Patients
We studied 68 male patients with sporadic PCT (range, 28-80 years;
median 61). PCT was diagnosed on the basis of typical clinical features
and urinary porphyrin excretion (urinary porphyrins range, 900-8,000 mg
per 24 hours; mean ± SD 3,700 ± 1,800). None of the
patients had a clinical picture or a family history of hemochromatosis.
Patients with clinical manifestations of PCT underwent phlebotomy with
removal of 300 mL of blood (equivalent to 150 mg of iron) every week,
even in the absence of evident iron overload. Iron depletion was
defined as transferrin saturation of < 20% and ferritin < 30
mg/L in the presence of mild anemia (hemoglobin < 11 g/dL in women
and 12 g/dL in men).
The iron status at the time of diagnosis was categorized in three
classes, as previously described.13 In brief, iron overload
was diagnosed in patients in the presence of a transferrin
saturation > 45%, iron removed to reach iron depletion > 2 g
or by liver iron concentration, available in 21 patients, > 28
µmol/g of dry tissue. Patients in whom the transferrin saturation was
45%, in whom the iron removed was 2 g, and in whom the liver
iron concentration was 28 µmol/g were included in class 0.
Patients in whom the transferrin saturation was > 45%, in whom
iron removed was > 2 g, or in whom the liver iron concentration
was > 28 µmol/g were included in class I, or in class II if
transferrin saturation was > 62% and the iron removed was >
4 g.
Markers of hepatitis B virus (HBV) and HCV infection were obtained in
all patients. Alcohol abuse was defined by present or past alcohol
intake > 80 g/day for more than 5 years. All diagnostic parameters,
with the exception of iron removed, were obtained before any
therapeutical intervention (phlebotomy or 
-interferon). No patient
had known environmental exposures to hepatotoxic or porphyrogenic
chemicals or toxins.
A control group of 128 subjects without any evidence of liver disease
or porphyria was formed by enrolling volunteer individuals among
hospital staff and medical students (group A). The frequency of HFE
mutations was also established in 50 patients who were infected with
HCV, who showed no evidence of porphyria or hemochromatosis, and who
had a histological diagnosis of chronic active hepatitis (group B).
Methods
After conversion to their methyl esters, urinary porphyrins were
fractionated by high performance liquid chromatography.25
Serum iron and total iron binding capacity were determined by standard
methods. Liver iron concentration was performed according to Barry’s
method.26 The presence of hepatitis C infection was
investigated by serology (EIAIII Ortho Diagnostic Systems, Raritan, NJ)
and HCV-RNA was detected as previously described.27 A
present or past HBV infection was investigated by hepatitis B surface
antigen and by the antibody to hepatitis B core antigen
(Abbott Laboratories, North Chicago, IL).
Genomic DNA was extracted from peripheral leukocytes by standard
procedures or from serum samples as described previously.28
The two mutations of HFE were detected after amplification by
polymerase chain reaction (PCR)14 and restriction with
RsaI for Cys282Tyr and BclI/MboI for His63Asp.
Polymorphic alleles of the D6S265 and D6S105 microsatellites were
analyzed by polyacrylamide gel electrophoresis after polymerase chain
reaction amplification with specific primers, as previously
described.29,30 The two microsatellite alleles D6S265-1 and
D6S105-8 are in strong linkage disequilibrium with the hemochromatosis
gene and are part of the ancestral haplotype. Allele D6S265-1 is
present only on chromosomes carrying HLA-A3.29,31 The
ancestral haplotype was not reported in a well chosen normal control
population in Italy,29 indicating that it is strictly
associated with the hemochromatosis gene.
Statistical comparison of data by the Fisher’s Exact test or by
2 was performed by the statistical package Instat 2.01
(Graphpad Software, San Diego, CA). All statistics were two-tailed.
The frequencies of HFE mutations and genotypes of patients and controls
are reported in Table 1
. The frequency of
Cys282Tyr mutation was not significantly different in PCT patients and
controls. The two patients carrying Cys282Tyr were heterozygous for the
mutation. The His63Asp mutation was found on 28.7% of chromosomes from
patients with PCT, a frequency significantly increased as compared with
12.9% of controls from the general population (group A) (P =
.0002). The frequency of the same mutation in controls with HCV chronic
hepatitis (group B) was not increased as compared with the general
population. Thirty-four of 68 PCT patients (50%) carried His63Asp in
the heterozygous or homozygous state versus 31 of 128 (24.1%) control
individuals (P = .0004). One patient was a compound
heterozygote for the two mutations.
