Download A new ferrochelatase mutation combined with low

Clinical Science (2002) 102, 501–506 (Printed in Great Britain)
A new ferrochelatase mutation combined with
low expression alleles in a Japanese patient
with erythropoietic protoporphyria
Yumiko YASUI*, Shikibu MURANAKA*, Tsuyoshi TAHARA*, Ryo SHIMIZU*,
Sonoko WATANABE*, Yutaka HORIE†, Eiji NANBA†, Hiroshi UEZATO‡,
Atsushi TAKAMIYAGI‡, Shigeru TAKETANI§ and Reiko AKAGI*
*Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja-shi 719-1197, Japan, †Faculty of
Medicine, Tottori University, Yonago 683-8503, Japan, ‡University of Ryukyus, Faculty of Medicine, Okinawa 903-0215,
Japan, and §Kyoto Institute of Technology, Kyoto 606-8585, Japan
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We investigated the molecular defect of the ferrochelatase gene in a Japanese patient with
erythropoietic protoporphyria (EPP), and identified a novel 16 base pair (574–589) deletion
within exon 5. This deletion resulted in a frame-shift mutation and created a premature stop
codon at amino acid position 198. The same molecular defect was also identified in his mother and
a brother who had symptomatic EPP, but not in his father who was asymptomatic. The subjects
with EPP were homozygous for the low expression haplotype, while his father was heterozygous
for this haplotype. These results indicate that the combination of a 16 base pair deletion and low
expression of the wild-type allelic variant is responsible for EPP in this pedigree.
INTRODUCTION
Erythropoietic protoporphyria (EPP) is known to occur
as an autosomal dominant disease, with a low clinical
penetrance, due to a reduced activity of ferrochelatase
(protoheme ferro-lyase ; EC 4.99.1.1) [1], the terminal
enzyme of the heme biosynthetic pathway, which catalyses the insertion of a ferrous iron into protoporphyrin
IX to form protoheme. Patients with EPP are clinically
characterized by painful photosensitivity due to accumulation of protoporphyrin in the skin, and high levels of
free protoporphyrin in erythrocytes, plasma and stool
[2]. While most EPP patients are associated with skin
photosensitivity only, some (5 % to 10 %) develop
hepatic failure due to massive hepatic accumulation of
protoporphyrin [3].
Since the sequence of ferrochelatase cDNA was published by Nakahashi et al. [4], more than 60 different
kinds of molecular defects of ferrochelatase have been
reported. In this study, we report the molecular cloning
and nucleotide sequencing of ferrochelatase cDNA from
a patient with EPP who was heterozygous for the gene
defect, a 16-base deletion within exon 5. This defect was
shown to be inherited from the proband’s mother, and
resulted in the production of a truncated protein.
Previously, it was reported that the low expression of
a wild-type allelic variant trans to a mutated ferrochelatase allele is generally required for clinical expression of EPP [5]. In this study, we also evaluated the
set of three intragenic polymorphisms [-251G, IVS1-23T,
IVS2µsatA9 ; G-T-A9] which were associated with the
low-expressed allele. The proband and his siblings with
EPP carried a deletion on an allele and three polymorphisms on both alleles.
METHODS
Patient and family members
The patient was a Japanese male, 27 years of age, with
cutaneous photosensitivity characteristic of EPP, which
started from early childhood. He was admitted to hospital
complaining of upper abdominal pain and jaundice,
additional to the cutaneous photosensitivity, at the age of
Key words: ferrochelatase, erythropoietic protoporphyria.
Abbreviations : EPP, erythropoietic protoporphyria ; EBV, Epstein-Barr virus.
Correspondence : Dr Reiko Akagi (e-mail akagirei!fhw.oka-pu.ac.jp).
# 2002 The Biochemical Society and the Medical Research Society
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mers. The entire coding region of ferrochelatase cDNA
was amplified by PCR, using the following two synthetic
oligonucleotides as primers. They are 5h-GAGGCTGCCCAGGCA-3h and 5h-TTTGCCTAACGCCACGGGGGT-3h, corresponding to the 5h- and 3h-untranslated
region of ferrochelatase cDNA respectively [4]. PCR was
carried out using a DNA Thermal Cycler (PerkinElmer
Cetus, Norwalk, CT, U.S.A.) with AmpliTaq DNA
polymerase (PerkinElmer Cetus), employing a thermal
cycle program, as described previously [8]. PCR was
performed three times in separate experiments, and
further steps were processed separately as well.
