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Two novel nonsense mutations of HFE gene in five unrelated Italian patients with hemochromatosis
GASTROENTEROLOGY 2000;119:441– 445
Two Novel Nonsense Mutations of HFE Gene in Five Unrelated Italian Patients With Hemochromatosis ` ,* PAOLA TROMBINI,* ALBERTO PIPERNO,* CRISTINA AROSIO,‡ LAURA FOSSATI,* MAURO VIGANO ANNA VERGANI,* and GIUSEPPE MANCIA* *Clinica Medica, Universita` degli Studi di Milano-Bicocca, Ospedale S. Gerardo, Monza; and ‡Istituto Auxologico Italiano, Ospedale S. Luca, Milan, Italy
Background & Aims: Most hemochromatosis patients of Northern European descent are homozygous for the C282Y mutation of HFE gene. In Italy, many patients with iron overload are not homozygous for C282Y, and the presence of other mutations or other genetic determinant has been suggested. Methods: Five unrelated Italian patients heterozygous for C282Y with the classic hemochromatosis phenotype were studied. The entire coding sequence and the exon-intron boundaries of the HFE gene were analyzed. Chromosome 6p haplotypes were defined in each patient by analysis of D6S265, D6S105, and D6S1281 microsatellites. Results: Two novel nonsense HFE mutations were identified in exon 3 in the C282Y negative chromosome. The first one, a G-to-T transition at codon 168, was detected in 3 probands; the second, a G-to-A transition at codon 169, was detected in the others. Conclusions: The 2 nonsense mutations in the compound heterozygous state with C282Y result in the classic hemochromatosis phenotype in several unrelated Italian patients. This confirms that hemochromatosis in Italy is not as homogeneous as in northern Europe and suggests that other mutations can exist in C282Y or H63D heterozygotes with iron overload. These findings have practical implications for diagnostic and screening strategies for hemochromatosis.
ereditary hemochromatosis (HH)1 is a common autosomal recessive disease characterized by increased iron absorption and progressive iron overload.2 In 1996, the HFE gene was identified and 2 mutations were originally described, C282Y and H63D.3 Most HH patients are homozygous for C282Y.3– 6 Among HH probands, compound heterozygotes C282Y/H63D account for approximately 5%–7%,3,7 H63D homozygotes are very rare, and both generally have a mild form of hemochromatosis.8 A minority of cases presenting with HH phenotype are C282Y or H63D heterozygotes or neither C282Y nor H63D. This appears to be more common in southern than in northern Europe.9,10 In a collaborative study of 188 Italian patients with HH-like phenotype, 28% were incompletely characterized with at least one
H
chromosome without an assigned mutation.10 In some of these patients, the presence of other HFE mutations or other 6p-linked genetic determinants or the existence of other forms of genetic iron overload unlinked to chromosome 6 has been suggested.10 –12 Three novel HFE mutations were discovered recently in the heterozygous state in association with C282Y in single cases with classical HH: a splice site mutation (IVS3 ⫹ 1G 3 T) causing obligate skipping of exon 313 and 2 missense mutations (I105T and G93R) located in the ␣1 domain that may affect the binding of HFE protein to the transferrin receptor.14 Moreover, the S65C missense mutation was found to be significantly enriched in HH chromosomes that were neither C282Y or H63D, suggesting that the S65C could be another variant contributing to a mild form of HH in association with C282Y or H63D.15 Other missense mutations have been described recently, but their role in the pathogenesis of HH is still unclear.16 Based on these findings, we reanalyzed Italian C282Y heterozygous probands with the classic HH phenotype to test the hypothesis that other HFE mutations in nonC282Y chromosomes could explain their fully expressed clinical phenotype. We report the analysis of 5 of these patients.
