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Human Molecular Genetics Advance Access originally published online on September 13, 2005
Human Molecular Genetics 2005 14(20):3089-3098; doi:10.1093/hmg/ddi342
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mutations in human CPO gene predict clinical expression of either hepatic hereditary coproporphyria or erythropoietic harderoporphyria

Caroline Schmitt1,2, Laurent Gouya1, Eva Malonova1,3, Jérôme Lamoril1, Jean-Michel Camadro4, Magali Flamme5, Christian Rose6, Said Lyoumi1, Vasco Da Silva1, Catherine Boileau2, Bernard Grandchamp1, Carole Beaumont1, Jean-Charles Deybach1,* and Hervé Puy1,2

1INSERM U656 and Centre Français des Porphyries, Université Paris VII, Hôpital Louis Mourier, 92701 Colombes, France, 2Laboratoire de Biochimie et Génétique Moléculaire, Hôpital Ambroise Paré, Faculté de Médecine Paris – Ile de France Ouest, 92100 Boulogne-Billancourt, France, 3Department of Pediatrics, Faculty of Medicine I, Charles University, Praha, Czech Republic, 4Laboratoire d'Ingénierie des Protéines et Contrôle Métabolique, Département de Biologie des Génomes, Institut Jacques Monod, CNRS UMR7592, Université Paris VI et VII, 75005 Paris, France, 5Service de Dermatologie, Cliniques Universitaires Saint-Luc, 1200 Bruxelles, Belgium and 6Service des Maladies du Sang, Hôpital Claude Huriez, 59037 Lille, France

* To whom correspondence should be addressed at: Centre Français des Porphyries – INSERM U656, Hôpital Louis Mourier, 92701 Colombes Cedex, France. Tel: +33 147606331; Fax: +33 147606703; Email: jean-charles.deybach{at}lmr.ap-hop-paris.fr

Received July 4, 2005; Accepted September 7, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Hereditary coproporphyria (HCP), an autosomal dominant acute hepatic porphyria, results from mutations in the gene that encodes coproporphyrinogen III oxidase (CPO). HCP (heterozygous or rarely homozygous) patients present with an acute neurovisceral crisis, sometimes associated with skin lesions. Four patients (two families) have been reported with a clinically distinct variant form of HCP. In such patients, the presence of a specific mutation (K404E) on both alleles or associated with a null allele, produces a unifying syndrome in which hematological disorders predominate: ‘harderoporphyria’. Here, we report the fifth case (from a third family) with harderoporphyria. In addition, we show that harderoporphyric patients exhibit iron overload secondary to dyserythropoiesis. To investigate the molecular basis of this peculiar phenotype, we first studied the secondary structure of the human CPO by a predictive method, the hydrophobic cluster analysis (HCA) which allowed us to focus on a region of the enzyme. We then expressed mutant enzymes for each amino acid of the region of interest, as well as all missense mutations reported so far in HCP patients and evaluated the amount of harderoporphyrin in each mutant. Our results strongly suggest that only a few missense mutations, restricted to five amino acids encoded by exon 6, may accumulate significant amounts of harderoporphyrin: D400–K404. Moreover, all other type of mutations or missense mutations mapped elsewhere throughout the CPO gene, lead to coproporphyrin accumulation and subsequently typical HCP. Our findings, reinforced by recent crystallographic results of yeast CPO, shed new light on the genetic predisposition to HCP. It represents a first monogenic metabolic disorder where clinical expression of overt disease is dependent upon the location and type of mutation, resulting either in acute hepatic or in erythropoietic porphyria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Human porphyrias are a group of inborn errors of heme biosynthesis that are classified as hepatic or erythropoietic according to clinical data and to the main site of expression of the specific enzymatic defect (Fig. 1). Of all autosomal dominant acute hepatic porphyria, hereditary coproporphyria (HCP) is the least common, with incomplete penetrance due to a partial deficiency of coproporphyrinogen III oxidase (CPO; EC 1.3.3.3 [EC] ). Human CPO is a mitochondrial enzyme encoded by a 14 kb CPO gene containing 7 exons located on chromosome 3q11.2 (1Go). The enzyme catalyses the stepwise oxidative decarboxylation of the heme precursor, coproporphyrinogen III to protoporphyrinogen IX, via a tricarboxylic intermediate known as ‘harderoporphyrinogen’ (Fig. 2A) (2Go). Two phenotypically distinct disorders related to the deficient enzyme have been described: HCP, including its homozygous variant form [MIM 121300 [OMIM] ] and harderoporphyria (3Go–6Go). The molecular relationship between the single CPO gene and these two different phenotypes have not yet been defined and the biochemical basis remains unexplained. In HCP, clinical penetrance is low and symptoms are very rare before puberty. Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often provoked by drugs, fasting, menstrual cycle or infectious diseases (7Go,8Go). Skin photosensitivity may also be present. Excretion of large amounts of coproporphyrin III, mostly in feces and urine, is observed. Over 44 mutations in the CPO gene have been identified in HCP families (Human Gene Mutation Database, http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html). So far, mutations are family specific without any hotspot or phenotype/genotype correlations (9Go).



