uncommon mutations and polymorphisms in the hemochromatosis gene

GENETIC TESTINGVolume 4, Number 2, 2000Mary Ann Liebert, Inc.

Uncommon Mutations and Polymorphisms in theHemochromatosis Gene

JENNIFER J. POINTON,1 DANIEL WALLACE,2 ALISON T. MERRYWEATHER-CLARKE,1

and KATHRYN J.H. ROBSON1

ABSTRACT

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron ab-sorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosisgene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine fora conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60–100% of patients.The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear butcompound heterozygotes for both these mutations have a significant risk of developing HH. Here we reviewother putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE in-trons.

INTRODUCTION

H ER ED ITA RY H EM OCHROM AT OSIS (HH) (OMIM 235200) isthe most common genetic disease in Caucasian popula-

tions; about 1 in 300 people are homozygous for the disease-causing mutation, but the degree of penetrance (i.e., how manyactually develop clinical disease) is unclear. The carrier fre-quency is 10–15% (Edwards et al., 1988; Leggett et al., 1990).It was shown to be heritable by Sheldon (1935), recessive, andlinked to the major histocom patibility complex (MHC) (Simonet al., 1976, 1977), which was localized to the short arm ofchromosome 6 (Morton et al., 1984).

HH is characterized by excessive iron absorption from theupper gastrointestinal tract that results in iron accumulation inthe parenchymal tissues (Bothwell et al., 1995). The onset ofthe disease is usually by the fourth decade in men and the fifthdecade in women with a clinical picture that can include anycombination of arthropathy, hypogonadism, diabetes, mellitus,cardiomyopathy , hyperm elanotic pigmentation, and liver fibro-sis and cirrhosis. Untreated hemochromato sis is fatal, with thepotential for massive iron stores in the range of 20–40 gramsin the parenchymal cells of many organs (Bomford andWilliams, 1976). The disease can be effectively treated by phle-botomy to reduce iron stores and maintain iron indices at nor-

mal levels. Early treatment prior to the development of livercirrhosis returns life expectancy to normal (Powell 1970;Niederau et al., 1996).

Feder et al. (1996) cloned the gene for HH by an impressivepositional cloning strategy. The gene was originally namedHLA-H but was renamed HFE (Bodmer et al., 1997; Mercieret al., 1997). Two missense mutations were identified, a gua-nine to adenine transition, 845G R A (OMIM 235200.0001),which results in a cysteine-to-tyrosin e substitution at amino acid282 (C282Y), and a cytosine to guanine transversion, 187C RG (OMIM 235200.0002), which results in a histidine-to-aspar-tic acid substitution at amino acid 63 (H63D). C282Y is themost prevalent mutation in HH patients. Between 60 and 100%of patients are homozygous for C282Y (Beutler et al., 1996;Feder et al., 1996; Jazwinska et al., 1996; Barton et al., 1997;Borot et al., 1997; Carella et al., 1997; Jouanolle et al., 1997;Mura et al., 1997; Worwood et al., 1997; Gottschalk et al.,1998). Of the HH patients who are not homozygous for C282Y,up to 4%, 6.7%, 4.3%, and 8.5% are either H63D homozygotes,compound heterozygotes, C282Y heterozygotes, or H63D het-erozygotes, respectively. Between 4% and 21% of clinically af-fected patients have neither of these two mutations in the HFEgene (Beutler et al., 1996; Feder et al., 1996; Adams et al.,1997; Barton et al., 1997; Borot et al., 1997; Carella et al.,

1MRC Molecular Haematology Unit, Institute Molecular Medicine, Headington, Oxford, OX3 9DS, UK.2Centre for Hepatology, Department of Medicine, Royal Free and University College Medical School, University College London, Royal Free

Campus, London, NW3 2PF, UK.

1997; Jouanolle et al., 1997; Mura et al., 1997; Worwood etal., 1997; Cardoso et al., 1998). The role and prevalence ofthese two mutations are reviewed by Merryweather-Cl arke etal. in this issue.

