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Genetic hemochromatosis in Italian patients with prophyria cutanea tarda: possible explanation for iron overload
Journal of Hepatology1996;24:564569 Printed in Denmark Allrights reserved Munksgaard
Copenhagen
Copyrrght Q European Association for the Study of the Liver 1996
.,OUrUal Of&QPtOh,gy ISSN0168.8278
Genetic hemochmmatosis in Itabn patients with porphyria cutanea tarda: possible explanation for iron overload Silvia Fargion, Anna Ludovica Fracanzani, Riccardo Romano, Maria Domenica Cappellini, Maristella Fare’, Michela Mattioli, Albert0 Pipemo2, Guido Ronchi3 and Gemino Fiorelli Institute of Internal Medicine and Medical Physiopathology, ‘Transfusional Cents, Institute G. Pini, Milan, ‘Institute of Biomedical Science, Monza, and ‘Institute of Internal Medicine, IRCCS Ospedale Maggiore, University of Milan, Milan, Italy
Background/Aims: Mild to moderate iron overload is found in most patients with porphyria cutanea tar-da. This study ahned to evaluate whether iron overload in patients with porphyria cutanea tarda is related to the presence of a coexistent genetic hemochromatosis gene. Methods: A cohort study of 94 Italian patients with porphyria cutanea tarda (90 men and 4 women) and 20 relatives of five patients with iron overload were studied. Diagnosis of iron overload was assessed by transferrin saturation, serum ferritin and iron removed by phlebotomy to reach depletion. HLA typing by microlymphocytotoxicity test and duodenal ferritin analysis by immunohistochemistry were performed in a smaller number of patients. The chi square test was used to compare means and prevalences.
Results: Iron overload was present in 62% of the patients. HLA-A3 prevalence was significantly higher (~~0.01)in subjects with iron overload than in those without. A lack of duodenal ferritin was observed in 14/18 patients with and in 6/12 without iron overload. Family studies showed the presence of iron overload but not of porphyria cutanea tarda in HLA identical or semi-identical relatives of the patients. Conclusions: Italian patients with porphyria cutanea tarda and iron overload appear to have one or even two genes for genetic hemochromatosis.
M
appearance of PCT cutaneous lesions and an improvement in hepatic biochemical tests (1 l), even in patients without evident iron overload, probably due to the presence of intra-hepatocytic toxic species of iron. Alcohol abuse, frequently present in these patients, could account for at least part of it. In addition, several authors have hypothesized that genetic hemochromatosis (GH) may coexist in patients with PCT and iron overload, but studies on the prevalence of HLA-A3, an antigen considered an independent marker of the GH allele, have provided conflicting results (12-16). GH is far more frequent than previously thought in Caucasian populations, with prevalences of homozygotes ranging from 2 to 5/1000 and of heterozygotes from 8 to 15/100, respectively (17-21). Thus, patients with PCT could also be carriers of one or more GH genes, and this could explain the mild to moderate iron overload found in many cases. The aim of this study was to evaluate the preva-
iron overload is a common finding in patients with porphyria cutanea tarda (PCT) and, according to some authors, it may trigger clinical manifestations of the disease (1,2). PCT is caused by reduced activity of uroporphyrinogen decarboxylase (URO-D), which in the sporadic form of the disease is confined to the liver, whereas in the familial form it extends to other tissues (3-S). It is unclear whether sporadic PCT is an acquired or hereditary recessive disease (6,7). In subjects with URO-D defect PCT is usually clinically silent until external factors precipitate symptoms. Such factors have been reported to include alcohol, estrogens, virus and iron overload (1, 2, 7-10). In fact, iron depletion therapy by weekly phlebotomy is usually followed by the disILD TO MODERATE
Received 12 May; revised 19 September; accepted 4 October 1995
Correspondence: Silvia Fargion, Institute of Internal Medicine and Medical Physiopathology, Ospedale Maggiore, Pad LI?TA, Via F. Sforza 35,20122 Milan, Italy.
564
Key words: Duodenal ferritin; Genetic hemochromatosis; HLA-A3; Iron overload; Porphyria cutanea tarda.
Genetic hemochromatosis TABLE 1 Sex, age and iron indexes in 94 patients with porphyria cutanea tarda Mean&SD (range) 9014 61f7.8 (40-78) 50.5+17.2 (32-100) 501f303.8 (42-1450) 2.5k1.58 (0.3-7.3)
Male/female Age (years) Transfertin saturation (%) Serum ferritin (pgll) Iron removed (g)
lence and severity of iron overload in a group of Italian patients with PCT and the possible coexistence of GH in those with increased iron stores. We determined the amount of iron removed by phlebotomy up to depletion and the prevalence of HLA-A3 in patients grouped according to body iron stores. The possible absence of ferritin in duodenal enterocytes, previously described in patients with GH (22), was investigated in a subgroup of our series. Family studies which aimed to verify, in siblings sharing one or two haplotypes with the proband, transmission of iron overload and PCT were also performed.
in porphyria cutanea tarda
patients. No patient had been exposed to other known hepatotoxins, with the exception of one woman who had taken estrogens 2 years before the onset of PCT. Hepatitis virus screening indicated that HBsAg was present in six (6%) and anti-HCV in 73 (78%) patients, including four of the five with familial PCT. Liver biopsy was performed in 60 patients. Histological findings were consistent with chronic liver disease in all patients, as previously reported in a smaller group of patients (10). Grade 2 or 3 siderosis was found in 44 of the 60 (23). Liver iron concentration was performed according to Barry’s method (24) in 28 patients. Patients with clinically manifest PCT underwent phlebotomy with removal of 300 ml of blood (equivalent to 180 mg of iron) every week, even in the absence of evident iron overload. Iron depletion was defined as transferrin saturation of < 20% and serum ferritin ~30 l.tg/l in presence of mild anemia (Hb ~11 g/d1 in women and < 12 g/d1 in men). The iron status of the patients was defined as normal when transferrin saturation was ~45% and/or iron removed (IR) < 2 g, and as increased when transferrin saturation was 245% and/or IR 22 g. Patients in the latter group were considered to have severe iron overload when transfenin saturation was 262% and/or IR 24 g.
