Am. J. Hum. Genet. 50:1203-1210, 1992
A Molecular Defect in Human Protoporphyria D. A. Brenner,* J. M. Didier,* F. Frasier,* S. R. Christensen,* G. A. Evans,t and H. A. Dailey$ *Department of Medicine and the Center for Molecular Genetics, University of California, San Diego, and tThe Salk Institute, La Jolla; and
$Department of Microbiology and the Center for Metalloenzyme Studies, University of Georgia, Athens
Summary
Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs, resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria. Introduction
Protoporphyria is generally a human autosomal dominant disease that is characterized biochemically by elevated protoporphyrin levels in the serum, erythrocytes, and feces and clinically by photosensitivity and hepatobiliary disease. A rare, but severe, clinical sequela of protoporphyria is liver failure leading to liver transplantation or death (Bloomer et al. 1975, 1989; Rademakers et al. 1990; Herbert et al. 1991). In patients with protoporphyria, activity of the enzyme ferrochelatase (protoheme ferro-lyase; E.C.4.99.1.1) is reduced to 15%-25% of normal in all tissues, although the concentration of immunoreactive ferrochelatase protein is unchanged (Straka et al. 1991b). Ferrochelatase catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron into protoporphyrin IX to form heme. In protoporphyria, the substrate for ferrochelatase, protoporphyrin, accumulates behind the partial enzyme block. The marked accumulation of protoporphyrin in the blood causes photosensitivity, and the clearance of excess protoporphyrin via the liver results in the formation of Received December 16, 1991; revision received February 10, 1992.
Address for correspondence and reprints: David A. Brenner, M.D., Department of Medicine, UCSD, La Jolla, CA 92093-0623. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/ 92/5006-0007$02.00
hepatic and biliary protoporphyrin crystals, cirrhosis, and liver failure. The molecular defect in protoporphyria is unknown. The molecular weight of purified mammalian ferrochelatase is ".40,000 daltons by SDS-PAGE (Taketani and Tokunaga 1981, 1982; Karr and Dailey 1988; Canepa and Llambias 1988; Nakahashi et al. 1990b), but the functional size by radiation inactivation is v80,000 daltons, consistent with a homodimer (Straka et al. 199 la). Recently the ferrochelatase gene from Saccharomyces cerevisiae (Labbe-Bois 1990) and ferrochelatase cDNAs from mouse (Taketani et al. 1990; Brenner and Frasier 1991) and human (Nakahashi et al. 1990a) liver have been cloned. In the present report we present data mapping the human ferrochelatase gene to chromosome 18q21.3. In addition, we identify a point mutation in the cDNAs from a patient with protoporphyria. Expression of this mutant ferrochelatase gene in Escherichia coli results in an enzyme with markedly reduced activity. Material and Methods Cell Culture
GM05008A, GMO5005A, and GM05007A fibroblast cell lines from protoporphyria patients were obtained from the NIGMS Human Genetic Mutant Cell 1203
Brenner et al.
1204 Repository. A deficiency in ferrochelatase activity was confirmed in these cell lines. MRC-5 and AF-2 normal control fibroblasts were obtained from the American Type Culture Collection and the University of California at San Diego's Core Tissue Culture Facility, respectively. Fibroblast cultures were grown in Earle's minimal essential medium with 20% FCS and supplement (10 ml containing 5,000 units penicillin/ml, 5,000 gg streptomycin/ml, 100 mM L-glutamine, 0.595 g HEPES, and 0.02 M PI-mercaptoethanol/500 ml of medium). Incubation was maintained at 38.41C in 5% CO2. Human-rodent somatic cell hybridomas were characterized and maintained according to a method described elsewhere (Grissmer et al. 1990). cDNA Preparation and PCR Total RNA was extracted from cultured cells by the guanidine isothiocyanate / phenol-chloroform method (Chomczynski and Sacchi 1987), and double-stranded cDNA was synthesized (Gubler and Hoffman 1983). Ferrochelatase cDNAs were amplified by PCR (Saiki et al. 1988). To clone the entire coding region of
mature-length human ferrochelatase, the forward primer 5'-CGTTCACTCGGCGCAAACATG-3' and the reverse primer 5'-GAGGTTGGGCATTTGCCTAACG-3' were chosen. To screen hybridomas for the presence of the human chromosome containing the ferrochelatase gene, the same 3'-terminus reverse primer was paired with a forward primer, 5'-ACATCAGAAGAGCTGAGTCTC-3', spanning bases 10912011 in the coding region (Nakahashi et al. 1990a). All primers were synthesized on a Cyclone Plus DNA synthesizer (Millipore). The PCR reaction mixture (50 Rl total) contained 1 Ril cDNA (from a SO-pl reversetranscriptase reaction starting with 10 ig of total RNA) plus 25 pmol of each primer, 200 tiM dNTPs, 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgC12, and 2.5 units TaqI polymerase (Perkin Elmer Cetus). Primers were annealed at 56°C for 1 min, extended at 720C for 2 min, and denatured at 940C for 1 min, through 25 cycles. Northern Blotting
Total RNA was extracted by the guanidine isothiocyanate/phenol-chloroform extraction method, as above. Twenty micrograms of RNA from normal and protoporphyric fibroblasts were electrophoresed on a formadehyde 1 % agarose gel, transferred to nitrocellulose filter by capillary blotting, and probed with cDNA for human ferrochelatase (obtained as above) that was [a-32P] dCTP radiolabeled by random primer
