      Population screening for hemochromatosis: a comparison of unbound iron-binding capacity, transferrin saturation, and C282Y genotyping in 5211 voluntary blood donors.
      Cross-sectional, n = 5211pNo dataNo data60 of 5211 (1.2%)4 of 60 (6.7%)Blood donors
      150 of 5211 (2.9%)9 of 150 (6%)
      278 of 5211 (5.3%)12 of 278 (4.3%)
      Adams et al.
      • Adams P.C.
      • Reboussin D.M.
      • Barton J.C.
      • McLaren C.E.
      • Eckfeldt J.H.
      • McLaren G.D.
      • et al.
      Hemochromatosis and iron-overload screening in a racially diverse population.
      Cross-sectional, n = 99,711aqNo dataNo dataNo dataNo dataHEIRS study
      Beutler et al.
      • Beutler E.
      • Felitti V.
      • Gelbart T.
      • Ho N.
      The effect of HFE genotypes on measurements of iron overload in patients attending a health appraisal clinic.
      Cross-sectional, n = 9650brNo dataNo data67% of males, 39% of females
      80% of males, 50% of females
      Barton et al.
      • Barton J.C.
      • Acton R.T.
      • Lovato L.
      • Speechley M.R.
      • McLaren C.E.
      • Harris E.L.
      • et al.
      Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in Native Americans and whites in the Hemochromatosis and Iron Overload Screening Study.
      Cross-sectional, n = 43,453 caucasianar9299 whites (21.4%)147 of 9299 (1.6%)2976 of 43,453 (6.8%)166 of 2976 (5.6%)
      Asberg et al.
      • Asberg A.
      • Hveem K.
      • Thorstensen K.
      • Ellekjter E.
      • Kannelonning K.
      • Fjosne U.
      • et al.
      Screening for hemochromatosis: high prevalence and low morbidity in an unselected population of 65,238 persons.
      Cross-sectional, n = 65,238mNo dataNo data2.7% of males, 2.5% of females269 of 1698 (15.8%)
      Gordeuk et al.
      • Gordeuk V.R.
      • Reboussin D.M.
      • McLaren C.E.
      • Barton J.C.
      • Acton R.T.
      • McLaren G.D.
      • et al.
      Serum ferritin concentrations and body iron stores in a multicenter, multiethnic primary-care population.
      Cross-sectional, n = 101,168ar2253 of 101,168 (2.2%)2253 of 101,168 (2.2%)155 of 2253 (6.9%)Primary care combination of TS and ferritin
      Ferritin [μg/L] cutoffs: a>300 males and postmenopausal females, >200 females, b>250 males and >200 females, c>300 males and females, d>250 males and >200 females, e>280 males >130 females, f>300 males and females, g>428 males >302 females, h>200 males and females, i95% percentile Transferrin saturation [%] cutoff: k>55: males >45: females, l>50, m>55: males and >50: females, n>45, o>55: males, >60: females, p>54 or >49 or >45, q>55: males >45: females, r>50 overall >45 overall.

       Transferrin saturation

      Elevated transferrin saturation was found in 1.2–7% of screened individuals in unselected populations [
      • Burt M.J.
      • George P.M.
      • Upton J.D.
      • Collett J.A.
      • Frampton C.M.
      • Chapman T.M.
      • et al.
      The significance of haemochromatosis gene mutations in the general population: implications for screening.
      ,
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
      ,
      • Olynyk J.K.
      • Cullen D.J.
      • Aquilia S.
      • Rossi E.
      • Summerville L.
      • Powell L.W.
      A population-based study of the clinical expression of the hemochromatosis gene.
      ,
      • Deugnier Y.
      • Jouanolle A.M.
      • Chaperon J.
      • Moirand R.
      • Pithois C.
      • Meyer J.F.
      • et al.
      Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.
      ,
      • Adams P.C.
      • Kertesz A.E.
      • McLaren C.E.
      • Barr R.
      • Bamford A.
      • Chakrabarti S.
      Population screening for hemochromatosis: a comparison of unbound iron-binding capacity, transferrin saturation, and C282Y genotyping in 5211 voluntary blood donors.
      ,
      • Barton J.C.
      • Acton R.T.
      • Lovato L.
      • Speechley M.R.
      • McLaren C.E.
      • Harris E.L.
      • et al.
      Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in Native Americans and whites in the Hemochromatosis and Iron Overload Screening Study.
      ,
      • Asberg A.
      • Hveem K.
      • Thorstensen K.
      • Ellekjter E.
      • Kannelonning K.
      • Fjosne U.
      • et al.
      Screening for hemochromatosis: high prevalence and low morbidity in an unselected population of 65,238 persons.
      ] (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) [
      • Bax L.
      • Yu L.M.
      • Ikeda N.
      • Tsuruta H.
      • Moons K.G.
      Development and validation of MIX: comprehensive free software for meta-analysis of causal research data.
      ,

      Bax L, Yu LM, Ikeda N, Tsuruta H, Moons KGM. MIX: comprehensive free software for meta-analysis of causal research data. Version 1.7., 2009.

      ].
      Figure thumbnail gr2
      Fig. 2Forest plot of studies on the penetrance of hemochromatosis. Studies are weighted on the inverse of the confidence interval. (For detailed information see ).

       Excess iron

      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.
      AuthorsRef.Study typeC282Y homozygotes (females)Definition of penetrant diseaseAffected individualsPenetranceComments
      Burt et al. (1998)
      • Burt M.J.
      • George P.M.
      • Upton J.D.
      • Collett J.A.
      • Frampton C.M.
      • Chapman T.M.
      • et al.
      The significance of haemochromatosis gene mutations in the general population: implications for screening.
      Cross-sectional5 (4)Hepatic iron index >1.9 upon liver biopsy360%No liver biopsy in unaffected individuals because of normal serum iron parameters
      Distante et al. (1999)
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      High prevalence of the hemochromatosis-associated Cys282Tyr HFE gene mutation in a healthy Norwegian population in the city of Oslo, and its phenotypic expression.