Material and Methods Patients Between January 1986 and June 1994, 94 patients with PCT were referred to our institution. PCT was diagnosed on the basis of typical clinical features and urinary porphyrin excretion (urinary porphyrins range 900-8000 pg/24 h, mean 3700 &1800). Sex, age and iron indexes of the patients are shown in Table 1. Five patients had a positive family history of PCT, Alcohol abuse defined as alcohol intake >80 g/ day for more than 5 years was present in 40% of
TABLE 2 Iron status in 94 patients with porphyria cutanea tarda Patients
Normal iron stores] Increased iron stones’ Moderate3 Severe4 i Transferrin * Transfetrin 3 Transferrin 4 Transferrin
saturation saturation saturation saturation
n
(%)
36 58 41 17
(38) (62) (44) (18)
< 45% and/or iron removed < 2 g 245% and/or iron removed 2 2 g 45-62% and /or iron removed 2-4 g 2 62% and/or iron removed 2 4 g
Methods Urinary porphyrins were fractionated, after conversion to their methyl esters, by high-performance liquid chromatography (25). Serum iron and total iron binding capacity were determined calorimetrically according to the International Committee for Standardization in Hematology (ICSH) (26,27). Serum ferritin was determined by radioimmunoassay (LisoPhase; Lepetit, Milan, Italy). HBsAg was determined by radioimmunoassay (RIA) (Abbott Laboratories, North Chicago, IL, USA) and anti-HCV detected on second generation ELISA II (Grtho Diagnostic System, Raritan, NJ, USA). HLA typing was performed by microlymphocytotoxicity tests in 56 patients with PCT. Control groups for HLA analysis included 80 patients with GH diagnosed according to standard criteria, and 700 normal subjects used as historical controls of our population. Duodenal biopsy analysis Thirty patients underwent duodenal biopsy and the results compared with those in 30 patients with homozygous GH, (some of whom have already been described (22)), four with heterozygous GH (sharing 565
S. Fargion et al. TABLE 3 HLA antigens frequency in patients with porphyria cutanea tarda compared with normal subjects and patients with genetic hemochromatosis Subjects
Normals
HLA N
A3
4,
Cases
n
700
150 22
%
B 35
B,
n
%
80 11
n
%
n
8011
%
2029
56 PCT all W/o iron overload 20 With iron overload 36
17 30 2*10 15*42
11 20 3 15 8 19
9 16 2 10 7 16
16 28 6 35 10 27
GH
55 69
8 10
20 26
23 29
80
*p
only one haplotype with a homozygous GH sibling), seven with secondary hemochromatosis (transfusion dependent anemias) and ten subjects who underwent endoscopy for dyspepsia. Duodenal biopsy specimens were processed as previously described (22). Briefly, paraffin-embedded tissue specimens were sliced and sections deparaffinized in xylene and then in absolute ethanol. Murine monoclonal antibody for L-chain ferritin was employed in the avidin-biotin immunoperoxidase technique. Endogenous peroxidase was blocked by incubation in a 0.3% H,O, solution. Family studies of five patients with PCT and iron overload were performed. A total of 20 relatives (16 siblings and four offsprings) were analyzed by HLA typing and iron parameters, and evaluated for silent or manifest PCT.
TABLE 4 Ferritin in duodenal enterocytes of patients with porphyria cutanea tarda (PCT), homozygous or heterozygous genetic hemochromatosis (GH), or secondary iron overload and of normal subjects Positive staining*
PCT Without iron overload With iron overload GH Homozygous GH Heterozygous Secondary iron overload Normals *By immunohistochemistry tin.
566
n
n
30 12 18
10 6 4
33 50 22
30
4
13
4 7 10
0 7 10
0 100 100
Statistical analysis Results are expressed as mean+S.D. The chi square test was used for comparisons of prevalences and means. The relationship between different variables was analyzed with correlation and regression analysis.
(%)
using monoclonal antibody against ferri-
Results Fifty-eight patients with PCT (62%) had iron overload, which was severe in 17 (18%) (Table 2). Liver iron concentration, performed in 28 patients including six with severe iron overload, ranged between 680-8870 pg/g dry tissue. Liver iron concentration/ age was > 2 in four of the latter patients. A significant positive correlation was found between uroporphyrin levels at the time of PCT diagnosis and IR @
Genetic hemochromatosis in porphyria cutanea tarda
Discussion The presence and severity of iron overload, the HLAA3 frequency, the absence of ferritin in duodenal enterocytes and the finding that HLA semi-identical siblings have iron overload but not PCT, suggest that Italian patients with PCT and iron overload carry one gene for GH or even two in a few cases. Analysis of the iron status of our patients with PCT showed that, according to the criteria used, 62% had increased iron stores, confirmed by liver siderosis grade 2 or 3 in the patients who underwent liver biopsy and by the high liver iron concentrations in most of the patients in whom the test was available. This figure fits with the incidence of iron overload in patients with PCT reported by other authors (1,2). The role of iron in the pathogenesis of PCT clinical manifestation is also supported by the highly significant correlation between uroporphyrin level at the time of PCT diagnosis and iron removed to reach depletion, one of the best indexes of iron overload. The possible coexistence of GH in PCT subjects is controversial (12-16,28,29). Beaumont et al. found no difference in HLA-A3 incidence between patients with PCT and controls (14). However, they did not have the possibility of separating patients into those with biochemical evidence of iron overload and those with normal iron stores, since most of their patients were in clinical remission at the time of HLA typing and previous biochemical data were not available. In fact, the HLA-A3 incidence in our PCT patients analyzed independently of their iron status did not differ from that found in controls. Kushner et al. (12), on the basis of HLA and URO-D analysis in a pedigree, proposed a two-locus causation model in the pathogenesis of sporadic PCT. They suggested that a single allele for GH might be responsible for hepatic siderosis in sporadic PCT and this siderosis, in turn, could exert an inhibitory effect on URO-D activity, thus making PCT clinically manifest. If, as they hypothesized, URO-D defect in sporadic PCT is transmitted as a recessive trait, it follows that two independent recessively transmitted factors are required for clinical manifestation of the sporadic form. This could explain the low incidence of the disease, and the common finding that in most families with PCT only a single individual is clinically detectable. In Elder’s editorial on Kushner et al.‘s hypothesis he concluded that the increased incidence of HLA-A3 in patients with PCT was not a demonstration of the two-locus model but an obvious consequence of the fact that GH heterozygosity is one of the most common causes of iron overload in the general population and thus a higher HLA-A3 frequency is to be expected in all
