uncommon mutations and polymorphisms in the hemochromatosis gene
TRANSCRIPT
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.
151
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).
POINTON ET AL.152
TA
BL
E1.
HF
EG
EN
EM
UT
AT
ION
SS
HO
WIN
GN
UC
LE
OT
IDE
CH
AN
GE
, L
OC
AT
ION
WIT
HIN
TH
EG
EN
E,
AN
DT
HE
INT
RO
DU
CT
ION
OR
EL
IMIN
AT
ION
OF
AR
ES
TR
ICT
ION
EN
ZY
ME
SIT
E
Res
tric
tion
Mut
atio
nL
ocat
ion
enzy
me
Com
men
tR
efer
ence
27
T ®
C59
UT
RN
ciI
Fou
nd i
n 1
pati
ent
wit
h dy
smet
abol
ic i
ron
synd
rom
eD
ouab
in e
t al
.(1
999)
H28
H 8
4C ®
TE
xon
2N
AF
ound
in
1 ob
liga
te C
282Y
car
rier
Lie
chti
-Gal
lati
et
al.
(199
9)V
53M
157
G ®
AE
xon
2M
aeII
Iden
tifi
ed i
n S
outh
Afr
ican
and
bus
hman
pop
ulat
ions
de V
illi
ers
et a
l.(1
999)
V59
M 1
75G
®A
Exo
n 2
Nla
IIId
enti
fied
in
1 ou
t of
102
con
trol
Cau
casi
ans
in S
outh
Afr
ica
de V
illi
ers
et a
l.(1
999)
H63
D 1
87C
®G
Exo
n 2
Mbo
IM
utat
ion
impl
icat
ion
in s
ome
case
s of
HH
; se
e M
erry
wea
ther
-Cla
rke
et a
l.,
this
iss
ueF
eder
et
al.
(199
6)H
63H
189
T ®
CE
xon
2M
boI
Sil
ent
mut
atio
n, i
ndis
ting
uish
able
fro
m H
63D
by
RF
LP
ana
lysi
sde
Vil
lier
s et
al.
(199
9)S
65C
193
A ®
TE
xon
2H
infI
Fir
st d
escr
ibed
in
a no
rmal
rel
ativ
e of
a C
282Y
hom
ozyg
ous
pati
ent
Hen
z et
al.
(199
7)V
68D
TE
xon
2M
woI
Iden
tifi
ed i
n a
C28
2Y h
eter
ozyg
ous
pati
ent
Lie
chti
-Gal
lati
et
al.
(199
9)
G93
R 2
77G
®C
Exo
n 2
NA
Des
crib
ed H
H a
ffec
ted
sibs
who
wer
e co
mpo
und
hete
rozy
gote
s fo
r C
282Y
and
G93
RB
arto
n et
al.
(199
9)I1
05T
314
T ®
CE
xon
2N
AD
escr
ibed
a f
amil
y in
whi
ch a
com
poun
d he
tero
zygo
te f
or I
105T
and
H63
D h
as H
HB
arto
n et
al.
(199
9)Q
127H
317
A ®
CE
xon
3N
AId
enti
fied
a c
ompo
und
hete
rozy
gote
in
seve
rely
aff
ecte
d va
riga
te p
orph
yria
pat
ient
de V
illi
ers
et a
l.(1
999)
P16
0DC
Exo
n 3
NA
Het
eroz
ygou
s H
H p
atie
nt w
ith
no o
ther
HF
Em
utat
ion
Poi
nton
et
al.
(199
7)IV
S31
1 G
®T
Intr
on 3
NA
Spl
ice
site
mut
atio
n fo
und
in a
sin
gle
Cau
casi
an f
amil
y. C
ause
s H
H i
n co
njun
ctio
nW
alla
ce e
t al
.(1
999)
wit
h C
282Y
het
eroz
ygos
ity
V21
2V 6
36G
®C
Exo
n 4
Hph
IS
ilen
t m
utat
ion
Bra
dbur
y et
al.
(199
9)V
272L
814
G ®
TE
xon
4R
saI
Iden
tifi
ed i
n on
e in
divi
dual
. D
estr
oys
Rsa
I co
ntro
l si
te i
n R
FL
P a
naly
sis
of C
282Y
mut
atio
n.W
orw
ood
et a
l.(1
999)
E27
7K 8
29G
®A
Exo
n 4
Mbo
IIId
enti
fied
in
1 di
abet
ic A
sian
mal
e w
ith
norm
al i
ron
indi
ces
Bra
dbur
y et
al.
(199
9)C
282Y
845
G ®
AE
xon
4R
saI
Mut
atio
n ca
usin
g m
ost
case
s of
HH
; se
e M
erry
wea
ther
-Cla
rke
et a
l.,
this
iss
ue.
Fed
er e
t al
.(1
996)
R33
0M 1
198G
®T
Exo
n 5
Nla
III,
Drd
IIId
enti
fied
in
one
C28
2Y a
nd H
63D
-neg
ativ
e H
H r
efer
ral
de V
illi
ers
et a
l.(1
999)
Exo
n 7
39U
TR
Bsm
IA
llel
e fr
eque
ncy
G 9
0.0%
, C
10.
