Vedr. VP

European Porphyria Initiative (EPI)
Porphyria and Porphirins Abstract:

HOMOZYGOUS VARIEGATE PORPHYRIA. FIRST ITALIAN CASE - LONG TERM FOLLOW UP - IDENTIFICATION OF NOVEL MUTATIONS IN PPOX GENE
Bonuglia M, D'Amato M, Sorge F, Griso D, Macri A, Biolcati G Porphyrias Center, San Gallicano Institute, IRCCS, Rome, Italy, 2003
Variegate Porphyria (VP) is a very rare autosomal dominant metabolic disorder of heme biosynthesis that results from partial deficiency of Protoporphyrinogen-oxidase enzyme activity caused by mutation in the PPOX gene. Homozygous VP (HVP) is a more than rare variant resulting from a mutation in both alleles of PPOX gene. Dermatological and neurological symptoms are severe and their age of onset is during first months of life. We describe a long term follow up of a HVP patient where severe photosensitivity leads to photomutilations; besides mental retardation aphasia and aggressivity developed in the course of years. Genetically he is a compoud heterozygotes: 1061C>T/397G>A NH N NH N OMe OMe NH O NH O NH N NH N H H

GENETIC ANALYSIS OF VARIEGATE PORPHYRIA IN ITALY: IDENTIFICATION OF EIGHT NOVEL MUTATIONS IN THE PROTOPORPHYRINOGEN OXIDASE GENE
D'Amato M, Bonuglia M, Barile S, Griso D, Macri A, Sorge F, Biolcati G Porphyrias Center, S. Gallicano Institute, IRCCS, Rome, Italy
Variegate Porphyria (VP) is one of the acute hepatic porphyrias, and is clinically characterised by skin lesions and acute neuropsychiatric/ visceral attacks that occur separately or together. The disorder is caused by a partial deficiency of protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, and a number of mutations have been described for the corresponding gene PPOX. We report a genetic analysis of VP in Italy, and the identification of eight novel and three previously characterised mutations from thirteen affected individuals. Among those newly identified, two mutations were small deletions (c.418_419delAA; c.759delA), leading to the formation of premature stop codons, two were splicing defects (IVS10+2T>G; IVS12+1G>C), two were nonsense (c.384G>A, c.1013C>G) and three missense mutations (c.848T>A, 202>A, 694>C). This is the first molecular genetics VP study on patients of Italian origin. Finding eight identified novel mutations in thirteen patients confirms the genetic heterogeneity observed for this disorder.