iron-precipitated disorders (13). Edwards et al. (28) extended Kushner’s studies and performed pedigree analyses based on HLA typing and iron indexes which provided evidence that GH coexisted in most of the patients studied and that six out of the 17 might even be homozygotes for GH. In addition, they found a high frequency of HLA-A3 in their PCT population, similar to that reported in homozygous GH (15,28). The coexistence of PCT and GH was described in another family studied by Seymour et al. (29). The present study carried out in a large Italian series supports the hypothesis that most of the patients with PCT carry one or even two genes for GH. Indeed, on the basis of transferrin saturation (higher than 62%) and the amount of iron removed to reach depletion (over 4 g), it is possible that a number of our patients are homozygotes for GH (30,3 1). HLA-A3 frequency, which was similar to that in normals when iron stores were not taken into account, was significantly higher in patients with iron overload than in those without or in normals. Given the expected HLA-A3 frequency in an Italian population of GH heterozygotes (32), as calculated by Simon et al. on the basis of HLA-A3 frequency in homozygous GH (33), our findings fit with the hypothesis that our PCT patients with increased iron stores may be heterozygous for GH. However, in the absence of a genetic marker for GH, the increased frequency of A3 in patients with PCT and iron overload only suggests but does not prove the coexistence of GH. Finally, the lack of duodenal ferritin in PCT patients with iron overload is in keeping with our previous data on GH homozygotes (22) and could account for the increased iron absorption reported in patients with PCT despite increased iron stores (34). In addition, since we found that also the few GH heterozygotes analyzed by us lacked fenitin in duodenal enterocytes (Table 4), our present results could provide further evidence that PCT patients are heterozygous for GH. The unexpected finding of some PCT patients without iron overload who did not have ferritin granules in duodenal cells might be explained by the presence of a few GH heterozygotes who, despite the PCT, did not develop iron overload. An altered expression of duodenal ferritin in patients with GH has also been reported by Pietrangelo et al. (35,36), who demonstrated a reduced expression of duodenal ferritin mRNA. Family studies supplied confirmatory evidence of the coexistence of GH in our patients with PCT, since siblings sharing one or two HLA haplotypes with the proband had iron overload but no evidence of PCT. 567
S. Fargion et al.
Alcohol abuse did not seem to cause marked modifications of the iron status of our PCT patients as shown by the lack of significant differences in the iron indexes between abusers and abstainers. However, we could not use ferritin values of PCT patients to discriminate between subjects with or without iron overload, since serum ferritin may overestimate the iron stores of these patients, probably due to liver damage (37) and/or to a possible induction of ferritin synthesis by alcohol. We previously reported that most Italian patients with PCT have an active HCV infection (10) although we were not able to establish whether such infection is a precipitating factor, which unmasks the underlying porphyrin metabolism disorder, or whether PCT patients are more susceptible to this virus. Whether there is relation between iron overload and HCV infection is still unclear, also in view of recent reports of iron overload and scanty response to interferon in patients with HCV positive chronic liver disease (38-40). However, the incidence of the virus is markedly lower in Italian patients with GH (24%) (41,42) than in those with PCT (78%) (lo), suggesting a complex interaction between iron, HCV and URO-D in the latter group. In patients with PCT, HCV could increase hepatocytic iron uptake by acting synergistically with a single GH gene, or, as demonstrated for other viruses (43), by interacting with transfer-i-inreceptors. Increased intra-hepatocytic iron could, in turn, trigger the metabolic defect underlying PCT. The relation between HCV and PCT remains however undefined.
7. 8.
9. 10.
11. 12.
13. 14.
15.
16.
17.
18.
19.
Acknowledgements The authors thank Maria Rita Balestrieri for her helpful technical assistance. This paper was partially supported by a grant from MURST 60% to Silvia Fargion.
20.
21.
References 1. Felsher BF, Kushner Jp. Hepatic siderosis and porphyria cutanea tarti relation of iron excess to the metabolic defect. Semin Hematol1977; 14: 243-5 1. 2. Lundvall 0, Weinfeld A, Lundin P. Iron storage in porphyria cutanea tarda. Acta Med Stand 1970; 188: 37-53. 3. Felsher BF, Norris ME, Shih JC. Red-cell uroporphyrinogen decarboxylase activity in porphyria cutanea tarda. N Engl J Med 1982; 306: 766-9. 4. De Vemeuil H, Aitken G, Nordmann Y. Familial and sporadic porphyria cutanea tarda: two different diseases. Hum Genet 1978; 44: 145-51. 5. Cohen PR, Deleo VA. Disorders of porphyrin metabolism. In: Alper JC, ed. Genetic Disorders of the Skin. Chicago: Year Book Medical Publishers, 1990: 79-104. 6. Elder GH, Urquhart AJ, De Salamanca RE, Munoz JJ, Bonk-
568
22.
23.
24. 25.