0%D
ouab
in e
t al
.(1
999)
nucl
eoti
des
124
G ®
CP
olyA
15
C ®
T39
UT
RR
saI
Roc
hett
e et
al.
(199
9)
Poi
nton
et
al.
(unp
ubli
shed
res
ults
)
Occ
urre
nce
and
freq
uenc
ies
(wit
h th
e nu
mbe
rs t
este
d in
bra
cket
s),
are
show
n w
here
fig
ures
are
ava
ilab
le.
NA
, no
t ap
plic
able
; N
D,
not
done
.
Con
trol
Cau
casi
ans
C 6
7.7%
(10
7),
T 3
2.3%
(51
)C
ontr
ol B
urm
ese
and
Kar
enC
63.
6% (
14),
T 3
6.4%
(8)
Con
trol
Sri
Lan
kans
C 5
4.5%
(36
), T
45.
5% (
30)
C28
2Y h
omoz
ygot
esC
85.
7% (
24),
T 1
4.3%
(4)
H63
D h
omoz
ygot
esC
100
% (
6)
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).
UNCOMMON HEMOCHROMATOSIS MUTATIONS 155
TA
BL
E2.
HF
E G
EN
EP
OL
YM
OR
PH
ISM
SS
HO
WIN
GN
UC
LE
OT
IDE
CH
AN
GE,
LO
CA
TIO
NW
ITH
INT
HE
GE
NE,
AN
DIN
TR
OD
UC
TIO
NO
RE
LIM
INA
TIO
NO
FA
RE
ST
RIC
TIO
NE
NZ
YM
ES
ITE
Res
tric
tion
Pol
ymor
phis
mL
ocat
ion
enzy
me
All
ele
freq
uenc
yC
omm
ent
Ref
eren
ce
212
06 G
®C
59N
onco
ding
Bbv
IC
ontr
ol C
auca
sian
sC
59.
2% (
129)
, G
40.
8% (
89)
Poi
nton
et
al.
Con
trol
Asi
ans
C 6
1.2%
(12
5),
G 3
8.8%
(79
)(u
npub
lish
ed r
esul
ts)
C28
2Y h
omoz
ygot
esC
19.
8% (
15),
G 8
0.2%
(76
)H
63D
hom
ozyg
otes
C 2
2.3%
(4)
, G
77.
7% (
14)
297
0 T
®G
59N
onco
ding
NA
ND
Fou
nd i
n on
e at
ypic
al H
H p
atie
ntP
oint
on e
t al
.(u
npub
lish
ed r
esul
ts)
282
5 G
®A
59N
onco
ding
Cvi
JIN
DF
ound
in
one
Asi
an p
atie
ntP
oint
on e
t al
.(u
npub
lish
ed r
esul
ts)
246
7 G
®C
59N
onco
ding
Mae
IIC
ontr
ol C
auca
sian
G 6
4.0%
(55
), C
36.
0% (
31)
Poi
nton
et
al.
Afl
III
C28
2Y h
omoz
ygot
esG
2.3
% (
1),
C 9
7.7%
(43
)(u
npub
lish
ed r
esul
ts)
H63
D h
omoz
ygot
esG
50.
0% (
4),
C 5
0.0%
(4)
237
0 A
®T
Intr
on 1
Mse
IF
ound
in
one
atyp
ical
HH
pat
ient
Poi
nton
et
al.
Afl
III
(unp
ubli
shed
res
ults
)IV
S1
111
2 A
®T
Intr
on 1
NA
ND
Fou
nd i
n 1
atyp
ical
HH
pat
ient
,P
oint
on e
t al
.1
affe
cted
Asi
an p
atie
nt a
nd(u
npub
lish
ed r
esul
ts)
2 re
late
d H
63D
het
eroz
ygou
s in
divi
dual
s
IVS
1 1
1156
G ®
TIn
tron
1B
mrI
ND
Fou
nd i
n 1
atyp
ical
HH
pat
ient
Poi
nton
et
al.
(unp
ubli
shed
res
ults
)IV
S1
- 68
0 A
®T
Intr
on 1
NA
ND
Fou
nd i
n 1
atyp
ical
HH
pat
ient
Poi
nton
et
al.
(unp
ubli
shed
res
ults
)IV
S2
14
T ®
CIn
tron
2R
saI
Con
trol
Cau
casi
ans
T 7
8.0%
(98
), C
22%
(28
)B
eutl
er a
nd W
est
Con
trol
Asi
ans
T 9
3.3%
(28
), C
6.7
% (
2)(1
977)
Con
trol
Bla
cks
T 6
9.2%
(18
), C
30.
8% (
8)C
282Y
hom
ozyg
otes
T 1
00%
(48
)O
ther
Cau
casi
an p
atie
nts
T 5
7.0%
(17
), C
43.
0% (
13)
Asi
an p
atie
nts
T 7
5.0%
(6)
, C
25.
0% (
2)H
63D
hom
ozyg
otes
C 1
00%
(8)
Con
trol
Cau
casi
ans
T 6
0.1%
(12
5),
C 3
9.9%
(83
)R
oche
tte
et a
l.(1
999)
Con
trol
Bur
mes
e &
Kar
enT
53.
1% (
17),
C 4
6.9%
(15
)C
ontr
ol S
ri L
anka
nsT
66.
