hereditary hemochromatosis: hfe mutation analysis in greeks reveals genetic heterogeneity

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G. Papanikolaou, et al. Blood Cells, Molecules, and Diseases (2000)26(2) April: 163–168

doi:10.1006/bcmd.2000.0292, available online at http://www.idealibrary.com on

Hereditary Hemochromatosis: HFE Mutation Analysis in GreeksReveals Genetic HeterogeneitySubmitted 03/25/00(Communicated by Ernest Beutler, M.D., 04/10/00)

George Papanikolaou,1 Marianna Politou,1 Evangelos Terpos,1 Stefanos Fourlemadis,2

Nikos Sakellaropoulos,1 and Dimitris Loukopoulos1,3

ABSTRACT: Hereditary hemochromatosis (HH) is common among Caucasians; reported disease frevary from 0.3 to 0.8%. Identification of a candidate HFE gene in 1996 was soon followed by the descritwo ancestral mutations, i.e., c.845G3A (C282Y) and c.187C3G (H63D). To these was recently addedmutation S65C, which may represent a simple polymorphism. The incidence of HH in Greece is unknclinical cases are rare. Also unknown is the carrier frequency of the two mutant alleles. A first estimate of tis given in the present report. It is based on data from the genetic analysis of 10 unrelated patients of Grewho were referred to our center for genotyping and 158 unselected male blood donors. The allele frequethe C282Y and H63D mutations were 0.003 and 0.145, respectively. The C282Y allele was detected in 50patients. This is considerably lower than the frequencies reported for HH patients in the U.S.A. (82%) an(91%) and closer to that reported in Italy (64%). Five patients did not carry any known HFE mutation; thrrepresent cases of juvenile hemochromatosis, given their early onset with iron overload, hypogonadism,disease. We suggest that genetic heterogeneity is more prominent in Southern Europe. It is also possibpenetrance of the responsible genes is different across the Mediterranean.© 2000 Academic Press

Key Words:HFE; hemochromatosis; iron; juvenile hemochromatosis.

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INTRODUCTION

Hereditary Hemochromatosis (HH) is tmost common genetic disorder of Northernropeans, having a carrier rate from 1:8 to 1and an overall incidence of approximat1:300 (1). In HH excessive intestinal iron asorption leads to iron overload and tissue dage; consequences of the latter include diabmellitus, arthropathy, cirrhosis, heart diseaand hypogonadism (2). The disease has bknown to be HLA-linked since 1976 (3);candidate gene (HFE) was identified on thshort arm of chromosome 6 (4). The HFE ghas undergone at least two ancestral mutati(a) the G3A transition at nucleotide 845 thhanges the invariant 282 cysteine residu

1 First Department of Medicine, University of Athens, Laikon Hospita2 Blood Bank, 251 Air Force Hospital, 11526 Athens, Greece.
3 Reprint requests to: Dimitris Loukopoulos.
163

the encoded protein to tyrosine. This is cleaassociated with HH and has been detecte83–100% of HH patients of North Europeorigin (4 – 6), and (b) the transition C3G atnucleotide 187, which changes the histidineto aspartic acid. Its association with iron ovload is less clear; however, compound heterogotes C282Y/H63D express hemochromatphenotype more often than C282Y heterogotes (7). A recently identified A3T transitionat nucleotide 193, expressed as a substitutiocysteine for serine at amino acid position #seems also to have an effect on iron metabo(8, 9).

The frequency of HH in Greece, a country10 millions Caucasian inhabitants at the southpart of the European Continent, is rather l

27 Athens, Greece.

1079-9796/00 $35.00Copyright© 2000 by Academic Press

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Blood Cells, Molecules, and Diseases (2000)26(2) April: 163–168 G. Papanikolaou, et al.doi:10.1006/bcmd.2000.0292, available online at http://www.idealibrary.com on

since patients with this condition are rarely admted to Greek hospitals. However, as underdnosing the disease or the presence of more beforms cannot be excluded, genetic analysis oaffected families and the general populationquire a primary importance for understandingspread of the HFE gene(s) across this countr

The present communication reports our fiings in 10 typical HH unrelated patients wregards to the presence of the C282Y, H63D,S65C mutations as well as the frequency offormer two mutations in 316 chromosomes frunrelated blood donors with no evidence of ioverload. Overall, only 50% of the patients’ chmosomes carried the C282Y mutation (3 homogotes and 2 compound C282Y/H63D heterogotes). The allele frequency for the C282Y mution was 0.003 and that of H63D 0.145.