DUAL PORPHYRIA - PORPHYRIA CUTANEA TARDA AND VARIEGATE PORPHYRIA - IN ARGENTINA
Melito V., Parera V.E., Rossetti M.V., Batlle A. CIPYP, Buenos Aires, Argentina
Porphyrias are hereditary and independent diseases due to a specific enzymatic failure in heme metabolism. Although the existence of two different porphyrias (Dual Porphyria) in one patient is unfrequent, cases of Dual Porphyria where Porphyria Cutanea Tarda (PCT) and Acute Intermitent Porphyria (AIP) and PCT and Variegate Porphyria (VP) coexisted, have been reported. Superimposition of both urinary and faecal porphyrin excretory patterns and clinical symptoms are observed in these patients. We report here a case of Dual Porphyria in a patient having both PCT and VP. In Argentine PCT and AIP are the most frequent porphyrias. We have already diagnosed a big family carrying PCT/AIP Dual Porphyria. Because VP is not very frequent in our country, its association with other porphyria is yet more rare. We present the case of two sisters showing cutaneous signs typical of PCT: blisters, photosensitivity, hyperpigmentation, hypertrichosis, cutaneous fragility, and no acute symptomatology. GW (age 28 years): urinary porphyrins (UP) 1975 gg/ 24 h exhibiting characteristic PCT pattern but with high coproporphyrin content (Uro: 36%, Hepta: 25%, Hexa: 4%, Penta: 10%, Copro: 25%). MW (age 32 years) UP: 256 gg/24 h showing abnormal but not PCT pattern (Uro: 4%, Hepta: not detectable (nd), Hexa: nd, Penta: 16%, Copro: 80%). In both patients urinary _- aminolevulinic acid (ALA) (GW: 1.0 mg/24 h; MW: 1.5 mg/24 h) was normal and porphobilinogen (PBG) was only slightly increased (GW: 3.2 mg/24 h; MW: 2.8 mg/24 h. Faecal porphyrins were elevated: GW: 270 gg/dry weight (Uro: 2%, Hepta: 1%, Hexa: 1%, Penta: 10%; Copro: 35%, Isocopro: 10%, Proto: 36%), MW: 896 gg/dry weight (Uro: 2%, Hepta: 1%, Hexa: 1%, Penta: 2 %; Copro: 35%, Isocopro: 10%, Proto: 49%). In this patient faecal porphyrin content was rather high for a PCT however characteristic for a VP pattern with predominance of Proto. Plasma porphyrin index (PPI) was above normal (<1.30) in both patients: GW: PPI was 4.33 with a wavelength (f) at 618 nm characteristic for PCT, MW: PPI was 6,20 with f at 628 nm characteristic for VP. According to these results patient GW presents a Dual Porphyria PCT/PV while her sister, up to date, has only developed VP. Genetic studies are been carrying out in the uroporphyrinogen decarboxylase and protoporphyrinogen oxydase. VISUALISATION OF PROTOPORPHYRINOGEN OXIDASE MITOCHONDRIAL TARGETING USING GREEN FLUORESCENT PROTEIN
Davids LM, Corrigall AV, Meissner PN Lennox Eales Porphyria Laboratories, Liver Centre, University of Cape Town, South Africa
Variegate porphyria (VP), an autosomal dominant disorder of haeme metabolism, results from defects in the protoporphyrinogen oxidase (PPOX) gene. The disease is characterised by photosensitivity and a propensity to develop acute neurological crises. Protoporphyrinogen oxidase, an inner mitochondrial (mt) membrane protein, does not have a reported mt targeting pre-sequence. This is in contrast the other mt located haeme biosynthetic proteins, in which targeting pre-sequences have been reported. It is therefore, of great interest to establish how PPO is targeted as it is conceivable that a particular PPOX defect(s) may cause VP by disrupting the targeting and translocation of PPO to the mitochondria. The targeting signals of mitochondrial preproteins are typically 17-35aa's in length, and exhibit several common features: i) presequences are rich in positively charged residues (mainly arginines); ii) they generally lack acidic amino acid residues; iii) in most cases they have a high content of hydroxylated residues; and iv) many show the tendency to fold in an amphiphilic \-helix. The amphipathic signals function as matrix targeting signals that can direct import of preproteins to receptors on the mitochondrial surface and subsequently across outer and inner membranes. In addition, some preproteins contain sorting signals that are typically more hydrophobic and direct their specific sorting to intra-mitochondrial compartments. Based on the (non)ability of clinically occurring mutant PPOs we propose that PPO has a recognisable mitochondrial presequence comprising the first 17aa at the N-terminus. We have further tested this hypothesis by creating different sized fragments of PPO-GFP fusion proteins and assessed mitochondrial targeting and translocation by transfection using a human hepatoma (HepG2) cell line followed by fluorescent microscopic analysis. By studying the mt (non)targeting of a number of VP-causing mutant PPOs we have shown that the N-terminal region is critical for efficient mt targeting. Results using the different sized N-terminus fragments further show that 17aa is the minimal length needed for efficient targeting and translocation of PPO to the mitochondria. The smaller fragments of 12, 14 and 15aa showed no targeting. Amino acid analyses and computer predictions of the 17aa pre-sequence show that it is a positively-charged fragment, and with intervening hydrophobic and neutral areas, has the ability to form an amphipathic \-helix.