synthesis. Conditions for prehybridization, hybridization, washing, and autoradiography have been described elsewhere (Brenner and Chojkier 1987; Brenner and Frasier 1991). DNA Sequencing
PCR products were electrophoresed on a 1 % agarose gel. The DNA fragment of the expected 1,300base size for the ferrochelatase cDNA was purified and directly ligated with the vector PCR 1000 (TA cloning kit; Invitrogen). Plasmid inserts containing cDNAs were sequenced by the dideoxy method, using primers taken from the published ferrochelatase coding sequence at various intervals and oriented in both directions (Hattori and Sakari 1986). Wild-type and mutant ferrochelatase cDNAs were sequenced in their entirety, with overlap in both orientations. Chromosomal Localization
A panel of human-rodent somatic cell hybridomas, in which human chromosomal content had been characterized by others (Grissmer et al. 1990), was probed by PCR amplification with primers specific for the human ferrochelatase gene. The chromosomal position of the ferrochelatase gene was mapped by chromosomal in situ suppression hybridization. Cosmid HCI containing part of the human ferrochelatase gene was obtained (D. A. Brenner, F. Frasier, J.-M. Didier, and S. Christensen, unpublished data) by screening a human genomic library cloned into the cosmid vector WE1S (Evans and Wahl 1987). The cosmid clone HCl was labeled with biotinylated UTP and hybridized to metaphase chromosomes prepared from human lymphocytes according to a method described elsewhere (Lichter et al. 1990). The position of hybridization was detected by using fluorescein isothiocyanate (FITC) streptavidin. The metaphase chromosomes were counterstained with propidium iodide. Expression of Recombinant Protein An Escherichia coli expression vector for ferrochelatase was constructed from pBTacl, a plasmid containing the tac promoter (Boehringer Mannheim) (Amann et al. 1988). cDNA for the mature-length mouse ferrochelatase was generated by PCR of a mouse cDNA (Brenner and Frasier 1991) by using a 5' oligonucleotide that yields an ATG start site just before the proposed processing site at gln 60 and thus expresses mature-length protein (Taketani et al.
1990). The engineered amino-terminal sequence then was MQPERR ... (with the Q corresponding to Q60
Molecular Defect in Protoporphyria
1205
of Taketani et al. 1990). This construct (pHDTF2) expressed high levels of ferrochelatase (H. A. Dailey and T. A. Dailey, unpublished data). The wild-type mouse and human ferrochelatase amino acid sequences are 88% identical, and within the first 65 residues of the mature (processed form) protein, there are only six amino acid differences and only one nonconservative amino acid replacement (Nakahashi et al. 1990a; Brenner and Frasier 1991). Because the aminoterminal portion of mouse and human ferrochelatases are highly conserved, pHDTF2 was employed to form a mouse-human chimeric cDNA for ferrochelatase. Human and mouse ferrochelatase cDNA sequences contain unique BamHI and PstI sites at identical positions. In pHDTF2 and the human ferrochelatase vectors sequences, unique HindIII sites exist just past the termination codon. To produce the chimeric cDNAs, the BamHI and HindIII sites were utilized to replace the mouse cDNA with the human ferrochelatase cDNAs. (As a reference point, the BamHI site of the pHDTF2 construct at bp 217 corresponds to this same restriction site in the full-length human ferrochelatase cDNA as published by Taketani et al. [1990], at bp 414.) All plasmids were expressed in E. coli DHS cells that had been grown for 14 h in Circlegrow medium (BIO 101). Enzymatic activity of the expressed ferrochelatase protein was assayed according to a method described elsewhere (Dailey 1986). Ferrochelatase activity is here reported as the average of three separate preparations. Results
As the first approach to identifying the molecular defect in protoporphyria, ferrochelatase mRNA levels
Control
were measured by northern blotting and were found to be equal in fibroblasts from patients with protoporphyria (i.e., patients GM05005A and GM05008A) and from normal controls (fig. 1). Thus, the defect does not appear to be at the level of expression of the ferrochelatase gene. In order to identify structural mutations in the ferrochelatase gene, ferrochelatase cDNAs from patients with protoporphyria and from normal controls were sequenced. As expected in an autosomal dominant disease, multiple clones obtained from both patients and normal controls were identical to the wild-type ferrochelatase cDNA. In addition, a patient with protoporphyria (i.e., patient GM05008A) had a single point mutation in some of the ferrochelatase cDNAs. Plasmids that contained this mutation were obtained by independent PCR and cloning procedures. This mutation consisted of a conversion of a T to a C, resulting in replacement of a phenylalanine (TTC) in the wild-type ferrochelatase by a serine (TCC) in the mutant ferrochelatase, at amino acid position 417 (Nakahashi et al. 1990a) in the carboxy-terminal end of the protein (fig. 2). This mutation was not identified in two patients with protoporphyria who were from an unrelated family (i.e., patients GMOSO0SA and GM05007A), reflecting the heterogeneous nature of the molecular defect in this condition.