2% (
86),
C 3
3.8%
(44
)C
ontr
ol C
auca
sian
sT
79.
0% (
402)
, C
21.
0% (
106)
Höh
ler
et a
l.(1
999)
C28
2Y h
omoz
ygot
esT
100
% (
40)
Con
trol
Cau
casi
ans
T 7
5.0%
(36
), C
25.
0% (
12)
de V
illi
ers
et a
l.R
efer
rals
for
C28
2Y t
esti
ngT
38.
0% (
10),
C 6
2.0%
(16
)(1
999)
Con
trol
Cau
casi
ans
T 7
3.5%
(75
), C
26.
5% (
27)
Agu
ilar
-Mar
tine
zC
ontr
ol A
fric
ans
T 5
8.3%
(28
), C
41.
7% (
20)
et a
l. (1
999)
C28
2Y h
omoz
ygot
esT
100
% (
50)
H63
D h
omoz
ygot
esC
100
% (
100)
Aty
pica
l G
Hpa
tien
tsT
32.
5% (
13),
C 6
7.5%
(27
)B
arto
n et
al.
(199
9)C
282Y
hom
ozyg
otes
T 1
00%
(74
)P
oint
on e
t al
.H
63D
hom
ozyg
otes
C 8
8.8%
(16
), T
12.
2% (
2)(u
npub
lish
ed r
esul
ts)
IVS
4 1
37 A
®G
Intr
on 4
Mse
IN
DD
ocum
ente
d, n
o fi
gure
s gi
ven
de V
illi
ers
et a
l.(1
999)
IVS
4 1
48 G
®A
Intr
on 4
Mse
IC
ontr
ol C
auca
sian
sG
91.
5%,
A 8
.5%
In F
eder
rev
erse
PC
R p
rim
erT
otar
o et
al.
(199
7)C
282Y
hom
ozyg
otes
G 9
3.9%
, A
6.1
%fo
r am
plif
ying
seq
uenc
e fl
anki
ngC
282Y
mut
atio
nH
H p
atie
nts
G
89.
4% (
102)
, A
10.
6% (
24)
Jeff
rey
et a
l.(1
999)
C28
2Y h
eter
ozyg
otes
G 8
1.4%
(70
), A
18.
6% (
16)
Som
ervi
lle
et a
l.(1
999)
Con
trol
Cau
casi
ans
G 8
6.8%
(54
0),
A 1
3.2%
(82
)M
erry
wea
ther
-Cla
rke
HH
ref
erra
lsG
91.
4% (
1003
), A
8.6
% (
87)
et a
l.(1
999)
Con
trol
Cau
casi
ans
G 8
9.5%
(28
1),
A 1
0.5%
(33
)G
omez
et
al.
(199
9)C
282Y
hom
ozyg
otes
G 9
9.55
% (
440)
, A
0.4
5% (
2)IV
S4-
50 A
®G
Intr
on 4
Fok
IN
DD
ocum
ente
d, n
o fi
gure
s gi
ven
de V
illi
ers
et a
l.(1
999)
HH
and
HH
ref
erra
lsG
100
% (
16)
Fou
nd w
hile
seq
uenc
ing
C28
2YP
oint
on e
t al
.ho
moz
ygou
s an
d at
ypic
al H
H(u
npub
lish
ed r
esul
ts)
pati
ents
IVS
4-44
T ®
CIn
tron
4Sa
u96I
Con
trol
Cau
casi
ans
T 8
7.1%
(61
), C
12.
9% (
9)B
eutl
er a
nd W
est
Con
trol
Bla
cks
T 8
4.6%
(22
), C
15.
4% (
4)(1
997)
Con
trol
Asi
ans
C
61.
0% (
33),
T 3
9.0%
(21
)C
282Y
hom
ozyg
otes
T 1
00%
(48
)N
on C
282Y
Cau
casi
anT
100
% (
30)
pati
ents
Non
C28
2Y A
sian
pat
ient
sC
62.
5% (
5),
T 3
7.5%
(3)
Con
trol
Cau
casi
ans
T 9
8.9%
, C
11.
1%B
arto
n et
al.
(19
99)
Aty
pica
l H
Hpa
tien
ts
T 9
5.0%
, C
5.0
%C
ontr
ol C
auca
sian
sT
91.
8% (
180)
, C
8.2
% (
16)
Roc
hett
e et
al.
(19
99)
Con
trol
Bur
mes
e &
Kar
en
T 6
7.9%
(19
), C
32.
1% (
9)C
ontr
ol S
ri L
anka
ns
T 7
9.8%
(99
), C
20.
2% (
25)
C28
2Y h
omoz
ygot
esT
97.
8% (
45),
C 2
.2%
(1)
Poi
nton
et
al.
H63
D h
omoz
ygot
esT
100
% (
12)
(unp
ubli
shed
res
ults
)N
DD
ocum
ente
d, n
o fi
gure
s gi
ven
de V
illi
ers
et a
l.(1
999)
IVS
5 1
14 A
®G
Intr
on 5
NA
ND
Fou
nd i
n at
ypic
al H
Hpa
tien
tB
arto
n et
al.
(19
99)
IVS
5-39
8 G
®T
Intr
on 5
Esp
IG
81.
0%,
T 1
9.0%
Dou
abin
et
al.