Patients and Methods Patients We studied 5 Italian probands with HH who were heterozygous for C282Y and negative for H63D. They all met the classic phenotypic criteria for homozygous HH: (1) increased transferrin saturation (repeatedly ⬎50% fasting) and increased serum ferritin levels; (2) hepatocellular hemosiderin Abbreviations used in this paper: HH, hereditary hemochromatosis; HIC, hepatic iron concentration; HII, hepatic iron index; IR, iron removed; PCR, polymerase chain reaction; RFLP, restriction length polymorphism analysis. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.9369
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deposits of grade III or IV according to Scheuer et al.17; and (3) hepatic iron index ⱖ2. Patients with iron-loading anemias, chronic viral hepatitis B or C, or a history of heavy alcohol intake or blood transfusions were excluded. All patients and relatives gave their informed consent to the study.
destroy a restriction endonuclease site, the portion of exon 3 containing the mutations was amplified by PCR using mismatched sense oligonucleotide specific for each mutation (Bria-F: 5⬘ TGGCCCACCAAGCTGGACT 3⬘; Dom-F: 5⬘ GCCTGGCCCACCAAACTG 3⬘) to create an informative restriction site for BsrSI in the wild-type HFE sequence. The antisense oligonucleotide was the same as the one used for sequencing.
Clinical Studies The presence of fibrosis and cirrhosis was determined at liver biopsy. All cases of cirrhosis were classified as Child’s class A. The presence of clinical complications related to HH was determined according to previously described criteria.18 Transferrin saturation and serum ferritin levels were measured by standard methods. Hepatic iron concentration (HIC) was determined by atomic absorption spectrophotometry (PerkinElmer S2380, Norwalk, CT), and the hepatic iron index (HII) (ratio of HIC [mol/g dry wt] and age [years]) was calculated. The total amount of iron removed by phlebotomy (IR) was estimated as previously reported.18 The geographic origin of each patient was determined to the fourth generation.
Results Table 1 reports iron and clinical data of the 5 probands. Three patients (1, 2, and 3) were from the same subalpine valley in the northwest (Ossola), and 2 (4 and 5) were from an area around the city of Monza (Brianza). Sequence analyses of the HFE gene in probands showed 2 novel nonsense mutations in exon 3 in the heterozygous state. The first, a G-to-T transition at codon 168 (GAG3 TAG, Glu3 Stop), was found in probands from the Ossola valley. The second, a G-to-A transition at codon 169 (TGG3 TAG, Trp3 Stop), was detected in probands from the Brianza region. The new variants were named HFE-Ossola and HFE-Brianza based on the origins of the patients. Figure 1 shows the direct sequence analyses of a portion of exon 3 containing the 2 mutations and the RFLP patterns of 2 probands with the HFE-Ossola and HFE-Brianza mutations and of controls. Figures 2 and 3 show the family pedigrees of the probands from the Ossola and Brianza regions, respectively, including iron data, chromosome 6p haplotypes, and HFE alleles. In family 1 we found an affected brother who shared the same chromosome 6p haplotypes and the same HFE mutations with the proband. His HIC was 176.2 mol/g and HII was 3.92. The 2 new variants were in complete linkage disequilibrium with the C282Y and H63D mutations. HFEOssola was associated with haplotype D6S265-3, HLAA24, D6S105-5, and D6S1281-6 in families 1 and 2, whereas in family 3 the haplotype changed at D6S265 and HLA-A loci, suggesting that a recombination event occurred between HLA-A and D6S105 (see Figure 2).
Molecular Studies Genomic DNA was extracted from peripheral blood leukocytes. C282Y and H63D mutations were detected using standard polymerase chain reaction (PCR) and restriction enzyme digestion with Rsa I and Bcl I, respectively.9 Microsatellites D6S265, D6S105, and D6S1281 were analyzed as previously described.19 HLA-A antigens were defined by the microlymphotoxicity test. Haplotypes were constructed manually on the basis of intrafamilial segregation of the marker alleles. The entire coding sequence and exon-intron boundaries of the HFE gene were amplified by PCR according to Carella et al.9 The nucleotide sequence of both DNA strands was independently determined in all probands and relatives with the C282Y-negative chromosome identical to the proband by using the dideoxy chain-termination reaction with Sequenase version 2.0 (USB Amersham, Cleveland, OH) and [␣-35S]deoxyadenosine triphosphate, according to the protocol recommended by the manufacturer. Reactions were separated on a 6% denaturing polyacrylamide gel and visualized by autoradiography. The primers used for sequencing were the same as those used for PCR. To further confirm the presence of the novel mutations (see Results), a restriction length polymorphism analysis (RFLP) was performed. Because the 2 mutations did not create or
Table 1. Data of the 5 Probands With Hemochromatosis
1 2 3 4 5
Sex
Age (yr)
TS (%)
SF (g/L)
Scheuer’s grade
HIC (mol/g)
HII
IR (g)
M M M M M
48 47 42 50 66
96 79 86 106 100
694 1206 608 2740 1351
4 4 3 4 4
238 359 144 425 421
4.9 7.6 3.4 8.5 5.4
4 23 3.6 21 —
Clinical complications None C None C, H, D C, H, A, D
TS, transferrin saturation; SF, serum ferritin; IR, iron removed; C, hepatic cirrhosis; H, hypogonadism; A, arthropathy; D, diabetes.