Next, the chromosomal location of the human ferrochelatase gene was identified. Human ferrochelatase sequences were specifically amplified by PCR from genomic DNA obtained from a panel of human-rodent somatic cell hybridomas, which had been previously characterized regarding human chromosomal content (Grissmer et al. 1990). There was 100% concordance for the presence of the human ferrochelatase PCR WT
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Molecular Defect in Protoporphyria product and human chromosome 18, in contrast to the discordance between human ferrochelatase and each of the other human chromosomes (table 1). Further, the chromosomal position of the ferrochelatase gene was localized by chromosomal in situ suppression hybridization. Fluorescent detection of a biotinylated human ferrochelatase genomic cDNA probe that was hybridized to propidium iodide G-banded chromosomes in metaphase (Lichter et al. 1990) demonstrated that the position of the human ferrochelatase gene was 18q21.3 (fig. 3). Analysis of 30 metaphases (60 chromosomes) demonstrated that more than 90% of chromosome 18's had two positions of hybridization at q21.3. The functional significance of the point mutation detected in the patient with protoporphyria was tested by expressing the wild-type and mutant ferrochelatase cDNAs in Escherichia coli by using a tac promoter expression vector (Amann et al. 1988) and assaying for ferrochelatase activity (Dailqy 1986) (fig. 4). The plasmid pHDTF2 contains the cDNA for the mature mouse ferrochelatase and generates a ferrochelatase protein with enzymatic activity of 0.80 nmol/mg/
1207 min. When the coding portion of the wild-type human cDNA was inserted into this expression vector, it generated an equally active recombinant protein with enzymatic activity of 0.77 nmol/mg/min (pHDTF6). However, when the same fragment from the mutant ferrochelatase was cloned into the expression plasmid, the resulting recombinant protein had markedly deficient enzymatic activity (pHDTF5). A chimeric protein containing the mutant ferrochelatase cDNA, but minus the sequence with the (Phe-Ser) mutation, had high enzymatic activity (pHDTF7), which demonstrated the absence of additional functional mutations. Finally, to further confirm that the identified mutation causes the functional defect in protoporphyria, the DNA fragments encoding the carboxy ends of the wild-type and mutant human ferrochelatases were cloned into the murine ferrochelatase expression vector. While the human wild-type carboxy-terminal end recreated a fully functional recombinant protein with high specific enzyme activity (pHDTF9), the recombinant ferrochelatase containing the human mutant carboxy-terminal end had markedly deficient activity (pHDTF8). Therefore, this single amino acid
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18 Figure 3 Mapping the human ferrochelatase gene by in situ hybridization. Left, Mapping of biotin-labeled cosmid HC-1, containing the human ferrochelatase gene, by chromosomal in situ suppression localization. Right, Diagram mapping cosmid HCl, containing the ferrochelatase gene, to position 18q21.3.
1208
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Composition of ferrochelatase expression vectors and corresponding enzyme activities. For the numbering above, base 4 corresponds to base 210 of the published full-length precursor form of the human sequence (Nakahashi et al. 1990a). Ferrochelatase enzyme activity is expressed as nanomoles mesoheme formed per milligram protein per minute. Sequences corresponding to wild-type mouse ferrochelatase are shown as unmarked boxes, wild-type human sequences are shown as hatched boxes, and protoporphyric mutant human sequences are shown as shaded boxes. The position of the point mutation, F417S, is designated by asterisk (*).
change in the ferrochelatase carboxy-terminal end translated into an enzyme with markedly deficient activity. Discussion
The present study identifies a molecular defect in protoporphyria. A single point mutation in the carboxy-terminal end of the ferrochelatase mRNA leads to the substitution of a serine for a phenylalanine at position 417. This point mutation renders recombinant ferrochelatase protein inactive. A very recent study (Lamoril et al. 1991) examined an unusual patient with a recessive form of protoporphyria and identified different point mutations on each of the patient's alleles. The functional significance of the mutations was not assessed. However, when the results of that study are combined with our result and with the highly variable clinical manifestations of pro-
toporphyria, it is likely that protoporphyria is a heterogenous disease resulting from various genetic defects. Using propidium iodide G-banding, we have identified the chromosomal localization of the human ferrochelatase gene to be 18q21.3. A recent study using R-banded chromosomes (Whitcombe et al. 1991) reported a very close, but not identical, location for the human ferrochelatase gene, at 1 8q22. In general, protoporphyria is not associated with any identifiable chromosomal abnormalities. However, a single case report describes a patient with mental retardation and protoporphyria. A deletion of chromosome arm 18q was detected in that patient by cytogenetic studies (Naylor et al. 1978). The chromosomal localization of the ferrochelatase gene is consistent with this case report.We suspect that in this unique patient the chromosomal break disrupted the ferrochelatase allele. Several complex models for the mode of inheritance and molecular pathogenesis in protoporphyria have
Molecular Defect in Protoporphyria
been proposed by others (Went and Klasen 1984; Norris et al. 1990). A simpler model to explain an autosomal dominant form of protoporphyria is that ferrochelatase is a functional dimer such that any complex containing the mutant protein would be inactive. That is, in heterozygous patients with protoporphyria, the mutant homodimers and mutant/wild-type heterodimers are inactive. Thus, the mutuant ferrochelatase protein would act as a "dominant-negative" by inactivating the wild-type ferrochelatase protein. This model explains many of the manifestations of protoporphyria, including its autosomal dominant mode of inheritance documented in many studies (Bonkowsky et al. 1975; Bottomley et al. 1975; de Goeij et al. 1975; Bloomer et al. 1976), the 15%-25% residual ferrochelatase activity in protoporphyric tissues (Bonkowsky et al. 1975; Bottomley et al. 1975; de Goeij et al. 1975), the functional dimer size of ferrochelatase determined by a functional assay (Straka et al. 1991a), the presence of normal amounts of immunoreactive ferrochelatase protein in protoporphyric tissues (Straka et al. 1991b), and the effect of a point mutation (F417S) in ferrochelatase cDNAs present in this protoporphyric patient. Future studies will determine the interaction between the wild-type and the mutant ferrochelatase proteins, in order to more completely characterize the molecular pathogenesis of protoporphyria, with the goal of developing novel therapies.