THE DIAGNOSTIC UTILITY OF PORPHYRIN ANALYSIS AND PLASMA FLUORESCENCE SCANNING IN VARIEGATE PORPHYRIA (VP): AN ACCURATE ASSESSMENT IN SUBJECTS IN WHOM THE PRESENCE OR ABSENCE OF A VP-ASSOCIATED MUTATION IS KNOWN
Hift RJ1, Davidson BP1, Van der Hooft C2, Meissner PN1 1Lennox Eales Porphyria Laboratories, Liver Centre, University of Cape Town, South Africa, 2Erasmus University Medical Centre, Rotterdam, The Netherlands
Variegate porphyria (VP) is the autosomal dominant disorder associated with deficiency of the enzyme protoporphyrinogen oxidase. Clinical features include a photodermatitis and susceptibility to the acute attack; an acute neurovisceral crisis which may be fatal. Early detection is important since with education and avoidance of dangerous medication, the acute attack can be avoided. The standard method for diagnosis of VP is porphyrin analysis of urine and stool. A newer technique is plasma porphyrin fluorescence scanning. The sensitivity and specificity of neither test is accurately known since absolute determination of the genotype was until very recently not possible, and ;surrogate markers such as clinical symptoms were used. We have now undertaken a study to determine the utility of faecal porphyrin analysis and plasma fluorescence scanning in subjects in whom the absolute presence or absence of porphyria is reliably known through mutation detection. Subjects for the study were all those referred to our routine laboratory in whom both the genotype and the plasma fluorescence scanning result and/or faecal porphyrin chromatographic analysis result were available. The majority of subjects carried the common South African R59W mutation. Plasma porphyrin fluorescence scanning was conducted according to published methods. Biochemical porphyrin analysis was by thin layer chromatography. Stool porphyrin profiles were analysed in 400 adults of whom 172 adults carried a VP-associated mutation. Construction of receiver operating characteristics curves and discriminant analysis indicate that stool coproporphyrin is the most sensitive predictor of VP; stool protoporphyrin is less predictive. The sensitivity does not however exceed 0.76 for a specificity of 0.9. Plasma fluorescence scanning was conducted in 676 subjects of whom 205 were positive for a VP-associated mutation. In adults, sensitivity is 0.891 with a specificity of 0.996. Sensitivity is poor below the age of 20, and declines beyond the fourth decade of life. This is the first study to determine accurately the sensitivity and specificity of commonly used diagnostic tests for VP, using the genotype to determine the presence or absence of VP. Our results show that traditional stool porphyrin analysis is poorly sensitive in detecting gene carriers. Furthermore, in contrast to existing practice, the stool coproporphyrin is more predictive of VP than the stool protoporphyrin. Plasma fluorescence scanning is considerably more sensitive than stool porphyrin analysis in detecting carriers. Neither test is however useful in children, and mutation detection remains the most appropriate test for the detection of VP in children.

VALIDATION OF A DENATURING HPLC ASSAY FOR MUTATION ANALYSIS IN THE HUMAN PROTOPORPHYRINOGEN OXIDASE GENE
Kaehne M1, J.C. Deybach2, Puy H2, Robreau A-M2, Viborg D1, Petersen NE1 1Department of Clinical Biochemistry and Clinical Genetics, Odense University Hospital, Denmark, 2Centre Francais des Porphyrias, Hopital Louis Mourier, France
A denaturing high pressure liquid chromatography (DHPLC) assay based on the formation of heteroduplexs, has been developed for mutation analysis of the PPOX gene. This method detects herozygotes for mutations in PPOX. Compared to other mutation analysis methods, the DHPLC method is much more automated and much faster to run. Exon 1 to exon 13 and the flanking intronic regions in the PPOX gene were blindly examined in DNA-samples from 14 patients with Variegate Porphyrias. The mutations in the 14 patients has previously been characterized in the laboratory of professor Jean-Charles Deybach by denaturing gradient gel electrophoresis (DGGE) and sequencing of abnormal band patterns. All the mutations previously found by DGGE were correctly identified using DHPLC. Futher information about the design of the assay and the results from comparison of the two methods will be presented.

