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Technical Briefs |
1
Zentrallabor, Stadtspital Triemli, Birmensdorferstrasse 497, CH-8063 Zurich, Switzerland
2
Porphyria Center, Institute of Hematology and Blood Transfusion, Chocimska 5, 00-957 Warsaw, Poland
3
Centre Francais des Porphyries, INSERM U 409, Hopital Louis Mourier, F-92701 Colombes, France
aauthor for correspondence: e-mail xiaoye.schneider{at}triemli.stzh.ch
Erythropoietic protoporphyria (EPP) is an inherited disorder
caused by a partial deficiency of the enzyme ferrochelatase.
Ferrochelatase catalyzes the insertion of ferrous ions into
protoporphyrin IX, the last step in heme biosynthesis. As a result of
ferrochelatase deficiency, protoporphyrin is accumulated in various
tissues; this accumulation is responsible for the clinical symptoms of
light sensitivity and, in rare cases, liver damage (1).
Several mutations have been identified in the human ferrochelatase
(FECH) gene among EPP patients (2). The results
indicate a possible link between the "null-allele" mutations (i.e.,
mutations that lead to the formation of a truncated enzyme) in the
FECH gene and liver complications in EPP (3).
Although EPP is considered an autosomal dominant disorder, only 
10%
of individuals with a defective enzyme develop clinical symptoms. A
recent study suggested that symptomatic patients differ from
asymptomatic gene carriers by the amount of mRNA output from the
nonmutated allele. This so-called "low expressed" wild-type
FECH allele, identified in EPP patients, is highly
associated with a specific haplotype at two single nucleotide
polymorphism (SNP) sites in the FECH gene namely, a G at the
-251 position in the promoter region and a T at IVS1-23
(4). These latest developments in the pathogenesis of EPP
allow prediction of the clinical course of the disease. We report here
the first study conducted in an EPP family from Poland.
The proband was a 15-year-old female patient from Masuria in the
northern part of Poland who had been suffering from photosensitivity
since early childhood. The diagnosis of EPP was established based on
the clinical symptom and laboratory analyses (Table 1
). Ferrochelatase activity in the proband was reduced to 25% of
the mean value, as is typical for EPP patients (5). A
massive increase of protoporphyrin concentration was measured in both
the red cells and feces. The patient had normal liver function.
Although asymptomatic, the proband’s father exhibited a >50%
reduction in ferrochelatase activity accompanied by a moderate increase
of protoporphyrin concentration in the red cells. Neither clinical nor
laboratory abnormalities were observed in the other two members of the
family.
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Peripheral blood samples were collected from all members of the family
for isolation of genomic DNA. All 11 coding exons as well as the
flanking intronic regions of the FECH gene were analyzed in
the genomic DNA from the proband by denaturing gradient gel
electrophoresis (DGGE) according to a method described previously
(2). An abnormal DGGE pattern was observed in exon 5 of
the FECH gene (Fig. 1A
). Direct sequencing of the PCR-amplified fragment containing
exon 5 revealed a missense mutation, T545G, which led to the conversion
of Leu-182 to Arg in ferrochelatase.
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Because T545G (L182R) was, to our knowledge, a novel missense mutation in the FECH gene, its effect on enzyme activity was examined in vitro. The L182R mutation was introduced into a wild-type FECH cDNA cloned into prokaryotic expression vector pGEX-2 (2). Ferrochelatase activity of the mutant in the crude bacterial cell extracts was 0.17 ± 0.02 U (an average of four independent clones), which corresponds to <6% of the activity of 2.85 ± 0.5 U observed with the wild-type FECH cDNA. This result demonstrated that L182R was indeed a causative missense mutation. Because no other sequence abnormalities were found in the FECH gene, L182R was likely the cause of the EPP condition in the patient. Because the patient is a carrier of a missense mutation rather than a null-allele mutation, she is highly unlikely to develop EPP-related liver complications later in life, based on current knowledge (3).
The other three members of the family were screened specifically for
the L182R mutation by DGGE. As shown in Fig. 1A
, T545
G (L182R) was
also present in the DNA sample from proband’s father but was absent in
samples from both her mother and her brother. The molecular analysis
confirmed that the proband’s father was an asymptomatic carrier of
mutation L182R.
As discussed earlier, two SNPs in the ferrochelatase gene, namely,
-251A/G in the promoter region and -23C/T in intron 1, play a role in
the pathogenesis of EPP (4). To verify the latest findings,
the two SNPs were studied in all four members of the family, using the
method described by Gouya et al. (4). Both the proband and
her mother were heterozygous for both SNPs, whereas the proband’s
father and brother were homozygous (-251A; IVS1-23C; Fig. 1B
).
Mutation L182R resided in one of the (-251A; IVS1-23C) alleles in the
father, which was passed to his daughter, the proband. From her mother,
the proband inherited a wild-type FECH allele featuring the
haplotype (-251G; IVS1-23T). According to the study of Gouya et al.
(4), the risk for a carrier of a FECH mutation to
develop EPP clinically is 60% if the person bears a wild-type
FECH allele featuring the haplotype (-251G; IVS1-23T),
which was the case in the proband. At the age of 40, the proband’s
father was asymptomatic, and he is likely to remain so throughout his
life: the likelihood that he will develop clinical symptoms is <2%
because he carries a wild-type FECH allele featuring the
haplotype (-251A; IVS1-23C). Our study has shown that molecular
analysis of the FECH gene is able not only to detect the
genetic defects underlining the EPP condition among patients and
thereby enables an accurate diagnosis of asymptomatic gene carriers,
but also is able to provide important clues on the clinical prognosis
of the affected individuals.
Acknowledgments
This work was supported by the Swiss National Science Foundation (Grant 31-53799.98).
References
The following articles in journals at HighWire Press have cited this article:
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  C. Herrero, J. To-Figueras, C. Badenas, M. Mendez, P. Serrano, R. Enriquez-Salamanca, and M. Lecha Clinical, Biochemical, and Genetic Study of 11 Patients With Erythropoietic Protoporphyria Including One With Homozygous Disease Arch Dermatol, September 1, 2007; 143(9): 1125 - 1129. [Abstract] [Full Text] [PDF]
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 S D Whatley, N G Mason, M Khan, M Zamiri, M N Badminton, W N Missaoui, T A Dailey, H A Dailey, W S Douglas, N J Wainwright, et al. Autosomal recessive erythropoietic protoporphyria in the United Kingdom: prevalence and relationship to liver disease J. Med. Genet., August 1, 2004; 41(8): e105 - e105. [Full Text] [PDF]
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, respectively. (A), DGGE
analysis of exon 5 of the FECH gene showing
abnormalities in samples from both the proband and her father.
N, control sample. (B), genotypic
analysis using two SNPs (-251A/G in the promoter region and
IVS1-23C/T). The mutation-bearing allele features the haplotype
(-251A; IVS1-23C) and is indicated by the boxed AC. The
allele that is responsible for the clinical manifestation of EPP
featuring the haplotype (-251G; IVS1-23T) is indicated by the
bold italic GT.