(199
9)D
deI
ND
Fou
nd i
n 1
atyp
ical
HH
pati
ent
Poi
nton
et
al.
(unp
ubli
shed
res
ults
)IV
S5-
47 A
®G
Intr
on 5
Ban
IC
ontr
ol C
auca
sian
sA
60.
0%(4
2),
G 4
0.0%
(28)
Beu
tler
and
Wes
tC
ontr
ol A
sian
s
A 6
9.6%
(39)
, G
30.
4%(1
7)(1
997)
Con
trol
Bla
cks
A
46.
2%(1
2),
G 5
3.8%
(14)
C28
2Y h
omoz
ygot
esG
100
% (
48)
Oth
er C
auca
sian
pat
ient
sA
56.
0% (
17),
G 4
4.0%
(13
)A
sian
pat
ient
sA
87.
5% (
7),
G 2
2.5%
(1)
H63
D h
omoz
ygot
esA
100
% (
8)C
ontr
ol C
auca
sian
sA
75.
0% (
3),
G 2
5.0%
(9)
Roc
hett
e et
al.
(19
99)
Con
trol
Bur
mes
e &
Kar
en
A 6
0.7%
(17
), G
39.
3% (
9)C
ontr
ol S
ri L
anka
ns
A 2
0.3%
(24
), G
79.
7% (
94)
(con
tinu
ed)
Nla
IC
auca
sian
sA
53.
0%,
G 4
7.0%
Dou
abin
et
al.
(199
9)C
ontr
ol C
auca
sian
sA
42.
2% (
43),
G 5
7.8%
(59
)A
guil
ar-M
arti
nez
et a
l.C
ontr
ol A
fric
ans
A
68.
7% (
33),
G 3
1.3%
(15
)(1
999)
C28
2Y h
omoz
ygot
esG
100
% (
50)
H63
D h
omoz
ygot
esA
100
% (
100)
C28
2Y h
omoz
ygot
esG
100
% (
46)
Poi
nton
et
al.
H63
D h
omoz
ygot
esA
75.
0% (
9),
G 2
5.0%
(3)
(unp
ubli
shed
res
ults
)IV
S6
142
6 A
®G
Intr
on 6
Bse
mJ
ND
Fou
nd i
n 1
atyp
ical
HH
pati
ent
Poi
nton
et
al.
(unp
ubli
shed
res
ults
)
Occ
urre
nce
and
freq
uenc
ies
(wit
h th
e nu
mbe
rs t
este
d in
bra
cket
s) a
re s
how
n w
here
fig
ures
are
ava
ilab
le.
NA
, N
ot a
ppli
cabl
e; N
D,
not
done
.
TA
BL
E2.
HF
E G
EN
EP
OL
YM
OR
PH
ISM
SS
HO
WIN
GN
UC
LE
OT
IDE
CH
AN
GE,
LO
CA
TIO
NW
ITH
INT
HE
GE
NE,
AN
DIN
TR
OD
UC
TIO
NO
RE
LIM
INA
TIO
NO
FA
RE
ST
RIC
TIO
NE
NZ
YM
ES
ITE
(CO
NT’D
)
Res
tric
tion
Pol
ymor
phis
mL
ocat
ion
enzy
me
All
ele
freq
uenc
yC
omm
ent
Ref
eren
ce
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.
REFERENCES
ADAMS, P.C., CAMPION, M.L., GANDON, G., LE GALL, J.-Y.,DAVID, V., and JOUANOLLE, A.-M. (1997). Clinical and familystudies in genetic hemochromato sis: microsatellite and HFE studiesin five atypical families. Hepatology 26, 986–990.
AGUILAR-MARTINEZ, P., THELCIDE, C., JEANJEAN, P., MAS-MEJEAN, C., GIANSILY, M., and SCHVED, J.-F. (1999). Haplo-type analysis of the HFE gene: implications for the origins of he-mochromatos is related mutations. Blood Cells Mol. Dis. 25,166–169.
BARTON, J.C., SHIH, W.W.H., SAWADA-HIRAI, R., ACTON, R.T.,HARMON, L., RIVERS, C., and ROTHENBERG, B.E. (1997). Ge-
netic and clinical description of hemochromatos is probands and het-erozygotes: evidence that multiple genes linked to the major histo-compatibility complex are responsible for hemochromato sis. BloodCells Mol. Dis. 23, 135–145.
BARTON, J.C., SAWADA-HIRAI, R., ROTHENBERG, B.E., andACTON, R. (1999). Two novel missense mutations of the HFE gene(I105C and G93R) and identification of the S65C mutation in Al-abama hemochromatosis probands. Blood Cells Mol. Dis. 25,146–154.
BENNETT, M.J., LEBRÓN, J.A., and BJORKMAN, P.J. (2000). Crys-tal structure of the hereditary haemochromatosis protein HFE com-plexed with transferrin receptor. Nature 403, 46–53.
BERNARD, P.S., AJIOKA, R.S., KUSHNER, J.P., and WITTWER,C.T. (1998). Homogeneous multiplex genotyping of hemochro-matosis mutations with fluorescent hybridization probes. Am. J.Pathol. 153, 1055–1061.
