Journal of Hepatology, 1992; 16:364-368 ©1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/92/$05.00
364
HEPAT 01137
Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection A. Piperno a, S. Fargion b, R. D'Alba a, L. RoftZia, A.L. Fracanzani a, L. Vecchi c, M. Failla ~ and G. Fiorelli" alnstitute of Biomedical Sciences S. Gerardo, Clinical Medicine, Monza, blnstitute oJ'Internal Medicine, University of Milan, Milan, and CDepartment of Clinical Chemistry, S. Gerardo Hospital, Monza. Italy (Received 12 December 1991)
We evaluated the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in 78 Italian patients with hereditary hemochromatosis as well as the relation between HCV antibody (anti-HCV) status, hepatitis B surface antigen (HBsAg) and liver histology. None of the patients had been transfused or ever consumed more than 60 g of alcohol per day. Eighteen showed histological signs of chronic hepatitis, active cirrhosis was present in 12, chronic active hepatitis in 4 and chronic persistent hepatitis in 2. Liver fibrosis or cirrhosis without inflammatory activity was observed in 31 subjects, whereas liver histology was normal except for iron overload in 18. The prevalence of HBsAg in the whole series was 5% and of anti-HCV was 20.5%. The prevalence of HBsAg and anti-HCV was significantly higher in the chronic hepatitis group than in the fibrosis/cirrhosis (p = 0.01) and the normal groups (p < 0.01). Fourteen of 18 hereditary hemochromatosis patients with chronic hepatitis were HBsAg (4) or anti-HCV (10) positive and all the latter subgroup had HCV-RNA in their serum as shown by the polymerase chain reaction. Although most of the patients with associated chronic hepatitis had cirrhosis, their serum ferritin levels and amount of mobilizable iron were significantly lower than those of the fibrosis/cirrhosis group (p < 0.01). This indicates that hepatitis viral infection acts synergistically with iron in accelerating the development of liver damage. K e y words." Iron overload; Iron damage; Chronic hepatitis; HCV-RNA
Patients with hereditary hemochromatosis develop liver damage due to iron overload. As iron stores increase, fibrosis causes expansion of the portal tract and eventually cirrhosis develops, but enlarged tracts usually show little or no inflammatory infiltration (1). However, we have previously observed that in some patients with hereditary hemochromatosis liver histological findings are consistent with viral-induced chronic hepatitis associated with the typical hereditary hemochromatosis pattern (2), which suggests that factors other than iron overload might be implicated in the genesis of liver damage in these patients. Iron overload may facilitate hepatitis B virus (HBV) infection and preliminary data indicate that hepatitis C virus (HCV) antibodies against C100-3 antigen are present in about onefourth of Italian patients with hereditary hemochromatosis (3,4). Bassett et al. (5) have demonstrated that patients with hereditary hemochromatosis and alcohol-
ism develop cirrhosis with unexpectedly low' iron overload. Similarly, HBV and HCV infections, which are widespread in Italy (6,7), might act with iron in inducing liver damage in patients with hereditary hemochromatosis. In this study we have evaluated the prevalence of HBV and HCV infection in 78 Italian patients with hereditary hemochromatosis as well as the relation between the presence of positive viral hepatitis markers and histological findings at liver biopsy.
Materials and Methods Subjects
Seventy-eight patients with hereditary hemochromatosis (51 males and 17 females), non-alcohol abusers,
Correspondence to: Dr. A. Piperno, Institute of BiomedicalSciences S. Gerardo, Clinical Medicine,Via Donizetti 106, 20052, Monza, Italy
HBV AND HCV INFECTION IN HEMOCHROMATOSIS were studied. Seventy were probands and 8 HLA-identical siblings. The mean age was 49.1 + 9.8 years (range 28-65 yr). The diagnosis of hereditary hemochromatosis was confirmed by liver biopsy showing parenchymal iron overload, graded according to Scheuer (I). None of the subjects had any known cause of secondary iron overload. Mean transferrin saturation and serum ferritin levels were 74.5+12.8% (range 44-95%) and 1373 #g/l (range 122-8000 /~g/I) respectively. Liver siderosis was Grade II in 4 patients, Grade III in 21 and Grade IV in 53. No patient had ever consumed more than 60 g of alcohol per day. None of the patients had a history of exposure to risk factors for hepatitis viral infections. Phlebotomy was performed in all the patients, and 50 had achieved complete iron depletion at the time of the study. The mean total iron removed to reach depletion was !1.6___9.1 g (range 3-35 g). Iron was always~>5 g in men and/> 3 g in women. All patients were tested for HBV markers and HCV antibodies (anti-HCV) and reverse transcription (RT)/ polymerase chain reaction (PCR) analysis was performed in 20 patients to detect the presence of the viral genome.