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  		Figure 1. Heme biosynthetic pathway. Hepatic porphyrias are written in white on a grey background; erythropoietic porphyrias are written in white on a black background. ALA, {delta}-aminolevulinic acid; PBG, porphobilinogen; URO'gen, uroporphyrinogen; COPRO'gen, coproporphyrinogen; PROTO'gen, protoporphyrinogen.

 


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  		Figure 2. (A) Stepwise decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX catalyzed by CPO. (B) High-performance liquid chromatography of porphyrins in stool of a healthy individual (N), an HCP patient and the harderoporphyric proband. P, protoporphyrin; H, harderoporphyrin; C, coproporphyrin.

 
Harderoporphyria is a rare erythropoietic variant form of HCP, characterized by neonatal hemolytic anemia, sometimes accompanied by skin lesions and accumulation of harderoporphyrin in feces (6Go,10Go,11Go). During childhood and adulthood, a mild residual anemia is chronically observed. The four affected individuals described so far are all homozygous or compound heterozygous, suggesting a recessive mode of inheritance of harderoporphyria. Clinically, harderoporphyric patients are quite different compared with homozygous HCP patients (hematological versus neurological and/or dermatological symptoms, respectively). In harderoporphyria, all patients reported to date are all homoallelic or heteroallelic for the same missense mutation in the CPO gene (K404E encoded by exon 6), suggesting that disruption of this region of CPO impairs the second stage of the conversion of coproporphyrinogen III to protoporphyrinogen IX (11Go). Interestingly, a heterozygous R401W mutation was found in a British patient with HCP. However, heterologous in vitro expression of the mutated protein induced a substantial 44% accumulation of harderoporphyrinogen rather than coproporphyrinogen (9Go). In this study, we investigated a new case of harderoporphyria in a Belgian family and evaluated iron metabolism in this patient as well as in one harderoporphyric patient previously reported. Furthermore, we performed expression study of all missense mutations reported so far in the CPO gene and of additional mutants generated by site-directed mutagenesis. On the basis of the results, we propose that amino acids D400–K404 encoded by exon 6 constitute the active site implicated in the second decarboxylation step and that only patients with mutation in this region will develop harderoporphyria.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
A new patient with harderoporphyria
The patient developed a neonatal icterus during ~3 months, resolving spontaneously. During adulthood, this evolved towards a mild- and well- tolerated anemia with episodic, spontaneous, photosensitive cutaneous lesions. An atypical profile of porphyrin excretion was found in feces with massive accumulation of harderoporphyrin (Table 1; Fig. 2B). Residual CPO activity of the patient was 18% of wild-type activity (Fig. 3). We identified a homozygous A to G transition at nucleotide 1210 in exon 6 of the CPO gene resulting in a lysine to glutamate substitution at position 404 in the abnormal protein (K404E). The brother and two children of this patient were heterozygous for the K404E mutation and were asymptomatic. This mutation has already been found in the first reported harderoporphyric patients (10Go).