      Cross-sectional2 (1)Iron removed >5 g or HII >1.9 or histological iron grade >2+150%Unaffected patient had Pearl’s stain Grade 2 and HII of 1.7
      McDonnell et al. (1999)
      • McDonnell S.M.
      • Hover A.
      • Gloe D.
      • Ou C.Y.
      • Cogswell M.E.
      • Grummer-Strawn L.
      Population-based screening for hemochromatosis using phenotypic and DNA testing among employees of health maintenance organizations in Springfield, Missouri.
      Cross-sectional4 (3)Iron removed >5 g or HII >1.9 or histological iron grade >2+375%One unaffected patient had elevated serum iron parameters
      Olynyk et al. (1999)
      • Olynyk J.K.
      • Cullen D.J.
      • Aquilia S.
      • Rossi E.
      • Summerville L.
      • Powell L.W.
      A population-based study of the clinical expression of the hemochromatosis gene.
      Cross-sectional16 (9)HII >1.9 or histological iron grade >2956.3%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
      Distante et al. (2000)
      • Distante S.
      • Berg J.P.
      • Lande K.
      • Haug E.
      • Bell H.
      HFE gene mutation (C282Y) and phenotypic expression among a hospitalised population in a high prevalence area of haemochromatosis.
      Cross-sectional & short term follow up14 (9)HII>1.9 or histological iron grade >2 or congestive heart failure + marked and persistent hyperferritinemia and TS >55%321.4%Liver biopsy available only in 5 patients; a total of 5 patients of whom 4 had no biopsy had persistent hyperferritinemia
      Bulaj et al. (2000)
      • Bulaj Z.J.
      • Ajioka R.S.
      • Phillips J.D.
      • LaSalle B.A.
      • Jorde L.B.
      • Griffen L.M.
      • et al.
      Disease-related conditions in relatives of patients with hemochromatosis.
      Cross-sectional – affected individuals184 (48)At least one disease-related condition (cirrhosis, fibrosis, elevated ALT or AST, arthropathy)13774.5%
      Cross-sectional – family members214 (101)3315.4%
      Cross-sectional – unselected107 (41)76.5%
      Barton et al. (1999)
      • Barton J.C.
      • Rothenberg B.E.
      • Bertoli L.F.
      • Acton R.T.
      Diagnosis of hemochromatosis in family members of probands: a comparison of phenotyping and HFE genotyping.
      Cross-sectional – family based25 (n.d.)Cirrhosis or diabetes attributable to iron overload6–2324–79%Ill-defined HC phenotype was present in a total of 23 patients
      Beutler et al. (2002)
      • Beutler E.
      • Felitti V.J.
      • Koziol J.A.
      • Ho N.J.
      • Gelbart T.
      Penetrance of 845G → A (C282Y) HFE hereditary haemochromatosis mutation in the USA.
      Cross-sectional152 (79)‘liver problems’ (assessed in 124)108.1%Signs and symptoms that would suggest a diagnosis of HC in only one patient
      Waalen et al. (2002)
      • Waalen J.
      • Felitti V.
      • Gelbart T.
      • Ho N.J.
      • Beutler E.
      Prevalence of hemochromatosis-related symptoms among individuals with mutations in the HFE gene.
      Cross-sectional141 (80)Only symptoms and serum iron parameters reported92 patients had elevated serum ferritin concentrations, disease-associated symptoms were equal in control group and C282Y homozygotes
      Deugnier et al. (2002)
      • Deugnier Y.
      • Jouanolle A.M.
      • Chaperon J.
      • Moirand R.
      • Pithois C.
      • Meyer J.F.
      • et al.
      Gender-specific phenotypic expression and screening strategies in C282Y-linked haemochromatosis: a study of 9396 French people.
      Cross-sectional54 (44)At least one disease-related symptom (fatigue, arthralgia, diabetes, increased ALT)3564.8%21 patients had increased serum iron parameters
      Phatak et al. (2002)
      • Phatak P.D.
      • Ryan D.H.
      • Cappuccio J.
      • Oakes D.
      • Braggins C.
      • Provenzano K.
      • et al.
      Prevalence and penetrance of HFE mutations in 4865 unselected primary care patients.
      Cross-sectional12 (8)Iron removed >5 g for males and >3 g for females542%Increased serum ferritin in 50% of patients
      Poullis et al. (2003)
      • Poullis A.
      • Moodie S.J.
      • Ang L.
      • Finlayson C.J.
      • Levin G.E.
      • Maxwell J.D.
      Routine transferrin saturation measurement in liver clinic patients increases detection of hereditary haemochromatosis.
      Cross-sectional12 (5)Histological iron grade >2758%Increased serum ferritin in 11 out of 12 patients, but coincidence of significant co-morbidities (HCV and iron in 5 patients)
      Olynyk et al. (2004)
      • Olynyk J.K.
      • Hagan S.E.
      • Cullen D.J.
      • Beilby J.
      • Whittall D.E.
      Evolution of untreated hereditary hemochromatosis in the Busselton population: a 17-year study.
      Longitudinal10 (6)Hepatic iron >25 μmol/g660%Gradual increase in TS over 10 year observation – no biopsy in 4 patients
      Andersen et al. (2004)
      • Andersen R.V.
      • Tybjaerg-Hansen A.
      • Appleyard M.
      • Birgens H.
      • Nordestgaard B.G.
      Hemochromatosis mutations in the general population: iron overload progression rate.
      Longitudinal23 (16)At least one disease-related condition (cirrhosis, fibrosis, elevated ALT or AST, arthropathy)313.0%Increased serum ferritin in 16 patients
      Gleeson et al. (2004)
      • Gleeson F.
      • Ryan E.
      • Barrett S.
      • Crowe J.
      Clinical expression of haemochromatosis in Irish C282Y homozygotes identified through family screening.
      Family based study71 (25)Histological iron grade >3+2636.6%Only 71 out of 209 C282Y homozygote patients who underwent liver biopsy were included
      Rossi et al. (2004)
      • Rossi E.
      • Kuek C.
      • Beilby J.P.
      • Jeffrey G.P.
