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Technical Briefs |
1
Clinical Biochemistry and Clinical Genetics and
2
Dermato-Venereology, Odense University Hospital, 5000 Odense C, Denmark;
3
Department of Dermato-Venereology, Bispebjerg Hospital, 2400 Copenhagen NV, Denmark;
a author for correspondence: fax 45-6541-1911, e-mail lec{at}imbmed.ou.dk
Porphyria cutanea tarda (PCT) is the most common form of the porphyria disorders. PCT is caused by a decreased activity of the fifth enzyme in the heme biosynthetic pathway, uroporphyrinogen decarboxylase (UROD; EC 4.1.1.37). In familial PCT (fPCT), the disease is associated with mutations in the gene encoding UROD, but the majority of PCT cases are apparently sporadic (sPCT). Although clinical manifestations are predominated by cutaneous lesions, various degrees of liver damage are often associated with PCT. Clinically manifest PCT usually is provoked by exogenic factors, including alcohol, estrogens, viral hepatitis infections, HIV, and iron (1).
A mild to moderate iron overload is common in PCT, and several studies have revealed that the frequency of either of the two known HFE gene mutations associated with hemochromatosis, H63D and C282Y, is substantially higher in PCT patients than in the general population (2)(3)(4)(5)(6)(7)(8). This suggests that the inheritance of these mutations predisposes individuals to development of PCT.
Recently, another HFE gene mutation, S65C, was characterized, and analysis of a large group of hemochromatosis probands suggested that S65C may also be associated with hemochromatosis (9)(10). The purpose of the present study was to examine the HFE gene in Danish PCT patients for sequence variations, including the C282Y, H63D, and S65C mutations.
Using denaturing gradient gel electrophoresis (DGGE), we screened the entire coding region of the HFE gene in 57 unrelated PCT patients (15 with fPCT and 42 with sPCT). PCT diagnoses were based on the clinical picture and verified by biochemical findings. fPCT and sPCT cases were discriminated by mutation analysis of the UROD gene (Christianson et al., unpublished data).
Band patterns corresponding to the detected sequence variations in
HFE are shown in Fig. 1
. The DGGE analysis and subsequent sequencing revealed the
presence of the C282Y, H63D, and S65C mutations. The frequencies of the
HFE mutations in both patient groups are presented in Table 1
. In addition, a T-to-C transition in intron 2 (IVS2+4T
C) was
found in 70% of the patients and was always present in either the
heterozygous or homozygous state in H63D and S65C mutants, giving rise
to multiple band patterns in exon 2 (Fig. 1
). The IVS2+4TC mutation
previously had been published by Douabin et al. (9). Reverse
transcription-PCR analysis demonstrated that this mutation had no
effect on the splicing of the HFE mRNA (results not shown),
and thus probably represents a highly frequent polymorphism.
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No other sequence variations were found in the HFE gene in the patients included in this study. However, because the DGGE method is capable of identifying only point mutations and small deletions/insertions, the existence of larger rearrangements cannot be excluded.
The frequencies of the C282Y and H63D mutations in the general Danish population have recently been investigated by Steffensen et al. (11). They found that the estimated frequency of the C282Y mutation was 0.46% [95% confidence interval (CI), 0.2–0.9] for C282Y homozygotes and 13% (95% CI, 8.6–17.5) for C282Y heterozygotes, whereas the estimated frequency of H63D was 1.6% (95% CI, 0.9–2.7) and 22.3% (95% CI, 17.4–27.4) for homozygotes and heterozygotes, respectively (11).
When these data were compared with the results obtained in this study
(Table 1
), only the homozygous state of the C282Y mutation was
significantly increased in the fPCT patients. Interestingly, there were
neither any H63D homozygotes nor any C282Y/H63D compound heterozygotes
in this group. Analysis of the sPCT patients revealed a substantial
increase in the frequency of both C282Y and H63D homozygotes compared
with the frequencies in the general Danish population. Furthermore,
there was a slight increase in C282Y heterozygotes in the sPCT group
compared with the general population as well as the fPCT group in this
study. Four of these were C282Y/H63D compound heterozygotes.
As shown in Table 1
, we also found that four (10%; 95% CI, 2.7–22.6)
of the sPCT patients were heterozygous for the S65C mutation. However,
this mutation was also present in 3 (9%; 95% CI, 1.9–24.3) of 33
control subjects (results not shown), indicating that S65C is merely a
genetic polymorphism. As has been suggested for H63D, however, S65C may
be associated with increased iron storage if inherited in compound
heterozygosity with either of the two other HFE mutations.
This is partly supported by the fact that two of the four sPCT patients
in the present study were either S65C/C282Y or S65C/H63D compound
heterozygotes.
The presented results are in accordance with data published by others demonstrating that the frequencies of HFE mutations are increased in PCT patients compared with the general population (2)(3)(4)(5)(6)(7)(8). The observed increased frequencies of C282Y heterozygotes and H63D homozygotes in the group of sPCT patients suggest that these mutations may contribute to the development of manifest, sporadic cases of PCT. The reported findings thus imply that mutations in the HFE gene could constitute, at least in part, a genetic basis of PCT in patients lacking mutations in the UROD gene.
Acknowledgments
This work was supported by grants from the Danish Medical Research Council and the Institute of Clinical Research, Odense University. We thank A. Jensen for technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  A. A. Killeen, J. W. Breneman III, A. R. Carillo, J. Liu, and C. S. Hixson Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations Clin. Chem., July 1, 2003; 49(7): 1050 - 1057. [Abstract] [Full Text] [PDF]
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 L. Bathum, L. Christiansen, H. Nybo, K. A. Ranberg, D. Gaist, B. Jeune, N. E. Petersen, J. Vaupel, and K. Christensen Association of Mutations in the Hemochromatosis Gene With Shorter Life Expectancy Arch Intern Med, November 12, 2001; 161(20): 2441 - 2444. [Abstract] [Full Text] [PDF]
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