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  		Table 1. Porphyrin data from harderoporphyric patients
 


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  		Figure 3. Family pedigree (solid symbols, patient). In parenthesis is the residual lymphocyte CPO activity expressed in percentage. NA, not available. Family 1 has previously been described in (6Go) and family 2 in (11Go).

 
Harderoporphyric patients have a moderate iron overload
In the course of our study, one of the four previously reported patients was studied in our porphyria centre. We noticed that, together with the new patient reported previously, they both presented with iron loading anemia with high serum ferritin level (Table 2). HFE genotype was normal, except for our new patient who was H63D homozygous. The clinical symptoms were reminiscent of hemochromatosis type 4 [MIM 606069 [OMIM] ] due to ferroportin/SLC40A1 mutations. We sequenced all the exons and intron–exon junctions of the ferroportin gene in both patients and found no mutation. The patient from reference (6Go) showed the most severe iron overload (serum ferritin=1438 µg/l), confirmed by the quantification of liver iron by MRI (107 µmol/g of liver tissue, N<36). Etiologies of iron loading anemia were explored for this caucasian patient. Hemoglobin electrophoresis was normal and no mutation in {alpha} and ß globin were found. Bone marrow examination showed dyserythropoiesis: increased erythroblasts (38%) with high morphologic abnormalities. Congenital dyserythropoietic anemia type I, II or III were discarded in the absence of specific morphologic features. Perls staining revealed increased sideroblasts (65%, without ringed sideroblasts) and highly active and iron overloaded macrophages. Erythrocyte survival half-time, determined by using autologous red blood cells labeled with chromium 51, was lower (21 days, N=28) with 2% hemolysis (N=0.83%). Ferrokinetic study with 59Fe revealed only 51% of red blood cell iron utilization (N=70%). We concluded that the iron overload observed is secondary to the marked dyserythropoiesis. The patient was treated with deferoxamine for 1 year, which normalized its serum ferritin and hepatic iron content (23 µmol/g of liver tissue).


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  		Table 2. Hematological data and iron investigation from harderoporphyric patients
 
Identification of the domain involved in the conversion of harderoporphyrinogen to protoporphyrinogen IX
Hydrophobic cluster analysis (HCA) is a method used to investigate the basis of protein stability and folding (12Go–14Go). First, the 1D-sequence of a globular protein is written in an {alpha}-helical bidimensional representation. Hydrophobic amino acids are not distributed randomly but form clusters which correspond to the positions of regular secondary structure ({alpha}-helices or ß-strands) (15Go). Therefore, the 2D-structure of a protein sequence can be predicted from the examination of the HCA plot. The HCA plot of CPO exhibited a region around the K404 amino acid which formed a hinge between two secondary structures (Fig. 4A). This region is highly conserved throughout evolution (Fig. 4B). We decided to study the five amino acids (D400, R401, G402, T403 and K404) of this region and three surrounding amino acids (F395, Y399 and F405).