MATERIALS AND METHODS

Patients

Ten adult unrelated individuals (7 men anwomen) of Greek origin, who had been diagnoas having hereditary hemochromatosis were sied. The diagnosis was based on serum iron, trferrin saturation, and ferritin levels along with testimation of the amount of iron mobilized

TA

Main Featu

No. Sex

Serumiron

(mg/dl)

Serumferritin(ng/dl)

Tranferrinsaturation

(%)Hep

1 M 270 2283 86 III–2 F 252 1750 65 III–3 F 186 4217 90 III–4 M 175 2500 85 III–5 F 273 — 79 Not6 M 175 2671 93 IV,7 M 178 1400 76 III–8 M 176 1342 78 III–9 M 121 1224 33 III–

10 M 292 890 — IV c

phlebotomy and that of stainable iron found on

164

liver biopsy. The latter was empirically gradfrom grade 0 to IV. Grade III–IV was consideras compatible with HH. Iron removed by phlebomy was calculated, estimating that 1 L of blocontains approximately 0.5 g of iron. On invegating the patients, causes of secondary iron oload were thoroughly excluded. The patientsa complete hematological and biochemical euation and assessment of all markers for hepaIn addition, alcohol consumption, transfusiohistory of supplementary iron intake were cafully reviewed. Two patients had alreaachieved a state of iron depletion (serum fer,25 ng/ml) by phlebotomy, while four others acurrently treated by this approach. The studyincludes two patients (#4 and #6) who have dduring the follow-up period from severe compcations of HH, heart failure and hepatocellucancer respectively (Table 1).

Unselected Blood Donors

One hundred and fifty-eight unrelated heamale blood donors of Greek origin were exained. Their work-up comprised a detailed histconcerning blood loss, symptoms and signstentially related to iron overload, a CBC and msurements of serum iron and ferritin. HFE mutions were identified by standard molecular te

1

HH Patients

iderosis)

Iron (g)removed byphlebotomy

Age atpresentation/

age atdiagnosis Genotype

osis 10.1 21/25 wt/wtosis 9.5 32/41 wt/wtosis .8.2 39/49 wt/wt

Death 25/32 wt/wt.21.2 20/21 wt/wt

sis HCC Death 62/68 C282Y/C28— —/51 C282Y/C282Y

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is No recordsavailable

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Mutation Analysis

DNA was isolated from blood leukocytes awas amplified with the PCR (polymerase chreaction). The PCR assay started with an indenaturation of 5 min at 95°C and was compleafter 30 cycles each comprising 1 min at 55°Cprimer annealing, 1 min at 72°C for primer etension and 1 min at 94°C for denaturation.

The PCR amplification was performed infinal volume of 100ml containing 300 ng o

NA, 1.5 U of Taq DNA polymerase (GibcoRL, U.S.A.), 20 mM Tris–HCl (pH 8.4), 50 mMCl, 1.5 mM MgCl2, 0.2 mM of each nucleotidnd 10 pmol of each primer. The primers used

he amplification are as follows: (a) Posit845, forward 59-TGGCAAGGGTAAACAGAT-9; reverse 59-GGTTGAGAGGAGTGCCTGAG9; (b) Position #187, forward 59-ACATGGTTA-GGCCTGTTGC-39, reverse 59-GGAAAAT-ACAACCACAGCAA-39.

The PCR amplification at position #845 gentes a 389-bp product. Digestion of the normal pct withRsaI generates two fragments, 250 andp long. In contrast, digestion of the mutant prodith the same enzyme yields a total of three fents of 250, 110, and 29 bp length becauseutation creates a new restriction site in the shormal fragment. As expected, the C282Y homootes display three fragments only, while thoseying one C282Y allele have four (250, 139, 1nd 29 bp). The PCR amplification at position #enerates a 208-bp product. Digestion of the lith the restriction enzymeMboI gives two fragents (138 and 70 bp) in the normal and one fent only (208 bp) in the mutant chromosomecause the mutation abolishes the restrictions expected, the homozygotes for the mutationlay one band only (208 bp), while heterozygohow three (208, 138, and 70 bp). For the detef the 193 A3T mutation the PCR amplificatioroduct of #187 was used. Digestion of the laith the restriction enzymeHinfI yields two frag-ents (147 and 61 bp) in the normal and one fent only (208 bp) in the mutant chromosomecause the mutation abolishes the restriction
s expected the S65C homozygotes display oneo
165

and (208 bp), while the heterozygotes have t208, 147, and 61 bp)

LA Typing

HLA typing was performed by the micro lymhocytotoxicity method described by Terasnd McClellaud.