Table 1. Allelic and Genotype Frequencies of Two Mutations of HFE in 68 Patients
With PCT, in 128 Control Subjects From the General Population (Group A)
and in 50 Patients With HCV Chronic Hepatitis and No Evidence of
Porphyria (Group B)
 | | Controls | |
| | Group A | Group B | |
| | PCT (n = 68) | (n = 128) | (n = 50) | P |
| Alleles |
| Cys282Tyr | 2/136 (1.5%) | 2/256 (0.7%) | 2/100 (2.0%) | NS |
| His63Asp | 39/136 (28.7%) | 33/256 (12.9%) | 12/100 (12.0%) | <0.001* |
| Genotypes |
| Cys282/Tyr282 | 2/68 (2.9%) | 2/128 (1.5%) | 2/50 (4.0%) | NS |
| Tyr282/Tyr282 | 0/68 | 0/128 | 0/50 | NS |
| His63/Asp63 | 29/68 (42.6%) | 29/128 (22.6%) | 8/50 (16.0%) | 0.005* |
| Asp63/Asp63 | 5/68 (7.3%) | 2/128 (1.5%) | 2/50 (4.0%) | 0.050* |
Abbreviation: NS, not significant.
*PCT patients vs. Group A controls. |
On the basis of criteria chosen to define the iron status, 16 patients
were classified as having a normal iron status (class 0); 39 patients
were included in iron overload class I; and 13 patients were included
in iron overload class II. The allelic and genotype frequencies of HFE
mutations in patients belonging to the three classes of iron status are
reported in Table 2
. The Cys282Tyr mutation
was reported in only two patients with iron overload, one in class I
and one in class II. The distribution of the His63Asp mutation was not
significantly different in the three classes of iron status.
Table 2. Allelic and Genotype Frequencies of Two Mutations of HFE in 68 Patients
With PCT Sorted According to Their Iron Status
| Class 0 (n = 16) | Class I (n = 39) | Class
II (n = 13) | P |
| Alleles |
| Cys282Tyr | 0/32 | 1/78 (3.3%) | 1/26 (3.8%) | NS |
| His63Asp | 12/32 (37.5%) | 23/78 (29.5%) | 6/26 (23.1%) | NS |
| Genotypes |
| Cys282/Tyr282 | 0/16 | 1/39 (2.6%) | 1/13 (7.7%) | NS |
| Tyr282/Tyr282 | 0/16 | 0/39 | 0/13 | NS |
| His63/Asp63 | 6/16 (37.5%) | 17/39 (43.6%) | 6/13 (46.1%) | NS |
| Asp63/Asp63 | 3/16 (18.7%) | 3/39 (7.7%) | 0/13 | NS |
NOTE. Iron class status as follows: class 0, normal iron status;
class I, mild to moderate iron overload; and class II, severe iron
overload.
Abbreviation: NS, not significant. |
The ancestral hemochromatosis haplotype was identified in five PCT
patients carrying D6S265-1 and D6S105-8 alleles, because of
homozygosity for one or both alleles or through a pedigree analysis; in
two patients, also heterozygous for both alleles, we were unable to
assign haplotypes. All the patients having the ancestral haplotype had
iron overload: two were in class I and three in class II. One of the
patients in class II was homozygous for the ancestral haplotype. No
patient had the ancestral hemochromatosis haplotype associated with HFE
mutations.
The geographical origin of patients with or without the His63Asp
mutation was similar, as follows: 88.4% and 87.5%, respectively, were
from Northern Italian regions. No correlation was found between the
presence of HCV infection or alcohol abuse and HFE mutations, as
follows: antibodies against HCV infection, usually associated with
viremia, were present in 82.3% of patients carrying the His63Asp
mutation versus 73.5% of those homozygous for the absence of the
mutation. Also alcohol abuse had a similar frequency in the two groups
(52.9% vs. 55.8%). The prevalence of HCV infection and alcohol abuse
in the three categories of iron status was not significantly different.