Cloning and sequencing of cDNA
Figure 1
Pedigree of the Japanese family with EPP
The proband is indicated by an arrow. #, normal ; $, cutaneous
photosensitivity ; *, hepatic disorder ; (age) in 1993.
21. Symptomatic EPP was also found in three generations
in the family (Figure 1). Protoporphyrin in red blood
cells (8779 µg\dl ; normal 30–86 µg\dl) and in stool
(6800 µg\dl ; normal
1830 µg\dl) were markedly elevated. Fluorescence examination of the proband’s blood,
urine and stool was positive for porphyrins, as were
blood samples from his mother and brother, who also
showed cutaneous signs of EPP. There were several
maternal relatives who had a history of hepatic disease.
The father of the proband and paternal relatives had no
signs of EPP, or liver disease. Informed consent for all
examinations, including genetic analysis, was obtained
from the patient and family members after full explanation of the purpose.
Cell cultures and Northern blot analysis
Isolation of lymphocytes, transformation of cells with
Epstein-Barr virus (EBV), and cultivation of lymphoblastoid cells were performed, as described previously
[6]. Total RNA was isolated from lymphoblastoid cells,
which were from the patient, the brother and an unrelated
control subject, according to the method of Chirgwin et
al. [7]. Total RNA (10 µg) was analysed by Northern
blot analysis, as described previously [8]. Cloned normal
human ferrochelatase cDNA was inserted into the PstI
site of pGEM 3Z vector (Promega, Madison, WI, U.S.A.).
A radioactive riboprobe of antisense ferrochelatase mRNA
was synthesized according to the method of Melton et al.
[9]. Hybridized Zeta probe filters were treated with
RNase A (1 µg\ml) for 7 min at 37 mC and then washed
under stringent conditions. Ferrochelatase mRNA was
visualized by exposing the membrane to X-ray film, and
its level was quantified using a Bio Image analyser.
Synthesis of ferrochelatase cDNA
The first strand of cDNA was synthesized using SuperScript RNaseH− reverse transcriptase (Gibco BRL,
Gaithersburg, MD, U.S.A.), primed with random hexa# 2002 The Biochemical Society and the Medical Research Society
The PCR products were purified using a Gene Clean kit
(BIO101 Inc., LaJolla, CA, U.S.A.), followed by cloning
into pGEM-T Easy Vectors (Promega, Madison, WI,
U.S.A.). DNA sequencing was carried out by the dideoxy
chain-termination method using a genetically engineered
T7 DNA polymerase (SequiThermTM Long-Read4
Cycle Sequencing Kits LC ; Epicentre Technologies,
Madison, WI, U.S.A.).
Nucleotide sequence analysis of amplified
ferrochelatase genomic DNA
Genomic DNA was isolated from lymphoblastoid cells
of the proband, the father, the mother and the brother,
and digested with EcoRI. Regions containing molecular
defects found in ferrochelatase cDNA were amplified by
PCR. The PCR was carried out using specific oligomers,
designed based on the reported sequence [10], which
were primer FC977 : 5h-tatatagGAAGCCGAAAACTGGAA-3h (intron 2–exon 3) and FC978 : 5h-caccatacCTGTGTTGGGGGA-3h (intron 4–exon 4). The PCR
primers used for the deletion are shown in Figure 3. The
region containing these intragenic polymorphisms was
amplified on genomic DNA from the proband, the father,
the mother and the brother by PCR. The primers for
-251G\A in the 5h promoter region are : 5h-AGGCAAACTGGAGAACTGA-3h and 5h-GGACAGACCCACTTACGCGGA-3h [10], 5h-GGATCCAGTGGGGAAGGAGGG-3h and 5h-GGAGGGCAGAAGGAGACTGATCG-3h for IVS1-23T\C [11] in intron 1,
and 5h-ATTGTGCCACCACACTCCAG-3h and 5hGCACCCTTAACATCAGGCAGA-3h for the dinucleotide repeat called IVS2µsatAn in intron 2 [12]
respectively. Cloning followed by sequence analysis was
carried out in the same way described above.