ovsky HL. Immunoreactive uroporphyrinogen decarboxylase in the liver in porphyria cutanea tarda. Lancet 1985; i: 229-32. Hindmarsh JT. Enzyme heterogeneity in the porphyria. Clin Biochem 1990; 23: 3714. Haberman HF, Rosemberg L, Menon LA. Porphyria cutanea tarda: comparison cases precipitated by alcohol and estrogens. Can Med Assoc J 1975; 113: 633-55. Nip-Sakamoto CJ, Wong RHW, Izumi AK. Porphyria cutanea tarda and AIDS. CUTIS 1989; 44: 47&l. Fargion S, Pipemo A, Cappellini MD, Sampietro M, Fracanzani AL, Roman0 R, Caldarelli R, Marcelli R, Vecchi L, Fiorelli G. Hepatitis C virus and porphyria cutanea tarda: evidence of a strong association. Hepatology 1992; 16: 1322-6. Lundvall 0. The effect of phlebotomy therapy in porphyria cutanea tarda. Acta Med Stand 197 1; 189: 33-49. Kushner JP, Edwards CQ, Dadone MM, Skolnick MH. Heterozygosity for HLA-linked hemochromatosis as a likely cause of hepatic siderosis associated with sporadic porphyria cutanea tarda. Gastroenterology 1985; 88: 1232-8. Elder GH. Porphyria cutanea tarda and HLA-linked hemochromatosis. Gastroenterology 1985; 88: 1276-9. Beaumont C, Fauchet R, Phung LN, Vemeuil HD, Gueguen M, Nordmann. Porphyria cutanea tarda and HLA-linked Hemochromatosis. Gastroenterology 1987; 92: 1833-8. Edwards CQ, Griffen LM, Kushner JP Increased frequency of HLA-A3 in subjects with sporadic porphyria cutanea tarda. Tissue Antigens 1988; 3 1: 250-l. Adams PC, Powell LW. Porphyria cutanea tarda and HLAlinked hemochromatosisall in the family? Gastroenterology 1987; 92: 2033-5. Edwards CQ, Griffen LM, Goldgar D, Drummond C, Skolnick MH, Kushner JP Prevalence of hemochromatosis among 11065 presumably healthy blood donors. N Engl J Med 1988; 318: 1355-62. Hallberg L, Bjom Rasmussen E, Jungmer I. Prevalence of hereditary hemochromatosis in two Swedish urban areas. J Intern Med 1989; 225: 249-55. Leggett BA, Halliday JW, Brown NN, Bryant S, Powell LW. Prevalence of hemochromatosis amongst asymptomatic Australians. Br J Haematol 1990; 74: 525-30. Velati C, Pipemo A, Fargion S, Colombo S, Fiorelli G. Prevalence of idiopathic hemochromatosis in Italy: study of 1301 blood donors. Haematologica 1990; 75; 309-12. Powell LW, Jazwinska E, Halliday JW. Primary iron overload. In: Brock JH, HaIliday JW, Pippard MJ, Powell LW eds. Iron Metabolism in Health and Disease. London, Philadelphia, Toronto, Sydney, Tokyo: Saunders Company Ltd, 1994; 227-70. Fracanzani AL, Fargion S, Roman0 R, Pipemo A, Arosio P, Ruggeri G, Ronchi G, Fiorelli G. Immunohistochemical evidence for a lack of ferritin in duodenal absorptive epithelial cells in idiopathic hemochromatosis. Gastroenterology 1989; 96: 1071-8. Scheuer PG, Williams R, Muiz AR Hepatic pathology in relatives of patients with hemochromatosis. J Path01 Bacterial 1962; 84: 53-64. Barry M, Sherlock S. Measurement of liver iron concentration in needle biopsy specimens. Lancet 197 1; i: 100-3. Elder GH, Lee GB, Tovey JA. Decrease activity of hepatic uroporphyrinogen decarboxylase in sporadic porphyria cutanea tarda. N Engl J Med 1978; 299: 274-8.
Genetic hemochromatosis 26. Iron Panel of the International Committee for Standardization in Haematology. The measurement of total and unsaturated iron- binding capacity in serum. Br J Haematol 1978; 38: 281-90. 27. Iron Panel of the International Committee for Standardization in Haematology. Recommendations for measurement of serum iron in human blood. Br J Haematol1978; 38: 291-4. 28. Edwards Q, Griffin LM, Skolnick MH, Kushner JP. Sporadic porphyria cutanea tarda in individuals with HLA-linked hemochromatosis allele(s). In: Weintraub LR, Edwards CQ, Krikker M, eds. Hemochromatosis. Proceedings of the First International Conference. Ann NY Acad Sci 1988: 526: 4753. 29. Seymour DG, Elder GH, Fryer A, Jacobs A, Williams GT. Porphyria cutanea tarda and haemochromatosis: a family study. Gut 1990; 31: 719-21. 30. Dadone MM, Khusner JP, Edwards CQ, Bishop DT, Skolnick MH. Hereditary hemochromatosis. Analysis of laboratory expression of the disease by genotype in 18 pedigrees. Am J Path01 1982; 78: 196-297. 31. Deugnier Y, Loreal 0, Turlin B, Guyader D, Jouanolle H, Moirand R, Jacquelinet C, Brissot P. Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations. Gastroenterology 1992; 102:2050-9. 32. Panajotopoulos N, Pipemo A, Conte D, Mandelli C, Cesana M, Mercuriali F, Fiorelli G, Bianchi PA, Fargion S. HLA typing in 67 Italian patients with idiopathic hemochromatosis and their relatives. Tissue Antigens 1989; 33: 431-6. 33. Simon M, LE Mignon L, Fauchet R, Yaouanq J, David V, Edan G, Bourel M. A study of 609 HLA haplotypes marking for the hemochromatosis gene: 1) mapping of the gene near the HLA-A locus and characters required to define a heterozygous population and 2) hypothesis concerning the underlying cause of hemochromatosis-HLA association. Am J Hum Genet 1987; 41: 89-105.
in porphyria cutanea tarda
34. Reizenstein P, Hoglund S, Landegren J, Carlmark B, Forsberg K. Iron metabolism in porphyria cutanea tarda. Acta Med Stand 1975; 198:95-9. 35. Pietrangelo A, Rocchi E, Rigo G, Ferrari AL, Perini M, Ventura E, Cairo G. Regulation of transfenin, transferrin receptor and ferritin gene expression in the duodenum of normal, anemic and siderotic subjects. Gastroenterology 1992; 102: 802-9. 36. Pietrangelo A, Gualdi R, Casalgrandi G, Conte D, Cesarini L, Ventura E, Cairo G. Molecular defects in the control of duodenal iron metabolism in genetic hemochromatosis. Gastroenterology 1993; 104: A272. 37. Lundin L, Hallgren R, Birgegard G, Wide L. Serum fenitin in alcoholics and the relation to liver damage, iron state and erythropoietic activity. Acta Med Stand 198 1; 209:327-3 1. 38. Di Bisceglie AM, Constantine AA, Hoofnagle JH, Bacon BR. Measurement of iron status in patients with chronic hepatitis. Gastroenterology 1992; 102: 2108-13. 39. Van Thiel DH, Friedlander L, Fagiuoli S, Wright HI, Irish W, Gavaler JS. Response to interferon a therapy is influenced by the iron content of the liver. J Hepatol 1994; 20:41&5. 40. Hayashi H, Takikawa T, Nishimura N, Yano M. Serum aminotransferase levels as an indicator of the effectiveness of venesection for chronic hepatitis C. J Hepatol 1995; 22:268-71. 41. Fargion S, Mandelli C, Pipemo A, Cesana B, Fracanzani AL, Fraquelli M, Bianchi PA, Fiorelli G, Conte D. Survival and prognostic factors in 212 Italian patients with genetic hemochromatosis. Hepatology 1992; 15: 655-9. 42. Pipemo A, Fargion S, D’Alba R, Roffi L, Fracanzani AL, Vecchi L, Failla M, Fiorelli G. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol1992;16: 364-8. 43. Beard P, Offord E, Paduwat N, Bruggmann H. SV40 activates transcription from transfertin receptor promoter by inducing a factor which binds to the CRE/AP-1 recognition sequence. Nucl Acid R 1991; 19: 7117-23.