The HFE gene has an open reading frame of 1,029 nucleotidesand is predicted to code for a 343-amino-acid protein that is highlyhomologous to the nonclassical MHC class I molecules. It is or-ganized into three extracellular domains (a 1, a 2, and a 3), a trans-membrane domain, and a short cytoplasmic tail. It contains fourhighly conserved cysteine residues that form two intramoleculardisulfide bridges, one in the a 2 domain and one in the a 3 do-main. These are essential for correct protein structure, for the non-covalent interaction with b 2-microglobulin, and cell-surface lo-calization. The C282Y mutation disrupts the disulfide bridge inthe a 3 domain, impairing the b 2-microglobulin interaction andcell-surface localization (Feder et al., 1997; Waheed et al., 1997).Functional studies and X-ray crystallography suggest that HFEinteracts with the transferrin receptor (TfR) at the cell surface andcompetes with transferrin for binding to TfR (Parkkila et al., 1997;Feder et al., 1998; Gross et al., 1998; Lebrón et al., 1998; Lebrónand Bjorkman 1999; Lebrón et al., 1999; Bennett et al., 2000).HFE with the H63D substitution interacts with b 2-microglobulinand is expressed normally at the cell surface. Recent evidencesuggests that its function in modulating the interaction of trans-ferrin with the TfR may be impaired (Parkkiila et al., 1997; Federet al., 1998; Lebrón et al., 1998).

THE SEARCH FOR OTHER CODINGMUTATIONS

The HFE gene from a number of HH patients who are nothomozygous for C282Y has been studied. In the majority ofcases, no further coding mutations has been found. Feder et al.(1996) sequenced 42 chromosomes and found that nine carriedthe H63D mutation; no other mutations were found. This rep-resents a considerable enrichment of H63D alleles in HH pa-tients compared with the control population (Beutler et al., 1996;Risch, 1997). Carella et al. (1997) studied 18 Italian HH patientsbut found no further mutations, although some were membersof families with demonstrable chromosome 6-linked inheritance.No further coding mutations were found in 10 HH patients(Beutler et al., 1997) or in 4 cases of juvenile HH (Kelly et al.,1998). In the Italian population, juvenile HH has now beenlinked to the long arm of chromosome 1 and, therefore, has adifferent etiology from chromosome 6-linked HH (Camaschellaet al., 1997; Roetto et al., 1999). The coding region of a further20 patients was investigated but revealed no mutations, (Muraet al., 1997). The cause of HH in this group of patients remainsunknown and demonstrates that HH is a heterogeneous diseaseand that further HFE mutations are rare. However, a number ofnew mutations have been found. Numbering of amino acids andnucleotides is from the start of translation.

AMINO ACID SUBSTITUTIONS CAUSED BYMISSENSE MUTATIONS

Other coding mutations have now been identified in the HFEgene; these are listed in Table 1. Some have been seen in iron

overload conditions, whereas others have no proven disease as-sociation and may be variants.

S65C (193A R T)

A second exon 2 missense mutation, an adenine-to-thym inetransversion, S65C (193A R T), results in the conservative sub-stitution of a cysteine for a serine in the HFE protein. It wasidentified in one of asymptomatic twin brothers of a C282Y ho-mozygous patient (Henz et al., 1997) and was thought to be apolymorphism not associated with disease. Bernard et al. (1998)reported allele frequencies of 5.5% for S65C in control chro-mosomes and 2.8% for patient chromosomes in a study of Cau-casian Americans. In another study of 20 HH patients who werenot C282Y or H63D homozygotes or compound heterozygotes,HFE gene sequencing identified two S65C heterozygotes (Bar-ton et al., 1999). One of these patients was heterozygous forC282Y with clinical evidence of HH and porphyria cutaneatarda. The second patient had severe iron overload and hered-itary stomatocytosis (Barton et al., 1999). S65C was found on2 of 176 (1.13%) controls and never on the same chromosomeas C282Y or H63D. All of 13 Caucasian HH referrals who wereC282Y- and H63D-negative , and 102 controls were negativefor the S65C mutation in a study of South Africans (de Villierset al., 1999).