BEUTLER, E., and WEST, C. (1997). New diallelic markers in theHLA region of chromosome 6. Blood Cells Mol. Dis. 23, 219–229.
BEUTLER, E., GELBART, T., WEST, C., LEE, P., ADAMS, M.,BLACKSTONE, R., POCKROS, P., KOSTY, M., VENDITTI, C.P.,PHATAK, P.D., SEESE, N.K., CHORNEY, K.A., TEN ELSHOF,A.E., GERHARD, G.S., and CHORNEY, M. (1996). Mutation analy-sis in hereditary hemochromato sis. Blood Cells Mol. Dis. 22,187–194.
BEUTLER, E., WEST, C., and GELBART, T. (1997). HLA-H and as-sociated proteins in patients with hemochromato sis. Mol. Med. 3,397–402.
BODMER, J.G., PARHAM, P., ALBERT, E.D., and MARSH, S.G.E.(1997). Putting a hold on “HLA-H”. Nature Genet. 15, 234–235.
BOMFORD, A., and WILLIAMS, R. (1976). Long term results of ve-nesection therapy in idiopathic haemochromatosis. Q. J. Med. 45,611–623.
BOROT, N., ROTH, M.-P., MALFOY, L., DEMANGEL, G., VINEL,J-P., PASCAL, J.-P., and COPPIN, H. (1997). Mutations in the MHCclass I-like candidate gene for hemochromato sis in French patients.Immunogeneti cs 45, 320–324.
BOSSERHOFF, A.-K., SEEGERS, S., HELLERBRAND, C.,SCHÖLMERICH, J., and BÜTTNER, R. (1999). Rapid geneticscreening for hemochromato sis using automated SSCP-based capil-lary electrophoresis (SSCP-CE). BioTechniques 26, 1105–1109.
BOTHWELL, T.H., CHARLTON, R.W., and MOTULSKY, A.G.(1995). Hemochromatosis. The Molecular Basis of Inherited Disease.C.R. Scriver, A.L. Beaudet, and W.S. Sly (eds.) (McGraw-Hill, NewYork) pp. 2237–2269.
BRADBURY, R., FAGAN, E., GOODSON, S., STEER, K., andPAYNE, S.J. (1999). New mutations in the HFE gene forhaemochromatosis. J. Med. Genet. 36, (Suppl. 1), pS96.
CAMASCHELLA, C., ROETTO, A., CICILANO, M., PASQUERO,P., BOSIO, S., GUBETTA, L., DI VITO, F., GIRELLI, D., TO-TARO, A., CARELLA, M., GRIFA, A., and GASPARINI, P. (1997).Juvenile and adult hemochromato sis are distinct genetic disorders.Eur. J. Hum. Genet. 5, 371–375.
CARDOSO, E.M., STÅL, P., HAGEN, K., CABEDA, J.M., ESIN, S.,DE SOUSA, M., and HULTCRANTZ, R. (1998). HFE mutations inpatients with hereditary hemochromato sis in Sweden. J. Intern. Med.243, 203–208.
CARELLA, M., D’AMBROSIO, L., TOTARO, A., GRIFA, A.,VALENTINO, M.A., PIPERNO, A., GIRELLI, D., ROETTO, A.,FRANCO, B., GASPARINI, P., and CAMASCHELLA, C. (1997).Mutation analysis of the HLA-H gene in Italian hemochromato sispatients. Am. J. Hum. Genet. 60, 828–832.
CRAWFORD, D.H., POWELL, L.W., LEGGETT, B.A., FRANCIS,J.S., FLETCHER, L.M., WEBB, S.I., HALLIDAY, J.W., andJAZWINSKA, E.C. (1995). Evidence that the ancestral haplotype inAustralian hemochromato sis patients may be associated with a com-mon mutation in the gene. Am. J. Hum. Genet. 57, 362–367.
UNCOMMON HEMOCHROMATOSIS MUTATIONS 159
DE VILLIERS, J.N.P., and KOTZE, M.J. (1999). Significance of link-age disequilibrium between mutation C282Y and MseI polymor-phism in population screening and DNA diagnosis of hemochro-matosis. Blood Cells Mol. Dis. 25, 250–252.
DE VILLIERS, J.N.P., HILLERMANN, R., LOUDSER, L., andKOTZE, M.J. (1999). Spectrum of mutation in the HFE gene im-plicated in haemochromatosis and porphyria. Hum. Mol. Genet. 8,1571–1522.
DOUABIN, V., MOIRAND, R., JOUANOLLE, A.-M., BRISSOTT,P., LE GALL, J.-Y., DEUGNIER, Y., and DAVID, V. (1999). Poly-morphisms in the HFE gene. Hum. Hered. 49, 21–26.
EDWARDS, C.Q., GRIFFEN, L.M., GOLDGAR, D., DRUMMOND,C., SKOLNICK, M.H., and KUSHNER, J.P. (1988). Prevalence ofhemochromatosis among 11,065 presumably healthy blood donors.N. Engl. J. Med. 318, 1355–1362.