The clinical manifestations of sporadic PCT seem to occur in
individuals following exposure to triggering agents, including iron
overload, which may cause a reduction of hepatic URO-D. However, the
nature of this predisposition and whether it is inherited are still a
matter for debate. A genetic contribution to the iron overload
frequently observed in PCT has been hypothesized in several studies and
it was suggested that the hypothetical inherited predisposition for PCT
could be coincidental to an inherited condition which causes iron
overload.10-13 A recent report describing the high
prevalence of a strong candidate causative mutation for hemochromatosis
in a novel major histocompatibility class I–like gene seemed to
confirm the relationship between PCT and hemochromatosis; inheritance
of one or two copies of the Cys282Tyr mutation of HFE is an important
susceptibility factor for sporadic PCT18 in British
patients.
Our data, obtained in a large group of Italian patients with sporadic
PCT, confirm a role for a HFE–linked determinant in PCT, but
surprisingly do not indicate a direct association between PCT and the
mutation responsible for the typical form of hemochromatosis. The
Cys282Tyr mutation, strongly associated with hemochromatosis in
Northern European countries and present in 44% of British patients
with PCT, is rare in Italian patients with PCT, while the second known
mutation of HFE, His63Asp, is highly prevalent in Italian patients with
PCT, as it is present in half of the patients. Because HCV
infection is frequent in Italian PCT patients and is rare in PCT
patients from Northern Europe, we examined a control group of patients
with HCV–chronic active hepatitis without PCT to rule out a possible
association between the His63Asp mutation and HCV infection. The
prevalence of HFE mutations in this group was almost identical to that
observed in controls from the general population, suggesting that, in
our series, the increased prevalence of His63Asp is, indeed, associated
with PCT.
The role of His63Asp in hemochromatosis has not been clearly shown.
Some authors suggest that His63Asp could be a polymorphism or a
polymorphic marker of another causative mutation of HFE which is
different from Cys282Tyr16,17; however, because the two
mutations are in complete linkage disequilibrium, an analysis that
considers only chromosomes “at risk,” i.e., those that do not
carry the Cys282Tyr, revealed also that the mutation His63Asp was
overrepresented in hemochromatosis and PCT patients,32
although homozygosity for His63Asp is rare in classic hemochromatosis
and the mutation is frequent in normal individuals.
Thus, several lines of evidence indicate that the His63Asp mutation
could cause a more subtle abnormality of iron metabolism than
Cys282Tyr. This alteration seems unable to induce the severe iron
overload that is typical of hemochromatosis, even when present in the
homozygous state; however, the alteration may result in the
hepatocellular accumulation of toxic iron species, which accelerates
the inactivation of hepatic uroporphyrinogen decarboxylase and the
development of the clinical manifestations of PCT. In our series, the
presence of His63Asp did not seem to correlate with the iron status of
PCT patients, as judged by transferrin saturation, by iron removal via
phlebotomy, and by liver iron concentration. This suggests the
inability of standard parameters of iron status to consistently
identify the abnormality of iron metabolism which is induced by
His63Asp. HCV and/or heavy alcohol intake, which is highly
prevalent in our population, might have a synergistic effect with the
His63Asp mutation in inducing a clinically manifest PCT. In contrast,
the Cys282Tyr mutation, which is more prevalent in Northern Europe and
which causes the typical iron-storage disease, could more efficiently
trigger PCT in the absence of viral liver disease.
At least a third unidentified HLA–linked genetic
determinant seems to influence the iron status of Italian patients with
PCT, as an increased frequency of the ancestral hemochromatosis
haplotype was observed in patients with iron overload. Interestingly,
none of the chromosomes bearing the ancestral haplotype carried the
Cys282Tyr HFE mutation. Roberts et al.18 also found four
chromosomes that carry the ancestral haplotype in the absence of the
Cys282Tyr mutation in patients with PCT. Previous studies in Australian
and Italian patients with hemochromatosis suggest a role for the
ancestral haplotype in determining the severity of phenotype
expression30,33 and the presence of 6 p-linked
modifying gene(s) that can explain the haplotype-related variability of
phenotype expression has been hypothesized.16
Our findings identify a state of morbidity which is associated with
heterozygosity or homozygosity for His63Asp in the absence of
Cys282Tyr. However, because the function of HFE and the range of
clinical effects of HFE mutations have not been clearly outlined, the
pathogenetic mechanism linking the His63Asp mutation with PCT remains
hypothetical. In particular, it is unclear in our population whether
the abnormality of iron metabolism induced by His63Asp might interfere
with URO-D activity directly, or indirectly through a synergistic
effect with the damage induced by viral hepatitis. The increased
frequency of the ancestral hemochromatosis haplotype in patients with
PCT and with iron overload suggests that, besides the existence of the
HFE His63Asp mutation, also a hemochromatosis determinant distinct from
Cys282Tyr could play a role in a proportion of Italian patients with
both PCT and iron overload. Because HFE is not an iron binding protein,
it is possible that other gene(s) that interact with HFE might be
involved in the regulation of iron absorption.