      • Devine A.
      • Prince R.L.
      Expression of the HFE hemochromatosis gene in a community-based population of elderly women.
      Cross-sectional200%No clinical symptoms
      Delatycki et al. (2005)
      • Delatycki M.B.
      • Allen K.J.
      • Nisselle A.E.
      • Collins V.
      • Metcalfe S.
      • du Sart D.
      • et al.
      Use of community genetic screening to prevent HFE-associated hereditary haemochromatosis.
      Cross-sectional51 (26)Disease-associated symptoms4588%45 patients had disease-associated symptoms (tiredness, abdominal pain, joint pain)
      Powell et al. (2006)
      • Powell L.W.
      • Dixon J.L.
      • Ramm G.A.
      • Purdie D.M.
      • Lincoln D.J.
      • Anderson G.J.
      • et al.
      Screening for hemochromatosis in asymptomatic subjects with or without a family history.
      Cross-sectional – family based401 (201)Histological iron grade >212832%At least one disease related condition 17%
      Cross-sectional – population based271 (112)Histological iron grade >213550%At least one disease related condition 27%
      Asberg et al. (2007)
      • Asberg A.
      • Hveem K.
      • Kannelonning K.
      • Irgens W.O.
      Penetrance of the C28Y/C282Y genotype of the HFE gene.
      Cross-sectional319 (0)Cirrhosis11–163.4–5%Predicted/calculated penetrance
      Allen et al. (2008)
      • Allen K.J.
      • Gurrin L.C.
      • Constantine C.C.
      • Osborne N.J.
      • Delatycki M.B.
      • Nicoll A.J.
      • et al.
      Iron-overload-related disease in HFE hereditary hemochromatosis.
      Longitudinal203 (108)Serum ferritin >1000 μg/L4019.7%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 [
      • Beutler E.
      • Felitti V.J.
      • Koziol J.A.
      • Ho N.J.
      • Gelbart T.
      Penetrance of 845G → A (C282Y) HFE hereditary haemochromatosis mutation in the USA.
      ].
      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 [
      • Gleeson F.
      • Ryan E.
      • Barrett S.
      • Crowe J.
      Clinical expression of haemochromatosis in Irish C282Y homozygotes identified through family screening.
      ]. Liver fibrosis was present in 18% of males and 5% in females homozygous for C282Y; cirrhosis was present in 6% of males and 2% of females [
      • Bell H.
      • Berg J.P.
      • Undlien D.E.
      • Distante S.
      • Raknerud N.
      • Heier H.E.
      • et al.
      The clinical expression of hemochromatosis in Oslo, Norway. Excessive oral iron intake may lead to secondary hemochromatosis even in HFE C282Y mutation negative subjects.
      ,
      • Powell L.W.
      • Dixon J.L.
      • Ramm G.A.
      • Purdie D.M.
      • Lincoln D.J.
      • Anderson G.J.
      • et al.
      Screening for hemochromatosis in asymptomatic subjects with or without a family history.
      ]. A recent meta-analysis concludes that 10–33% of C282Y homozygotes eventually would develop hemochromatosis-associated morbidity [
      • Whitlock E.P.
      • Garlitz B.A.
      • Harris E.L.
      • Beil T.L.
      • Smith P.R.
      Screening for hereditary hemochromatosis: a systematic review for the U.S. Preventive Services Task Force.
      ].
      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 [
      • Olynyk J.K.
      • Hagan S.E.
      • Cullen D.J.
      • Beilby J.
      • Whittall D.E.
      Evolution of untreated hereditary hemochromatosis in the Busselton population: a 17-year study.
      ,
      • Andersen R.V.
      • Tybjaerg-Hansen A.
      • Appleyard M.
      • Birgens H.
      • Nordestgaard B.G.
      Hemochromatosis mutations in the general population: iron overload progression rate.
      ,
      • Allen K.J.
      • Gurrin L.C.
      • Constantine C.C.
      • Osborne N.J.
      • Delatycki M.B.
      • Nicoll A.J.
      • et al.
      Iron-overload-related disease in HFE hereditary hemochromatosis.
      ]. 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 [
      • Whitlock E.P.
      • Garlitz B.A.
      • Harris E.L.
      • Beil T.L.
      • Smith P.R.
      Screening for hereditary hemochromatosis: a systematic review for the U.S. Preventive Services Task Force.
      ]. The proportion of C282Y homozygotes with iron overload-related disease is substantially higher for men than for women (28% vs. 1%) [
      • Allen K.J.
      • Gurrin L.C.
      • Constantine C.C.
      • Osborne N.J.
      • Delatycki M.B.
      • Nicoll A.J.
      • et al.
      Iron-overload-related disease in HFE hereditary hemochromatosis.
      ].

       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 [
      • Asberg A.
      • Hveem K.
      • Thorstensen K.
      • Ellekjter E.
      • Kannelonning K.
      • Fjosne U.
      • et al.
      Screening for hemochromatosis: high prevalence and low morbidity in an unselected population of 65,238 persons.
      ,
      • Asberg A.
      • Hveem K.
      • Kannelonning K.
      • Irgens W.O.
      Penetrance of the C28Y/C282Y genotype of the HFE gene.
      ].
      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 [
      • Asberg A.
      • Hveem K.
      • Kannelonning K.
      • Irgens W.O.
      Penetrance of the C28Y/C282Y genotype of the HFE gene.
      ].

      Recommendations for genetic testing:

      General population:
      • 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 [
      • Somerville M.J.
      • Sprysak K.A.
      • Hicks M.
      • Elyas B.G.
      • Vicen-Wyhony L.
      An HFE intronic variant promotes misdiagnosis of hereditary hemochromatosis.
      ]. The common S65C polymorphism may complicate interpretation of real-time PCR and melting curve analysis tests [
      • Klaassen C.H.
      • van Aarssen Y.A.
      • van der Stappen J.W.
      Improved real-time detection of the H63D and S65C mutations associated with hereditary hemochromatosis using a SimpleProbe assay format.
      ]. Finally, in cis inheritance of rare genetic variants [
      • Schranz M.