S65C was found in 3% of patients with porphyria who werenegative for the common mutation (R59W) in the protopor-phyrinoge n oxidase gene (PPOX) (Meissner et al., 1996;Warnich et al., 1996), and one of 73 R59W-positive patientsalso had the S65C variant (de Villiers et al., 1999). An S65Cheterozyg ote who has dysmetabolic iron overload has beendescribed by Douabin et al. (1999). An allele frequency of3.2% for both normal and non-C 282Y homozygous patientchrom osomes was found in this study. However, only a smallnumber of patient chrom osomes were examined , 16 HH chro-mosomes from non-C 282Y homozygous men (4 H63D ho-mozygotes, 2 C282Y heterozyg otes, 1 compound heterozy-gote, and 1 with no known mutations), and 16 from men withdysmetabolic iron overload with wild-type alleles for the HHmutations. The S65C mutation was not observed on any of96 chrom osomes carrying the C282Y mutation (Douabin etal., 1999). A female, who is heterozyg ous for both S65C andH63D, and who is iron deficient has been described (Wor-wood et al., 1999).

There is a significant enrichm ent of S65C-carrying alleles inHH patients who have one chromosome that does not carry theC282Y or H63D mutations (Mura et al., 1999). Of the controlchromosomes that did not carry C282Y or H63D, 2.49% car-ried S65C. Of the patients that were not homozygous for C282Yor H63D or compound heterozygotes, then 7.8% carried S65C.S65C may be implicated in the development of a mild form ofHH that seems to affect men more than women (Mura et al.,1999). Again, S65C was not found on chromosomes that al-ready carried either C282Y or H63D. C282Y and H63D havebeen found in cis only once (Spriggs et al., 1999), suggestingthat these three mutations are nearly always mutually exclusive.In normal Southeast Asians (28) and Sri Lankans (126), S65Cwas not detected but was found on 1.6% of normal Caucasianchromosomes (Rochette et al., 1999).

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G93R (277G R C).

The missense mutation G93R (277G R C) is caused by a gua-nine-to-cytosine transversion and results in the nonsynonymoussubstitution of an arginine for a glycine. It was identified in aCaucasian proband who is heterozygous for C282Y. He was di-agnosed at age 40 and is being treated for HH. His father, aC282Y homozygote who is G93R negative, is being treated forHH. The proband has inherited C282Y on the HLA-B7 and HLA-A3 haplotype and G93R on HLA-A2 and HLA-B62 haploytpe.He has a haploidentical sister who was diagnosed at 37 years andis being treated for HHC (Barton et al., 1999).

I105T (314T R C).

In a study of 20 HH patients who lacked homozygosity forC282Y and H63D and who were not compound heterozygotesfor these two mutations, two new missense mutations were iden-tified by sequencing (Barton et al., 1999). The first is a non-synonymous substitution of tyrosine for isoleucine caused by athymine-to-cyto sine transition, I105T (314T R C). It wasfound in a male proband of European extraction who is het-erozygous for H63D. A family study showed that these two mu-tations segregated independently. The sister of the proband isalso a heterozygote for I105T, but is H63D negative. She hashyperferritinemi a but normal transferrin saturation. A 31-year-old daughter of the proband has inherited the I105T mutationand has normal iron indices. In this family, H63D is carried ona haplotype carrying the HLA antigens HLA-B7 and HLA-A2.In population studies, H63D is most often associated with HLA-A29 and HLA-B44, both in patients and controls (review ed byPorto and de Sousa, 2000). I105T is on a haplotype carryingthe HLA antigens HLA-A3 and HLA-B7. Interestingly, theseHLA antigens are associated with the HH ancestral haplotype(Crawford et al., 1995). HLA-A3 occurs in between 55–83%of unrelated patients, but only 20–32% controls. HLA-B7 isalso found more frequently in HH patients than controls whenassociated with HLA-A3: 34–60% and 19–28%, respectively(review ed by Milman, 2000). However, in the area from whichthe proband comes (Alabama, USA), the HLA-A3, B7 haplo-type is more common in C282Y-negativ e patients than controls(Barton et al., 1997).