Acknowledgment
We are grateful to J. S. Wainscoat and Y-M. D. Lo for their precious
advice in setting up DNA extraction from serum samples; and to F.
Martinez di Montemuros and V. Molteni for their help in collecting
cases.
1. Kappas A, Sassa S, Galbraith RA, Nordmann Y. The porphyrias. In: Scriver CL, Beaudet AR, Sly VS, Valle D, eds. The Molecular and Metabolic Basis of Inherited Disease. New York: McGraw Hill. 1995;2103-2159.
2. De Verneuil H, Aitken G, Nordmann Y. Familial and sporadic porphyria cutanea tarda: two different diseases. Hum Genet 1978;44:145-151.
3. Lefkowitch JK, Grossman ME. Hepatic pathology in porphyria cutanea tarda. Liver 1983;3:19-29.
4. Lundvall O, Weinfeld A, Lundin P. Iron storage in porphyria cutanea tarda. Acta Med Scand 1970;188:37-53.
5. Felsher BF, Kushner JP. Hepatic siderosis and porphyria cutanea tarda: relation of iron excess to the metabolic defect. Semin Hematol 1977;14:243-251.
6. Fargion S, Piperno A, Cappellini MD, Sampietro M, Fracanzani AL, Romano R, Caldarelli R, et al. Hepatitis C virus and Porphyria Cutanea Tarda: evidence of a strong association. Hepatology 1992;16:1322-1326.
7. Elder GH, Roberts AG. Uroporphyrinogen decarboxylase. J Biomembr Bioenerg 1995;27:207-214.
8. Lundvall O. The effect of phlebotomy therapy in porphyria cutanea tarda. Acta Med Scand 1971;189:33-49.
9. Adams PC, Powell LW. Porphyria cutanea tarda and HLA-linked hemochromatosis - all in the family? Gastroenterology 1987;92:2033-2035.
10. Kuntz BME, Goerz G, Merk H, Strohmeyer G, Bruster HT. HLA-A3 and B7 in porphyria cutanea tarda. Tissue Antiges 1984;24:67-69.
11. Edwards CQ, Griffen LM, Kushner JP. Increased frequency of HLA-A3 in subjects with sporadic porphyria cutanea tarda. Tissue Antiges 1988;31:250-251.
12. Edwards CQ, Griffen LM, Goldgar DE, Skolnick MH, Kushner JP. HLA-linked hemochromatosis alleles in sporadic porphyria cutanea tarda. Gastroenterology 1989;97:972-981.
13. Fargion S, Fracanzani AL, Romano R, Cappellini MD, Faré M, Mattioli M, Piperno A, et al. Genetic hemochromatosis in Italian patients with porphyria cutanea tarda: possible explanation for iron overload. J Hepatol 1996;24:564-569.
14. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399-408.
15. Beutler E, Gelbart T, West C, Lee P, Adams M, Blackstone R, Pockros P, et al. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 1996;22:187-194.
16. Jazwinska EC, Cullen LM, Busfield F, Pyper WR, Webb SI, Powell LW, Morris CP, et al. Haemochromatosis and HLA-H. Nat Genet 1996;14:249-251.
17. Jouanolle AM, Gandon G, Jézéquel P, Blayau M, Campion ML, Yaouanq J, Mosser J, et al. Haemochromatosis and HLA-H. Nat Genet 1996;14:251-252.
18. Roberts AG, Whatley SD, Morgan RR, Worwood M, Elder GH. Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda. Lancet 1997;349:321-323.