Acknowledgments This work was supported by the following NIH grants: DK-39996 (to D.A.B.), DK-32303 and DK-35898 (to H.A.D.), and HG-00202 and GM-33868 (to G.A.E.) and by the March of Dimes (support to D.A.B.). J.-M.D. was supported by a student fellowship from the A.G.A. We also wish to gratefully acknowledge both the invaluable assistance of Caryn Wagner-McPherson in performing the fluorescence in situ hybridization and the technical help of Scott Magness.
References Amann E, Ochs B, Avel K-J (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301-315 BloomerJR, Bonkowsky HL, Ebert PS, Mahoney MJ (1976) Inheritance in protoporphyria. Lancet 2:226-228 Bloomer JR, Phillips MJ, Davidson DL, Klatskin G (1975) Hepatic disease in erythropoietic protoporphyria. Am J Med 58:869-882
1209 Bloomer JR, Weiner MK, Bossenmaier IC, Snover DC, Payne WD, Ascher NL (1989) Liver transplantation in a patient with protoporphyria. Gastroenterology 97:188194 Bonkowsky HL, BloomerJR, Ebert PS, Mahoney MJ (1975) Heme synthetase deficiency in human protoporphyria. J Clin Invest 56:1139-1148 Bottomley SS, Tanaka M, Everett MA (1975) Diminished erythroid ferrochelatase activity in protoporphyria. J Lab Clin Med 86:126-131 Brenner DA, Chojkier M (1987) Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. J Biol Chem 262:17690-17695 Brenner DA, Frasier F (1991) Cloning of murine ferrochelatase. Proc Natl Acad Sci USA 88:849-853 Canepa ET, Llambias EBC (1988) Purification and characterization of ferro- and cobalto-chelatases. Biochem Cell Biol 66:32-39 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 Dailey HA (1986) Purification and characterization of bacterial ferrochelatase. Methods Enzymol 123:408-415 de Goeij AFPM, Chistianse K, van Steveninck I (1975) Decreased heme synthetase activity in blood cells of patients with erythropoietic protoporphyria. Eur J Clin Invest 5: 397-400 Evans GA, Wahl GM (1987) Cosmid vectors for genomic walking and rapid restriction mapping. Methods Enzymol 152:604-610 Grissmer S, Dethlefs B, Wasmuth JJ, Goldin AL, Gutman GA, Cahalan MD, Chandy KG (1990) Expression and chromosomal localization of a lymphocyte K+ channel gene. Proc Natl Acad Sci USA 87:9411-9415 Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263-269 Hattori M, Sakari Y (1986) Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152: 232-238 Herbert A, Corbin D, Williams A, Thompson D, Buckels J, Elias E (1991) Erythropoietic protoporphyria: unusual skin and neurological problems after liver transplantation. Gastroenterology 100:1753-1757 Karr SR, Dailey HA (1988) The synthesis of murine ferrochelatase in vitro and in vivo. Biochem J 254:799-803 Labbe-Bois R (1990) The ferrochelatase from Saccharomyces cerevisiae. J Biol Chem 265:7278-7283 Lamoril J, Boulechfar S, de Verneuil H, Grandchamp B, Nordmann Y, Deybach J (1991) Human erythropoietic protoporphyria: two point mutations in the ferrochelatase gene. Biochem Biophys Res Commun 181:594-599 Lichter P, Tang CC, Call K, Hermanson G, Evans GA, Housman D, Ward D (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64-69 Nakahashi Y, Taketani S, Okuda M, Inoue K, Tokunaga
1210 R (1 990a) Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase. Biochem Biophys Res Commun 173:748-755 Nakahashi Y, Taketani S, Samesshima Y, Tokunaga R (1990b) Characterization of ferrochelatase in kidney and erythroleukemia cells. Biochim Biophys Acta 1037:321327 Naylor EW, Murphey WH, Domoszlai El, Guthrie R (1978) Erythropoietic protoporphyria, heterozygous cystinuria, and reduced peptidase A activity in a patient with 46,xx/ 46,xx,18q - mosaicism. J Med Genet 15:157-159 Norris PG, Nunn AV, Hawk JLM (1990) Genetic heterogeneity in eyrthropoietic protoporphyria: a study of the enzymatic defect in nine affected families. Invest Dermatol 95:260-263 Rademakers LH, Cleton MI, Kooijman C, Baart de la Faille H, van Hattum J (1990) Early involvement of hepatic parenchymal cells in erythrohepatic protoporphyria. Hepatology 11:449-457 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491
Brenner et al. StrakaJG, BloomerJR, Kempner ES (199la) The functional size of ferrochelatase determined in situ by radiation inactivation. J Biol Chem 266:24637-24641 Straka JG, Hill HD, Krikava JM, Kools AM, Bloomer JR (1991b) Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria. Am J Hum Genet 48:72-78 Taketani S, Nakahashi Y, Osumi T, Tokunaga R (1990) Molecular cloning, sequencing, and expression of mouse ferrochelatase. J Biol Chem 265:19377-19380 Taketani S, Tokunaga R (1981) Rat liver ferrochelatase. J Biol Chem 256:12748-12753 (1982) Purification and substrate specificity of bovine liver-ferrochelatase. Eur J Biochem 127:443-447 Went LN, Klasen EC (1984) Genetic aspects of erythropoietic protoporphyria. Ann Hum Genet 48:105-117 Whitcombe D, Carter N, Albertson D, Smith SJ, Rhodes DA, Cox TM (1991) Assignment of the human ferrochelatase gene (FECH) and a locus for protoporphyria to chromosome 18q22. Genomics 11:1152-1154
A Molecular Defect in Human Protoporphyria D. A. Brenner,* J. M. Didier,* F. Frasier,* S. R. Christensen,* G. A. Evans,t and H. A. Dailey$ *Department of Medicine and the Center for Molecular Genetics, University of California, San Diego, and tThe Salk Institute, La Jolla; and
$Department of Microbiology and the Center for Metalloenzyme Studies, University of Georgia, Athens
Summary