Expression of mutant ferrochelatase cDNA
The VspI–BamHI or BamHI–EcoRI fragment of wild-type
mature ferrochelatase cDNA in prokaryotic expression
vector pAR-HF [13] was replaced with the O1 or O4
mutant cDNA fragment respectively. They were transformed into Escherichia coli BL21 (ER3) for expression
of ferrochelatase proteins, as described previously [13].
Novel molecular defect in ferrochelatase
Western blot analysis and ferrochelatase
assay
Ferrochelatase protein expressed in E. coli was analysed
by Western blot analysis, as described previously [11].
Detection of the specific immune complex was carried
out using the ECL2 Western blotting detection system
(Amersham Life Sciences, Bucks., U.K.). Ferrochelatase
activity in the E. coli transformed with human ferrochelatase cDNA was determined by the method of Rossi
et al. [15], as described previously [13].
RESULTS
Cloning and sequencing analysis of
ferrochelatase cDNAs from the patient
Nucleotide sequence analysis of the cloned ferrochelatase
cDNA of the proband revealed three point mutations
(O1, O2 and O3) and a 16 base pair (574–589) deletion,
termed the O4 deletion. The O1 mutation (A#)( G),
which resulted in the amino acid substitution Gln*'
Arg, was detected in all the clones analysed. Both the O2
mutation (G(*) C) and the O3 mutation (G*#" A)
were present on the same allele, termed allele 1, and were
silent mutations (Figure 2). The other allele (allele 2)
contained the O4 deletion (Figure 3), which introduced a
frame-shift mutation and created a premature stop codon
at amino acid position 198 (Figure 2). The frequency
between allele 1 and allele 2 from randomly picked clones
was approximately 9 : 1 (Table 1), suggesting that the O4
deletion results in a ferrochelatase mRNA with a very
short half-life.
Pedigree studies
The O1 and O4 deletions were also confirmed in PCRamplified genomic DNA from the proband. The O4
mutation was additionally found in genomic DNA from
the proband’s mother and brother (Table 2). The same
Figure 2
mutation was also identified in an unrelated Caucasian
without EPP (results not shown). The frequency between
the normal sequence and the O4 deletion was approximately 1 : 1, in randomly picked clones from the proband,
his mother and brother (Table 2). The O4 deletion was
absent in the father (Table 2). These findings indicated
that the O4 deletion has been transmitted to the proband
from his mother.
Northern blot analysis
Since the transcript from allele 2 was suspected to be
significantly lower than that from allele 1, we carried out
Northern blot analysis to measure ferrochelatase mRNA
levels in the proband and his brother, in comparison with
a normal control. Two species of ferrochelatase mRNAs
of 2.5 kb and 1.6 kb, consistent with the two polyadenylation sites in this mRNA [4], were detected in total
RNA isolated from EBV-transformed lymphoblasts
from the proband and his brother, as well as a normal
control. The levels of the major 1.6 kb ferrochelatase
mRNAs, quantified after standardizing the rRNA level,
in the proband and his brother were approx. 10 % and
25 % compared with the normal control respectively
(Figure 4).
Identification of three intragenic
polymorphisms
Since the ferrochelatase mRNA level in the proband and
his brother was significantly lower than half of the
normal, the expression of both alleles was suspected to be
decreased. We therefore carried out an investigation of
the polymorphisms, which were reported to be strongly
associated with the low-expressed allelic variant (Figure
5) [5]. The set of three intragenic polymorphisms [G-TA9] was identified as a homozygous trait not only in the
proband but also in his mother and brother. The father
Molecular defects detected in the proband cDNAs
Nucleotide sequence analysis of ferrochelatase cDNA from the proband revealed four kinds of molecular defects. They are termed the O1, O2 and O3 mutations,
and the O4 deletion. The O1 mutation (A287 G) was detected in all clones. One allele, termed allele 1, carried the O2 and O3 mutations, which were silent
mutations. The O4 deletion was detected on the other allele, termed allele 2.
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Table 2 Detection of the O4 deletion in genomic DNA from
family members
The number of clones, of which sequence analysis confirmed the O4 deletion, versus
clones analysed is shown.
