Variegate Porphyria: Identification of a Nonsense Mutation in The

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MUTATION REPORTS Variegate Porphyria: Identiﬁcation of a Nonsense Mutation in the Protoporphyrinogen Oxidase Gene

Jorge Frank, Frank K. Jugert,* Katrin Kalka,† Gu¨nter Goerz,† Hans F. Merk,* and Angela M. Christiano Department of Dermatology, Columbia University, New York, New York, U.S.A.; *Department of Dermatology, University Clinic of the RWTH, Aachen, Germany; †Department of Dermatology, Heinrich Heine University, Du¨sseldorf, Germany

The porphyrias are disorders of porphyrin metabolism protoporphyrinogen oxidase gene, resulting in the substi- that result from inherited or acquired aberrations in the tution of glutamic acid by a nonsense codon, designated control of the heme biosynthetic pathway. Variegate E133X. Our investigation establishes that a nonsense porphyria is characterized by a partial reduction in the mutation in the protoporphyrinogen oxidase gene is activity of protoporphyrinogen oxidase. In this study, we the underlying mutation in this family with variegate identiﬁed the ﬁrst nonsense mutation in a family with porphyria. Key words: bullous diseases/genodermatosis/photo- variegate porphyria. The mutation consisted of a previ- sensitivity/porphyrin-heme biosynthetic pathway. J Invest ously unreported G-to-T transversion in exon 5 of the Dermatol 110:449–451, 1998

he porphyrias are disorders of heme metabolism resulting in the PPO gene in the clinically affected patient and one of her sons from the inherited or acquired dysregulation of one of by heteroduplex analysis, automated sequencing, and allele specific the eight enzymes that control the porphyrin-heme oligonucleotide (ASO) hybridization. The mutation consisted of a biosynthetic pathway. Variegate porphyria (VP), one previously unreported G-to-T transversion in exon 5 of the PPO of the acute hepatic porphyrias, is characterized by gene, resulting in the substitution of glutamic acid by a premature Tdeficiencies in protoporphyrinogen oxidase (PPO), the seventh enzyme termination codon. Our study establishes this nonsense mutation as in the pathway of heme biosynthesis (Frank and Christiano, 1997). the underlying defect in the PPO gene in this patient with VP. Biochemically, increased levels of porphyrin precursors, raised values of protoporphyrin, coproporphyrin, and pentacarboxyporphyrin in the MATERIALS AND METHODS feces, and, possibly, increased porphyrins in the urine and plasma can be found (Day, 1986). VP is usually inherited as an autosomal dominant Clinical material and biochemical analyses We investigated the proband, three of her family members, and 50 unrelated, unaffected control individuals. trait displaying incomplete penetrance, as not all persons carrying a The proband is a 35 y old female of German ancestry who was diagnosed with mutation in the PPO gene develop the clinical phenotype (Frank and VP clinically and by biochemical porphyrin analysis (Table I) in 1992. Her Christiano, 1997). In heterozygotes, PPO activity is decreased by mother and her two sons were clinically unaffected with VP. Blood samples µ50% (Eales et al, 1980). from the family were collected in tubes containing ethylenediamine tetraacetic The cutaneous manifestations of VP include increased photo- acid. All individuals provided informed consent for inclusion in the study, in sensitivity, blistering, skin fragility with chronic scarring of sun-exposed accordance with guidelines set forth by the local institutional review board. areas, and postinflammatory hyperpigmentation (Bickers et al, 1993). Genomic DNA was isolated according to standard techniques (Sambrook The photosensitivity may occur alone or in combination with acute et al, 1989). symptoms such as abdominal pain, the passage of dark urine, and neuropsychiatric crises, which characterize the acute hepatic porphyrias. PCR amplification and mutation detection A mutation detection strategy In heterozygotes, the disease does not usually present before puberty was developed for PCR amplification of all PPO exons using PCR primers (Kauppinen and Mustajoki, 1992). that were published recently (Warnich et al, 1996). For amplification of exon The human PPO cDNA and gene were recently cloned and mapped 5 of the PPO gene in this specific case, the following primers were used, as to chromosome 1q22–23 (Nishimura et al, 1995; Roberts et al, 1995; described recently (Warnich et al, 1996): PPO Exon 5L, 5Ј GAGGTATGT- Taketani et al, 1995a), and we and others have recently identified CAGGAGCTTCC 3Ј; and PPO Exon 5R, 5Ј GATTTGAACAGGGAGCT- CTG 3Ј. pathogenic mutations in several families with VP (Deybach et al, 1996; PCR was carried out on genomic DNA from all family members according Meissner et al, 1996; Warnich et al, 1996; Frank and Christiano, 1997; to the following program: 95°C for 10 min; followed by 40 cycles of 95°C for Lam et al, 1997). 45 s, 57°C for 45 s, and 72°C for 1 min; followed by 72°C for 15 min, in an In this study, we investigated a proband with VP and three of her OmniGene thermal cycler (Marsh Scientific, Rochester, NY). For mutation clinically unaffected family members. We identified a nonsense mutation detection, PCR products were subjected to conformation sensitive gel electrophoresis (CSGE) analysis as described previously (Ganguly et al, 1993). PCR products displaying a heteroduplex on CSGE analysis were sequenced using an ABI Prism 310 Genetic Analyzer from Applied Biosystems. Manuscript received April 21, 1997; revised November 12, 1997; accepted For confirmation of the G-to-T transversion in exon 5 at the genomic DNA for publication November 24, 1997. level, ASO hybridization was carried out in an independently generated PCR Reprint requests to: Dr. Angela M. Christiano, Department of Dermatology, product according to standard techniques (Sambrook et al, 1989). In this Columbia University, College of Physicians and Surgeons, 630 West 168th case, the following oligonucleotides were used: Oligo exon 5 wild-type, 5Ј Street, VC-1526, New York, New York 10032. GGGCTGAGGGAGCTGACC 3Ј; Oligo exon 5 mutant, 5Ј Abbreviations: PPO, protoporphyrinogen oxidase; VP, variegate porphyria. GGCTGAGGTAGCTGACCA 3Ј.