A STUDY OF THE POSSIBLE ROLE OF LOW EXPRESSED FUNCTIONAL PPOX ALLELES IN THE VARIABLE PENETRANCE OF VARIEGATE PORPHYRIA
Kimberg M, Warnich L University of Stellenbosch, Stellenbosch, South Africa
The protoporphyrinogen oxidase (PPOX) gene is responsible for the penultimate step of the heme biosynthesis pathway where protoporphyrinogen-IX is oxidized to protoporphyrin. Mutations in the PPOX gene are associated with a diseased state known as variegate porphyria (VP). The term was chosen to describe the low penetrance and varied expression of the autosomal dominant inherited state, including the development of severe abdominal and neurological crises as well as cutaneous photosensitivity, with up to three-quarters of affected individuals remaining asymptomatic. South Africa has the highest incidence of VP in the world due to a founder effect. However, despite decreasing PPOX activity, the R59W founder mutation seems to play no role in the variable penetrance of the VP phenotype. It has recently been shown that where gene mutations are insufficient to convey a disease phenotype, symptoms may penetrate when the mutated allele is coinherited with a functional, yet low-expressed wild-type allele. This study investigates the possibility of a similar modulating mechanism for symptomatic penetrance in VP. Five conserved PPOX haplotypes have been identified based on four single nucleotide polymorphisms (SNPs) in the gene. Despite observed differences in haplotype frequencies between symptomatic, asymptomatic and control patients for at least two of the haplotypes, analysis of variance and association studies yielded negative results. However, due to the limited size of the sample population used the effects of varied expression of wild type haplotypes cannot be excluded. We are now focussing on a more causative approach to identify regulatory elements and possible low-expressed wild type alleles. The -1081G>A sequence variant in the promoter area of the PPOX gene has already been shown to reduce transcriptional activity relative to wild type alleles in in vitro expression assays, and allele specific quantification is now being used to confirm these results. Results from this study will additionally provide a better understanding of the mechanisms involved in PPOX gene regulation for future applications and possible therapies.

KINETIC AND PHYSICAL CHARACTERISATION OF RECOMBINANT WILD TYPE AND MUTANT HUMAN PROTOPORPHYRINOGEN OXIDASES
Maneli MH1, Corrigall AV1, Davids LM1, Klump H2, Kirsch RE3, Meissner PN1 1Lennox Eales Porphyria Laboratories, 2Dept of Molecular and Cell Biology, 3Dept. of Medicine, Liver Centre, University of Cape Town, South Africa
Variegate porphyria (VP) is an autosomal dominant disorder of haem metabolism resulting from defects in the protoporphyrinogen oxidase (PPOX) gene. World wide heterogeneity for VP exists and over 100 mutations have been described to date. In this study, the effects of various PPOX mutations (R59W, H20P, R168C and Y348C) responsible for VP in South Africa, the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, on catalysis and/or cofactor binding, were examined. Wild type recombinant human PPOX and a selection of mutants were generated, expressed and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The Kms for the arginine-59 mutants suggested a substrate-binding problem. T1/2 indicated that arginine 59 is required for the integrity of the active site. The dominant \ helical content was decreased in the mutants. The degree of a helix did not correlate linearly with T1/2 nor Tm values, supporting the suggestion that arginine 59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two state transition suggesting formation of a multi-domain structure. Generally all mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C. This work illustrates the use of studying expressed, purified mutant PPOXs in elucidating the importance of specific amino acid residues to fully understand the structure-function relationship underlying both normal and impaired PPOX activity.