Protoporphyria is generally an autosomal dominant disease that is characterized clinically by photosensitivity and hepatobiliary disease and that is characterized biochemically by elevated protoporphyrin levels. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is deficient in all tissues of patients with protoporphyria. In this study, sequencing of ferrochelatase cDNAs from a patient with protoporphyria revealed a single point mutation in the cDNAs, resulting in the conversion of a Phe(TTC) to a Ser(TCC) in the carboxy-terminal end of the protein, F417S. Further, the human ferrochelatase gene was mapped to chromosome 18q21.3 by chromosomal in situ suppression hybridization. Finally, expression of recombinant ferrochelatase in Escherichia coli demonstrated a marked deficiency in activity of the mutant ferrochelatase protein and of mouse-human mutant ferrochelatase chimeric proteins. Therefore, a point mutation in the coding region of the ferrochelatase gene is the genetic defect in some patients with protoporphyria. Introduction
Protoporphyria is generally a human autosomal dominant disease that is characterized biochemically by elevated protoporphyrin levels in the serum, erythrocytes, and feces and clinically by photosensitivity and hepatobiliary disease. A rare, but severe, clinical sequela of protoporphyria is liver failure leading to liver transplantation or death (Bloomer et al. 1975, 1989; Rademakers et al. 1990; Herbert et al. 1991). In patients with protoporphyria, activity of the enzyme ferrochelatase (protoheme ferro-lyase; E.C.4.99.1.1) is reduced to 15%-25% of normal in all tissues, although the concentration of immunoreactive ferrochelatase protein is unchanged (Straka et al. 1991b). Ferrochelatase catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron into protoporphyrin IX to form heme. In protoporphyria, the substrate for ferrochelatase, protoporphyrin, accumulates behind the partial enzyme block. The marked accumulation of protoporphyrin in the blood causes photosensitivity, and the clearance of excess protoporphyrin via the liver results in the formation of Received December 16, 1991; revision received February 10, 1992.
Address for correspondence and reprints: David A. Brenner, M.D., Department of Medicine, UCSD, La Jolla, CA 92093-0623. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/ 92/5006-0007$02.00
hepatic and biliary protoporphyrin crystals, cirrhosis, and liver failure. The molecular defect in protoporphyria is unknown. The molecular weight of purified mammalian ferrochelatase is ".40,000 daltons by SDS-PAGE (Taketani and Tokunaga 1981, 1982; Karr and Dailey 1988; Canepa and Llambias 1988; Nakahashi et al. 1990b), but the functional size by radiation inactivation is v80,000 daltons, consistent with a homodimer (Straka et al. 199 la). Recently the ferrochelatase gene from Saccharomyces cerevisiae (Labbe-Bois 1990) and ferrochelatase cDNAs from mouse (Taketani et al. 1990; Brenner and Frasier 1991) and human (Nakahashi et al. 1990a) liver have been cloned. In the present report we present data mapping the human ferrochelatase gene to chromosome 18q21.3. In addition, we identify a point mutation in the cDNAs from a patient with protoporphyria. Expression of this mutant ferrochelatase gene in Escherichia coli results in an enzyme with markedly reduced activity. Material and Methods Cell Culture
GM05008A, GMO5005A, and GM05007A fibroblast cell lines from protoporphyria patients were obtained from the NIGMS Human Genetic Mutant Cell 1203
Brenner et al.
1204 Repository. A deficiency in ferrochelatase activity was confirmed in these cell lines. MRC-5 and AF-2 normal control fibroblasts were obtained from the American Type Culture Collection and the University of California at San Diego's Core Tissue Culture Facility, respectively. Fibroblast cultures were grown in Earle's minimal essential medium with 20% FCS and supplement (10 ml containing 5,000 units penicillin/ml, 5,000 gg streptomycin/ml, 100 mM L-glutamine, 0.595 g HEPES, and 0.02 M PI-mercaptoethanol/500 ml of medium). Incubation was maintained at 38.41C in 5% CO2. Human-rodent somatic cell hybridomas were characterized and maintained according to a method described elsewhere (Grissmer et al. 1990). cDNA Preparation and PCR Total RNA was extracted from cultured cells by the guanidine isothiocyanate / phenol-chloroform method (Chomczynski and Sacchi 1987), and double-stranded cDNA was synthesized (Gubler and Hoffman 1983). Ferrochelatase cDNAs were amplified by PCR (Saiki et al. 1988). To clone the entire coding region of
mature-length human ferrochelatase, the forward primer 5'-CGTTCACTCGGCGCAAACATG-3' and the reverse primer 5'-GAGGTTGGGCATTTGCCTAACG-3' were chosen. To screen hybridomas for the presence of the human chromosome containing the ferrochelatase gene, the same 3'-terminus reverse primer was paired with a forward primer, 5'-ACATCAGAAGAGCTGAGTCTC-3', spanning bases 10912011 in the coding region (Nakahashi et al. 1990a). All primers were synthesized on a Cyclone Plus DNA synthesizer (Millipore). The PCR reaction mixture (50 Rl total) contained 1 Ril cDNA (from a SO-pl reversetranscriptase reaction starting with 10 ig of total RNA) plus 25 pmol of each primer, 200 tiM dNTPs, 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgC12, and 2.5 units TaqI polymerase (Perkin Elmer Cetus). Primers were annealed at 56°C for 1 min, extended at 720C for 2 min, and denatured at 940C for 1 min, through 25 cycles. Northern Blotting