Q127H (371A R C).

The Q127H (371A R C) occurs in exon 3 of the HFE gene.It is an adenine-to-cytosin e transversion that results in a non-synonymous substitution, histidine being substituted for a glu-tamine. Q127H was found with H63D in a severely affectedporphyria patient who also carried the R59W mutation in thePPOX gene. The R59W mutation is the most common of thePPOX mutations and causes variegate porphyria, a dominantlyinherited disease (Meissner et al., 1996; Warnich et al., 1996).

V53M (157G R A) and V59M (175G R A)

V53M (157G R A) and V59M (175G R A) are conserva-tive substitutions of a methionine for a valine caused by a gua-nine-to-adenine transitions in exon 2. V53M was identified in8 out of 458 control South African black and bushman popu-lations, and V59M was found in only one of 102 Caucasianscontrols in a South African study (de Villiers et al., 1999).

It is not yet established if either mutation has any role in thedisturbance of iron metabolism, but they both occur at con-served amino acids in human, mouse, and rat (de Villiers et al.,1999).

V272L (814G R T).

The exon 4 mutation, V272L (814G R T), is a guanine-to-thymine transversion resulting in the conservative substitutionof a leucine for a valine was identified by heteroduplex analy-sis (Worwood et al., 1999). This mutation abolishes the con-trol RsaI site used to check for complete digestion in the com-monly used restriction fragm ent length polymorphism (RFLP)analysis of PCR products for the determ ination of C282Y sta-tus (Worwood et al., 1997). In reviewing the results of the di-agnostic tests performed by one of us (ATM-C), we have foundno evidence for the presence of this mutation in our cohort of253 HH referrals. We conclude that this is a rare mutation.

E277K (829G R A).

An exon 4 mutation, E277K (829G R A) is a guanine-to-adenine transition that results in the nonsynonymous substitu-tion of lysine for a glutamic acid. It was found in one of over400 diabetic patients being screened for C282Y and H63D. Thepatient is a 58-year-old Asian man with no evidence of ironoverload (Bradbury et al., 1999).

R330M 1198G R T).

The exon 4 mutation, R330M (1198G R T), is the nonsyn-onymous substitution of methionine for an arginine at an evo-lutionary conserved site caused by a guanine-to-thym ine trans-version. The mutation was identified in a patient referred formolecular diagnosis of HH who was found to be negative forH63D and C282Y mutations. It was not detected in 40 controls(de Villiers et al., 1999). Again the role of this mutation in aber-rant iron metabolism remains to be investigated.

SYNONYMOUS MUTATIONS

Three synonymous mutations have been identified. H28H(84C R T), a cytosine-to-thym ine transition in exon 2 wasfound in a C282Y heterozygote (Liechti-Gallat i et al., 1999).The synonym ous mutation H63H (189T R C), a thymine-to-cytosine transition affects the same codon as H63D (de Villierset al., 1999; Bradbury et al., 1999). This mutation is indistin-guishable from H63D by RFLP analysis of PCR products, asH63D is detected by the loss of a restriction enzyme site thatis also lost by H63H. The exon 4 mutation V212V (636G RC), a guanine-to-cytosin e transversion occurred one of over 400diabetics (Liechti-G allati et al., 1999). There is no evidence thatthese mutations have any role in HH.

FRAMESHIFT MUTATIONS

Two frameshift mutations have been seen, both associatedwith HH. A single cytosine deletion (P160 D C) exon 3 of theHFE gene in a patient appears to cause HH in one UK patient

POINTON ET AL.154

(Pointon et al., 1997). This patient had the diagnosis of HHconfirmed by liver biopsy examination. P160 D C causes aframeshift and introduces a premature termination codon 50amino acids downstream of the deletion. This patient has noother coding mutations in the HFE gene identified to date, soit appears that P160 D C may act in a dominant manner. Screen-ing 206 Caucasians and 110 Asians failed to identify any fur-ther cases. The proband has only two blood relatives, two sons;only one son was available for testing and was negative for thismutation. It is probable that this mutation is private to this in-dividual.