      • Talasz H.
      • Graziadei I.
      • Winder T.
      • Sergi C.
      • Bogner K.
      • et al.
      Diagnosis of hepatic iron overload: a family study illustrating pitfalls in diagnosing hemochromatosis.
      ] must be considered when gene tests are interpreted.
      Table 7Methods for HFE genotyping.
      MethodSimultaneous detection of multiple mutationsbf Detection of novel/rare genetic variationsSpecialized equipment requiredAmenable for high throughputRef.
      RFLPPCR amplification followed by restriction fragment length polymorphism+/−
      • Feder J.N.
      The hereditary hemochromatosis gene (HFE): a MHC class I-like gene that functions in the regulation of iron homeostasis.
      ,
      • Jazwinska E.C.
      • Powell L.W.
      Hemochromatosis and ‘HLA-H’: definite!.
      ,
      • Koeken A.
      • Cobbaert C.
      • Quint W.
      • van Doorn L.J.
      Genotyping of hemochromatosis-associated mutations in the HFE gene by PCR-RFLP and a novel reverse hybridization method.
      ,
      • Cunat S.
      • Giansily Blaizot M.
      • Bismuth M.
      • Blanc F.
      • Dereure O.
      • Larrey D.
      • et al.
      Global sequencing approach for characterizing the molecular background of hereditary iron disorders.
      ,
      • Cukjati M.
      • Koren S.
      • Curin Serbec V.
      • Vidan-Jeras B.
      • Rupreht R.
      A novel homozygous frameshift deletion c.471del of HFE associated with hemochromatosis.
      ,
      • Steiner M.
      • Ocran K.
      • Genschel J.
      • Meier P.
      • Gerl H.
      • Ventz M.
      • et al.
      A homozygous HFE gene splice site mutation (IVS5+1 G/A) in a hereditary hemochromatosis patient of Vietnamese origin.
      ,
      • Barton J.C.
      • Sawada Hirai R.
      • Rothenberg B.E.
      • Acton R.T.
      Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands.
      ,
      • de Kok J.B.
      • Wiegerinck E.T.
      • Giesendorf B.A.
      • Swinkels D.W.
      Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).
      ,
      • Behrens M.
      • Lange R.
      A highly reproducible and economically competitive SNP analysis of several well characterized human mutations.
      ,
      • Alsmadi O.A.
      • Al Kayal F.
      • Al Hamed M.
      • Meyer B.F.
      Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.
      ,
      • Castley A.
      • Higgins M.
      • Ivey J.
      • Mamotte C.
      • Sayer D.C.
      • Christiansen F.T.
      Clinical applications of whole-blood PCR with real-time instrumentation.
      ,
      • Walburger D.K.
      • Afonina I.A.
      • Wydro R.
      An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis.
      ,
      • Cheng J.
      • Zhang Y.
      • Li Q.
      Real-time PCR genotyping using displacing probes.
      ,
      • Bach V.
      • Barcelo M.J.
      • Altes A.
      • Remacha A.
      • Felez J.
      • Baiget M.
      Genotyping the HFE gene by melting point analysis with the LightCycler system: Pros and cons.
      ,
      • Moyses C.B.
      • Moreira E.S.
      • Asprino P.F.
      • Guimaraes G.S.
      • Alberto F.L.
      Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR.
      ,
      • Biasiotto G.
      • Belloli S.
      • Ruggeri G.
      • Zanella I.
      • Gerardi G.
      • Corrado M.
      • et al.
      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.
      ,
      • Smillie D.
      A PCR-SSP method for detecting the Cys282Tyr mutation in the HFE gene associated with hereditary haemochromatosis.
      ,
      • Smillie D.
      A PCR-SSP method for detecting the His63Asp mutation in the HFE gene associated with hereditary haemochromatosis.
      ,
      • Steffensen R.
      • Varming K.
      • Jersild C.
      Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.
      ,
      • Wenz H.M.
      • Baumhueter S.
      • Ramachandra S.
      • Worwood M.
      A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).
      ,
      • Guttridge M.G.
      • Carter K.
      • Worwood M.
      • Darke C.
      Population screening for hemochromatosis by PCR using sequence-specific primers.
      ,
      • Guttridge M.G.
      • Thompson J.
      • Worwood M.
      • Darke C.
      Rapid detection of genetic mutations associated with haemochromatosis.
      ,
      • Kaur G.
      • Rapthap C.C.
      • Xavier M.
      • Saxena R.
      • Choudhary V.P.
      • Reuben S.K.
      • et al.
      Distribution of C282Y and H63D mutations in the HFE gene in healthy Asian Indians and patients with thalassaemia major.
      ,
      • Turner M.S.
      • Penning S.
      • Sharp A.
      • Hyland V.J.
      • Harris R.
      • Morris C.P.
      • et al.
      Solid-phase amplification for detection of C282y and H63D hemochromatosis (HFE) gene mutations.
      ,
      • Bosserhoff A.K.
      • Seegers S.
      • Hellerbrand C.
      • Scholmerich J.
      • Buttner R.
      Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).
      ,
      • Simonsen K.
      • Dissing J.
      • Rudbeck L.
      • Schwartz M.
      Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR-SSCP: detection of a new polymorphic mutation.
      ,
      • Kim S.
      • Edwards J.R.
      • Deng L.
      • Chung W.
      • Ju J.
      Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry.
      ,
      • Bernacki S.H.
      • Farkas D.H.
      • Shi W.
      • Chan V.
      • Liu Y.
      • Beck J.C.
      • et al.
      Bioelectronic sensor technology for detection of cystic fibrosis and hereditary hemochromatosis mutations.
      ,
      • Footz T.
      • Somerville M.J.
      • Tomaszewski R.
      • Elyas B.
      • Backhouse C.J.
      Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis.
      ,
      • Bosserhoff A.K.
      • Buettner R.
      • Hellerbrand C.
      Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview.
      ,
      • Devaney J.M.
      • Pettit E.L.
      • Kaler S.G.
      • Vallone P.M.
      • Butler J.M.
      • Marino M.A.
      Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography.
      ,
      • Lubin I.M.
      • Yamada N.A.
      • Stansel R.M.
      • Pace R.G.
      • Rohlfs E.M.
      • Silverman L.M.
      HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis.
      ,
      • Kotze M.J.
      • de Villiers J.N.
      • Bouwens C.S.
      • Warnich L.
      • Zaahl M.G.
      • van der Merwe S.
      • et al.
      Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.
      ,
      • Oberkanins C.
      • Moritz A.
      • de Villiers J.N.
      • Kotze M.J.
      • Kury F.
      A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.
      ,
      • Rivers C.A.
      • Barton J.C.
      • Acton R.T.
      A rapid PCR-SSP assay for the hemochromatosis-associated Tyr250Stop mutation in the TFR2 gene.
      ,
      • Somerville M.J.
      • Sprysak K.A.
      • Hicks M.
      • Elyas B.G.
      • Vicen-Wyhony L.
      An HFE intronic variant promotes misdiagnosis of hereditary hemochromatosis.
      ,
      • Klaassen C.H.
      • van Aarssen Y.A.
      • van der Stappen J.W.
      Improved real-time detection of the H63D and S65C mutations associated with hereditary hemochromatosis using a SimpleProbe assay format.
      ,
      • Schranz M.
      • Talasz H.
      • Graziadei I.
      • Winder T.
      • Sergi C.
      • Bogner K.
      • et al.
      Diagnosis of hepatic iron overload: a family study illustrating pitfalls in diagnosing hemochromatosis.
      ,
      • Sebastiani G.
      • Wallace D.F.
      • Davies S.E.
      • Kulhalli V.
      • Walker A.P.
      • Dooley J.S.
      Fatty liver in H63D homozygotes with hyperferritinemia.
      ,
      • Jackson H.A.
      • Carter K.
      • Darke C.
      • Guttridge M.G.
      • Ravine D.
      • Hutton R.D.
      • et al.
      HFE mutations, iron deficiency and overload in 10,500 blood donors.
      ,
      • de Villiers J.N.
      • Hillermann R.
      • Loubser L.
      • Kotze M.J.
      Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria.
      ,
      • Bradbury R.
      • Fagan E.
      • Payne S.J.
      Two novel polymorphisms (E277K and V212V) in the haemochromatosis gene HFE.
      ,
      • Floreani A.
      • Navaglia F.
      • Basso D.
      • Zambon C.F.
      • Basso G.
      • Germano G.
      • et al.
      Intron 2 [IVS2, T-C +4] HFE gene mutation associated with S65C causes alternative RNA splicing and is responsible for iron overload.
      ,
      • Piperno A.
      • Arosio C.
      • Fossati L.
      • Vigano M.
      • Trombini P.
      • Vergani A.
      • et al.
      Two novel nonsense mutations of HFE gene in five unrelated italian patients with hemochromatosis.
      ,
      • Wallace D.F.
      • Dooley J.S.
      • Walker A.P.
      A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote.
      ,
      • Gochee P.A.
      • Powell L.W.
      • Cullen D.J.
      • Du Sart D.
      • Rossi E.
      • Olynyk J.K.
      A population-based study of the biochemical and clinical expression of the H63D hemochromatosis mutation.
      ,
      • Wallace D.F.
      • Walker A.P.
      • Pietrangelo A.
      • Clare M.
      • Bomford A.B.
      • Dixon J.L.
      • et al.
      Frequency of the S65C mutation of HFE and iron overload in 309 subjects heterozygous for C282Y.
      ,
      • Aguilar Martinez P.
      • Biron C.
      • Blanc F.
      • Masmejean C.
      • Jeanjean P.
      • Michel H.
      • et al.
      Compound heterozygotes for hemochromatosis gene mutations: may they help to understand the pathophysiology of the disease?.
      ,
      • Gandon Y.
      • Olivie D.
      • Guyader D.
      • Aube C.
      • Oberti F.
      • Sebille V.
      • et al.
      Non-invasive assessment of hepatic iron stores by MRI.
      ,
      • St Pierre T.G.
      • Clark P.R.
      • Chua-Anusorn W.
      • Fleming A.J.
      • Jeffrey G.P.
      • Olynyk J.K.
      • et al.
      Noninvasive measurement and imaging of liver iron concentrations using proton magnetic resonance.
      ,
      • Ernst O.
      • Sergent G.
      • Bonvarlet P.
      • Canva-Delcambre V.
      • Paris J.C.
      • L’Hermine C.
      Hepatic iron overload: diagnosis and quantification with MR imaging.
      ,
      • Brittenham G.M.
      • Farrell D.E.
      • Harris J.W.
      • Feldman E.S.
      • Danish E.H.
      • Muir W.A.
      • et al.
      Magnetic-susceptibility measurement of human iron stores.
      ,
      • Nielsen P.
      • Engelhardt R.
      • Duerken M.
      • Janka G.E.
      • Fischer R.
      Using SQUID biomagnetic liver susceptometry in the treatment of thalassemia and other iron loading diseases.
      ,
      • Fischer R.
      • Piga A.
      • Harmatz P.
      • Nielsen P.
      Monitoring long-term efficacy of iron chelation treatment with biomagnetic liver susceptometry.
      ,
      • Guyader D.
      • Jacquelinet C.
      • Moirand R.
      • Turlin B.
      • Mendler M.H.
      • Chaperon J.
      • et al.
      Noninvasive prediction of fibrosis in C282Y homozygous hemochromatosis.
      ,
      • Morrison E.D.
      • Brandhagen D.J.
      • Phatak P.D.
      • Barton J.C.
      • Krawitt E.L.
      • El-Serag H.B.
      • et al.
      Serum ferritin level predicts advanced hepatic fibrosis among U.S. patients with phenotypic hemochromatosis.
      ,
      • Crawford D.H.
      • Murphy T.L.
      • Ramm L.E.
      • Fletcher L.M.
      • Clouston A.D.
      • Anderson G.J.
      • et al.