569
Copenhagen
Copyrrght Q European Association for the Study of the Liver 1996
.,OUrUal Of&QPtOh,gy ISSN0168.8278
Genetic hemochmmatosis in Itabn patients with porphyria cutanea tarda: possible explanation for iron overload Silvia Fargion, Anna Ludovica Fracanzani, Riccardo Romano, Maria Domenica Cappellini, Maristella Fare’, Michela Mattioli, Albert0 Pipemo2, Guido Ronchi3 and Gemino Fiorelli Institute of Internal Medicine and Medical Physiopathology, ‘Transfusional Cents, Institute G. Pini, Milan, ‘Institute of Biomedical Science, Monza, and ‘Institute of Internal Medicine, IRCCS Ospedale Maggiore, University of Milan, Milan, Italy
Background/Aims: Mild to moderate iron overload is found in most patients with porphyria cutanea tar-da. This study ahned to evaluate whether iron overload in patients with porphyria cutanea tarda is related to the presence of a coexistent genetic hemochromatosis gene. Methods: A cohort study of 94 Italian patients with porphyria cutanea tarda (90 men and 4 women) and 20 relatives of five patients with iron overload were studied. Diagnosis of iron overload was assessed by transferrin saturation, serum ferritin and iron removed by phlebotomy to reach depletion. HLA typing by microlymphocytotoxicity test and duodenal ferritin analysis by immunohistochemistry were performed in a smaller number of patients. The chi square test was used to compare means and prevalences.
Results: Iron overload was present in 62% of the patients. HLA-A3 prevalence was significantly higher (~~0.01)in subjects with iron overload than in those without. A lack of duodenal ferritin was observed in 14/18 patients with and in 6/12 without iron overload. Family studies showed the presence of iron overload but not of porphyria cutanea tarda in HLA identical or semi-identical relatives of the patients. Conclusions: Italian patients with porphyria cutanea tarda and iron overload appear to have one or even two genes for genetic hemochromatosis.
M
appearance of PCT cutaneous lesions and an improvement in hepatic biochemical tests (1 l), even in patients without evident iron overload, probably due to the presence of intra-hepatocytic toxic species of iron. Alcohol abuse, frequently present in these patients, could account for at least part of it. In addition, several authors have hypothesized that genetic hemochromatosis (GH) may coexist in patients with PCT and iron overload, but studies on the prevalence of HLA-A3, an antigen considered an independent marker of the GH allele, have provided conflicting results (12-16). GH is far more frequent than previously thought in Caucasian populations, with prevalences of homozygotes ranging from 2 to 5/1000 and of heterozygotes from 8 to 15/100, respectively (17-21). Thus, patients with PCT could also be carriers of one or more GH genes, and this could explain the mild to moderate iron overload found in many cases. The aim of this study was to evaluate the preva-
iron overload is a common finding in patients with porphyria cutanea tarda (PCT) and, according to some authors, it may trigger clinical manifestations of the disease (1,2). PCT is caused by reduced activity of uroporphyrinogen decarboxylase (URO-D), which in the sporadic form of the disease is confined to the liver, whereas in the familial form it extends to other tissues (3-S). It is unclear whether sporadic PCT is an acquired or hereditary recessive disease (6,7). In subjects with URO-D defect PCT is usually clinically silent until external factors precipitate symptoms. Such factors have been reported to include alcohol, estrogens, virus and iron overload (1, 2, 7-10). In fact, iron depletion therapy by weekly phlebotomy is usually followed by the disILD TO MODERATE
Received 12 May; revised 19 September; accepted 4 October 1995
Correspondence: Silvia Fargion, Institute of Internal Medicine and Medical Physiopathology, Ospedale Maggiore, Pad LI?TA, Via F. Sforza 35,20122 Milan, Italy.
564
Key words: Duodenal ferritin; Genetic hemochromatosis; HLA-A3; Iron overload; Porphyria cutanea tarda.
Genetic hemochromatosis TABLE 1 Sex, age and iron indexes in 94 patients with porphyria cutanea tarda Mean&SD (range) 9014 61f7.8 (40-78) 50.5+17.2 (32-100) 501f303.8 (42-1450) 2.5k1.58 (0.3-7.3)
Male/female Age (years) Transfertin saturation (%) Serum ferritin (pgll) Iron removed (g)
lence and severity of iron overload in a group of Italian patients with PCT and the possible coexistence of GH in those with increased iron stores. We determined the amount of iron removed by phlebotomy up to depletion and the prevalence of HLA-A3 in patients grouped according to body iron stores. The possible absence of ferritin in duodenal enterocytes, previously described in patients with GH (22), was investigated in a subgroup of our series. Family studies which aimed to verify, in siblings sharing one or two haplotypes with the proband, transmission of iron overload and PCT were also performed.
in porphyria cutanea tarda
patients. No patient had been exposed to other known hepatotoxins, with the exception of one woman who had taken estrogens 2 years before the onset of PCT. Hepatitis virus screening indicated that HBsAg was present in six (6%) and anti-HCV in 73 (78%) patients, including four of the five with familial PCT. Liver biopsy was performed in 60 patients. Histological findings were consistent with chronic liver disease in all patients, as previously reported in a smaller group of patients (10). Grade 2 or 3 siderosis was found in 44 of the 60 (23). Liver iron concentration was performed according to Barry’s method (24) in 28 patients. Patients with clinically manifest PCT underwent phlebotomy with removal of 300 ml of blood (equivalent to 180 mg of iron) every week, even in the absence of evident iron overload. Iron depletion was defined as transferrin saturation of < 20% and serum ferritin ~30 l.tg/l in presence of mild anemia (Hb ~11 g/d1 in women and < 12 g/d1 in men). The iron status of the patients was defined as normal when transferrin saturation was ~45% and/or iron removed (IR) < 2 g, and as increased when transferrin saturation was 245% and/or IR 22 g. Patients in the latter group were considered to have severe iron overload when transfenin saturation was 262% and/or IR 24 g.