Methods
Serum samples were taken for liver function tests and iron indexes including serum iron, total iron-binding capacity (TIBC) and serum ferritin, at the time of diagnosis. These samples were stored at - 2 0 ° C until tested for HBV markers and anti-HCV. For RT/PCR analysis samples were taken and stored in guanidine thiocyanate at - 8 0 ° C until RNA extraction was performed. Serum iron, TIBC and liver function tests were performed using standard methods. Serum ferritin was determined by radioimmunoassay (RIA). HBV markers were detected by RIA (Sorin Antony, France) and antiHCV by an enzyme-linked immunoadsorbent assay (ELISA II, Ortho Diagnostic System, U.S.A.) and confirmed by recombinant immunoblot assay (RIBA II, Ortho Diagnostic System, U.S.A.). For RT/PCR analysis, RNA was extracted from I ml
365 of serum according to Chomczynski et al. (8). RNA was copied into cDNA by RT transcription (cDNA Synthesis System Plus 17, Amersham International, Amersham, U.K.) and amplified by PCR (Perkin Elmer Cetus PCR kit Z808-909) according to the manufacturer's instructions. Primers for RT and PCR were those described by Weiner et al. (9), derived from cDNA clones from the non-structural region of the HCV genome. Forty cycles of PCR were performed as follows: (1) denaturation for 4 min at 94 °C in cycle 1 and for I min at 92 °C in cycles 2-40; (2) annealing for l min at 42 °C; and (3) extension for 2 min at 72 °C in cycles 1-39 and for 5 min at 72 °C in cycle 40 (Biostar Thermal Cycler, Violet, Rome, Italy). An aliquot of the PCR product was electrophoresed on i.8% agarose gel and stained with ethidium bromide. Liver sections were stained with standard stains for histological evaluation and Perrs Prussian blue for hemosiderin deposits. Histological diagnosis and siderosis grading was done by one of us (L.R.), without knowledge of any clinical data. These series were subdivided into three groups, one including patients with normal histological findings apart from iron overload, one with patients with fibrosis or cirrhosis without inflammatory activity and one with patients with signs o f chronic hepatitis, i.e. enlarged portal tracts with inflammatory infiltration associated with piecemeal or other forms of necrosis with or without cirrhosis, and associated with the typical hereditary hemochromatosis pattern. Statistical analysis was performed using the chi-square test, one-tailed Fisher's exact test and Student's t-test for paired data.
Results
Table 1 shows the prevalence of HBsAg and antiHCV in the whole series and in the subgroups. The mean serum ALT is also reported. Of the 4 HBsAgpositive patients, 3 were also HBeAg-positive. Twelve of the 18 patients with histological signs of
TABLE I Relation between HBV and HCV infectionand liver histologyin patients with hereditary hemochromatosis Liver histology No. of p a t i e n t s Normal 29 Fibrosis/cirrhosis 31 Chronic hepatitis 18 Total 78 'p=0.01, *p
364
HEPAT 01137
Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection A. Piperno a, S. Fargion b, R. D'Alba a, L. RoftZia, A.L. Fracanzani a, L. Vecchi c, M. Failla ~ and G. Fiorelli" alnstitute of Biomedical Sciences S. Gerardo, Clinical Medicine, Monza, blnstitute oJ'Internal Medicine, University of Milan, Milan, and CDepartment of Clinical Chemistry, S. Gerardo Hospital, Monza. Italy (Received 12 December 1991)
We evaluated the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in 78 Italian patients with hereditary hemochromatosis as well as the relation between HCV antibody (anti-HCV) status, hepatitis B surface antigen (HBsAg) and liver histology. None of the patients had been transfused or ever consumed more than 60 g of alcohol per day. Eighteen showed histological signs of chronic hepatitis, active cirrhosis was present in 12, chronic active hepatitis in 4 and chronic persistent hepatitis in 2. Liver fibrosis or cirrhosis without inflammatory activity was observed in 31 subjects, whereas liver histology was normal except for iron overload in 18. The prevalence of HBsAg in the whole series was 5% and of anti-HCV was 20.5%. The prevalence of HBsAg and anti-HCV was significantly higher in the chronic hepatitis group than in the fibrosis/cirrhosis (p = 0.01) and the normal groups (p < 0.01). Fourteen of 18 hereditary hemochromatosis patients with chronic hepatitis were HBsAg (4) or anti-HCV (10) positive and all the latter subgroup had HCV-RNA in their serum as shown by the polymerase chain reaction. Although most of the patients with associated chronic hepatitis had cirrhosis, their serum ferritin levels and amount of mobilizable iron were significantly lower than those of the fibrosis/cirrhosis group (p < 0.01). This indicates that hepatitis viral infection acts synergistically with iron in accelerating the development of liver damage. K e y words." Iron overload; Iron damage; Chronic hepatitis; HCV-RNA
Patients with hereditary hemochromatosis develop liver damage due to iron overload. As iron stores increase, fibrosis causes expansion of the portal tract and eventually cirrhosis develops, but enlarged tracts usually show little or no inflammatory infiltration (1). However, we have previously observed that in some patients with hereditary hemochromatosis liver histological findings are consistent with viral-induced chronic hepatitis associated with the typical hereditary hemochromatosis pattern (2), which suggests that factors other than iron overload might be implicated in the genesis of liver damage in these patients. Iron overload may facilitate hepatitis B virus (HBV) infection and preliminary data indicate that hepatitis C virus (HCV) antibodies against C100-3 antigen are present in about onefourth of Italian patients with hereditary hemochromatosis (3,4). Bassett et al. (5) have demonstrated that patients with hereditary hemochromatosis and alcohol-
ism develop cirrhosis with unexpectedly low' iron overload. Similarly, HBV and HCV infections, which are widespread in Italy (6,7), might act with iron in inducing liver damage in patients with hereditary hemochromatosis. In this study we have evaluated the prevalence of HBV and HCV infection in 78 Italian patients with hereditary hemochromatosis as well as the relation between the presence of positive viral hepatitis markers and histological findings at liver biopsy.
