Identification of mutations in the uroporphyrinogen III cosynthase gene in German patients with congenital erythropoietic porphyria.

The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes along the heme biosynthetic pathway. Congenital erythropoietic porphyria is a very rare disease that is inherited as an autosomal recessive trait and results from a profound deficiency of uroporphyrinogen III cosynthase, the fourth enzyme in heme biosynthesis. The degree of severity of clinical symptoms mainly depends on the amount of residual uroporphyrinogen III cosynthase activity. In this study, we sought to characterize the molecular basis of congenital erythropoietic porphyria in Germany by studying four patients with congenital erythropoietic porphyria and their families. Using PCR-based techniques, we identified four different mutations: C73R, a well-known hotspot mutation, the promoter mutation -86A that was also described previously, and two novel missense mutations, designated G236V and L237P, the latter one encountered in the homozygous state in one of the patients. Our data from the German population further emphasize the molecular heterogeneity of congenital erythropoietic porphyria as well as the advantages of molecular genetic techniques as a diagnostic tool and for the detection of clinically asymptomatic heterozygous mutation carriers within families.

[Acral necroses after therapy with quinine sulfate for calf cramps]. / Akrale Nekrosen nach Therapie mit Chininsulfat wegen Wadenkrämpfen.

The most common cause of acral cyanosis is vascular spasm which can be induced by several drugs. An 87-year-old woman developed red and livid skin lesions on the fingers of both hands and several toes one month after beginning treatment with quinine sulfate 200 mg daily. The skin lesions progressed to necrosis in some areas. Quinine sulfate is a widely prescribed drug for nocturnal cramps. The following side effects may develop, particularly in the elderly: exanthems, pruritus, urticaria, erythema multiforme, purpura and photosensitivity. Our case points to the possibility of acral necrosis and demonstrates the efficacy of vasodilator treatment.

Treatment of porphyria cutanea tarda with oral thalidomide.

Eight male patients with overt clinical and biochemical features of porphyria cutanea tarda (PCT) were orally treated with 300 mg/day thalidomide for 1 week and with 200 mg/day for 3 more weeks. Already after the first week of treatment no new vesicles and/or bullae could be observed. Spontaneous blisters completely disappeared, increased skin fragility subsided and skin hyperpigmentation receded about 2 months after completion of therapy, whereas hypertrichosis persisted. There was a rapid decrease in the urinary total porphyrin excretion which reached normal levels in all patients by the end of the fourth week of therapy, whereas the posttreament chromatographic pattern of urinary porphyrins revealed a slight reduction of higher carboxylated porphyrin metabolites and an increase in the amount of the excreted coproporphyrin, as compared to the pretreatment period. Somnolence, intermittent constipation and dry mouth occurred in all patients, 2 patients additionally experienced dizziness. No evidence of peripheral neuropathy could be detected and laboratory investigations revealed no abnormalities, as compared to the pretreatment period. During the 16- to 28-month follow-up of the patients, no clinical or biochemical relapse was observed. In view of the encouraging results of the present investigation, further studies are now warranted in order to definitely answer the question whether oral thalidomide may be regarded as an effective alternative approach to the treatment of PCT.

Alterations of rat liver phospholipid composition induced by oral thalidomide.

In an attempt to elucidate the effects of oral thalidomide on liver phospholipid composition, doses of 1 and 3 mg/kg/day of thalidomide were orally administered to two groups of female Wistar rats (7 animals each), respectively, over a period of 60 days. Control animals (n = 7) received corresponding quantities of the vehicle alone. Chromatographic analysis and quantitative determination of the isolated phospholipid classes revealed statistically significant alterations of phospholipid fractions in the liver of the animals treated with the higher thalidomide dose (3 mg/kg/day). These alterations may be associated with changes in the metabolic activity, ionic transport and cell-cell interactions of the hepatic cellular components.

Erythropoietic protoporphyria: identification of novel mutations in the ferrochelatase gene and comparison of biochemical markers versus molecular analysis as diagnostic strategies.

BACKGROUND: Erythropoietic protoporphyria (EPP) results from an inherited deficiency of the last enzyme of the heme biosynthetic pathway, ferrochelatase (FC). EPP is usually inherited in an autosomal dominant fashion, and the mutations in the FC gene on chromosome 18q21.3 detected in EPP patients are heterogeneous. METHODS: In this study, we screened the FC gene for mutations in 12 patients from 10 unrelated families with EPP and their family members using heteroduplex analysis, automated sequencing, and restriction enzyme digestion. RESULTS: We detected 8 different mutations in these patients, including 1 missense mutation, 5 frameshift mutations, and 2 splice site mutations, 6 of which are previously undescribed. CONCLUSIONS: We have established the molecular basis of EPP in 10 unrelated families, thereby providing further evidence for the heterogeneity in this disorder. Importantly, molecular diagnosis allowed revisions in the status of several clinically unaffected silent mutation carriers within the families. We compare the value of genetic research strategies with the combination of biochemical data and clinical phenotype as diagnostic tools to confirm a putative diagnosis in EPP.

Multiple basal cell carcinomas associated with hairy cell leukaemia.