Material and Methods Patients Between January 1986 and June 1994, 94 patients with PCT were referred to our institution. PCT was diagnosed on the basis of typical clinical features and urinary porphyrin excretion (urinary porphyrins range 900-8000 pg/24 h, mean 3700 &1800). Sex, age and iron indexes of the patients are shown in Table 1. Five patients had a positive family history of PCT, Alcohol abuse defined as alcohol intake >80 g/ day for more than 5 years was present in 40% of
TABLE 2 Iron status in 94 patients with porphyria cutanea tarda Patients
Normal iron stores] Increased iron stones’ Moderate3 Severe4 i Transferrin * Transfetrin 3 Transferrin 4 Transferrin
saturation saturation saturation saturation
n
(%)
36 58 41 17
(38) (62) (44) (18)
< 45% and/or iron removed < 2 g 245% and/or iron removed 2 2 g 45-62% and /or iron removed 2-4 g 2 62% and/or iron removed 2 4 g
Methods Urinary porphyrins were fractionated, after conversion to their methyl esters, by high-performance liquid chromatography (25). Serum iron and total iron binding capacity were determined calorimetrically according to the International Committee for Standardization in Hematology (ICSH) (26,27). Serum ferritin was determined by radioimmunoassay (LisoPhase; Lepetit, Milan, Italy). HBsAg was determined by radioimmunoassay (RIA) (Abbott Laboratories, North Chicago, IL, USA) and anti-HCV detected on second generation ELISA II (Grtho Diagnostic System, Raritan, NJ, USA). HLA typing was performed by microlymphocytotoxicity tests in 56 patients with PCT. Control groups for HLA analysis included 80 patients with GH diagnosed according to standard criteria, and 700 normal subjects used as historical controls of our population. Duodenal biopsy analysis Thirty patients underwent duodenal biopsy and the results compared with those in 30 patients with homozygous GH, (some of whom have already been described (22)), four with heterozygous GH (sharing 565
S. Fargion et al. TABLE 3 HLA antigens frequency in patients with porphyria cutanea tarda compared with normal subjects and patients with genetic hemochromatosis Subjects
Normals
HLA N
A3
4,
Cases
n
700
150 22
%
B 35
B,
n
%
80 11
n
%
n
8011
%
2029
56 PCT all W/o iron overload 20 With iron overload 36
17 30 2*10 15*42
11 20 3 15 8 19
9 16 2 10 7 16
16 28 6 35 10 27
GH
55 69
8 10
20 26
23 29
80
*p
only one haplotype with a homozygous GH sibling), seven with secondary hemochromatosis (transfusion dependent anemias) and ten subjects who underwent endoscopy for dyspepsia. Duodenal biopsy specimens were processed as previously described (22). Briefly, paraffin-embedded tissue specimens were sliced and sections deparaffinized in xylene and then in absolute ethanol. Murine monoclonal antibody for L-chain ferritin was employed in the avidin-biotin immunoperoxidase technique. Endogenous peroxidase was blocked by incubation in a 0.3% H,O, solution. Family studies of five patients with PCT and iron overload were performed. A total of 20 relatives (16 siblings and four offsprings) were analyzed by HLA typing and iron parameters, and evaluated for silent or manifest PCT.
TABLE 4 Ferritin in duodenal enterocytes of patients with porphyria cutanea tarda (PCT), homozygous or heterozygous genetic hemochromatosis (GH), or secondary iron overload and of normal subjects Positive staining*
PCT Without iron overload With iron overload GH Homozygous GH Heterozygous Secondary iron overload Normals *By immunohistochemistry tin.
566
n
n
30 12 18
10 6 4
33 50 22
30
4
13
4 7 10
0 7 10
0 100 100
Statistical analysis Results are expressed as mean+S.D. The chi square test was used for comparisons of prevalences and means. The relationship between different variables was analyzed with correlation and regression analysis.
(%)
using monoclonal antibody against ferri-
Results Fifty-eight patients with PCT (62%) had iron overload, which was severe in 17 (18%) (Table 2). Liver iron concentration, performed in 28 patients including six with severe iron overload, ranged between 680-8870 pg/g dry tissue. Liver iron concentration/ age was > 2 in four of the latter patients. A significant positive correlation was found between uroporphyrin levels at the time of PCT diagnosis and IR @
Genetic hemochromatosis in porphyria cutanea tarda
Discussion The presence and severity of iron overload, the HLAA3 frequency, the absence of ferritin in duodenal enterocytes and the finding that HLA semi-identical siblings have iron overload but not PCT, suggest that Italian patients with PCT and iron overload carry one gene for GH or even two in a few cases. Analysis of the iron status of our patients with PCT showed that, according to the criteria used, 62% had increased iron stores, confirmed by liver siderosis grade 2 or 3 in the patients who underwent liver biopsy and by the high liver iron concentrations in most of the patients in whom the test was available. This figure fits with the incidence of iron overload in patients with PCT reported by other authors (1,2). The role of iron in the pathogenesis of PCT clinical manifestation is also supported by the highly significant correlation between uroporphyrin level at the time of PCT diagnosis and iron removed to reach depletion, one of the best indexes of iron overload. The possible coexistence of GH in PCT subjects is controversial (12-16,28,29). Beaumont et al. found no difference in HLA-A3 incidence between patients with PCT and controls (14). However, they did not have the possibility of separating patients into those with biochemical evidence of iron overload and those with normal iron stores, since most of their patients were in clinical remission at the time of HLA typing and previous biochemical data were not available. In fact, the HLA-A3 incidence in our PCT patients analyzed independently of their iron status did not differ from that found in controls. Kushner et al. (12), on the basis of HLA and URO-D analysis in a pedigree, proposed a two-locus causation model in the pathogenesis of sporadic PCT. They suggested that a single allele for GH might be responsible for hepatic siderosis in sporadic PCT and this siderosis, in turn, could exert an inhibitory effect on URO-D activity, thus making PCT clinically manifest. If, as they hypothesized, URO-D defect in sporadic PCT is transmitted as a recessive trait, it follows that two independent recessively transmitted factors are required for clinical manifestation of the sporadic form. This could explain the low incidence of the disease, and the common finding that in most families with PCT only a single individual is clinically detectable. In Elder’s editorial on Kushner et al.‘s hypothesis he concluded that the increased incidence of HLA-A3 in patients with PCT was not a demonstration of the two-locus model but an obvious consequence of the fact that GH heterozygosity is one of the most common causes of iron overload in the general population and thus a higher HLA-A3 frequency is to be expected in all
iron-precipitated disorders (13). Edwards et al. (28) extended Kushner’s studies and performed pedigree analyses based on HLA typing and iron indexes which provided evidence that GH coexisted in most of the patients studied and that six out of the 17 might even be homozygotes for GH. In addition, they found a high frequency of HLA-A3 in their PCT population, similar to that reported in homozygous GH (15,28). The coexistence of PCT and GH was described in another family studied by Seymour et al. (29). The present study carried out in a large Italian series supports the hypothesis that most of the patients with PCT carry one or even two genes for GH. Indeed, on the basis of transferrin saturation (higher than 62%) and the amount of iron removed to reach depletion (over 4 g), it is possible that a number of our patients are homozygotes for GH (30,3 1). HLA-A3 frequency, which was similar to that in normals when iron stores were not taken into account, was significantly higher in patients with iron overload than in those without or in normals. Given the expected HLA-A3 frequency in an Italian population of GH heterozygotes (32), as calculated by Simon et al. on the basis of HLA-A3 frequency in homozygous GH (33), our findings fit with the hypothesis that our PCT patients with increased iron stores may be heterozygous for GH. However, in the absence of a genetic marker for GH, the increased frequency of A3 in patients with PCT and iron overload only suggests but does not prove the coexistence of GH. Finally, the lack of duodenal ferritin in PCT patients with iron overload is in keeping with our previous data on GH homozygotes (22) and could account for the increased iron absorption reported in patients with PCT despite increased iron stores (34). In addition, since we found that also the few GH heterozygotes analyzed by us lacked fenitin in duodenal enterocytes (Table 4), our present results could provide further evidence that PCT patients are heterozygous for GH. The unexpected finding of some PCT patients without iron overload who did not have ferritin granules in duodenal cells might be explained by the presence of a few GH heterozygotes who, despite the PCT, did not develop iron overload. An altered expression of duodenal ferritin in patients with GH has also been reported by Pietrangelo et al. (35,36), who demonstrated a reduced expression of duodenal ferritin mRNA. Family studies supplied confirmatory evidence of the coexistence of GH in our patients with PCT, since siblings sharing one or two HLA haplotypes with the proband had iron overload but no evidence of PCT. 567