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  		Figure 4. Secondary structure and sequence alignment of CPO. (A) HCA plot of human CPO between amino acids 340 and 454. The CPO sequence is written in an {alpha}-helix that is duplicated to restore the full environment of each amino acid. Hydrophobic clusters of amino acids are in grey and predicted secondary structure elements in the protein. The arrow indicates how to read the 1D-sequence in HCA representation. (B) Comparison of amino acid sequences from human (H. sapiens), mouse (M. musculus), Glycine max (G. max), Nicotiana tabacum (N. tabacum), Hordeum vulgare (H. vulgare), Escherichia coli (E. coli), Salmonella typhimurium (S. typhi.) and Saccharomyces cerevisiae (S. cerevisiae). Highlighted in green are amino acids tested by site-directed mutagenesis and resulting in mutant proteins expressed in E. coli that accumulated high level of harderoporphyrin. H, position where a mutation has previously been described in patients and resulting in mutant enzyme that accumulate harderoporphyrin in vitro (9Go,10Go). (C) Secondary structure of yeast CPO (carboxyterminal end). H, {alpha}-helix; S, ß-strand. Adapted from Phillips et al. (23Go). (D) Stereo view ribbon diagram showing closed conformation of yeast CPO. Helices H2 and H8 move to enclose the active-site cavity. Compared with human CPO, H8 contains amino acids 400–408. Adapted from Phillips et al. (23Go).

 
Heterologous expression of mutated CPO
The eight amino acids of interest located in the region encoded by exon 6 of the CPO gene (F395, Y399, D400, R401, G402, T403, K404 and F405) were individually mutated by site directed mutagenesis using pGEX-2T:CPO bacterial expression vectors and the mutant proteins were expressed in E. coli. It has already been shown that when CPO is expressed as a GST-fusion protein, its enzymatic activity is not affected significantly (16Go). For the six amino acids Y399–K404, we introduced mutations that modified the polarity and/or the charge of the amino acid. This selection was made with the help of the Dayhoff's mutation odds matrix (17Go,18Go). The Y399L mutant showed only a minor defect (81% of residual activity compared with wild-type CPO) (Table 3). The mutation of the five amino acids in the region of interest (D400–K404) resulted in variable loss of enzymatic activity with major harderoporphyrin accumulation. F395 and F405 were replaced by a glycine which was expected to destabilize secondary structure. This resulted in loss of enzymatic activity (4 and 1% of residual activity, respectively) without significant harderoporphyrin accumulation. To further document whether harderoporphyrin accumulation may be restricted to mutation introduced in this region, pGEX-2T:CPO expression vectors were constructed for each mutant allele reported so far in human patients and expressed in E. coli. Table 4 shows that the CPO activities of the mutant proteins, determined by measuring protoporphyrin(ogen) formation, were 1–66% of the wild-type activity, for all the mutations. Protoporphyrin(ogen) was the main reaction product in all cases (>80% compared with harderoporphyrin ≤12%), with the notable exception of the two missense mutations R401W and K404E encoded by exon 6 which caused substantial accumulation of the tricarboxylic intermediate harderoporphyrinogen (44 and 28%, respectively; Table 4).


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  		Table 3. E. coli expression of mutant in exon 6 of CPO cDNAs
 