We report the case of a caucasian woman who, between the ages of 49 and 51 years, developed multiple (> 20) basal cell carcinomas (BCC). There was no family history of BCC. No abnormalities in the human homologue of the Drosophila segment polarity gene patched (PTCH), glutathione S-transferases T1 and M1, or cytochrome P450 1A1 were detected by polymerase chain reaction (PCR)-based molecular analysis. There was, however, actinic damage of the skin in sun-exposed areas. The patient was diagnosed as having hairy cell leukaemia (HCL) at the age of 51 years, based upon leucocyte morphology as assessed by light and electron microscopy, tartrate-resistant acid leucocyte phosphatase (TRAP) staining, fluorescence activated cell scanning of peripheral blood leucocytes and bone marrow histology. As the leukaemia slowly progressed over a 3-month period, the patient developed four further BCCs. Given that HCL is characterized by a profound defect in T-cell function, it is conceivable that T-cell immune dysregulation can contribute to the pathogenesis of BCC, possibly enhancing the aetiological effect of ultraviolet irradiation.

C73R is a hotspot mutation in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from profoundly deficient activity of the fourth enzyme of the haeme biosynthetic pathway, uroporphyrinogen III synthase (UROIIIS). CEP is a rare, recessively inherited disorder, and mutations in the UROIIIS gene detected in CEP patients are heterogeneous. The notable exception to this rule is a single missense mutation, designated C73R, which represents over 40% of all mutant UROIIIS alleles. In this study, we investigated three separate families with CEP from different ethnic backgrounds. We performed haplotype analysis using two microsatellite markers that closely flank the UROIIIS gene on chromosome 10q24, spanning a region of 4 cM on the GB4 linkage panel. Haplotype analysis revealed the occurrence of C73R on different haplotypes in four out of four disease chromosomes studied. The results are consistent with the hypothesis that C73R is a hotspot mutation for CEP, and does not represent wide dispersion of a single ancestral mutant C73R allele.

Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria.

The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Efficacy and safety of systemic recombinant interferon-alpha in Behçet's disease.

OBJECTIVES: To evaluate the therapeutic efficacy and safety of systemic recombinant interferon alpha-2a (IFN-alpha) in patients with Behcet's disease (BD) and to determine the incidence of episodes in complete responders during the one-year pretreatment period and follow-up. DESIGN: An open clinical study. SETTING: Departments of Dermatology and Ophthalmology, University of Patras, Greece and Department of Dermatology, Heinrich-Heine University of Düsseldorf, Germany. SUBJECTS: Twelve patients (aged 23-52 years) with active BD who had previously been unsuccessfully treated with systemic steroids and/or immunosuppressives. INTERVENTIONS: IFN-alpha was administered subcutaneously at a dose of 6 X 10(6) IU per day 3 times per week for 2 months. MAIN OUTCOME MEASURES: Change of area or number of mucocutaneous lesions, grading score for thrombophlebitis and ocular inflammation, haematological and biochemical parameters and number of episodes during the pretreatment period and the follow-up. Evaluation of IFN-alpha side effects. RESULTS: Nine patients (75.0%) revealed a complete remission, two (16.6%) a partial remission and one patient (8.3%) showed no response. During the follow-up in five out of the nine complete responders (55.5%) no episodes of BD were seen, whereas, the other four patients (44.5%) had 1-2 episodes, as compared to 5-8 and 5-12 episodes, respectively, during the pretreatment period. An influenza-like syndrome (fever, nausea and myalgias) appeared during the early phase of therapy in all (but one) patients. No patient had to discontinue IFN-alpha because of intolerance. CONCLUSIONS: Subcutaneous human recombinant interferon alpha-2a appears to be an effective and fairly well tolerated therapy for BD.

[Extracorporeal photopheresis in therapy-refractory disseminated discoid lupus erythematosus]. / Extrakorporale Photopherese bei therapieresistentem disseminiert diskoidem Lupus erythematodes (DDLE).

Lupus erythematosus (LE) is a disease with a wide spectrum of cutaneous and systemic manifestations. Discoid LE (DLE) is the most common form of cutaneous LE; the disseminated form of DLE is rare. We report an encouraging response to treatment with extracorporeal photopheresis (ECP) in a single patient with disseminated DLE who did not respond to conventional therapy. To the best of our knowledge this is the first successful use of ECP in the management of such a patient. Extracorporeal photopheresis is a therapeutic modality that has been under investigation for more than 12 years. Although originally developed for the treatment of cutaneous T-cell lymphoma, ECP has recently been used for the management of autoimmune diseases including systemic scleroderma, pemphigus vulgaris and SLE, as well as prevent organ rejection in patients with cardiac or kidney transplants and graft versus host disease after bone marrow transplantation.

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence.

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of >120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

Variegate porphyria: identification of a nonsense mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders of porphyrin metabolism that result from inherited or acquired aberrations in the control of the heme biosynthetic pathway. Variegate porphyria is characterized by a partial reduction in the activity of protoporphyrinogen oxidase. In this study, we identified the first nonsense mutation in a family with variegate porphyria. The mutation consisted of a previously unreported G-to-T transversion in exon 5 of the protoporphyrinogen oxidase gene, resulting in the substitution of glutamic acid by a nonsense codon, designated E133X. Our investigation establishes that a nonsense mutation in the protoporphyrinogen oxidase gene is the underlying mutation in this family with variegate porphyria.