S. Fargion et al.
Alcohol abuse did not seem to cause marked modifications of the iron status of our PCT patients as shown by the lack of significant differences in the iron indexes between abusers and abstainers. However, we could not use ferritin values of PCT patients to discriminate between subjects with or without iron overload, since serum ferritin may overestimate the iron stores of these patients, probably due to liver damage (37) and/or to a possible induction of ferritin synthesis by alcohol. We previously reported that most Italian patients with PCT have an active HCV infection (10) although we were not able to establish whether such infection is a precipitating factor, which unmasks the underlying porphyrin metabolism disorder, or whether PCT patients are more susceptible to this virus. Whether there is relation between iron overload and HCV infection is still unclear, also in view of recent reports of iron overload and scanty response to interferon in patients with HCV positive chronic liver disease (38-40). However, the incidence of the virus is markedly lower in Italian patients with GH (24%) (41,42) than in those with PCT (78%) (lo), suggesting a complex interaction between iron, HCV and URO-D in the latter group. In patients with PCT, HCV could increase hepatocytic iron uptake by acting synergistically with a single GH gene, or, as demonstrated for other viruses (43), by interacting with transfer-i-inreceptors. Increased intra-hepatocytic iron could, in turn, trigger the metabolic defect underlying PCT. The relation between HCV and PCT remains however undefined.
7. 8.
9. 10.
11. 12.
13. 14.
15.
16.
17.
18.
19.
Acknowledgements The authors thank Maria Rita Balestrieri for her helpful technical assistance. This paper was partially supported by a grant from MURST 60% to Silvia Fargion.
20.
21.
References 1. Felsher BF, Kushner Jp. Hepatic siderosis and porphyria cutanea tarti relation of iron excess to the metabolic defect. Semin Hematol1977; 14: 243-5 1. 2. Lundvall 0, Weinfeld A, Lundin P. Iron storage in porphyria cutanea tarda. Acta Med Stand 1970; 188: 37-53. 3. Felsher BF, Norris ME, Shih JC. Red-cell uroporphyrinogen decarboxylase activity in porphyria cutanea tarda. N Engl J Med 1982; 306: 766-9. 4. De Vemeuil H, Aitken G, Nordmann Y. Familial and sporadic porphyria cutanea tarda: two different diseases. Hum Genet 1978; 44: 145-51. 5. Cohen PR, Deleo VA. Disorders of porphyrin metabolism. In: Alper JC, ed. Genetic Disorders of the Skin. Chicago: Year Book Medical Publishers, 1990: 79-104. 6. Elder GH, Urquhart AJ, De Salamanca RE, Munoz JJ, Bonk-
568
22.
23.
24. 25.