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  		Table 4. Expression of all missense mutant CPO cDNAs in E. coli and overall mutations in the exon/intron 6 CPO gene reported so far in human
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this study, we report clinical and molecular investigations in a third family with harderoporphyria, presenting a proband homozygous for the K404E mutation. The patient displayed symptoms and signs of anemia and chronic skin lesions. The pattern of porphyrin excretion showed that the major part of fecal porphyrin was harderoporpyhrin. In addition, total porphyrins were increased in plasma. Enzymatic studies of CPO activity in lymphocytes showed a 18% residual activity compatible with a homozygous deficient CPO gene. Clinical and biological patterns were similar to that observed in the four harderoporphyric patients previously reported (6Go,10Go,11Go). It must be emphasized that abdominal pain and neurological symptoms, suggestive of acute hepatic porphyrias, have not been seen in harderoporphyric patients. For this reason, these patients have never been treated by heme arginate injections, which is the main therapy used in acute hepatic porphyrias. For the first time, we report that harderoporphyric patients are iron overloaded. It is likely that harderoporphyria is an iron loading anemia. Two mechanisms could contribute to the hematological phenotype observed. Firstly, dyserythropoiesis occurring in bone marrow could be related to harderoporphyrin accumulation and/or heme deficiency in red blood cell progenitors. Furthermore, maturation arrest of erythroid precursors is known to stimulate intestinal iron-absorption. Secondly, overproduction of porphyrins may account for hemolytic symptoms. Indeed, hemolysis of erythrocytes could result from photolysis of porphyrin-laden cells exposed to light in the dermal capillaries (11Go). This intravascular hemolysis should worsen the anemia, but not participate to the iron overload, because intravascular hemolysis does not stimulate intestinal iron-absorption (19Go). In our study, molecular analysis of the new patient showed a homozygous point mutation, resulting in a lysine to a glutamic acid substitution (K404E), previously reported in harderoporphyria at the homozygous state or associated with a null allele (10Go,11Go). The neighbouring mutation R401W has been identified at the heterozygous state in a British woman with a typical HCP. However, the R401W mutant enzyme expressed in a prokaryote system showed a 75% residual activity (compared with wild-type CPO) and a 44% harderoporphyrin accumulation. Both mutations lie in a highly conserved region of CPO, encoded by exon 6, that may be adjacent or form part of its active site (11Go) and may impair decarboxylation of harderoporphyrinogen (9Go,20Go). So far, only four other mutations in exon 6 have been found in HCP: two lead to frameshift mutations resulting in a probable truncated protein and two point mutations result in exon 6 skipping which leads to a null activity in in vitro expression study (11Go). These observations lead us to investigate the bidimensional structure of the CPO domain encoded by exon 6. Secondary structure prediction by HCA revealed a hinge zone between two secondary structures extending from amino acids 400 to 404. The systematic analysis, by site directed mutagenesis, of the five amino acids of this region (D400–K404) resulted in variable loss of enzymatic activity with major harderoporphyrin accumulation, whereas only mild harderoporphyrin accumulation was observed when the surrounding amino acids were mutated. Moreover, we have expressed in vitro, all the 21 missense mutations reported so far in HCP and harderoporphyric patients and measured the porphyrins produced. R401W and K404E are the only two that mainly affect the second decarboxylation reaction during the conversion of coproporphyrinogen III to protoporphyrinogen IX. Our data strongly support the hypothesis that the short region extending from amino acids 400 to 404 predicted by HCA and encoded by exon 6 is important for catalysis of the oxidative decarboxylation of harderoporphyrinogen. This region may be important in preventing diffusion of harderoporphyrinogen away from the catalytic site during the sequential reaction.

Human CPO is a 76 kDa protein that is active as a homodimer in the mitochondrial intermembrane space (16Go). However, because of the lack of crystallographic data, little structural information about the human CPO enzyme is available. A catalytic model has first been proposed by Elder et al. (2Go) and has recently been refined by Lash et al. (21Go,22Go). The active site of CPO is presented with three individualized sites: one as a binding site for a propionate side chain, one representing the catalytic site where oxidative decarboxylation of a propionate occurs and a steric site for a small non-polar unit (methyl or vinyl). In this model, both oxidative decarboxylations of coproporphyrinogen occur at the same active site after a rapid 90° anti-clockwise rotation of the reaction intermediate, harderoporphyrinogen. The substrate, interacting with the enzyme surface, does not leave the enzyme cleft. This model could explain why the first decarboxylation step is rate-limiting. However, a loss of harderoporphyrinogen from the cleft could occur when an excess of coproporphyrinogen is present. Kinetic studies have shown that in harderoporphyric patients, the abnormal CPO has a reduced affinity for harderoporphyrinogen which may leave the enzyme cleft more easily and this may account for its accumulation in patients (6Go). Recently, Phillips et al. (23Go) published the crystal structure of Saccharomyces cerevisiae CPO. Whereas the yeast enzyme resides in the cytosol, it shares 52% homology with human CPO. This study supports the catalytic model of Elder et al. refined by Lash et al. (2Go,21Go,22Go). The yeast enzyme adopts a dimeric conformation with two independent active sites, one per monomer. In yeast, two different crystal forms have been isolated depending on the opened or closed conformation of the active site. The closed conformation is induced by binding of the substrate. This substrate-induced conformational change is mediated by the movement of two helices (H2 and H8) which form a lid over the active site resulting in a buried substrate-sized cavity. Alignment of the amino acid sequences of yeast and human CPO reveals that our region of interest between amino acids D400–K404 is located in the H8 helix (Fig. 4C and D). Consequently, we can speculate that mutations affecting H8 helix could decrease the affinity of CPO for harderoporphyrinogen, as observed in harderoporphyric patients (6Go). Indeed, Philips et al. (23Go) hypothesised that the K404E mutation could destabilize the binding site by H-bonds CO groups at C-terminus of the helix and that the R401 in human (R275 in yeast) and D400 (D274 in yeast) could be involved in the active site.