Proportion of clones with the O4 deletion
Proband
Father
Mother
Brother
Figure 4
4/8
0/6
2/6
2/5
Ferrochelatase mRNA from lymphoblastoid cells
Ferrochelatase mRNA was examined by Northern blot analysis, as described in the
Materials and methods section. (A) Ferrochelatase mRNA. (B) Ethidium bromidestained agarose gel showing rRNA profiles. Lane 1, proband ; lane 2, brother ; lane
3, normal control.
Figure 3
O4 deletion in genomic DNA
(A) Partial sequence of ferrochelatase from proband’s genomic DNA is shown.
Bold letters in the square show the position of the 16 base pairs (574–589). (B)
DNA sequence of ferrochelatase exons 5 and 6 (upper-case letters) and intron 5
(lower-case letters), surrounding the O4 deletion. Primer FC971 (570 " 559 in
exon 5) and primer FC972 (629 " 674 in exon 6) were used for the first PCR
using genomic DNA from the normal subject as a template. On the basis of the
nucleotide sequence obtained from the first PCR, primer FC979 (k1 "k233
in intron 5) was designed as the primer for PCR on genomic DNA.
Table 1 Proportion of two allelic products detected in cDNA
from the proband
The number of two allelic products detected in the proband ’s cDNAs is shown for
each separate library obtained by three separate experiments. Characteristics of
allele 1 and allele 2 are shown in Figure 2.
Proportion of two allelic products detected
Allele
Library 1
Library 2
Library 3
Total
1
2
5/6
1/6
12/12
0/12
14/17
3/17
31/35 (89 %)
4/35 (11 %)
was heterozygous for the set of polymorphisms, indicating that the proband and his brother inherited a wildtype allele with low expression from their father, and a
mutant allele from their mother.
# 2002 The Biochemical Society and the Medical Research Society
Figure 5 The co-inheritance of a low-expressed ferrochelatase haplotype and a mutant allele
Haplotypes that are associated with the low-expressed alleles are in the dotted
box, and those associated with the O4 mutant are in the black box. EPP patients
are indicated with arrows.
Ferrochelatase activity expressed by the
mutant cDNAs
To determine the effect of the O1 and O4 defects on
enzyme activity, mutant ferrochelatase cDNA was
expressed by transfection into E. coli cells. The expressed
ferrochelatase activities were compared with the activities
obtained by transfection of the normal ferrochelatase
cDNA. Human ferrochelatase protein expressed in E.
Novel molecular defect in ferrochelatase
Expression of human ferrochelatase in E. coli
Transfection and expression of normal, O1 and O4 mutant ferrochelatase cDNAs in
E. coli were carried out as described in the Material and methods section. Upper
panel : the amount of ferrochelatase expressed was determined by Western blot
analysis. Lower panel : enzyme activity was expressed as the activity/mass ratio,
standardized by the normal control. ‘ Mock ’ indicates E. coli transfected with
plasmid only.
Figure 6
coli cells was specifically detected and quantified by
Western blot analysis using a polyclonal antibody, which
reacts with the human ferrochelatase but not with E. coli
ferrochelatase. Human-specific ferrochelatase activity
was calculated as an activity versus mass ratio (A\M).
The O1 mutant showed 91.8 % of the A\M ratio
compared with that expressed by the normal ferrochelatase cDNA (Figure 6). Mutant ferrochelatase carrying the O4 deletion expressed no significant immunological protein, with null activity.
DISCUSSION
In this study, we demonstrated a novel molecular defect
of the ferrochelatase gene in a Japanese male with EPP. A
total of four mutations in the ferrochelatase gene have
been identified in this subject. While the O2 and O3
mutations were silent, thus representing sequence polymorphisms, the O1 mutation and O4 deletion accompanied an amino acid substitution and frame-shift mutation respectively.
The O1 mutation, which resulted in an amino acid
substitution, has been identified not only in the symptomatic proband, his mother and brother, but also in the
asymptomatic father. It was also found in an unrelated
healthy Caucasian without any symptoms. Thus this
mutation is unlikely to be related to EPP in this family. In
fact, mutant cDNA containing the O1 mutation expressed in E. coli showed a significant amount of
ferrochelatase protein with normal activity (Figure 6).