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443
Figure 1. (A) Direct sequencing of sense strand of PCR products of exon 3 in patients with HFE-Ossola and HFEBrianza mutations compared with wild type. The sequence is for codons of amino acids numbered 167–170. Arrow indicates the single base substitution in the heterozygous state in probands. (B) Restriction endonuclease analysis of the PCR products obtained with specific mismatched primers creating a BsrSI site in the wild-type sequence. In the heterozygous affected patients, both the digested (wild-type) and undigested (mutated) bands can be seen. Lane M, marker; lane 1, wild-type; lane 2, patient.
HFE-Brianza was associated with haplotype D6S265-3, HLA-A24, D6S105-6, and D6S1281-6.
Discussion We describe 5 Italian patients with classic HH who are compound heterozygotes for C282Y and 2 novel nonsense HFE mutations. The 2 mutations determine a stop codon at nucleotide 502 and 506 of the open reading frame (nucleotides 502 GAG3 TAG and 506 TGG3 TAG), respectively, leading to proteins that lack the ␣3 domain, transmembrane domain, and cytoplasmic tail. These mutations probably produce, differently from C282Y, a complete disruption of the function of HFE similar to that induced by the partial deletion of exon 4 in HFE-deficient mice.20,21 Thus, these mutated alleles may behave as a null allele. It is expected that the combination of these mutations in the compound heterozygous state with C282Y leads to severe phenotype expression. Three of the 5 probands (2, 4, and 5) had severe disease characterized by the presence of high amounts of iron overload and HH-related clinical complications. Proband 3 had a mild phenotype, probably because he had been a blood donor for the past 10 years;
proband 1 and his affected brother had a relatively mild phenotype and no other obvious protective factors such as blood loss or malabsorption. Thus, phenotype variability exists in these patients and could be related to both environmental and genetic factors. The existence of modifying genes located around the D6S105 region that influence phenotypic expression of C282Y homozygous HH patients has been proposed recently,18,22 but further studies are needed to clarify genotype-phenotype correlations in HH patients. Heterozygotes for HFE-Ossola and HFE-Brianza mutations did not have increased serum iron indices, indicating that neither mutation was able to produce iron overload in the heterozygous state. Also, the 3 compound heterozygotes for either HFE-Ossola or HFE-Brianza and H63D did not show evidence of iron overload. Because they were female and/or young, it is possible that, as for C282Y, the combination of the 2 nonsense mutations with H63D or other mild HFE mutations produces a HH phenotype with low penetrance.8 This is the first reported finding of nonsense mutations of the HFE gene. This is also the first description of several unrelated probands affected by HH who are com-
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pound heterozygotes for C282Y and a mutation other than H63D. The 2 mutations we report probably originated in the 2 northern Italian regions and increased in frequency as a result of a local founder effect. Because no consanguinity is present up to the fourth generations between the affected families within the 2 geographical areas, the 2 mutations likely arose more than 100 years ago. Analysis of the frequency of HFE genotypes in HH probands originating from the 2 Italian regions of Ossola and Brianza shows that compound heterozygotes for C282Y and HFE-Ossola or HFE-Brianza are found more frequently than or at least as frequently as C282Y/H63D compound heterozygotes (data not shown). In fact, C282Y/HFE-Ossola compound heterozygotes account for 25% of HH probands in the Ossola region (compared
Figure 3. Family pedigrees of the 2 HH proband compound heterozygotes for C282Y and HFE-Brianza. The ages and iron indices of the probands are shown in Table 1. Figure includes age and serum iron indices of the relatives and chromosome 6p haplotypes of all individuals. Chromosome 6p haplotypes of the HH patients are boxed, and the haplotype carrying the novel mutation is shown in bold. Arrows indicate probands; black squares indicate patients with hemochromatosis.