MOLECULAR ANALYSIS OF THE PPOX GENE IN ITALIAN PATIENTS WITH VARIEGATE PORPHYRIA (VP): IDENTIFICATION OF 3 NOVEL MUTATIONS
Patti E, Martinez di Montemuros F, Di Pierro E, Tavazzi D, Cappellini MD Maggiore Policlinico Hospital, IRCCS, University of Milan, Italy
Variegate porphyria (VP; MIM 176200) is a low-penetrance, autosomal dominant disorder resulting from the partial deficiency of protoporphyrinogen oxidase (PPOX; EC 1.3.3.4), the 7th enzyme of the heme biosynthesis. VP is clinically characterized by photosensitivity and occasional acute neurovisceral attacks. Among the diagnostic criteria, the most powerful is the detection of a plasma fluorescent peak at 630 nm, present in roughly 70 % of the symptomatic VP patients. More than 100 molecular abnormalities have been so far identified in the PPOX gene as responsible for VP showing a high molecular heterogeneity. Few data are presently available on the Italian population. Aim of this study was to identify the molecular defect in the PPOX gene in 9 Italian unrelated subjects (1 male, 8 females) with suspected diagnosis of VP. The plasma peak is detected by fluorometric plasma scan from 580 to 650 nm. The coding region of PPOX gene was amplified by PCR in six fragments and directly sequenced. The plasma 20S Physiol. Res. 2003 Vol. 52 peak at 630 nm was markedly positive in 8 subjects and slightly positive in one. Eight different mutations have been identified among the 9 VP patients and summarized in the following Table. Pt. Sex Mutation Exon Protein change Pl. peak (630 nm) Ref. 1 M 218 T>C 3 L73P + Whatley 1999 2 F 306 insC 4 FS>stop+41 ++ This study 3 F 532 C>G 6 L178V + De Siervi 2000 4 F 694 G>C 7 G232R + Deybach 1996 5 F 745 insG 7 FS>stop+31 ++ Deybach 1996 6 F 851 G>T 8 S248I +/- This study 7 F 1013 C>G 10 S338X ++ This study 8 F 1013 C>G 10 S338X ++ This study 9 F 1082 insC 10 FS>stop+19 ++ Whatley 1999 The 1013 C>G mutations was identified in two unrelated subjects. The 306 insC, (FS>stop+41), 851 G>T (S284I) and 1013 C>G (S338X) mutations have been identified for the first time in this study and cause truncated or unstable proteins. 1082 insC seems to be quite common being found in 12% of VP patients in France and in another Italian study. The molecular analysis has been extended to 10 probands' family members with the finding of 7 mutation carriers. These results, suggest a high molecular heterogeneity for VP in Italy as described for other countries.

MITOCHONDRIAL TARGETING MECHANISMS OF PROTOPORPHYRINOGEN OXIDASE: IDENTIFICATION AND CHARACTERIZATION OF INTERNAL TARGETING SEQUENCES
von und zu Fraunberg M, Kauppinen R Department of Medicine, Division of Endocrinology, University Central Hospital of Helsinki, Biomedicum Helsinki, Finland
Recent studies have revealed that a relatively short 28-amino acid segment in the amino terminus of protoporphyrinogen oxidase (PPOX) contains information, which is sufficient to direct the protein into mitochondria. Furthermore, our experiments with amino-terminally truncated green fluorescent protein (GFP)-fusion polypeptides showed that amino acids 25-477 of PPOX contained an additional mitochondrial targeting signal(s), which could direct the reporter protein into mitochondria even in the absence of the amino-terminal signal. We have investigated the internal signal sequences for mitochondrial transport of PPOX by performing a secondary structure analysis of the polypeptide and studying the intracellular localization of mutated PPOX-GFP-fusion proteins in COS-1 cells. Since the model for the interaction between the amino terminus of PPOX and the putative mitochondrial receptor 28S Physiol. Res. 2003 Vol. 52 protein Tom20 suggested that leucine and isoleucine residues forming an alpha-helical hydrophobic motif LXXXIXXL were crucial for the recognition of the targeting signal, we searched for helical, leucine-rich segments with net positive charge and chose four most promising candidate segments. Three of the segments could direct PPOX into mitochondria when replacing the authentic amino-terminal targeting signal. Substituting polar glutamine for non-polar leucine residues of these segments could impair the mitochondrial transport in the absence of the amino-terminal targeting signal indicating that these regions contribute to internal mitochondrial targeting signaling of PPOX. The importance of leucine and isoleucine residues for the mitochondrial targeting suggests that hydrophobic interactions are involved also in the recognition of internal targeting signals, possibly through interaction with Tom20 or other mitochondrial receptors.