      Serum hyaluronic acid with serum ferritin accurately predicts cirrhosis and reduces the need for liver biopsy in C282Y hemochromatosis.
      ,
      • Adhoute X.
      • Foucher J.
      • Laharie D.
      • Terrebonne E.
      • Vergniol J.
      • Castera L.
      • et al.
      Diagnosis of liver fibrosis using FibroScan and other noninvasive methods in patients with hemochromatosis: a prospective study.
      ,
      • El Serag H.B.
      • Inadomi J.M.
      • Kowdley K.V.
      Screening for hereditary hemochromatosis in siblings and children of affected patients. A cost-effectiveness analysis.
      ,
      • Houglum K.
      • Ramm G.A.
      • Crawford D.H.
      • Witztum J.L.
      • Powell L.W.
      • Chojkier M.
      Excess iron induces hepatic oxidative stress and transforming growth factor beta1 in genetic hemochromatosis.
      ,
      • Bassett M.L.
      • Halliday J.W.
      • Powell L.W.
      Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron level associated with fibrosis.
      ,
      • Le Lan C.
      • Loreal O.
      • Cohen T.
      • Ropert M.
      • Glickstein H.
      • Laine F.
      • et al.
      Redox active plasma iron in C282Y/C282Y hemochromatosis.
      ,
      • Bomford A.
      • Williams R.
      Long term results of venesection therapy in idiopathic haemochromatosis.
      ,
      • Milman N.
      • Pedersen P.
      • a Steig T.
      • Byg K.E.
      • Graudal N.
      • Fenger K.
      Clinically overt hereditary hemochromatosis in Denmark 1948–1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients.
      ,
      • Falize L.
      • Guillygomarc’h A.
      • Perrin M.
      • Laine F.
      • Guyader D.
      • Brissot P.
      • et al.
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      ,
      • Powell L.W.
      • Kerr J.F.
      Reversal of ‘cirrhosis’ in idiopathic haemochromatosis following long-term intensive venesection therapy.
      ,
      • Niederau C.
      • Fischer R.
      • Purschel A.
      • Stremmel W.
      • Haussinger D.
      • Strohmeyer G.
      Long-term survival in patients with hereditary hemochromatosis.
      ,
      • Fracanzani A.L.
      • Fargion S.
      • Romano R.
      • Conte D.
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      • et al.
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      ,
      • Adams P.C.
      Factors affecting the rate of iron mobilization during venesection therapy for genetic hemochromatosis.
      ,
      • Hicken B.L.
      • Tucker D.C.
      • Barton J.C.
      Patient compliance with phlebotomy therapy for iron overload associated with hemochromatosis.
      ,
      • Adams P.C.
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      • Valberg L.S.
      Rate of iron reaccumulation following iron depletion in hereditary hemochromatosis. Implications for venesection therapy.
      ,
      • Hutchinson C.
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      • Powell J.J.
      • Bomford A.
      Proton pump inhibitors suppress absorption of dietary non-haem iron in hereditary haemochromatosis.
      ,
      • Kaltwasser J.P.
      • Werner E.
      • Schalk K.
      • Hansen C.
      • Gottschalk R.
      • Seidl C.
      Clinical trial on the effect of regular tea drinking on iron accumulation in genetic haemochromatosis.
      ,
      • Milward E.A.
      • Baines S.K.
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      • Divitini M.L.
      • Ravine D.G.
      • et al.
      Noncitrus fruits as novel dietary environmental modifiers of iron stores in people with or without HFE gene mutations.
      ,
      • Nienhuis A.W.
      Vitamin C and iron.
      ,
      • Schofield R.S.
      • Aranda Jr., J.M.
      • Hill J.A.
      • Streiff R.
      Cardiac transplantation in a patient with hereditary hemochromatosis: role of adjunctive phlebotomy and erythropoietin.
      ,
      • Barton J.C.
      • Grindon A.J.
      • Barton N.H.
      • Bertoli L.F.
      Hemochromatosis probands as blood donors.
      ,
      • Fletcher L.M.
      • Dixon J.L.
      • Purdie D.M.
      • Powell L.W.
      • Crawford D.H.
      Excess alcohol greatly increases the prevalence of cirrhosis in hereditary hemochromatosis.
      ,
      • Harrison-Findik D.D.
      • Klein E.
      • Crist C.
      • Evans J.
      • Timchenko N.
      • Gollan J.
      Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol.
      ,
      • Chapman R.W.
      • Morgan M.Y.
      • Boss A.M.
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      Acute and chronic effects of alcohol on iron absorption.
      ,
      • Ioannou G.N.
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      • Heagerty P.J.
      • Kowdley K.V.
      The effect of alcohol consumption on the prevalence of iron overload, iron deficiency, and iron deficiency anemia.
      ,
      • Ioannou G.N.
      • Weiss N.S.
      • Kowdley K.V.
      Relationship between transferrin-iron saturation, alcohol consumption, and the incidence of cirrhosis and liver cancer.
      ,
      • Barton J.C.
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      • et al.
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      ,
      • Kowdley K.V.
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      • et al.
      Survival after liver transplantation in patients with hepatic iron overload: the national hemochromatosis transplant registry.
      ,
      • Yu L.
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      ,
      • Edwards C.Q.
      • Kelly T.M.
      • Ellwein G.
      • Kushner J.P.
      Thyroid disease in hemochromatosis. Increased incidence in homozygous men.
      ,
      • Guggenbuhl P.
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      • et al.
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      Direct sequencingPCR amplification followed by direct sequencing++
      • Cunat S.
      • Giansily Blaizot M.
      • Bismuth M.
      • Blanc F.
      • Dereure O.
      • Larrey D.
      • et al.
      Global sequencing approach for characterizing the molecular background of hereditary iron disorders.
      ,
      • Cukjati M.
      • Koren S.
      • Curin Serbec V.
      • Vidan-Jeras B.
      • Rupreht R.
      A novel homozygous frameshift deletion c.471del of HFE associated with hemochromatosis.
      ,
      • Steiner M.
      • Ocran K.
      • Genschel J.
      • Meier P.
      • Gerl H.