with 8.3% of C282Y/H63D), and C282Y/HFE-Brianza and C282Y/H63D compound heterozygotes each account for 8.4% of HH probands in Brianza region. The low frequency of C282Y/H63D compound heterozygotes is attributable to the very low penetrance of this genotype (from 0.44% to 1.5% in different populations),8 but it is expected that C282Y/HFE-Ossola or HFE-Brianza compound heterozygotes have a 100% penetrance because of the severity of both mutations. These results confirm that HH in Italy is not as homogeneous as in northern Europe and underline the possibility of other mutations of HFE that may have different relevance in different countries. The recognition of these mutations may have practical implications in diagnostic and screening strategies for HH.
References Figure 2. Family pedigrees of the 3 HH proband compound heterozygotes for C282Y and HFE-Ossola. The ages and iron indices of the probands are shown in Table 1. Figure shows age and serum iron indices of the relatives and chromosome 6p haplotypes of all individuals studied. Chromosome 6p haplotypes of the HH patients are boxed, and the haplotype carrying the novel mutation is shown in bold. Arrows indicates probands; black squares indicate patients with hemochromatosis.
1. Bacon BR, Powell LW, Adams PC, Kresina TF, Hoofnagle JH. Molecular medicine and hemochromatosis: at the crossroads. Gastroenterology 1999;116:193–207. 2. Camaschella C, Piperno A. Hereditary hemochromatosis: recent advances in molecular genetics and clinical management. Haematologica 1997;82:77– 84. 3. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo R, Ellis MC Fullan A, Hinton LM, Jones
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NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, MoriKang E, Prass CE, Quintana L, Starnes SM, Schatzman RC, Brunke KJ, Drayana DT, Risch NJ, Bacon BR, Wolff RK. A novel MHC class I–like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399 – 408. Jouanolle AM, Gandon G, Jezeque P, Blayau M, Campion ML, Yaouanq Y, Mosser J, Fergelot P, Chauvel B, Bouric P, Carn G, Andrieux N, Gicquel I, Le Gall JY, David V. Haemochromatosis and HLA-H. Nat Genet 1996;14:251–252. Beutler E, Gelbert T, West C, Lee P, Adams M, Blackston R, Pockros P, Kosty M, Venditti CP, Phatak PD, Seese NK, Chorney KA, Ten Elshof AE, Gerhard GS, Chorney M. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 1996;22:187– 194. Pietrangelo A, Camaschella C. Molecular genetics and control of iron metabolism in hemochromatosis. Haematologica 1998;83: 456 – 461. Beutler E. Genetic iron beyond haemocromatosis: clinical effects of HLA-H mutations. Lancet 1997;349:296 –297. Beutler E. The significance of 187G (H63D) mutation in hemochromatosis. Am J Hum Genet 1997;61:762–764. Carella M, D’Ambrosio L, Totaro A, Grifa A, Valentino MA, Piperno A, Girelli D, Roetto A, Franco B, Gasparini P, Camaschella C. Mutation analysis of HLA-H gene in Italian hemochromatosis patients. Am J Hum Genet 1997;60:828 – 832. Piperno A, Sampietro M, Pietrangelo A, Arosio C, Lupica L, Montosi G, Vergani A, Fraquelli M, Girelli D, Pasquero P, Roetto A, Gasparini P, Fargion S, Conte D, Camaschella C. Heterogeneity of hemochromatosis in Italy. Gastroenterology 1998;114:996 –1002. Camaschella C, Fargion S, Sampietro M, Roetto A, Bosio S, Garozzo G, Arosio C, Piperno A. Inherited HFE-unrelated hemochromatosis in Italian families. Hepatology 1999;29:1563–1564. Pietrangelo A, Montosi G, Totaro A, Garuti C, Conte D, Cassanelli S, Fraquelli M, Sardini C, Vasta F, Gasparini P. Hereditary hemochromatosis in adults without pathogenic mutations in the hemochromatosis gene. N Engl J Med 1999;341:725–732. Wallace DF, Dooley JS, Walker AP. A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote. Gastroenterology 1999;116:1409 –1412. Barton JC, Sawada-Hirai R, Rothenberg BE, Acton RT. Two novel missense mutations of the HFE gene (I105T and G93R) and identification of the S65C mutation in Alabama hemochromatosis probands. Blood Cells Mol Dis 1999;25:146 –154.