VARIEGATE PORPHYRIA IN SOUTH AFRICA: A POPULATION-BASED STUDY TO DETERMINE THE FREQUENCY OF THE FOUNDER GENE MUTATION (R59W)
Warnich L, Koegelenberg AJ University of Stellenbosch, Stellenbosch, South Africa
The incidence of variegate porphyria (VP) in the South African population of European descent is the highest in the world because of a founder gene effect and the gene frequency was estimated to be 0.3%. However, this estimate is questionable on the grounds of genealogical and biochemical methods used and as indicated by more recent records from diagnostic laboratories. According to earlier reports more than 80% of the estimated 10 000-20 000 South African individuals with VP remain undetected and are therefore at risk of potentially lethal acute neurological attacks. This poses a serious question regarding the actual frequency of VP, and specifically the founder gene mutation, in South Africa. Are there thousands of undiagnosed VP carriers in South Africa as genealogical studies and population growth curves have predicted, or is the allele frequency much lower than 0.3%? The aim of this study was thus to determine the prevalence of the founder mutation (R59W) with a highly specific DNA test. Blood samples were obtained at blood transfusion clinics, pathology clinics and maternity wards. For the initial screening we used SSCP analysis, which proved to be consistent and cost-effective. All R59W positive samples were re-tested using restriction enzyme analysis. The R59W mutation was detected in five of the 4637 samples collected to date. The estimated frequency of 0.15% (5/3311) for the founder mutation in South Africans of European descent is much lower than the frequency of 0.3% estimated previously. However, different frequencies were obtained within the various population samples and we suggest that a newborn baby population sample be used in the future, as it is the most unbiased sample source. Interestingly, the highest frequency for the R59W mutation was obtained in this sample. One of the two adult R59W positive participants was unaware of her carrier status, in accordance with the incomplete penetrance of the trait. Two of the mothers of the three newborn babies found to be R59W positive were also not aware of VP in their families, indicating that ignorance regarding VP status in South Africa is a matter of some concern.

NEUROPSYCHIATRIC PORPHYRIA IN PATIENTS WITH REFRACTORY EPILEPSY: REPORT OF THREE CASES
Winkler AS1, Peters TJ2, Elwes RDC1 1King's College Hospital, 2King's College, London, UK
Acute symptomatic seizures are a well recognised symptom of acute porphyria in relapse. We report three patients with a long history of chronic refractory epileptic seizures who were subsequently found to suffer from one of the neuropsychiatric porphyrias. Patient 1 had an eight-year history of seizures since the age of 12, probably due to primary generalised epilepsy. Past medical history showed chronic constipation requiring enemas. The seizure frequency increased despite high doses of Sodium Valproate and the patient developed abdominal pains when Lamotrigine was added which caused a marked worsening of the seizures. A diagnosis of acute intermittent porphyria was established. Patient 2 had a three-year history of intractable generalised convulsions. Carbamazepine and Phenytoin were unable to control the seizures. Blood tests showed abnormal liver toxicity tests and hyponatraemia. Revisiting her medical history she admitted to frequent abdominal pains with constipation and sun sensitive skin. The patient was found to suffer from variegate porphyria. Patient 3 had an 18-year history of mainly simple partial sensory and motor seizures and then atonic seizures. She was unsuccessfully tried on a variety of antiepileptic drugs and after starting Topiramate developed an acute neurological disorder with reduced level of consciousness, bulbar disturbance, an asymmetric spastic quadriparesis, negative imaging with only partial recovery. At this time her urine was noted to be dark and a diagnosis of variegate porphyria was established. Porphyria may be an aetiological factor in some cases of chronic refractory partial or generalised epilepsy. Porphyria should also be considered if addition of a new antiepileptic medication causes a major deterioration in epilepsy.