FEDER, J.N., GNIRKE, A., THOMAS, W., TSUCHIHASHI, Z.,RUDDY, D.A., BASAVA, A., DORMISHIAN, F., DOMINGO, JR.R., ELLIS, M.C., FULLAN, A., HINTON, L.M., JONES, N.L., KIM-MEL, B.E., KRONMAL, G.S., LAUER, P., LEE, V.K., LOEB, D.B.,MAPA, F.A., MCCLELLAND, E., MEYER, N.C., MINTIER, G.A.,MOELLER, N., MOORE, T., MORIKANG, E., PRASS, C.E.,QUINTANA, L., STARNES, S.M., SCHATZMAN, R.C.,BRUNKE, K.J., DRAYNA, D.T., RISCH, N.J., BACON, B.R., andWOLFF, R.K. (1996). A novel MHC class I-like gene is mutated inpatients with hereditary haemochromatosis. Nature Genet. 13,399–408.
FEDER, J.N., TSUCHIHASHI, S., IRRINKI, A., LEE, V.K., MAPA,F.A., MORIKANG, E., PRASS, C.E., STARNES, S.M., WOLFF,R.K., PARKKILA, S., SLY, W.S., and SCHATZMAN, R.C. (1997).The hemochromatos is founder mutation in HLA-H disrupts b 2-microglobulin interaction and cell surface expression. J. Biol. Chem.272, 14025– 14028.
FEDER, J.N., PENNY, D.N., IRRINKI, A., LEE, V.K., LEBRÓN, J.A.,WATSON, N., TSUCHIHASHI, Z., SIGAL, E., BJORKMAN, P.J.,and SCHATZMAN, R.C. (1998). The hemochromato sis gene prod-uct complexes with the transferrin receptor and lowers its affinity forligand binding. Proc. Natl. Acad. Sci. USA 95, 1472–1477.
GOMEZ, P.S., PARKS, S., RIES, R., TRAN, T.C., GOMEZ, P.F., andPRESS, R.D. (1999). Polymorphism in intron 4 of HFE does notcompromise haemochromatosis mutation results. Nature Genet. 23,272.
GOTTSCHALK, R., SEIDL, C., LÖFFLER, T., SEIFRIED, E.,HOELZER, D., and KALTWASSER, J.P. (1998). HFE codon 63/282(H63D/C282Y) dimorphism in German patients with genetic he-mochromatos is. Tiss. Antigens 51, 270–275.
GROSS, C.N., IRRINKI, A., FEDER, J.N., and ENNS, C.A. (1998).Co-trafficking of HFE, a nonclassical major histocompatibility com-plex class I protein, with the transferrin receptor implies a role in in-tracellular iron regulation. J. Biol. Chem. 273, 22068– 22074.
HENZ, S., REICHEN, J., and LIECHTI-GALLATI, S. (1997). HLA-H gene mutations and haemochromatosis: the likely association ofH63D with mild phenotype and the detection of S65C, a novel vari-ant in exon 2. J. Hepatol. 26, (Suppl. 1), 57.
HÖHLER, T., LEININGER, S., and SCHNEIDER, P.M. (1999). Apolymorphism in the human HFE gene. Immunogenet ics 49, 823–824.
JAZWINSKA, E.C., CULLEN, L.M., BUSFIELD, F., PYPER, W.R.,WEBB, S.I., POWELL, L.W., MORRIS C.P., and WALSH, T.P.(1996). Haemochromatosis and HLA-H. Nature Genet. 14, 249–251.
JEFFREY, G.P., CHAKRABARTI, S., HEGELE, R.A., and ADAMS,P.C. (1999). Polymorphism in intron 4 of HFE may cause overesti-mation of C282Y homozygote prevalence in haemochromatosis. Na-ture Genet. 22, 325–326.
JOUANOLLE, A-M., GANDON, G., JÉZÉQUEL P., BLAYAU, M.,CAMPION, M.L., YAOUANQ, J., MOSSER, J., FERGELOT, P.,CHAUVEL, B., BOURIC, P., CARN, G., ANDRIEUX, N., GIC-
QUEL, I., LE GALL, J-Y., and DAVID, V. (1996). Haemochro-matosis and HLA-H. Nature Genet. 14, 251–252.
JOUANOLLE, A.M., FERGELOT, P., GANDON, G., YAOUANQ, J.,LE GALL, J-Y., and DAVID, V. (1997). A candidate gene for he-mochromatos is: frequency of the C282Y and H63D mutations. Hum.Genet. 100, 544–547.
KELLY, A.L., RHODES, D.A., ROLAND, J.M., SCHOFIELD, P., andCOX, T.M. (1998). Hereditary juvenile haemochromatosis: a genet-ically heterogeneous life-threatening iron-storage disease. Q. J. Med.91, 607–618.
LEBRÓN, J.A., and BJORKMAN, P.J. (1999). The transferrin recep-tor binding site on HFE, the class I MHC-related protein mutated inhereditary hemochromato sis. J. Mol. Biol. 289, 1109–1118.
LEBRÓN, J.A., BENNETT, M.J., VAUGHN, D.E., CHIRINO, A.J.,SNOW, P.M., MINTIER, G.A., FEDER, J.N., and BJORKMAN, P.J.(1998). Crystal structure of the hemochromato sis protein HFE andcharacterisatio n of its interaction with transferrin receptor. Cell 93,111–123.
LEBRÓN, J.A., WEST, JR. A.P., and BJORKMAN, P.J. (1999). Thehemochromatosis protein HFE competes with transferrin for bindingto the transferrin receptor. J. Mol. Biol. 294, 239–245.