      • Ventz M.
      • et al.
      A homozygous HFE gene splice site mutation (IVS5+1 G/A) in a hereditary hemochromatosis patient of Vietnamese origin.
      ,
      • Barton J.C.
      • Sawada Hirai R.
      • Rothenberg B.E.
      • Acton R.T.
      Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands.
      Allelic discrimination PCRReal time PCR (TaqMan®) with displacing probes and modifications+/−+/−
      • de Kok J.B.
      • Wiegerinck E.T.
      • Giesendorf B.A.
      • Swinkels D.W.
      Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).
      ,
      • Behrens M.
      • Lange R.
      A highly reproducible and economically competitive SNP analysis of several well characterized human mutations.
      ,
      • Alsmadi O.A.
      • Al Kayal F.
      • Al Hamed M.
      • Meyer B.F.
      Frequency of common HFE variants in the Saudi population: a high throughput molecular beacon-based study.
      ,
      • Castley A.
      • Higgins M.
      • Ivey J.
      • Mamotte C.
      • Sayer D.C.
      • Christiansen F.T.
      Clinical applications of whole-blood PCR with real-time instrumentation.
      ,
      • Walburger D.K.
      • Afonina I.A.
      • Wydro R.
      An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis.
      ,
      • Cheng J.
      • Zhang Y.
      • Li Q.
      Real-time PCR genotyping using displacing probes.
      Melting curve analysis(Light Cycler®)+++/−+/−
      • Bach V.
      • Barcelo M.J.
      • Altes A.
      • Remacha A.
      • Felez J.
      • Baiget M.
      Genotyping the HFE gene by melting point analysis with the LightCycler system: Pros and cons.
      ,
      • Moyses C.B.
      • Moreira E.S.
      • Asprino P.F.
      • Guimaraes G.S.
      • Alberto F.L.
      Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR.
      D-HPLCDenaturing HPLC+++/−+
      • Biasiotto G.
      • Belloli S.
      • Ruggeri G.
      • Zanella I.
      • Gerardi G.
      • Corrado M.
      • et al.
      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.
      SSPSequence specific priming PCR+
      • Smillie D.
      A PCR-SSP method for detecting the Cys282Tyr mutation in the HFE gene associated with hereditary haemochromatosis.
      ,
      • Smillie D.
      A PCR-SSP method for detecting the His63Asp mutation in the HFE gene associated with hereditary haemochromatosis.
      ,
      • Steffensen R.
      • Varming K.
      • Jersild C.
      Determination of gene frequencies for two common haemochromatosis mutations in the Danish population by a novel polymerase chain reaction with sequence-specific primers.
      ,
      • Wenz H.M.
      • Baumhueter S.
      • Ramachandra S.
      • Worwood M.
      A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).
      ,
      • Guttridge M.G.
      • Carter K.
      • Worwood M.
      • Darke C.
      Population screening for hemochromatosis by PCR using sequence-specific primers.
      ,
      • Guttridge M.G.
      • Thompson J.
      • Worwood M.
      • Darke C.
      Rapid detection of genetic mutations associated with haemochromatosis.
      ,
      • Kaur G.
      • Rapthap C.C.
      • Xavier M.
      • Saxena R.
      • Choudhary V.P.
      • Reuben S.K.
      • et al.
      Distribution of C282Y and H63D mutations in the HFE gene in healthy Asian Indians and patients with thalassaemia major.
      SPASolid-phase amplification+
      • Turner M.S.
      • Penning S.
      • Sharp A.
      • Hyland V.J.
      • Harris R.
      • Morris C.P.
      • et al.
      Solid-phase amplification for detection of C282y and H63D hemochromatosis (HFE) gene mutations.
      SSCPSingle strand conformation polymorphism analysis+++/−
      • Bosserhoff A.K.
      • Seegers S.
      • Hellerbrand C.
      • Scholmerich J.
      • Buttner R.
      Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).
      ,
      • Simonsen K.
      • Dissing J.
      • Rudbeck L.
      • Schwartz M.
      Rapid and simple determination of hereditary haemochromatosis mutations by multiplex PCR-SSCP: detection of a new polymorphic mutation.
      OLAOligonucleotide ligation assay+
      • Feder J.N.
      The hereditary hemochromatosis gene (HFE): a MHC class I-like gene that functions in the regulation of iron homeostasis.
      SCAIPSingle-condition amplification with internal primer+/−+/−+
      • Cunat S.
      • Giansily Blaizot M.
      • Bismuth M.
      • Blanc F.
      • Dereure O.
      • Larrey D.
      • et al.
      Global sequencing approach for characterizing the molecular background of hereditary iron disorders.
      Advanced read-outMass spectrometry based, capillary electrophoresis, chip basedn/an/an/a+
      • Kim S.
      • Edwards J.R.
      • Deng L.
      • Chung W.
      • Ju J.
      Solid phase capturable dideoxynucleotides for multiplex genotyping using mass spectrometry.
      ,
      • Bernacki S.H.
      • Farkas D.H.
      • Shi W.
      • Chan V.
      • Liu Y.
      • Beck J.C.
      • et al.
      Bioelectronic sensor technology for detection of cystic fibrosis and hereditary hemochromatosis mutations.
      ,
      • Footz T.
      • Somerville M.J.
      • Tomaszewski R.
      • Elyas B.
      • Backhouse C.J.
      Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis.
      ,
      • Bosserhoff A.K.
      • Buettner R.
      • Hellerbrand C.
      Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview.
      ,
      • Devaney J.M.
      • Pettit E.L.
      • Kaler S.G.
      • Vallone P.M.
      • Butler J.M.
      • Marino M.A.
      Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography.
      ,
      • Lubin I.M.
      • Yamada N.A.
      • Stansel R.M.
      • Pace R.G.
      • Rohlfs E.M.
      • Silverman L.M.
      HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis.
      Reverse hybridization assayMultiplex PCR amplification followed by reverse hybridizationn/an/an/a+
      • Beutler E.
      • Felitti V.J.
      • Koziol J.A.
      • Ho N.J.
      • Gelbart T.