Figure 2. Alignment of deduced amino acid sequences from codons 131 to 140 in exon 5 of human PPO (hPPO), mouse PPO (mPPO), and the yeast Saccharomyces cerevisiae (SC hem 14). The identical residues in codon 133 and the substitution derived from the described nonsense mutation are shaded and bold.

Table II. Reported mutations in the PPO gene

Location Designation Consequence Reference

Dominant VP exon 3 165insAG Frameshift mutation Lam et al, 1997 exon 7 1022insG Frameshift mutation Deybach et al, 1996 exon 10 1083deIT Frameshift mutation Frank and Christiano, 1997 exon 11 1144delGT Frameshift mutation Frank and Christiano, Figure 1. Mutation analysis in the VP patient in this study and her 1997 family members. (A) Pedigree of the nuclear family with regard to the exon 2 H20P Missense mutation Warnich et al, 1996 occurrence of VP; the proband (II/1) is indicated by a half-ﬁlled circle. The exon 3 R59W Missense mutation Warnich et al, 1996 other family member carrying the E133X mutation (III/1) is indicated by a Meissner et al, 1996 half-ﬁlled square. (B) Heteroduplex analysis of the family and a control (C); exon 6 R168C Missense mutation Warnich et al, 1996 heteroduplex formation in the proband (II/1) and one of her sons (III/1) in Meissner et al, 1996 exon 5 of the PPO gene. (C) ASO hybridization results: in exon 5, the proband exon 7 G232R Missense mutation Deybach et al, 1996 (II/1) and her son (III/1) show the mutant E133X allele (lower panel) and the exon 13 S450P Missense mutation Frank and Christiano, wild-type allele, whereas the other family members (I/1 and III/2) and the 1997 control (C) only show the wild-type allele (upper panel). (D) Automated exon 5 E133X Nonsense mutation This study sequence of the forward strand of the PCR fragment of exon 5; G-to-T Recessive VP transversion in exon 5 of the proband (lower panel, indicated by an arrow) exon 6/10 G169E/G358R Compound Frank et al, 1998 compared with the wild-type sequence of control C (upper panel). heterozygote missense mutation