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15. Mura C, Raguenes O, Ferec C. HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis. Blood 1999;93:2502–2505. 16. Villiers JN, Hillermann R, Loubser L, Kotze MJ. Spectrum of mutations in the HFE gene implicated in haemochromatosis and porphyria. Hum Mol Genet 1999;8:1517–1522. 17. Scheuer PJ, Williams R, Muir AR. Hepatic pathology in relatives of patients with hemochromatosis. J Pathol Bacteriol 1962;84:53– 64. 18. Piperno A, Arosio C, Fargion S, Roetto A, Nicoli C, Girelli D, Sbaiz L, Gasparini P, Boari G, Sampietro M, Camaschella C. The ancestral hemochromatosis haplotype is associated with a severe phenotype expression in italian patients. Hepatology 1996;24: 43– 46. 19. Camaschella C, Roetto A, Ciciliano M, Pasquero P, Bosio S, Gubetta L, Di Vito F, Girelli D, Totaro A, Carella M, Grifa A, Gasparini P. Juvenile and adult hemochromatosis are distinct genetic disorders. Eur J Hum Genet 1997;5:371–375. 20. Zhou XY, Tomatsu S, Fleming RE, Parkkila S, Waheed A, Jiang J, Fei Y, Brunt EM, Ruddy DA, Prass CE, Schatzman RC, O’Neill R, Britton RS, Bacon BR, Sly WS. HFE gene knockout produces mouse model of hereditary hemochromatosis. Proc Natl Acad Sci U S A 1998;95:2492–2497. 21. Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC. The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999;94:9 –11. 22. Pratiwi R, Fletcher LM, Pyper WR, Do KA, Crawford D, Powell LW, Jazwinska EC. Linkage disequilibrium analysis in Australian haemochromatosis patients indicates bipartite association with clinical expression. J Hepatol 1999;31:39 – 46.
Received November 4, 1999. Accepted March 15, 2000. Address requests for reprints to: Alberto Piperno, M.D., Clinica Medica, Ospedale S. Gerardo, via Donizetti 106, 20052, Monza, Italy. e-mail: [email protected]; fax: (39) 039-322274. Supported by a grant from the Association for the Study of Hemochromatosis and Iron Overload Disorders, Monza, Italy (to L.S.). The authors thank the probands and families and Dr. P. Cerutti, on behalf of the blood bank of Domodossola, for their cooperation, Dr. A. Roetto for kindly providing the D6S1281 microsatellite primers, and Dr. G. Cazzaniga for technical support.
Two Novel Nonsense Mutations of HFE Gene in Five Unrelated Italian Patients With Hemochromatosis ` ,* PAOLA TROMBINI,* ALBERTO PIPERNO,* CRISTINA AROSIO,‡ LAURA FOSSATI,* MAURO VIGANO ANNA VERGANI,* and GIUSEPPE MANCIA* *Clinica Medica, Universita` degli Studi di Milano-Bicocca, Ospedale S. Gerardo, Monza; and ‡Istituto Auxologico Italiano, Ospedale S. Luca, Milan, Italy
Background & Aims: Most hemochromatosis patients of Northern European descent are homozygous for the C282Y mutation of HFE gene. In Italy, many patients with iron overload are not homozygous for C282Y, and the presence of other mutations or other genetic determinant has been suggested. Methods: Five unrelated Italian patients heterozygous for C282Y with the classic hemochromatosis phenotype were studied. The entire coding sequence and the exon-intron boundaries of the HFE gene were analyzed. Chromosome 6p haplotypes were defined in each patient by analysis of D6S265, D6S105, and D6S1281 microsatellites. Results: Two novel nonsense HFE mutations were identified in exon 3 in the C282Y negative chromosome. The first one, a G-to-T transition at codon 168, was detected in 3 probands; the second, a G-to-A transition at codon 169, was detected in the others. Conclusions: The 2 nonsense mutations in the compound heterozygous state with C282Y result in the classic hemochromatosis phenotype in several unrelated Italian patients. This confirms that hemochromatosis in Italy is not as homogeneous as in northern Europe and suggests that other mutations can exist in C282Y or H63D heterozygotes with iron overload. These findings have practical implications for diagnostic and screening strategies for hemochromatosis.