In human porphyrias, we establish the existence of a phenotype/genotype relationship in the human CPO gene; we support the catalytic model of Lash et al. and demonstrate that harderoporphyrin accumulation is restricted to five amino acid substitutions at position 400–404. Our results, supported by recent crystallographic data of yeast CPO, demonstrate that the type and location of the mutations in the CPO gene modulate the phenotype. This molecular and protein relationship strongly suggests that mutations in human CPO gene predict the clinical outcome of the disease, with either hepatic hereditary coproporphyria or hematological manifestations of erythropoietic harderoporphyria.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Case report
Procedures involving human subjects were performed in accordance with the 1983 revised version of the Declaration of Helsinki and informed consent was obtained from all subjects prior to their inclusion in the study. The patient, a 39-year-old woman, was born at term of healthy, non-consanguineous Belgium parents. Shortly after birth, she developed a severe icterus which disappeared spontaneously at 3 months of age; she remained slightly icteric afterwards. No diagnosis was established. Hepatic porphyria was suspected when the patient was 33 years old because of skin lesions associated with chronic anemia. The cutaneous lesions were characterized by prurigo, hyperpigmentation and milia localized on light-exposed areas of the backs of the hands and on the arms and face. On these locations, she complained of itching for 6 months which was worsened by alcohol intake. The patient had increased skin fragility, but no hypertrichosis, alopecia or porphyrin-rich gall stones were found. She was taking oral contraceptive which was halted consequently. Microscopic examination from a cutaneous biopsy showed no specific lesions, only hyperplasic epidermis, hypergranulosis, hyperkeratosis with irregular acanthosis, hypoderm and derm inflammation with predominant polynuclear cells. Immunofluorescence microscopy was negative. The porphyrin and hematological data are summarized in Tables 1 and 2. The patient is the second of two siblings and has two healthy children (Fig. 3). In her both parents and in her brother, hematological data were normal (data not shown). The proband and her healthy relatives never exhibited abdominal and/or neurological symptoms typical of acute hepatic porphyrias.

Iron metabolism investigations
Serum ferritin, transferrin and iron levels were determined by automated turbidimetry/nephelemetry or spectrophotometry using standard procedures (Dade Behring) on a RXL or an ACS analyzer (Bayer). Genomic DNA was extracted from peripheral blood. Each of the 8 exons of the ferroportin gene was amplified and sequenced with intronic primers, as previously described (24Go). The cysteine 282 tyrosine (C282Y) HFE mutation encoded by exon 4 and the histidine 63 aspartic acid and serine 65 cysteine (H63D, S65C) mutations encoded by exon 2 were detected using PCR amplification with primers and restriction enzyme digestion, as already described (25Go).

Porphyrin synthesis investigations
Erythrocyte, plasma, urinary and fecal porphyrins were determined using standard methods (26Go,27Go). Lymphocyte CPO activity was measured as described (28Go). CPO protein has a low stability in lymphocytes so that decay of enzyme activity could be observed if a more than a 1 h delay occurred between blood sampling, lymphocyte isolation and storage at –80°C (20Go).