A second case of a single base deletion in exon 2 has beenreported V68 D T causes a frameshift leading to a premature ter-mination codon 19 amino acids downstream. In this case theframshift is in an HH patient who is also a C282Y heterozy-gote (Liechti-G allati et al., 1999).

In both these cases, the frameshift occurs in the a 2 domainof the protein so that cell-surface expression of the truncatedprotein would not be expected because the a 3 domain is miss-ing and with it the ability to interact with b 2-microglobulin.

SPLICE SITE MUTATION

A novel splice site mutation has been described in a C282Yheterozygous patient who has liver cirrhosis, grade 4 1 hepa-tocellular siderosis, and a hepatic iron index of 5.0 (Wallace etal., 1999). This mutation, IVS3 1 1G R T, a guanine-to-thymine transversion, is inherited through one family with theproband and his sister both affected and both found to be com-pound heterozygotes. A family study showed that C282Y andIVS3 1 1G R T segregate independently. A daughter of theproband has inherited IVS3 1 1G R T in the absence of C282Yand has normal iron indices at age 22. The mutation causes exonskipping, with exon 2 being spliced to exon 4. Three hundredand sixty-five Europeans, including 50 selected C282Y het-erozygotes, were negative for the mutation. This mutation alsoappears to be exclusive to this family.

MUTATIONS IN NONCODING REGIONS OF EXONS

Three mutations in noncoding exonic regions have been ob-served; but their relevance to iron metabolism is unknown. Athymine-to-cytosine transition 7 nucleotides upstream of thetranslation initiation codon ( 2 7T R C) is in the 5 9 untranslatedregion (UTR) (Douabin et al., 1999). A patient with dysmeta-bolic iron overload syndrome was found to be heterozygous forthis mutation, but it was not present in a control group of 88 chro-mosomes or in 18 C282Y homozygous HH patients and 18C282Y heterozygous patients with dysmetabolic iron syndrome.Two mutations have been found in the noncoding exon 7 (3 9UTR). The first is a poly A 1 5 (nucleotide 2,484) cytosine-to-thymine transition (Rochette et al., 1999) that occurs with an al-lele frequency of 32% in unaffected Caucasians and 16% inC282Y mutant chromosomes (Pointon, unpublished results). Thesecond is a guanine-to-cytosine transversion at nucleotide 124 ofexon 7 (nucleotide 2186) (Douabin et al., 1999) that has an al-lele frequency of 10% in both patients and controls.

SUMMARY

Nine missense mutations have been documented; five are theresult of a transversions and four the result of transitions. Apartfrom S65C, these are largely confined to individual families ora handful of individuals. Three synonymous mutations havebeen identified; two are the result of base transitions and onethe result of a transversion. The two frameshift mutations arethe result of single base deletions and again are confined to in-dividual families. The one splice site mutation so far identifiedis also exclusive to one family: this is the result of a single basetransversion.

Finally, three mutations in nontranslated exons have beenseen, two single base transitions and a single base transversion.Eight of these mutational events have been found in exon 2(264 bps), compared to three in exon 3 (277 bps), five in exon4 (276 bps), and one in exon 5 (113 bps). A possible explana-tion is that exons 2 and 4 have been subjected to extensiveanalysis by various forms of single-stranded conformationalpolymorphism analysis to screen for H63D and C282Y: thismethod can highlight the presence of other mutational eventsin the target sequence (Simonsen et al., 1999; Wenz et al.,1999).