ereditary hemochromatosis (HH)1 is a common autosomal recessive disease characterized by increased iron absorption and progressive iron overload.2 In 1996, the HFE gene was identified and 2 mutations were originally described, C282Y and H63D.3 Most HH patients are homozygous for C282Y.3– 6 Among HH probands, compound heterozygotes C282Y/H63D account for approximately 5%–7%,3,7 H63D homozygotes are very rare, and both generally have a mild form of hemochromatosis.8 A minority of cases presenting with HH phenotype are C282Y or H63D heterozygotes or neither C282Y nor H63D. This appears to be more common in southern than in northern Europe.9,10 In a collaborative study of 188 Italian patients with HH-like phenotype, 28% were incompletely characterized with at least one
H
chromosome without an assigned mutation.10 In some of these patients, the presence of other HFE mutations or other 6p-linked genetic determinants or the existence of other forms of genetic iron overload unlinked to chromosome 6 has been suggested.10 –12 Three novel HFE mutations were discovered recently in the heterozygous state in association with C282Y in single cases with classical HH: a splice site mutation (IVS3 ⫹ 1G 3 T) causing obligate skipping of exon 313 and 2 missense mutations (I105T and G93R) located in the ␣1 domain that may affect the binding of HFE protein to the transferrin receptor.14 Moreover, the S65C missense mutation was found to be significantly enriched in HH chromosomes that were neither C282Y or H63D, suggesting that the S65C could be another variant contributing to a mild form of HH in association with C282Y or H63D.15 Other missense mutations have been described recently, but their role in the pathogenesis of HH is still unclear.16 Based on these findings, we reanalyzed Italian C282Y heterozygous probands with the classic HH phenotype to test the hypothesis that other HFE mutations in nonC282Y chromosomes could explain their fully expressed clinical phenotype. We report the analysis of 5 of these patients.
Patients and Methods Patients We studied 5 Italian probands with HH who were heterozygous for C282Y and negative for H63D. They all met the classic phenotypic criteria for homozygous HH: (1) increased transferrin saturation (repeatedly ⬎50% fasting) and increased serum ferritin levels; (2) hepatocellular hemosiderin Abbreviations used in this paper: HH, hereditary hemochromatosis; HIC, hepatic iron concentration; HII, hepatic iron index; IR, iron removed; PCR, polymerase chain reaction; RFLP, restriction length polymorphism analysis. © 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.9369
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deposits of grade III or IV according to Scheuer et al.17; and (3) hepatic iron index ⱖ2. Patients with iron-loading anemias, chronic viral hepatitis B or C, or a history of heavy alcohol intake or blood transfusions were excluded. All patients and relatives gave their informed consent to the study.
destroy a restriction endonuclease site, the portion of exon 3 containing the mutations was amplified by PCR using mismatched sense oligonucleotide specific for each mutation (Bria-F: 5⬘ TGGCCCACCAAGCTGGACT 3⬘; Dom-F: 5⬘ GCCTGGCCCACCAAACTG 3⬘) to create an informative restriction site for BsrSI in the wild-type HFE sequence. The antisense oligonucleotide was the same as the one used for sequencing.
Clinical Studies The presence of fibrosis and cirrhosis was determined at liver biopsy. All cases of cirrhosis were classified as Child’s class A. The presence of clinical complications related to HH was determined according to previously described criteria.18 Transferrin saturation and serum ferritin levels were measured by standard methods. Hepatic iron concentration (HIC) was determined by atomic absorption spectrophotometry (PerkinElmer S2380, Norwalk, CT), and the hepatic iron index (HII) (ratio of HIC [mol/g dry wt] and age [years]) was calculated. The total amount of iron removed by phlebotomy (IR) was estimated as previously reported.18 The geographic origin of each patient was determined to the fourth generation.