ovsky HL. Immunoreactive uroporphyrinogen decarboxylase in the liver in porphyria cutanea tarda. Lancet 1985; i: 229-32. Hindmarsh JT. Enzyme heterogeneity in the porphyria. Clin Biochem 1990; 23: 3714. Haberman HF, Rosemberg L, Menon LA. Porphyria cutanea tarda: comparison cases precipitated by alcohol and estrogens. Can Med Assoc J 1975; 113: 633-55. Nip-Sakamoto CJ, Wong RHW, Izumi AK. Porphyria cutanea tarda and AIDS. CUTIS 1989; 44: 47&l. Fargion S, Pipemo A, Cappellini MD, Sampietro M, Fracanzani AL, Roman0 R, Caldarelli R, Marcelli R, Vecchi L, Fiorelli G. Hepatitis C virus and porphyria cutanea tarda: evidence of a strong association. Hepatology 1992; 16: 1322-6. Lundvall 0. The effect of phlebotomy therapy in porphyria cutanea tarda. Acta Med Stand 197 1; 189: 33-49. Kushner JP, Edwards CQ, Dadone MM, Skolnick MH. Heterozygosity for HLA-linked hemochromatosis as a likely cause of hepatic siderosis associated with sporadic porphyria cutanea tarda. Gastroenterology 1985; 88: 1232-8. Elder GH. Porphyria cutanea tarda and HLA-linked hemochromatosis. Gastroenterology 1985; 88: 1276-9. Beaumont C, Fauchet R, Phung LN, Vemeuil HD, Gueguen M, Nordmann. Porphyria cutanea tarda and HLA-linked Hemochromatosis. Gastroenterology 1987; 92: 1833-8. Edwards CQ, Griffen LM, Kushner JP Increased frequency of HLA-A3 in subjects with sporadic porphyria cutanea tarda. Tissue Antigens 1988; 3 1: 250-l. Adams PC, Powell LW. Porphyria cutanea tarda and HLAlinked hemochromatosisall in the family? Gastroenterology 1987; 92: 2033-5. Edwards CQ, Griffen LM, Goldgar D, Drummond C, Skolnick MH, Kushner JP Prevalence of hemochromatosis among 11065 presumably healthy blood donors. N Engl J Med 1988; 318: 1355-62. Hallberg L, Bjom Rasmussen E, Jungmer I. Prevalence of hereditary hemochromatosis in two Swedish urban areas. J Intern Med 1989; 225: 249-55. Leggett BA, Halliday JW, Brown NN, Bryant S, Powell LW. Prevalence of hemochromatosis amongst asymptomatic Australians. Br J Haematol 1990; 74: 525-30. Velati C, Pipemo A, Fargion S, Colombo S, Fiorelli G. Prevalence of idiopathic hemochromatosis in Italy: study of 1301 blood donors. Haematologica 1990; 75; 309-12. Powell LW, Jazwinska E, Halliday JW. Primary iron overload. In: Brock JH, HaIliday JW, Pippard MJ, Powell LW eds. Iron Metabolism in Health and Disease. London, Philadelphia, Toronto, Sydney, Tokyo: Saunders Company Ltd, 1994; 227-70. Fracanzani AL, Fargion S, Roman0 R, Pipemo A, Arosio P, Ruggeri G, Ronchi G, Fiorelli G. Immunohistochemical evidence for a lack of ferritin in duodenal absorptive epithelial cells in idiopathic hemochromatosis. Gastroenterology 1989; 96: 1071-8. Scheuer PG, Williams R, Muiz AR Hepatic pathology in relatives of patients with hemochromatosis. J Path01 Bacterial 1962; 84: 53-64. Barry M, Sherlock S. Measurement of liver iron concentration in needle biopsy specimens. Lancet 197 1; i: 100-3. Elder GH, Lee GB, Tovey JA. Decrease activity of hepatic uroporphyrinogen decarboxylase in sporadic porphyria cutanea tarda. N Engl J Med 1978; 299: 274-8.
Genetic hemochromatosis 26. Iron Panel of the International Committee for Standardization in Haematology. The measurement of total and unsaturated iron- binding capacity in serum. Br J Haematol 1978; 38: 281-90. 27. Iron Panel of the International Committee for Standardization in Haematology. Recommendations for measurement of serum iron in human blood. Br J Haematol1978; 38: 291-4. 28. Edwards Q, Griffin LM, Skolnick MH, Kushner JP. Sporadic porphyria cutanea tarda in individuals with HLA-linked hemochromatosis allele(s). In: Weintraub LR, Edwards CQ, Krikker M, eds. Hemochromatosis. Proceedings of the First International Conference. Ann NY Acad Sci 1988: 526: 4753. 29. Seymour DG, Elder GH, Fryer A, Jacobs A, Williams GT. Porphyria cutanea tarda and haemochromatosis: a family study. Gut 1990; 31: 719-21. 30. Dadone MM, Khusner JP, Edwards CQ, Bishop DT, Skolnick MH. Hereditary hemochromatosis. Analysis of laboratory expression of the disease by genotype in 18 pedigrees. Am J Path01 1982; 78: 196-297. 31. Deugnier Y, Loreal 0, Turlin B, Guyader D, Jouanolle H, Moirand R, Jacquelinet C, Brissot P. Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations. Gastroenterology 1992; 102:2050-9. 32. Panajotopoulos N, Pipemo A, Conte D, Mandelli C, Cesana M, Mercuriali F, Fiorelli G, Bianchi PA, Fargion S. HLA typing in 67 Italian patients with idiopathic hemochromatosis and their relatives. Tissue Antigens 1989; 33: 431-6. 33. Simon M, LE Mignon L, Fauchet R, Yaouanq J, David V, Edan G, Bourel M. A study of 609 HLA haplotypes marking for the hemochromatosis gene: 1) mapping of the gene near the HLA-A locus and characters required to define a heterozygous population and 2) hypothesis concerning the underlying cause of hemochromatosis-HLA association. Am J Hum Genet 1987; 41: 89-105.
in porphyria cutanea tarda
34. Reizenstein P, Hoglund S, Landegren J, Carlmark B, Forsberg K. Iron metabolism in porphyria cutanea tarda. Acta Med Stand 1975; 198:95-9. 35. Pietrangelo A, Rocchi E, Rigo G, Ferrari AL, Perini M, Ventura E, Cairo G. Regulation of transfenin, transferrin receptor and ferritin gene expression in the duodenum of normal, anemic and siderotic subjects. Gastroenterology 1992; 102: 802-9. 36. Pietrangelo A, Gualdi R, Casalgrandi G, Conte D, Cesarini L, Ventura E, Cairo G. Molecular defects in the control of duodenal iron metabolism in genetic hemochromatosis. Gastroenterology 1993; 104: A272. 37. Lundin L, Hallgren R, Birgegard G, Wide L. Serum fenitin in alcoholics and the relation to liver damage, iron state and erythropoietic activity. Acta Med Stand 198 1; 209:327-3 1. 38. Di Bisceglie AM, Constantine AA, Hoofnagle JH, Bacon BR. Measurement of iron status in patients with chronic hepatitis. Gastroenterology 1992; 102: 2108-13. 39. Van Thiel DH, Friedlander L, Fagiuoli S, Wright HI, Irish W, Gavaler JS. Response to interferon a therapy is influenced by the iron content of the liver. J Hepatol 1994; 20:41&5. 40. Hayashi H, Takikawa T, Nishimura N, Yano M. Serum aminotransferase levels as an indicator of the effectiveness of venesection for chronic hepatitis C. J Hepatol 1995; 22:268-71. 41. Fargion S, Mandelli C, Pipemo A, Cesana B, Fracanzani AL, Fraquelli M, Bianchi PA, Fiorelli G, Conte D. Survival and prognostic factors in 212 Italian patients with genetic hemochromatosis. Hepatology 1992; 15: 655-9. 42. Pipemo A, Fargion S, D’Alba R, Roffi L, Fracanzani AL, Vecchi L, Failla M, Fiorelli G. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol1992;16: 364-8. 43. Beard P, Offord E, Paduwat N, Bruggmann H. SV40 activates transcription from transfertin receptor promoter by inducing a factor which binds to the CRE/AP-1 recognition sequence. Nucl Acid R 1991; 19: 7117-23.
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