Total RNA was extracted by the guanidine isothiocyanate/phenol-chloroform extraction method, as above. Twenty micrograms of RNA from normal and protoporphyric fibroblasts were electrophoresed on a formadehyde 1 % agarose gel, transferred to nitrocellulose filter by capillary blotting, and probed with cDNA for human ferrochelatase (obtained as above) that was [a-32P] dCTP radiolabeled by random primer
synthesis. Conditions for prehybridization, hybridization, washing, and autoradiography have been described elsewhere (Brenner and Chojkier 1987; Brenner and Frasier 1991). DNA Sequencing
PCR products were electrophoresed on a 1 % agarose gel. The DNA fragment of the expected 1,300base size for the ferrochelatase cDNA was purified and directly ligated with the vector PCR 1000 (TA cloning kit; Invitrogen). Plasmid inserts containing cDNAs were sequenced by the dideoxy method, using primers taken from the published ferrochelatase coding sequence at various intervals and oriented in both directions (Hattori and Sakari 1986). Wild-type and mutant ferrochelatase cDNAs were sequenced in their entirety, with overlap in both orientations. Chromosomal Localization
A panel of human-rodent somatic cell hybridomas, in which human chromosomal content had been characterized by others (Grissmer et al. 1990), was probed by PCR amplification with primers specific for the human ferrochelatase gene. The chromosomal position of the ferrochelatase gene was mapped by chromosomal in situ suppression hybridization. Cosmid HCI containing part of the human ferrochelatase gene was obtained (D. A. Brenner, F. Frasier, J.-M. Didier, and S. Christensen, unpublished data) by screening a human genomic library cloned into the cosmid vector WE1S (Evans and Wahl 1987). The cosmid clone HCl was labeled with biotinylated UTP and hybridized to metaphase chromosomes prepared from human lymphocytes according to a method described elsewhere (Lichter et al. 1990). The position of hybridization was detected by using fluorescein isothiocyanate (FITC) streptavidin. The metaphase chromosomes were counterstained with propidium iodide. Expression of Recombinant Protein An Escherichia coli expression vector for ferrochelatase was constructed from pBTacl, a plasmid containing the tac promoter (Boehringer Mannheim) (Amann et al. 1988). cDNA for the mature-length mouse ferrochelatase was generated by PCR of a mouse cDNA (Brenner and Frasier 1991) by using a 5' oligonucleotide that yields an ATG start site just before the proposed processing site at gln 60 and thus expresses mature-length protein (Taketani et al.
1990). The engineered amino-terminal sequence then was MQPERR ... (with the Q corresponding to Q60
Molecular Defect in Protoporphyria
1205
of Taketani et al. 1990). This construct (pHDTF2) expressed high levels of ferrochelatase (H. A. Dailey and T. A. Dailey, unpublished data). The wild-type mouse and human ferrochelatase amino acid sequences are 88% identical, and within the first 65 residues of the mature (processed form) protein, there are only six amino acid differences and only one nonconservative amino acid replacement (Nakahashi et al. 1990a; Brenner and Frasier 1991). Because the aminoterminal portion of mouse and human ferrochelatases are highly conserved, pHDTF2 was employed to form a mouse-human chimeric cDNA for ferrochelatase. Human and mouse ferrochelatase cDNA sequences contain unique BamHI and PstI sites at identical positions. In pHDTF2 and the human ferrochelatase vectors sequences, unique HindIII sites exist just past the termination codon. To produce the chimeric cDNAs, the BamHI and HindIII sites were utilized to replace the mouse cDNA with the human ferrochelatase cDNAs. (As a reference point, the BamHI site of the pHDTF2 construct at bp 217 corresponds to this same restriction site in the full-length human ferrochelatase cDNA as published by Taketani et al. [1990], at bp 414.) All plasmids were expressed in E. coli DHS cells that had been grown for 14 h in Circlegrow medium (BIO 101). Enzymatic activity of the expressed ferrochelatase protein was assayed according to a method described elsewhere (Dailey 1986). Ferrochelatase activity is here reported as the average of three separate preparations. Results
As the first approach to identifying the molecular defect in protoporphyria, ferrochelatase mRNA levels
Control
were measured by northern blotting and were found to be equal in fibroblasts from patients with protoporphyria (i.e., patients GM05005A and GM05008A) and from normal controls (fig. 1). Thus, the defect does not appear to be at the level of expression of the ferrochelatase gene. In order to identify structural mutations in the ferrochelatase gene, ferrochelatase cDNAs from patients with protoporphyria and from normal controls were sequenced. As expected in an autosomal dominant disease, multiple clones obtained from both patients and normal controls were identical to the wild-type ferrochelatase cDNA. In addition, a patient with protoporphyria (i.e., patient GM05008A) had a single point mutation in some of the ferrochelatase cDNAs. Plasmids that contained this mutation were obtained by independent PCR and cloning procedures. This mutation consisted of a conversion of a T to a C, resulting in replacement of a phenylalanine (TTC) in the wild-type ferrochelatase by a serine (TCC) in the mutant ferrochelatase, at amino acid position 417 (Nakahashi et al. 1990a) in the carboxy-terminal end of the protein (fig. 2). This mutation was not identified in two patients with protoporphyria who were from an unrelated family (i.e., patients GMOSO0SA and GM05007A), reflecting the heterogeneous nature of the molecular defect in this condition.