Results Table 1 reports iron and clinical data of the 5 probands. Three patients (1, 2, and 3) were from the same subalpine valley in the northwest (Ossola), and 2 (4 and 5) were from an area around the city of Monza (Brianza). Sequence analyses of the HFE gene in probands showed 2 novel nonsense mutations in exon 3 in the heterozygous state. The first, a G-to-T transition at codon 168 (GAG3 TAG, Glu3 Stop), was found in probands from the Ossola valley. The second, a G-to-A transition at codon 169 (TGG3 TAG, Trp3 Stop), was detected in probands from the Brianza region. The new variants were named HFE-Ossola and HFE-Brianza based on the origins of the patients. Figure 1 shows the direct sequence analyses of a portion of exon 3 containing the 2 mutations and the RFLP patterns of 2 probands with the HFE-Ossola and HFE-Brianza mutations and of controls. Figures 2 and 3 show the family pedigrees of the probands from the Ossola and Brianza regions, respectively, including iron data, chromosome 6p haplotypes, and HFE alleles. In family 1 we found an affected brother who shared the same chromosome 6p haplotypes and the same HFE mutations with the proband. His HIC was 176.2 mol/g and HII was 3.92. The 2 new variants were in complete linkage disequilibrium with the C282Y and H63D mutations. HFEOssola was associated with haplotype D6S265-3, HLAA24, D6S105-5, and D6S1281-6 in families 1 and 2, whereas in family 3 the haplotype changed at D6S265 and HLA-A loci, suggesting that a recombination event occurred between HLA-A and D6S105 (see Figure 2).
Molecular Studies Genomic DNA was extracted from peripheral blood leukocytes. C282Y and H63D mutations were detected using standard polymerase chain reaction (PCR) and restriction enzyme digestion with Rsa I and Bcl I, respectively.9 Microsatellites D6S265, D6S105, and D6S1281 were analyzed as previously described.19 HLA-A antigens were defined by the microlymphotoxicity test. Haplotypes were constructed manually on the basis of intrafamilial segregation of the marker alleles. The entire coding sequence and exon-intron boundaries of the HFE gene were amplified by PCR according to Carella et al.9 The nucleotide sequence of both DNA strands was independently determined in all probands and relatives with the C282Y-negative chromosome identical to the proband by using the dideoxy chain-termination reaction with Sequenase version 2.0 (USB Amersham, Cleveland, OH) and [␣-35S]deoxyadenosine triphosphate, according to the protocol recommended by the manufacturer. Reactions were separated on a 6% denaturing polyacrylamide gel and visualized by autoradiography. The primers used for sequencing were the same as those used for PCR. To further confirm the presence of the novel mutations (see Results), a restriction length polymorphism analysis (RFLP) was performed. Because the 2 mutations did not create or
Table 1. Data of the 5 Probands With Hemochromatosis
1 2 3 4 5
Sex
Age (yr)
TS (%)
SF (g/L)
Scheuer’s grade
HIC (mol/g)
HII
IR (g)
M M M M M
48 47 42 50 66
96 79 86 106 100
694 1206 608 2740 1351
4 4 3 4 4
238 359 144 425 421
4.9 7.6 3.4 8.5 5.4
4 23 3.6 21 —
Clinical complications None C None C, H, D C, H, A, D
TS, transferrin saturation; SF, serum ferritin; IR, iron removed; C, hepatic cirrhosis; H, hypogonadism; A, arthropathy; D, diabetes.
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Figure 1. (A) Direct sequencing of sense strand of PCR products of exon 3 in patients with HFE-Ossola and HFEBrianza mutations compared with wild type. The sequence is for codons of amino acids numbered 167–170. Arrow indicates the single base substitution in the heterozygous state in probands. (B) Restriction endonuclease analysis of the PCR products obtained with specific mismatched primers creating a BsrSI site in the wild-type sequence. In the heterozygous affected patients, both the digested (wild-type) and undigested (mutated) bands can be seen. Lane M, marker; lane 1, wild-type; lane 2, patient.
HFE-Brianza was associated with haplotype D6S265-3, HLA-A24, D6S105-6, and D6S1281-6.