19. Carella M, D’Ambrosio L, Totaro A, Grifa A, Valentino MA, Piperno A, Girelli D, et al. Mutation analysis of the HLA-H gene in Italian hemochromatosis patients. Am J Hum Genet 1997;4:828-832.
20. Borot N, Roth MP, Malfroy L, Demangel C, Vinel JP, Pascal JP, Coppin H. Mutation in the MHC class I-like candidate gene for hemochromatosis. Immunogenetics 1997;45:320-324.
21. Herrero C, Vicente A, Bruguera M, Ercilla MG, Barrera JM, Vidal J, Teres J. Is Hepatitis C virus infection a trigger of porphyria cutanea tarda? Lancet 1993;341:788-789.
22. Lacour JP, Bodokh I, Castanet J, Bekri S, Ortonne JP. Porphyria cutanea tarda and antibodies to hepatitis C virus. British Journal of Dermatology 1993;128:121-123.
23. Murphy A, Dooley S, Hillary IB, Murpy GM. HCV infection in porphyria cutanea tarda. Lancet 1993;341:1534-1535.
24. Stölzel U, Kostler E, Koszka C, Stoffler-Meilicke M, Schuppan D, Somasundaram R, Doss M, et al. Low prevalence of hepatitis virus infection in Porphyria Cutanea Tarda in Germany. Hepatology 1995;21:1500-1503.
25. Elder GH, Lee GB, Tovey JA. Decreased activity of hepatic uroporphyrinogen decarboxylase in sporadic porphyria cutanea tarda. N Engl J Med 1978;299:274-278.
26. Barry M, Sherlock S. Measurement of liver iron concentration in needle biopsy specimens. Lancet 1971;1:100-103.
27. Sampietro M, Salvadori S, Corbetta N, Badalamenti S, Graziani G, Fiorelli G. Single-tube reverse transcription and heminested polymerase chain reaction of hepatitis C virus RNA to detect viremia in serologically negative hemodialysis patients. Int J Clin Lab Res 1995;25:52-54.
28. Lo Y-MD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CWG, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485-487.
29. Camaschella C, Roetto A, Gasparini P, Piperno A, Fortina R, Surrey S, Rappaport E. Allelic association of microsatellites of 6p in Italian hemochromatosis patients. Hum Genet 1996;97:476-481.
30. Piperno A, Arosio C, Fargion S, Roetto A, Nicoli C, Girelli D, Sbaiz L, et al. The ancestral hemochromatosis haplotype is associated with a severe phenotype expression in Italian patients. Hepatology 1996;24:43-46.
31. Worwood M, Dorak MT, Nicklin S, Stone C, Pointon J, Darke C. The frequency of the haemochromatosis-associated genotype D6S265-1:D6S105-8 in blood donors. Brit J Haematol 1996;93:838-840.
32. Beutler E. Genetic irony beyond haemochromatosis: clinical effects of HLA-H mutations. Lancet 1997;349:296-297.
33. Crawford DH, Powell LW, Leggett BA, Francis JS, Fletcher LM, Webb SI, Halliday JW, et al. Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene. Am J Hum Genet 1995;57:362-367.
- Supported by MURST 60% contributions to M.S., M.D.C., S.F., and G.F.
and by intramural funds at IRCCS Ospedale Maggiore, Milano. C.A. is the
holder of a grant from Centro Auxologico, Ospedale S.Luca, Milano;
L.L., N.C., and M.M. are supported by grants from IRCCS Ospedale
Maggiore, Milano
- From the 1Istituto di Medicina Interna e Fisiopatologia
Medica, Università di Milano and IRCCS Ospedale
Maggiore, Milano; and 2Cattedra di Medicina Interna,
Divisione di Medicina I, Ospedale S.Gerardo, Monza, Italy
- Received June 26, 1997
- Accepted October 6, 1997
- Address reprint requests to: Maurizio Sampietro, M.D., Istituto di
Medicina Interna e Fisiopatologia Medica, Istituto di Ricovero e Cura a
Carattere Scientifico Ospedale Policlinico, Padiglione Granelli,
via F. Sforza, 35, 20122 Milano, Italy. Fax:
39-2-5518-0241.
Copyright © 1998 by the American Association for the Study of Liver Diseases
- 1270-9139/98/2701-0028$3.00/0
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