Table I. Diagnostic porphyrin analysis of the proband DISCUSSION Porphyrin analysis Proband Reference ranges We report the identification of a nonsense mutation in a patient with Urine VP and her unaffected son by CSGE analysis, automated sequencing, Total urinary porphyrins 848 µg/l Ͻ150 µg/l and ASO hybridization. To date, 12 different mutations have been Urinary ALA 12.2 mg/l Ͻ5.0 mg/l detected in the PPO gene on chromosome 1q22–23 (Table II), Urinary porphobilinogen 9.9 mg/l Ͻ9.9 mg/l demonstrating the heterogeneity of VP (Deybach et al, 1996; Meissner Stool et al, 1996; Warnich et al, 1996; Frank and Christiano, 1997; Lam et al, Total fecal porphyrins 1084 nmol/g Ͻ200 nmol/g 1997). These mutations reflect a wide spectrum of possible types of mutations, including single base substitutions (transitions and transver- sions) as well as deletions and insertions in the PPO gene. The mutation found in this study consisted of a previously unreported G-to-T transversion in exon 5 of the PPO gene, leading to the RESULTS substitution of a glutamic acid residue by a nonsense codon (E133X). Heteroduplex analysis of exon 5 of the PPO gene in this family This mutation was the only sequence variant detected in this family. (Fig 1A), revealed a heteroduplex in the proband and one son (Fig 1B). Interestingly, a comparison of nucleotide deduced amino acid sequences Automated sequence analysis of this PCR product in all family members (Fig 2) revealed that this conversion occurred in a region of PPO that revealed the same sequence variant in the patient and her son (Fig 1D). is strictly conserved in human (Nishimura et al, 1995), in mouse over The mutation consisted of a G-to-T transversion at nucleotide position 90 million evolutionary years (Taketani et al, 1995a), and over 500 396 of the PPO cDNA (numbered according to GenBank #D38537, million evolutionary years in the yeast Saccharomyces cerevisiae (Camadro counting the first base of the initiating methionine as #1), leading to et al, 1996), although their entire amino acid sequence identity is less a previously unreported nonsense mutation consisting of an amino acid than 20%. Therefore, this glutamic acid residue is believed to be highly conversion from glutamic acid to a nonsense codon at position 133 important for proper functioning of PPO. The mutation may cause (E133X) in the deduced amino acid sequence (Fig 2). Heteroduplex disturbances of specific regions in PPO and thereby impair the integrity analysis of exon 5 of the PPO gene was also performed in 50 unrelated of the protein. Although we did not measure mRNA levels in this control individuals and showed no heteroduplex formation indicative study, there is evidence that nonsense mutations are associated with of the mutation (data not shown). Because the mutation did not dramatically reduced amounts of cytoplasmic mRNA, which presum- introduce a restriction site for any known restriction endonuclease, ably leads to the elimination of the production of protein that is ASO hybridization was carried out to verify the sequence variation. potentially deleterious to cell viability (Cooper, 1993; Maquat, 1996). In the proband and one son, the normal as well as the mutant Previous studies have shown that VP displays a high variability in the allele was observed, indicating that they are heterozygous for the clinical expression of defects in the PPO gene within family members mutation (Fig 1C). carrying the same mutation (Deybach et al, 1996), suggesting the VOL. 110, NO. 4 APRIL 1998 PPO MUTATION IN VARIEGATE PORPHYRIA 451 influence of genetic background on the expression of the disease Deybach JC, Puy H, Robreau AM, Lamoril J, Da Silva V, Grandchamp B, Nordmann Y: Mutations in the protoporphyrinogen oxidase gene in patients with variegate phenotype. The reasons for the different patterns of inheritance and porphyria. Hum Mol Genet 5:407–410, 1996 differences in phenotypic severity in VP are not well understood, and Eales L, Day RS, Blekkenhorst GH: The clinical and biochemical features of variegate require further investigation. porphyria: An analysis of 300 cases studied at Groote Schuur Hospital, Cape Town. Once a mutation has been identified in a VP patient, the detection Int J Biochem 12:837–853, 1980 Frank J, Christiano AM: Genetic research strategies: a review of the acute porphyrias. of asymptomatic carriers within the patient’s family is easily accomp- Retinoids 13:88–92, 1997 lished using molecular biologic techniques. The identification of these Frank J, McGrath J, Lam H, Graham RM, Hawk JLM, Christiano AM: Homozygous so-called ‘‘silent carriers’’ is the first and most important step in the variegate porphyria: identification of mutations on both alleles of the prevention of acute attacks. protoporphyrinogen oxidase gene in a severely affected proband. J Invest Dermatol 110:452–455, 1998 This study underscores the phenotypic variability in VP in a family Ganguly A, Rock MJ, Prockop DJ: Conformation-sensitive gel electrophoresis for rapid with carriers of the same nonsense mutation in the PPO gene. There detection of single-base differences in double-stranded PCR products and DNA is little doubt that DNA analysis will be the method of choice to fragments: Evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl confirm putative diagnosis of VP, and consequently, to prevent acute Acad Sci USA 90:10325–10329, 1993 Kauppinen R, Mustajoki P: Prognosis of acute porphyria: Occurrence of acute attacks, attacks in ‘‘silent’’ carriers of mutations. Further investigations are precipitating factors, and associated diseases. Med 71:1– 13, 1992 underway to establish genotype-to-phenotype correlations in VP by Lam H, Dragan L, Tsou HC, et al: Molecular basis of variegate porphyria: A de novo the expression of mutant alleles, and to understand additional and insertion mutation in the protoporphyrinogen oxidase gene. Hum Genet 99:126– unusual mechanisms of inheritance in this disease. 129, 1997 Maquat LE: Defects in RNA splicing and the consequence of shortened translational reading frames. Am J Hum Genet 59:279–286, 1996 Meissner PN, Dailey TA, Hift RJ, et al: A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with We are especially grateful to the patient and her family for their interest and cooperation variegate porphyria. Nat Genet 13:95–97, 1996 in this study. 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