Discussion We describe 5 Italian patients with classic HH who are compound heterozygotes for C282Y and 2 novel nonsense HFE mutations. The 2 mutations determine a stop codon at nucleotide 502 and 506 of the open reading frame (nucleotides 502 GAG3 TAG and 506 TGG3 TAG), respectively, leading to proteins that lack the ␣3 domain, transmembrane domain, and cytoplasmic tail. These mutations probably produce, differently from C282Y, a complete disruption of the function of HFE similar to that induced by the partial deletion of exon 4 in HFE-deficient mice.20,21 Thus, these mutated alleles may behave as a null allele. It is expected that the combination of these mutations in the compound heterozygous state with C282Y leads to severe phenotype expression. Three of the 5 probands (2, 4, and 5) had severe disease characterized by the presence of high amounts of iron overload and HH-related clinical complications. Proband 3 had a mild phenotype, probably because he had been a blood donor for the past 10 years;
proband 1 and his affected brother had a relatively mild phenotype and no other obvious protective factors such as blood loss or malabsorption. Thus, phenotype variability exists in these patients and could be related to both environmental and genetic factors. The existence of modifying genes located around the D6S105 region that influence phenotypic expression of C282Y homozygous HH patients has been proposed recently,18,22 but further studies are needed to clarify genotype-phenotype correlations in HH patients. Heterozygotes for HFE-Ossola and HFE-Brianza mutations did not have increased serum iron indices, indicating that neither mutation was able to produce iron overload in the heterozygous state. Also, the 3 compound heterozygotes for either HFE-Ossola or HFE-Brianza and H63D did not show evidence of iron overload. Because they were female and/or young, it is possible that, as for C282Y, the combination of the 2 nonsense mutations with H63D or other mild HFE mutations produces a HH phenotype with low penetrance.8 This is the first reported finding of nonsense mutations of the HFE gene. This is also the first description of several unrelated probands affected by HH who are com-
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pound heterozygotes for C282Y and a mutation other than H63D. The 2 mutations we report probably originated in the 2 northern Italian regions and increased in frequency as a result of a local founder effect. Because no consanguinity is present up to the fourth generations between the affected families within the 2 geographical areas, the 2 mutations likely arose more than 100 years ago. Analysis of the frequency of HFE genotypes in HH probands originating from the 2 Italian regions of Ossola and Brianza shows that compound heterozygotes for C282Y and HFE-Ossola or HFE-Brianza are found more frequently than or at least as frequently as C282Y/H63D compound heterozygotes (data not shown). In fact, C282Y/HFE-Ossola compound heterozygotes account for 25% of HH probands in the Ossola region (compared
Figure 3. Family pedigrees of the 2 HH proband compound heterozygotes for C282Y and HFE-Brianza. The ages and iron indices of the probands are shown in Table 1. Figure includes age and serum iron indices of the relatives and chromosome 6p haplotypes of all individuals. Chromosome 6p haplotypes of the HH patients are boxed, and the haplotype carrying the novel mutation is shown in bold. Arrows indicate probands; black squares indicate patients with hemochromatosis.
with 8.3% of C282Y/H63D), and C282Y/HFE-Brianza and C282Y/H63D compound heterozygotes each account for 8.4% of HH probands in Brianza region. The low frequency of C282Y/H63D compound heterozygotes is attributable to the very low penetrance of this genotype (from 0.44% to 1.5% in different populations),8 but it is expected that C282Y/HFE-Ossola or HFE-Brianza compound heterozygotes have a 100% penetrance because of the severity of both mutations. These results confirm that HH in Italy is not as homogeneous as in northern Europe and underline the possibility of other mutations of HFE that may have different relevance in different countries. The recognition of these mutations may have practical implications in diagnostic and screening strategies for HH.
References Figure 2. Family pedigrees of the 3 HH proband compound heterozygotes for C282Y and HFE-Ossola. The ages and iron indices of the probands are shown in Table 1. Figure shows age and serum iron indices of the relatives and chromosome 6p haplotypes of all individuals studied. Chromosome 6p haplotypes of the HH patients are boxed, and the haplotype carrying the novel mutation is shown in bold. Arrows indicates probands; black squares indicate patients with hemochromatosis.
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Received November 4, 1999. Accepted March 15, 2000. Address requests for reprints to: Alberto Piperno, M.D., Clinica Medica, Ospedale S. Gerardo, via Donizetti 106, 20052, Monza, Italy. e-mail: [email protected]; fax: (39) 039-322274. Supported by a grant from the Association for the Study of Hemochromatosis and Iron Overload Disorders, Monza, Italy (to L.S.). The authors thank the probands and families and Dr. P. Cerutti, on behalf of the blood bank of Domodossola, for their cooperation, Dr. A. Roetto for kindly providing the D6S1281 microsatellite primers, and Dr. G. Cazzaniga for technical support.