ESULTS

andom Population

Of the group of 158 unselected male bloonors 1 individual was found to be heterozyg

or the C282Y mutant allele. This 34-year-oale had normal serum iron and ferritin valut had already voluntarily donated 10 blood uver the last five years. The C282Y allele fuency in this limited population sample is th.003. In the same group 40 individuals weterozygous and 3 homozygous carriers of63D allele; the H63D allele frequency is thstimated at 0.145. Compound C282Y/H63Drozygotes were not found in the control groomparison of the serum iron and ferritin valmong blood donors with different genotypes (t, H63D/wt, H63D/H63D) failed to reveal antatistically significant differences (Table 2).

H Patients

Three of the hereditary hemochromatosisients (30%) were C282Y homozygotes, an20%) compound C282Y/H63D heterozygohe remaining 5 patients (50%) did not carry

TABLE 2

Genotypes and Mean Serum Iron and Ferritin Values in thUnselected Individuals of the Present Study

GenotypeNumber of

subjectsSerum iron

(mg/dl)Serum ferritin

(ng/ml)

wt/wt 114 89.2 76.1C282Y/wt 1 80.0 92.0H63D/wt 40 97.7 77.4H63D/H63D 3 129.7 73.7

ne of the above HFE associated mutations. The

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mutation S65C was never identified amongpatients. Results are summarized in Table 3.

Features of the HH Patients with No DetectaHFE Mutations

The clinical records of the 5 patients not crying HFE mutations are summarized in TablePatient #1 presented with ejaculation failure atage of 21 and was diagnosed at the age owhen evaluated for unexplained fatigue, arthgias, and altered liver function tests. Patientwas examined for infertility at the age of 25 areceived hormone replacement therapy forpogonadotrophic hypogonadism for a long pewith no etiologic diagnosis. He was diagnose

TA

Genotypes of Control

C282Y1/1H63D2/2

C282Y1/2H63D2/2

HH patients (n 5 10) 3(30%)

0

Controls (n 5 158) 0 1(0.6%)

TA

Clinical Characteristics of P

Clinical feature #1

ex/age at diagnosis M/25ge at presentation 21

resenting symptoms

Failure ofejaculation

Sa

ypogonadism 111 1eart disease Absentkin hyperpigmentation 1 1rthralgias/arthritis 1 1

ron removed with phlebotomies 10.1 g .

FE mutations C282Y, H63D, and S65C

LA antigens A2, B44,CW2

AC

A32, B49 B35, C

166

the age of 34 because of diabetes mellitusskin pigmentation. Laboratory evaluation wcompatible with hereditary hemochromatosisboth of them. The latter patient was inadequatreated with desferrioxamine and died at theof 48 because of cardiac failure due to hemocmatosis. Patient #5 is a female who presentedamenorrhea at the age of 21; her younger spresented also amenorrhea at the age of 19.have unequivocal laboratory evidence of hertary hemochromatosis. Besides, their elder shas died of cardiac failure due to hemochromsis at the age of 30. Over the past yearssisters have been on an intensive phlebottreatment and remain asymptomatic. Accordin

3

lation and Patient Groups

C282Y1/2H63D1/2

C282Y2/2H63D1/1

C282Y2/2H63D1/2

C282Y2/2H63D2/2

2(20%)

0 0 5(50%)

0 3(1.8%)

40(25.3%)

114(72.1%)

4

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Patient

#3 #4 #5

/49 F/41 M/32 F/2139 32 25 20

Hypogonadism

daryrrhea

Oligomenorrhea Infertility,loss of libido

Secondaryamenorrhe

111 111 111ent 11 111 Death Absent

111 111 11Absent 11 Absent9.5 g .4.0 g 21.2 g

Not detected

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the generally accepted classification, the abpatients should be considered as cases of juvhemochromatosis because they all presenteddisease onset (i.e., before the age of 30),clinical signs of hypogonadism (10).

Three of five patients do not display any ofHLA antigens known to be associated with heritary hemochromatosis.

DISCUSSION

Numerous studies have demonstrated thareditary hemochromatosis occurs ratherquently in Northwestern Europe, with a genequency varying from 0.06 to 0.1 (11). It is posble that the main hemochromatosis mutaC282Y has arisen relatively recently in a Celor a Viking or a German population. (12, 13).this survey the C282Y mutant allele was fouonly once in 316 chromosomes from normGreek individuals (estimated frequency: 0.00This finding is in keeping with earlier observtions that hemochromatosis is rare in the EMediterranean countries (11). According toHardy–Weinberg equilibrium the predicted hmozygosity for the C282Y allele is 0.00000This low probability along with the fact that tpenetrance of the gene may be different ingeneral population may sufficiently explainrarity of clinical diagnoses of HH in Greece (1Of course, these calculations are only indicaand require genotyping of a significantly larpopulation sample.