LEGGETT, B.A., HALLIDAY, J.W., BROWN, N.N., BRYANT, S.,and POWELL, L.W. (1990). Prevalence of haemochromatosisamongst asymptomatic Australians. Br. J. Haematol. 74, 525–530.
LIECHTI-GALLATI, S., VARGA, D., and REICHEN, J. (1999).Screening for haemochromatosis in Switzerland: detection of a newpathogenic mutation and two additional variants in exon 2 of the HFEgene. European Society of Human Genetics Meeting, Geneva,Switzerland. May 29 th–June 1st 1999, p. 122.
MEISSNER, P.N., DAILEY, T.A., HIFT, R.J., ZIMAN, J., CORRI-GALL, A.V., ROBERTS, A.G., MEISSNER, D.M., KIRSCH, R.E.,and DAILEY, H.A. (1996). A R59W mutation in human protopor-phyringen oxidase results in decreased enzyme activity and is preva-lent in South Africans with variegate porphyria. Nature Genet. 13,95–97.
MERCIER, B., MURA, C., and FEREC, C. (1997). Putting a hold on“HLA-H”. Nature Genet. 15, 234.
MERRYWEATHER-CLARKE, A.T., POINTON, J.J., SHEARMAN,J.D., ROBSON, K.J.H., JOUANOLLE, A.M., MOSSER, A.,DAVID, V., LE GALL, J.-Y., HALSALL, D.J., ELSEY, T.S.,KELLY, A., COX, T.M., CLARE, M., BOMFORD, A., VAND-WALLE, J.L., ROCHETTE, J., BOROT, N., COPPIN, H., ROTH,M.-P., RYAN, E., CROWE, J., TOTARO, A., GASPARINI, P.,ROETTO, A., CAMASCHELLA, C., DARKE, C., WALLACE,D.F., SAEB-PARSY, K., DOOLEY, J.S., WORWOOD, M., andWALKER, A.P. (1999). Polymorphism in intron 4 of HFE does notcompromise haemochromatosis mutation results. Nature Genet. 23,271.
MILMAN, N. (2000). Inheritance of hemochromato sis: family studies.In Hemochromatosis. J.C. Barton and C.Q. Edwards (eds.) (Cam-bridge University Press, Cambridge, UK) pp. 15–41.
MORTON, C.C., KIRSCH, I.R., NANCE, W.E., EVANS, G.A., KOR-MAN, A.J., and STROMINGER, J.L. (1984). Orientation of lociwithin the human major histocompatibility complex by chromoso-mal in situ hybridization. Proc. Natl. Acad. Sci. USA 81, 2816–2820.
MURA, C., NOUSBAUM, J.-B., VERGER, P., MOALIC, M.-T.,RAGUENES, O., MERCIER, A.-Y., and FÉREC, C. (1997). Phe-notype-genotype correlation in haemochromatosis subjects. Hum.Genet. 101, 271–276.
MURA, C., RAGUENES, O., and FÉREC, C. (1999). HFE mutationsanalysis in 711 hemochromato sis probands: evidence for S65C im-plication in mild form of hemochromatosis. Blood 93, 2502–2505.
NIEDERAU, C., FISCHER, R., PURSCHEL, A., STREMMEL, W.,HAUSSINGER, D., and STROHMEYER, G. (1996). Long-term sur-vival in patients with hereditary hemochromato sis. Gastroenterology110, 1107–1119.
POINTON ET AL.160
NOLL, W.W., BELLONI, D.R., STENZEL, T.T., and GRODY, W.W.(1999). Polymorphism in intron 4 of HFE does not compromisehaemochromatosis mutation results. Nature Genet. 23, 271–272.
PARKKILA, S., WAHEED, A., BRITTON, R.S., BACON, B.R.,ZHOU, X.Y., TOMATSU, S., FLEMING, R.E., and SLY, W.S.(1997). Association of the transferrin receptor in human placenta withHFE, the protein defective in hereditary hemochromato sis. Proc.Natl. Acad. Sci. USA 94, 13198– 13202.
POINTON, J.J., SHEARMAN, J.D., MERRYWEATHER-CLARKE,A.T., and ROBSON, K.J.H. (1997). A single nucleotide deletion inthe putative haemochromatosis gene in a patient who is negative forthe C282Y and H63D mutations. Proceedings of the InternationalSymposium on iron and biology and medicine, Saint Malo, France,16 th–20 th June 1997, p. 268.
PORTO, G., and DE SOUSA, M. (2000). Variation of hemochromato sisprevalence and genotype in national groups. In Hemochromatosis .J.C. Barton and C.Q. Edwards (eds.) (Cambridge University Press,Cambridge, UK, pp. 51–62.
POWELL, L.W. (1970). Changing concepts in haemochromatosis.Postgrad. Med. J. 46, 200–209.
RISCH, N. (1997). Haemochromatosis, HFE and genetic complexity.Nature Genet. 17, 375–376.