      Penetrance of 845G → A (C282Y) HFE hereditary haemochromatosis mutation in the USA.
      ,
      • Koeken A.
      • Cobbaert C.
      • Quint W.
      • van Doorn L.J.
      Genotyping of hemochromatosis-associated mutations in the HFE gene by PCR-RFLP and a novel reverse hybridization method.
      ,
      • Kotze M.J.
      • de Villiers J.N.
      • Bouwens C.S.
      • Warnich L.
      • Zaahl M.G.
      • van der Merwe S.
      • et al.
      Molecular diagnosis of hereditary hemochromatosis: application of a newly-developed reverse-hybridization assay in the South African population.
      ,
      • Oberkanins C.
      • Moritz A.
      • de Villiers J.N.
      • Kotze M.J.
      • Kury F.
      A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.
      Novel extraction methodsDried blood spots, whole-blood PCRn/an/an/a++
      • Hoppe C.
      • Watson R.M.
      • Long C.M.
      • Lorey F.
      • Robles L.
      • Klitz W.
      • et al.
      Prevalence of HFE mutations in California newborns.
      ,
      • Castley A.
      • Higgins M.
      • Ivey J.
      • Mamotte C.
      • Sayer D.C.
      • Christiansen F.T.
      Clinical applications of whole-blood PCR with real-time instrumentation.
      ,
      • Rivers C.A.
      • Barton J.C.
      • Acton R.T.
      A rapid PCR-SSP assay for the hemochromatosis-associated Tyr250Stop mutation in the TFR2 gene.
      Sequencing of the HFE gene in C282Y heterozygotes presenting with a phenotype compatible with hemochromatosis has revealed the existence of other rare HFE mutations. Among these, the S65C mutation has been more intensively studied [
      • Mura C.
      • Raguenes O.
      • Ferec C.
      HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.
      ]. It may contribute – but only when inherited in trans with the C282Y mutation – to the development of mild iron overload with no clinical expression in the absence of co-morbid factors.
      Homozygosity for H63D is not a sufficient genetic cause of iron overload and when H63D homozygosity is found in association with hyperferritinemia, co-morbid factors are usually present and do not reflect true iron overload [
      • Sebastiani G.
      • Wallace D.F.
      • Davies S.E.
      • Kulhalli V.
      • Walker A.P.
      • Dooley J.S.
      Fatty liver in H63D homozygotes with hyperferritinemia.
      ]. In a population based study of blood donors, homozygosity for H63D was associated with higher transferrin saturation [
      • Jackson H.A.
      • Carter K.
      • Darke C.
      • Guttridge M.G.
      • Ravine D.
      • Hutton R.D.
      • et al.
      HFE mutations, iron deficiency and overload in 10,500 blood donors.
      ].
      In rare selected pedigrees, private mutations have also been reported (V59M [
      • de Villiers J.N.
      • Hillermann R.
      • Loubser L.
      • Kotze M.J.
      Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria.
      ], R66C [
      • Biasiotto G.
      • Belloli S.
      • Ruggeri G.
      • Zanella I.
      • Gerardi G.
      • Corrado M.
      • et al.
      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.
      ], G93R, I105T [
      • Barton J.C.
      • Sawada Hirai R.
      • Rothenberg B.E.
      • Acton R.T.
      Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands.
      ,
      • de Villiers J.N.
      • Hillermann R.
      • Loubser L.
      • Kotze M.J.
      Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria.
      ], E168Q [
      • Oberkanins C.
      • Moritz A.
      • de Villiers J.N.
      • Kotze M.J.
      • Kury F.
      A reverse-hybridization assay for the rapid and simultaneous detection of nine HFE gene mutations.
      ], R224G [
      • Biasiotto G.
      • Belloli S.
      • Ruggeri G.
      • Zanella I.
      • Gerardi G.
      • Corrado M.
      • et al.
      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.
      ], E277K & V212V [
      • Bradbury R.
      • Fagan E.
      • Payne S.J.
      Two novel polymorphisms (E277K and V212V) in the haemochromatosis gene HFE.
      ], and V295A [
      • Jones D.C.
      • Young N.T.
      • Pigott C.
      • Fuggle S.V.
      • Barnardo M.C.
      • Marshall S.E.
      • et al.
      Comprehensive hereditary hemochromatosis genotyping.
      ]) as well as intronic HFE variant frame shift mutations c.340+4 T>C (also referred to as IVS2, T-C +4) [
      • Floreani A.
      • Navaglia F.
      • Basso D.
      • Zambon C.F.
      • Basso G.
      • Germano G.
      • et al.
      Intron 2 [IVS2, T-C +4] HFE gene mutation associated with S65C causes alternative RNA splicing and is responsible for iron overload.
      ], c.1008+1 G>A (also referred to as IVS5+1G/A) [
      • Steiner M.
      • Ocran K.
      • Genschel J.
      • Meier P.
      • Gerl H.
      • Ventz M.
      • et al.
      A homozygous HFE gene splice site mutation (IVS5+1 G/A) in a hereditary hemochromatosis patient of Vietnamese origin.
      ], and c.471del [
      • Cukjati M.
      • Koren S.
      • Curin Serbec V.
      • Vidan-Jeras B.
      • Rupreht R.
      A novel homozygous frameshift deletion c.471del of HFE associated with hemochromatosis.
      ]. Some of these may result in a severe HC phenotype when present in the homozygous state [
      • Steiner M.
      • Ocran K.
      • Genschel J.
      • Meier P.
      • Gerl H.
      • Ventz M.
      • et al.
      A homozygous HFE gene splice site mutation (IVS5+1 G/A) in a hereditary hemochromatosis patient of Vietnamese origin.
      ] or in the compound heterozygote state with C282Y [
      • Piperno A.
      • Arosio C.
      • Fossati L.
      • Vigano M.
      • Trombini P.
      • Vergani A.
      • et al.
      Two novel nonsense mutations of HFE gene in five unrelated italian patients with hemochromatosis.
      ,
      • Wallace D.F.
      • Dooley J.S.
      • Walker A.P.
      A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a