Materials and Methods Subjects
Seventy-eight patients with hereditary hemochromatosis (51 males and 17 females), non-alcohol abusers,
Correspondence to: Dr. A. Piperno, Institute of BiomedicalSciences S. Gerardo, Clinical Medicine,Via Donizetti 106, 20052, Monza, Italy
HBV AND HCV INFECTION IN HEMOCHROMATOSIS were studied. Seventy were probands and 8 HLA-identical siblings. The mean age was 49.1 + 9.8 years (range 28-65 yr). The diagnosis of hereditary hemochromatosis was confirmed by liver biopsy showing parenchymal iron overload, graded according to Scheuer (I). None of the subjects had any known cause of secondary iron overload. Mean transferrin saturation and serum ferritin levels were 74.5+12.8% (range 44-95%) and 1373 #g/l (range 122-8000 /~g/I) respectively. Liver siderosis was Grade II in 4 patients, Grade III in 21 and Grade IV in 53. No patient had ever consumed more than 60 g of alcohol per day. None of the patients had a history of exposure to risk factors for hepatitis viral infections. Phlebotomy was performed in all the patients, and 50 had achieved complete iron depletion at the time of the study. The mean total iron removed to reach depletion was !1.6___9.1 g (range 3-35 g). Iron was always~>5 g in men and/> 3 g in women. All patients were tested for HBV markers and HCV antibodies (anti-HCV) and reverse transcription (RT)/ polymerase chain reaction (PCR) analysis was performed in 20 patients to detect the presence of the viral genome.
Methods
Serum samples were taken for liver function tests and iron indexes including serum iron, total iron-binding capacity (TIBC) and serum ferritin, at the time of diagnosis. These samples were stored at - 2 0 ° C until tested for HBV markers and anti-HCV. For RT/PCR analysis samples were taken and stored in guanidine thiocyanate at - 8 0 ° C until RNA extraction was performed. Serum iron, TIBC and liver function tests were performed using standard methods. Serum ferritin was determined by radioimmunoassay (RIA). HBV markers were detected by RIA (Sorin Antony, France) and antiHCV by an enzyme-linked immunoadsorbent assay (ELISA II, Ortho Diagnostic System, U.S.A.) and confirmed by recombinant immunoblot assay (RIBA II, Ortho Diagnostic System, U.S.A.). For RT/PCR analysis, RNA was extracted from I ml
365 of serum according to Chomczynski et al. (8). RNA was copied into cDNA by RT transcription (cDNA Synthesis System Plus 17, Amersham International, Amersham, U.K.) and amplified by PCR (Perkin Elmer Cetus PCR kit Z808-909) according to the manufacturer's instructions. Primers for RT and PCR were those described by Weiner et al. (9), derived from cDNA clones from the non-structural region of the HCV genome. Forty cycles of PCR were performed as follows: (1) denaturation for 4 min at 94 °C in cycle 1 and for I min at 92 °C in cycles 2-40; (2) annealing for l min at 42 °C; and (3) extension for 2 min at 72 °C in cycles 1-39 and for 5 min at 72 °C in cycle 40 (Biostar Thermal Cycler, Violet, Rome, Italy). An aliquot of the PCR product was electrophoresed on i.8% agarose gel and stained with ethidium bromide. Liver sections were stained with standard stains for histological evaluation and Perrs Prussian blue for hemosiderin deposits. Histological diagnosis and siderosis grading was done by one of us (L.R.), without knowledge of any clinical data. These series were subdivided into three groups, one including patients with normal histological findings apart from iron overload, one with patients with fibrosis or cirrhosis without inflammatory activity and one with patients with signs o f chronic hepatitis, i.e. enlarged portal tracts with inflammatory infiltration associated with piecemeal or other forms of necrosis with or without cirrhosis, and associated with the typical hereditary hemochromatosis pattern. Statistical analysis was performed using the chi-square test, one-tailed Fisher's exact test and Student's t-test for paired data.
Results
Table 1 shows the prevalence of HBsAg and antiHCV in the whole series and in the subgroups. The mean serum ALT is also reported. Of the 4 HBsAgpositive patients, 3 were also HBeAg-positive. Twelve of the 18 patients with histological signs of
TABLE I Relation between HBV and HCV infectionand liver histologyin patients with hereditary hemochromatosis Liver histology No. of p a t i e n t s Normal 29 Fibrosis/cirrhosis 31 Chronic hepatitis 18 Total 78 'p=0.01, *p