In our study, 5 of 10 patients with HH wenot shown to carry any of the C282Y, H63DS65C mutations. Three of those undoubtedly mthe clinical criteria for juvenile hemochromatosi.e., age at presentation,30 years, hypogonaism, cardiac impairment and severe iron overl(Table 4) (10).

The percentage of wt/wt Greek HH patientrelatively high when compared with the onesported for HH patients from North Ameri(6.8%) (5) and France (0%) (15), but is closethat recently reported from Italy (21%) (1Moreover, in another study where HH patiefrom Northern Italy were compared to HH p
tients from the South, the respective percentage of
167

wt/wt HH patients was 11.3% in Northern a36.7% in Southern Italy (17). Our results arekeeping with the observation of a decreasingquency of the C282Y allele from North to SouEurope and stress that HH is a heterogengenetic disease. The large proportion of HHtients with no detectable HFE mutation in Soueast Europe as well as in this study raisesquestion whether many of these patients belonthe so-called “juvenile form” (17, 18). Howeveas other as yet unidentified mutations mayunderlie the HH defect in our patients, thorougenotype studies are definitely indicated.

Taking into account that HH is a heteroneous genetic disease and that its’ prevalencGreek population is not known, diagnosis ofclinical condition and carrier identification in thcountry should be still based on transferrin sration and serum ferritin measurements. Liveropsy remains the “gold standard” to diagncases where the HFE gene analysis is not inmative, and has a unique prognostic significasince it is the only way to accurately defineextent of liver damage. Genotyping forHFE mu-tations may then intervene as a second stepcould make liver biopsy unnecessary in the estages of the disease.

ACKNOWLEDGMENT

This study was supported by the Universitythens (Grant 4/70/4303).

REFERENCES

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2. Cartwright, G. E., Edwards, C. Q., Kravitz, K.,et al.(1979) Hereditary hemochromatosis: Phenotypicpression of the disease.New Engl. J. Med.301,175–179.

3. Simon, M., Bourel, R., Fauchet, R., and Genete(1976) Association of HLA-A3 and HLA-B14 angens with idiopathic hemochromatosis.Gut 17, 322–334.

4. Feder, J. N.,Gnirke, A., Thomas, W.,et al. (1996) A
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6. Jazwinska, E. C., Cullen, L. M., Busfield, F.,et al.(1996) Hemochromatosis and HLA-H.Nat. Genet.13,399–408.

7. Beutler, E. (1997) The significance of187G(H63D) mutation in hemochromatosis.Am. JHum. Genet.61, 762–764.

8. Mura, C., Raguenes, O., and Ferec, C. (1999)mutation analysis in 711 hemochromatosis probaEvidence for S65 C implication in mild form of hemchromatosis.Blood 93, 2502–2505.

9. Beutler, E., Felitti, V. J., Ho, N. J., and Gelbart,(1999) Commentary on: HFE S65C variant isassociated with increased transferrin saturation inuntary blood donors by Naveen Arya, SubrChakrabrati, Robert A. Hegele, Paul C. Adams.BloodCells Mol. Dis.25, 358–360.

10. Cazzola, M., Ascari, E., Barosi, G.,et al. (1983)Juvenile idiopathic haemochromatosis: A life-threening disorder presenting a hypogonadotropic hgonadism.Hum. Genet.65, 149–154.

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12. Simon, M., Alexandre, J. L., Fauchet, R.,et al. (1980)The genetics of hemochromatosis.Prog. Med. Gene4, 135–168.

13. Fairbanks, V. F. (2000) Hemochromatosis: Populagenetics.In Hemochromatosis (Barton, J. C., andwards, C. Q., Eds.), pp. 42–50. Cambridge UPress, UK..

14. Olynyk, J. K., Cullen, D. J., Aquilia, S.,et al. (1999)A population-based study of the clinical expressiothe hemochromatosis gene.New Engl. J. Med.341,718–724.

15. Jouanolle, A. M., Gandon, G., Jezequel, P.,et al.(1996) Hemochromatosis and HLA-H.Nat. Genet.14,251–252.

16. Carella, M. D., Ambrosio, L., Totaro, A.,et al. (1997)Mutation analysis of the HLA-H gene in Italian hemchromatosis patients.Am. J. Hum. Genet.60, 828–832.

17. Piperno, A., Sampietro, M., Pietrangelo, A.,et al.(1998) Heterogeneity of hemochromatosis in ItGastroenterology114,996–1002.

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hereditary hemochromatosis: hfe mutation analysis in greeks reveals genetic heterogeneity

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