Next, the chromosomal location of the human ferrochelatase gene was identified. Human ferrochelatase sequences were specifically amplified by PCR from genomic DNA obtained from a panel of human-rodent somatic cell hybridomas, which had been previously characterized regarding human chromosomal content (Grissmer et al. 1990). There was 100% concordance for the presence of the human ferrochelatase PCR WT
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Molecular Defect in Protoporphyria product and human chromosome 18, in contrast to the discordance between human ferrochelatase and each of the other human chromosomes (table 1). Further, the chromosomal position of the ferrochelatase gene was localized by chromosomal in situ suppression hybridization. Fluorescent detection of a biotinylated human ferrochelatase genomic cDNA probe that was hybridized to propidium iodide G-banded chromosomes in metaphase (Lichter et al. 1990) demonstrated that the position of the human ferrochelatase gene was 18q21.3 (fig. 3). Analysis of 30 metaphases (60 chromosomes) demonstrated that more than 90% of chromosome 18's had two positions of hybridization at q21.3. The functional significance of the point mutation detected in the patient with protoporphyria was tested by expressing the wild-type and mutant ferrochelatase cDNAs in Escherichia coli by using a tac promoter expression vector (Amann et al. 1988) and assaying for ferrochelatase activity (Dailqy 1986) (fig. 4). The plasmid pHDTF2 contains the cDNA for the mature mouse ferrochelatase and generates a ferrochelatase protein with enzymatic activity of 0.80 nmol/mg/
1207 min. When the coding portion of the wild-type human cDNA was inserted into this expression vector, it generated an equally active recombinant protein with enzymatic activity of 0.77 nmol/mg/min (pHDTF6). However, when the same fragment from the mutant ferrochelatase was cloned into the expression plasmid, the resulting recombinant protein had markedly deficient enzymatic activity (pHDTF5). A chimeric protein containing the mutant ferrochelatase cDNA, but minus the sequence with the (Phe-Ser) mutation, had high enzymatic activity (pHDTF7), which demonstrated the absence of additional functional mutations. Finally, to further confirm that the identified mutation causes the functional defect in protoporphyria, the DNA fragments encoding the carboxy ends of the wild-type and mutant human ferrochelatases were cloned into the murine ferrochelatase expression vector. While the human wild-type carboxy-terminal end recreated a fully functional recombinant protein with high specific enzyme activity (pHDTF9), the recombinant ferrochelatase containing the human mutant carboxy-terminal end had markedly deficient activity (pHDTF8). Therefore, this single amino acid
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18 Figure 3 Mapping the human ferrochelatase gene by in situ hybridization. Left, Mapping of biotin-labeled cosmid HC-1, containing the human ferrochelatase gene, by chromosomal in situ suppression localization. Right, Diagram mapping cosmid HCl, containing the ferrochelatase gene, to position 18q21.3.
1208
Brenner et al.
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Composition of ferrochelatase expression vectors and corresponding enzyme activities. For the numbering above, base 4 corresponds to base 210 of the published full-length precursor form of the human sequence (Nakahashi et al. 1990a). Ferrochelatase enzyme activity is expressed as nanomoles mesoheme formed per milligram protein per minute. Sequences corresponding to wild-type mouse ferrochelatase are shown as unmarked boxes, wild-type human sequences are shown as hatched boxes, and protoporphyric mutant human sequences are shown as shaded boxes. The position of the point mutation, F417S, is designated by asterisk (*).
change in the ferrochelatase carboxy-terminal end translated into an enzyme with markedly deficient activity. Discussion
The present study identifies a molecular defect in protoporphyria. A single point mutation in the carboxy-terminal end of the ferrochelatase mRNA leads to the substitution of a serine for a phenylalanine at position 417. This point mutation renders recombinant ferrochelatase protein inactive. A very recent study (Lamoril et al. 1991) examined an unusual patient with a recessive form of protoporphyria and identified different point mutations on each of the patient's alleles. The functional significance of the mutations was not assessed. However, when the results of that study are combined with our result and with the highly variable clinical manifestations of pro-
toporphyria, it is likely that protoporphyria is a heterogenous disease resulting from various genetic defects. Using propidium iodide G-banding, we have identified the chromosomal localization of the human ferrochelatase gene to be 18q21.3. A recent study using R-banded chromosomes (Whitcombe et al. 1991) reported a very close, but not identical, location for the human ferrochelatase gene, at 1 8q22. In general, protoporphyria is not associated with any identifiable chromosomal abnormalities. However, a single case report describes a patient with mental retardation and protoporphyria. A deletion of chromosome arm 18q was detected in that patient by cytogenetic studies (Naylor et al. 1978). The chromosomal localization of the ferrochelatase gene is consistent with this case report.We suspect that in this unique patient the chromosomal break disrupted the ferrochelatase allele. Several complex models for the mode of inheritance and molecular pathogenesis in protoporphyria have
Molecular Defect in Protoporphyria
been proposed by others (Went and Klasen 1984; Norris et al. 1990). A simpler model to explain an autosomal dominant form of protoporphyria is that ferrochelatase is a functional dimer such that any complex containing the mutant protein would be inactive. That is, in heterozygous patients with protoporphyria, the mutant homodimers and mutant/wild-type heterodimers are inactive. Thus, the mutuant ferrochelatase protein would act as a "dominant-negative" by inactivating the wild-type ferrochelatase protein. This model explains many of the manifestations of protoporphyria, including its autosomal dominant mode of inheritance documented in many studies (Bonkowsky et al. 1975; Bottomley et al. 1975; de Goeij et al. 1975; Bloomer et al. 1976), the 15%-25% residual ferrochelatase activity in protoporphyric tissues (Bonkowsky et al. 1975; Bottomley et al. 1975; de Goeij et al. 1975), the functional dimer size of ferrochelatase determined by a functional assay (Straka et al. 1991a), the presence of normal amounts of immunoreactive ferrochelatase protein in protoporphyric tissues (Straka et al. 1991b), and the effect of a point mutation (F417S) in ferrochelatase cDNAs present in this protoporphyric patient. Future studies will determine the interaction between the wild-type and the mutant ferrochelatase proteins, in order to more completely characterize the molecular pathogenesis of protoporphyria, with the goal of developing novel therapies.