INTRONIC POLYMORPHISMS

A number of diallelic intronic polymorphisms have now beenfound; these are listed in Table 2, with allele frequencies, if avail-able. It is not thought that these are implicated in any way withthe HH. The presence of the polymorphism IVS4 1 48A R G(Totaro et al., 1997) in the commonly used reverse primer forPCR (Feder et al., 1996) for the detection of the C282Y muta-tion has focused attention on the potential pitfalls such poly-morphisms can cause (de Villiers et al., 1999; Gomez et al.,1999; Jeffrey et al., 1999; Merryweather-Clarke et al., 1999;Noll et al., 1999; Somerville et al., 1999). For this reason, it isnecessary to be aware of their locations so that inappropriate se-quences are not used as PCR primers. This is even more crucialwhen one allele of a polymorphism is in linkage disequilibriumwith the mutation being detected: this is the case for some ofthese polymorphisms (see Table 2).

In the case of IVS2 1 4T R C, for example, C282Y is foundexclusively on chromosom es that are thymine at this locus butthymine is found on between 60.1% and 79% of Caucasian con-trols. Thymine is also associated with H63D at this locus in be-tween 88.8% and 100% of chromosomes. At IVS4 2 44T RC, C282Y and H63D are strongly associated with chrom osomesthat are thymine positive (97.8–100% ). Wild-type Caucasianchromosomes are also associated with thymine-bearing chro-mosomes (87.1–98.9% ), but for South African blacks this dropsto 84.6% and for Asians to 61–79.8% . At IVS5 2 47A R G,C282Y is found exclusively on chrom osomes that contain gua-nine at this position, but guanine is only found on between 25and 57.8% of normal Caucasian chromosomes. H63D is foundpredom inantly on chromosomes bearing adenine at this locus.Some of these polymorphism s have been used to establish hap-lotypes within the HFE gene; this type of study can aid in theunderstanding of the origins of the mutations (Beutler and West,1997; Aguilar-Martin ez, et al., 1999; Rochette et al., 1999).

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NT’D

IN CONCLUSION

Two main mutations in the HFE gene, C282Y and H63D,have both been implicated in HH. A high percentage of HH pa-tients are homozygous for C282Y, with the disease being causedby H63D homozygosity or compound heterozygosity some ofthe time. Now S65C appears to have a role in disease when oc-curring in the compound heterozygous state with C282Y. Wedo not understand yet why there is incom plete penetrance associated with these two compound-hetero zygous states(H63D/C282Y; S65C/C282Y) and what the factors are that leadto disease for some patients and a healthy life for others. Theseobservations suggest that HH is a heterogenous disease.

Eleven other novel mutations have been reviewed here. Threeare not associated with disease (V53M, V59M, and E277K), buthave only been described in the heterozygous state. One,R330M, was identified in a HH referral, but we do not knowthe clinical status of the individual concerned. Three missensemutations (G93R, I105T, and Q127H) were found to cause dis-ease when found in combination with either C282Y or H63D.The splice site mutation (IVS3 1 1G R T) and one of the twoframeshift mutations (V68 D C) occur with C282Y. In only onecase, the frameshift P160 D C, where only this mutation has beenidentified in a patient with HH, the possibility that it is actingin a dominant manner can be considered. Interestingly, all theamino acid substitutions are at sites that are conserved betweenman, mouse, and rat, with the exception of Q127H.

There is evidence that another gene is involved in the HHphenotype, namely juvenile hemochromato sis is linked tochromosome 1, therefore, it will not be surprising if furthergenes are found to be involved in HH. Iron overload has notbeen thoroughly investigated in populations that are not ofnorthern European descent; the study of such populations maywell reveal further HFE mutations or genes associated with ironmetabolism.

Major advances in the understanding of HH and iron me-tabolism have been made since the identification of the HFEgene, but there is still much to be uncovered.

ACKNOWLEDGMENTS

We thank Professor Sir David Weatherall and ProfessorMark Worwood for helpful comment and Milly Graver forpreparation of the tables.

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Address reprint requests to:Ms. Jennifer J. Pointon

MRC Molecular Haematology UnitInstitute Molecular Medicine

Headley WayHeadington, Oxford, OX3 9DS

United Kingdom

E-mail: jenny@ hammer.imm .ox.ac.uk

Received for publication March 2, 2000; accepted March 28,2000.

uncommon mutations and polymorphisms in the hemochromatosis gene

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