ROCHETTE, J., POINTON, J.J., FISHER, C.A., PERERA, G., ARAM-BEPOLA, M., KODIKARA ARICHCHI, D.S., DE SILVA, S.,VANDWALLE, J.L., MONTI, J.P., OLD, J.M., MERRY-WEATHER-CLARKE, A.T., WEATHERALL, D.J., and ROBSON,K.J.H. (1999). Multicentric origin of hemochromato sis gene (HFE)mutations. Am. J. Hum. Genet. 64, 1056–1062.
ROETTO, A., TOTARO, A., CAZZOLA, M., CICILANO, M., BO-SIO, M., D’ASCOLA, G., CARELLA, M., ZELANTE, L., KELLY,A.L., COX, T.M., GASPARINI, P., and CAMASCHELLA, C.(1999). Juvenile hemochromato sis locus maps to chromosome 1q.Am. J. Hum. Genet. 64, 1388–1393.
SALTER-CID, L., BRUNMARK, A., LI, Y., LETURCQ, D., PETER-SON, P.A., JACKSON, M.R., and YANG, Y. (1999). Transferrin re-ceptor is negatively modulated by the hemochromatosis protein HFE:implications for cellular iron homeostasis. Proc. Natl. Acad. Sci. USA96, 5434–5439.
SHELDON, J.H. (1935). Haemochromatosis . (Oxford University Press,London).
SIMON, M., BOUREL, R., FAUCHET, R., and GENETET, B. (1976).Association of HLA-A3 and HLA-B14 antigens with idiopathichaemochromatosis. Gut 17, 332–334.
SIMON, M., BOUREL, M., GENETET, B., and FAUCHET, R. (1977).Idiopathic hemochromato sis. Demonstration of recessive transmis-sion and early detection by family HLA typing. N. Engl. J. Med.297, 1017–1021.
SIMONSEN, K., DISSING, J., RUDBECK, L., and SCHWARTZ, M.(1999). Rapid and simple determination of hereditary haemochro-matosis mutations by multiplex PCR-SSCP: detection of a new poly-morphic mutation. Ann. Hum. Genet. 63, 193–197.
SOMERVILLE, M.J., SPRYSAK, K.A., HICKS, M., ELYAS, B.G.,and VICEN-WYHONY, L. (1999). An HFE intronic variant pro-motes misdiagnosis of hereditary hemochromato sis. Am. J. Hum.Genet. 65, 924–926.
SPRIGGS, E.L., HARRIS, P.E., and BEST, L.G. (1999). Hemochro-matosis mutations C282Y and H63D in “cis” phase. Am. J. Hum.Genet. 65 (Suppl.), A492.
TOTARO, A., GRIFA, A., CARELLA, M., D’AMBROSIO, L.,VALENTINO, M., ROTH, M.P., BOROT, N., COPPIN, H.,ROETTO, A., CAMASCHELLA, C., and GASPARINI, P. (1997).Hereditary hemochromatosis: a HpaI polymorphism within the HLA-H gene. Mol. Cell. Probes 11, 229–230.
WAHEED, A., PARKKILA, S., ZHOU, X.Y., TOMATSU, S.,TSUCHIHASHI, Z., FEDER, J.N., SCHATZMAN, R.C., BRIT-TON, R.S., BACON, B.R., and SLY, W.S. (1997). Hereditary he-mochromatos is: effects of C282Y and H63D mutations on associa-tion with b 2-microglobulin, intracellular processing, and cell surfaceexpression of HFE protein in COS-7 cells. Proc. Natl. Acad. Sci.USA 94, 12384–12389.
WALLACE, D.F., DOOLEY, J.S., and WALKER, A.P. (1999). Anovel mutation of HFE explains the classical phenotype of genetichemochromatosis in a C282Y heterozygote. Gastroenterology 116,1409–1412.
WARNICH, L., KOTZE, M.J., GROENWALD, I.M., GROENWALD,J.Z., VAN BRAKEL, M.G., VAN HEERDEN, C.J., DE VILLIERS,J.N., VAN DE VEN, W.J., SCHOEMAKERS, E.F., TAKETANI, S.,and RETIEF, A.E. (1996). Identification of three mutations and as-sociated haplotypes in the protoporphyrino gen oxidase gene in SouthAfrican families with variegate porphyria. Hum. Mol. Genet. 5,981–984.
WENZ, H.M., BAUMHUETER, S., RAMACHANDRA, S., and WOR-WOOD, M. (1999). A rapid automated SSCP multiplex capillaryelectrophoresis protocol that detects the two common mutations im-plicated in hereditary hemochromato sis (HH). Hum. Genet. 104,29–35.
WORWOOD, M., SHEARMAN, J.D., WALLACE, D.F., DOOLEY,J.S., MERRYWEATHER-CLARKE, A.T., POINTON, J.J.,ROSENBERG, W.M.C., BOWEN, D.J., BURNETT, A.K., JACK-SON, H.A., LAWLESS, S., RAHA-CHOWDHURY, R., PAR-TRIDGE, J., WILLIAMS, R., BOMFORD, A., WALKER, A.P., andROBSON, K.J.H. (1997). A simple genetic test identifies 90% of UKpatients with haemochromatosis. Gut 41, 841–844.
WORWOOD, M., JACKSON, H.A., FEENEY, G.P., EDWARDS, C.,and BOWEN, D.J. (1999). A single tube heteroduplex PCR for thecommon HFE genotypes. Blood 94 (S1), 405a.
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.
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