Acknowledgments This work was supported by the following NIH grants: DK-39996 (to D.A.B.), DK-32303 and DK-35898 (to H.A.D.), and HG-00202 and GM-33868 (to G.A.E.) and by the March of Dimes (support to D.A.B.). J.-M.D. was supported by a student fellowship from the A.G.A. We also wish to gratefully acknowledge both the invaluable assistance of Caryn Wagner-McPherson in performing the fluorescence in situ hybridization and the technical help of Scott Magness.
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1209 Bloomer JR, Weiner MK, Bossenmaier IC, Snover DC, Payne WD, Ascher NL (1989) Liver transplantation in a patient with protoporphyria. Gastroenterology 97:188194 Bonkowsky HL, BloomerJR, Ebert PS, Mahoney MJ (1975) Heme synthetase deficiency in human protoporphyria. J Clin Invest 56:1139-1148 Bottomley SS, Tanaka M, Everett MA (1975) Diminished erythroid ferrochelatase activity in protoporphyria. J Lab Clin Med 86:126-131 Brenner DA, Chojkier M (1987) Acetaldehyde increases collagen gene transcription in cultured human fibroblasts. J Biol Chem 262:17690-17695 Brenner DA, Frasier F (1991) Cloning of murine ferrochelatase. Proc Natl Acad Sci USA 88:849-853 Canepa ET, Llambias EBC (1988) Purification and characterization of ferro- and cobalto-chelatases. Biochem Cell Biol 66:32-39 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 Dailey HA (1986) Purification and characterization of bacterial ferrochelatase. Methods Enzymol 123:408-415 de Goeij AFPM, Chistianse K, van Steveninck I (1975) Decreased heme synthetase activity in blood cells of patients with erythropoietic protoporphyria. Eur J Clin Invest 5: 397-400 Evans GA, Wahl GM (1987) Cosmid vectors for genomic walking and rapid restriction mapping. Methods Enzymol 152:604-610 Grissmer S, Dethlefs B, Wasmuth JJ, Goldin AL, Gutman GA, Cahalan MD, Chandy KG (1990) Expression and chromosomal localization of a lymphocyte K+ channel gene. Proc Natl Acad Sci USA 87:9411-9415 Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263-269 Hattori M, Sakari Y (1986) Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152: 232-238 Herbert A, Corbin D, Williams A, Thompson D, Buckels J, Elias E (1991) Erythropoietic protoporphyria: unusual skin and neurological problems after liver transplantation. Gastroenterology 100:1753-1757 Karr SR, Dailey HA (1988) The synthesis of murine ferrochelatase in vitro and in vivo. Biochem J 254:799-803 Labbe-Bois R (1990) The ferrochelatase from Saccharomyces cerevisiae. J Biol Chem 265:7278-7283 Lamoril J, Boulechfar S, de Verneuil H, Grandchamp B, Nordmann Y, Deybach J (1991) Human erythropoietic protoporphyria: two point mutations in the ferrochelatase gene. Biochem Biophys Res Commun 181:594-599 Lichter P, Tang CC, Call K, Hermanson G, Evans GA, Housman D, Ward D (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64-69 Nakahashi Y, Taketani S, Okuda M, Inoue K, Tokunaga
1210 R (1 990a) Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase. Biochem Biophys Res Commun 173:748-755 Nakahashi Y, Taketani S, Samesshima Y, Tokunaga R (1990b) Characterization of ferrochelatase in kidney and erythroleukemia cells. Biochim Biophys Acta 1037:321327 Naylor EW, Murphey WH, Domoszlai El, Guthrie R (1978) Erythropoietic protoporphyria, heterozygous cystinuria, and reduced peptidase A activity in a patient with 46,xx/ 46,xx,18q - mosaicism. J Med Genet 15:157-159 Norris PG, Nunn AV, Hawk JLM (1990) Genetic heterogeneity in eyrthropoietic protoporphyria: a study of the enzymatic defect in nine affected families. Invest Dermatol 95:260-263 Rademakers LH, Cleton MI, Kooijman C, Baart de la Faille H, van Hattum J (1990) Early involvement of hepatic parenchymal cells in erythrohepatic protoporphyria. Hepatology 11:449-457 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491
Brenner et al. StrakaJG, BloomerJR, Kempner ES (199la) The functional size of ferrochelatase determined in situ by radiation inactivation. J Biol Chem 266:24637-24641 Straka JG, Hill HD, Krikava JM, Kools AM, Bloomer JR (1991b) Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria. Am J Hum Genet 48:72-78 Taketani S, Nakahashi Y, Osumi T, Tokunaga R (1990) Molecular cloning, sequencing, and expression of mouse ferrochelatase. J Biol Chem 265:19377-19380 Taketani S, Tokunaga R (1981) Rat liver ferrochelatase. J Biol Chem 256:12748-12753 (1982) Purification and substrate specificity of bovine liver-ferrochelatase. Eur J Biochem 127:443-447 Went LN, Klasen EC (1984) Genetic aspects of erythropoietic protoporphyria. Ann Hum Genet 48:105-117 Whitcombe D, Carter N, Albertson D, Smith SJ, Rhodes DA, Cox TM (1991) Assignment of the human ferrochelatase gene (FECH) and a locus for protoporphyria to chromosome 18q22. Genomics 11:1152-1154