In contrast, the O4 deletion, which is a 16 base pair
deletion involving nucleotide base pairs 574–589, and
results in translation into a truncated protein, is exclusively found in subjects with EPP, but not in the
asymptomatic father in this family. Thus it is highly
likely that the O4 deletion is responsible for EPP in this
family. Our expression studies in E. coli cells clearly
showed that the mutant ferrochelatase with the O4
deletion was translated as no immunologically detectable
protein or enzyme activity (Figure 6). The O4 deletion,
which results in six abnormal amino acid residues after
amino acid 192, caused a frame-shift mutation and formed
a premature stop codon at amino acid position 198.
As a result, allele 2 is translated as an abnormally short
protein lacking a large portion of its C-terminal region
(Figure 2). This C-terminal region is known to contain
various residues, or sites essential for the function
of ferrochelatase. It has been reported that His#'$ in
ferrochelatase is essential for metal binding, and that
Asp#%' and Tyr#%) are also involved in metal binding
in a synergistic manner [14]. His$)) was reported to
contribute to the maintenance of the structure of the
protein molecule [16]. Besides, it is known that human
ferrochelatase contains critical cysteine residues at
positions 403, 406 and 411, which comprise a [2Fe–2S]
cluster, essential for enzyme activity [17,18]. This information suggests that the truncated protein, due to the
O4 deletion in allele 2, does not have significant ferrochelatase activity because of the lack of a number of
amino acids which are essential for enzyme activity.
It was reported that ferrochelatase mRNA levels
decreased by haem depletion [19], thus impaired haem
biosynthesis in the EPP patient might cause the failure to
maintain ferrochelatase mRNA levels. Besides, the existence of a premature stop codon in the O4-derived mRNA
is likely to reduce its stability. Ferrochelatase mRNA in
the patient transcribed from allele 2 is unstable, because
(i) the frequency of allele 2-derived transcript from
the proband cDNA is approximately 10 % compared
with allele 1 (Table 1), and (ii) Northern blot analysis
of total RNA prepared from lymphoblastoid cells
showed a significant decrease in ferrochelatase mRNA
in the proband and his brother (Figure 4). Although the
proband and his brother were suspected to be heterogeneous in the molecular status, ferrochelatase mRNA
levels, which were expected to be decreased to half, were
about 10 % and 25 % compared with that of cells from a
normal control (Figure 4). The result suggests that the
low expression is not only due to the allele 2 having the
O4 deletion, but also due to the normal allele 1. Recently,
it has been reported that clinical expression required
# 2002 The Biochemical Society and the Medical Research Society
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coinheritance of a normal ferrochelatase allele with low
expression together with a mutant ferrochelatase allele
[5]. The proband, his mother and brother with EPP
symptoms had the three polymorphisms [G-T-A9] of
ferrochelatase, which were shown to bring low expression of the ferrochelatase allele (Figure 5). The father
was heterozygous for the three polymorphisms, indicating that it was inherited from the father to the sons,
because they carried the mutant allele from their mother.
It was characteristic for the patient and his symptomatic
brother to develop this severe hepatic disorder. The
molecular defects leading to a null allele were reported to
be a common feature among EPP pedigrees with liver
complications [20]. Since the novel mutant ferrochelatase
found in this family expressed null enzyme activity in E.
coli (Figure 6), it might be reasonable to suppose that the
massive accumulation of protoporphyrin caused by
markedly decreased ferrochelatase activity induced progressive liver failure.
Although the mechanism of low expression remains
unclear, the frequency of the low expression ferrochelatase allele in the asymptomatic Caucasian population is estimated to be from 6.5 to 11.5 % [5]. The
extensive analysis of the relationship between clinical
consequences and ferrochelatase deficiency by
Schneider-Yin et al. [21] clearly showed that the combination of a mutated allele and a low expressed normal
allele is responsible for the clinical complications observed in the patient with EPP. Moreover about 1 % of
the population was supposed to be homozygous. This is
the first report of the three polymorphisms being
identified in a homozygous trait, together with a unique
frame-shift mutation caused by a 16 base pair deletion
within exon 5 of the ferrochelatase gene from Japanese
patients with EPP.
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ACKNOWLEDGMENT
We thank Dr Shigeru Sassa (The Rockefeller University,
New York, U.S.A.) for critical reading of the manuscript.
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Received 19 July 2001/8 October 2001; accepted 14 January 2002
# 2002 The Biochemical Society and the Medical Research Society