DNA preparation, amplification by PCR, DGGE analysis and DNA sequencing
Genomic DNA from the proband, her children and brother were extracted from peripheral blood according to a standard protocol. Genomic DNA fragments of interest were amplified by PCR using primers selected from the published CPO sequence (1Go). Briefly, 20 pmol of each set of primers was mixed in 50 µl of PCR solution containing 1 U of Taq polymerase (Beckmann Inc., Fullerton, CA, USA), 50 mmol/l KCl, 10 mmol/l Tris–HCl pH 8.5, 1.5 mmol/l MgCl2, 200 mmol/l of each dNTP. Reactions were performed in a DNA thermocycler (Hybaid, Teddington, UK) as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at specific temperature for 30 s and elongation at 72°C for 1 min. Mutation screening was realized by denaturing gradient gel electrophoresis (DGGE) and abnormal patterns sequenced to identify mutations and polymorphisms as already described (20Go). For the DNA sequencing, CPO gene was amplified using primers different from those used for the screening analysis (20Go).

Structure of CPO protein and identification of the domain involved in the conversion of harderoporphyrinogen to protoporphyrinogen IX
To predict the secondary structure of the CPO enzyme, we performed the sensitive 2D-method HCA (reviewed in 12Go) which highlights the correspondence between amino acid sequence and secondary structures, whether {alpha}-helices or ß-strands (15Go). HCA plot of the enzyme was generated with the DrawHCA program (available at smi.snv.jussieu.fr/hca/hca-form.html).

Construction and prokaryotic expression of normal and mutated human CPO cDNA
Normal human CPO was expressed using the pGEX-2T:CPO expression vector (Pharmacia Biotech, Uppsala, Sweden) as already described (29Go). Point mutations were introduced into the pGEX-2T:CPO vector, using the QuickChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Each expression construct was sequenced to ensure that only the desired mutation had been introduced and that the remainder of the sequence was correct.

CPO assay
The recombinant bacteria (Epicurian Coli® XL-1 blue) were grown and CPO activities of controls and mutant enzymes were determined in bacteria lysates as previously described (10Go). Specific activity was expressed in pmol of protoporphyrin/h/mg protein at 37°C and results are the mean of three duplicate experiments. Residual activity was determined by the formula:

Some lysates were purified with the RediPack GST purification module (Amersham Pharmacia Biotek, Uppsala, Sweden) according to the manufacturer's protocol. To control for the purification, eluates were checked by SDS-PAGE, followed by Coomassie blue staining (30Go) (Supplementary Material). No significant differences were observed before or after purification concerning the residual activities or percentages of harderoporphyrin accumulation between mutated and normal enzymes.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
This research was supported by Contrat Barrande 2004 to 2005 no. 09149ZE Programme d'actions intégrées Ministère de la Recherche, by GIS-Maladie rares 2005 research network on rare disorders and C.S. was supported by a grant from the FRM (Fondation pour la Recherche Médicale). The authors would like to thank Sylvie Simonin for her excellent technical work, Samantha Parker and Nadine Lemaire for their help in preparing the manuscript and John Phillips for his kind permission of reproducing a previously published figure (Fig. 4D), order 1322284 at the Republication Licensing Service, Copyright Clearance Center.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Delfau-Larue, M.H., Martasek, P. and Grandchamp, B. (1994) Coproporphyrinogen oxidase: gene organization and description of a mutation leading to exon 6 skipping. Hum. Mol. Genet., 3, 1325–1330.[Abstract/Free Full Text]

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  5. Nordmann, Y. and Deybach, J.C. (1990) Human hereditary porphyrias. In Dailey, H.A. (ed.), Biosynthesis of Heme and Chlorophylls. McGraw-Hill, NY, pp. 491–502.

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