Carbon dioxide laser treatment of extramammary Paget's disease guided by photodynamic diagnosis.

Extramammary Paget's disease (EPD) is a rare malignancy occurring mainly in apocrine gland-bearing regions. Surgical excision is the treatment of choice. This may be very difficult or even impossible if the disease is widespread or located in a critical anatomical site. We report on the successful treatment of a 71-year-old man with EPD in the suprapubic region with CO2 laser guided by photodynamic diagnosis.

Photodynamic therapy in dermatology.

Photodynamic therapy (PDT) uses exogenously administered or endogenously formed photosensitizers activated by light to induce cell death via formation of singlet oxygen and other free radicals. Photodynamic therapy is increasingly used for the treatment of skin cancers and other indications. The efficacy of PDT depends on the structure of the photosensitizer, the administration modality, the light source, and the treatment procedure. We reviewed the most recent clinical and experimental developments in PDT research related to dermatology. The substrate under most intense investigation in PDT research is delta-aminolevulinic acid (ALA). Photodynamic therapy with topically applied ALA has been shown to be highly efficient in the treatment of cutaneous neoplasms by using intralesionally formed porphyrins as photosensitizers. For solar keratoses, best response rates have been described. delta-Aminolevulinic-PDT is also efficient in the treatment of superficial basal cell and squamous cell carcinomas. In addition, the fluorescence of ALA-induced porphyrins under a Wood light is highly selective in neoplastic cutaneous tissue and offers a useful technique in detecting and delineating skin tumors with ill-defined borders.

Successful surgery of multiple recurrent basal cell carcinomas guided by photodynamic diagnosis.

We describe a 56-year-old Caucasian man with history of multiple regressed basal cell carcinomas. During the last 20 years approximately 200 histologically proven basal cell carcinomas preferentially localized on the face were surgically treated. Several large skin grafts were necessary to cover the extensive tissue defects on the face and scalp. Although all excised tissues were histologically proven to be basal cell carcinomas with tumor-free margins, new tumors developed in proximity to the skin graft margins. The dissemination of the new tumors made it difficult to perform additional invasive operation procedures without influencing the cosmetic result. Thus, we used photodynamic diagnosis to improve detection and demarcation of the neoplastic tissues. This procedure facilitated surgical planning and enabled primary in toto excisions. Surgical trauma and a number of interventions were thus minimized with the consequence of improved cosmetic and functional results.

Treatment of condylomata acuminata with oral isotretinoin.

PURPOSE: The aim of our study was to evaluate the efficacy and safety of oral isotretinoin in the treatment of condylomata acuminata. MATERIALS AND METHODS: A total of 56 male patients with a history of condylomata acuminata refractory to at least 1 standard therapeutic regimen was treated orally with isotretinoin (1 mg./kg. daily) during a 3-month period. RESULTS: At the end of treatment 21 of the 53 evaluated patients (39.6%) had complete response, 7 (13.2%) had partial response and 25 (47.1%) had no response. A statistically significant inverse relationship was found between age and area of treated lesions and response to medication. Two complete responders (9.5%) revealed recurrence during the 1-year followup. CONCLUSIONS: Oral isotretinoin may be regarded as an effective, fairly well tolerated and noninvasive alternative form of therapy for immature and small condylomata acuminata.

Photodynamic therapy with 5-aminolaevulinic acid-induced porphyrins of an amelanotic melanoma in vivo.

Of particular interest for photodynamic therapy (PDT) are the endogenously formed and photodynamically active porphyrins produced following topical or systemic application of 65-aminolaevulinic acid (ALA), a haem precursor. Having determined the pharmacokinetics and wavelength dependence of PDT with ALA-induced porphyrins, we analysed the porphyrin metabolites in tumour and surrounding skin. The therapeutic efficacy of PDT using ALA-induced porphyrins was investigated. Amelanotic melanomas (A-Mel-3) were implanted subcutaneously in the back of Syrian golden hamsters (body weight (b.w.), 70-80 g). After 5-7 days, tumours with a volume of approximately 150 mm3 were used for PDT (n = 36). ALA (500 mg kg-1 b.w., pH 6.5) was injected intravenously 45, 90, 150 and 300 min before light irradiation (635 nm, 100 mW cm-2, 100 J cm-2). Tumours with light irradiation only served as controls. The tumour volume was measured after PDT for 28 days. The total porphyrin content was determined in the tumours, the surrounding skin and erythrocytes prior to and 45, 90, 180, 240, 300 and 480 min and 24 h following intravenous injection of ALA (500 mg kg-1 b.w.; n = 32). Porphyrin metabolites were separated by high pressure liquid chromatography (HPLC). Tumour growth was significantly delayed when PDT with ALA was performed 45, 90 or 150 min following intravenous administration. At that time, protoporphyrin (1.8 +/- 0.4 nmol g-1), coproporphyrin (2.2 +/- 0.5 nmol g-1) and uroporphyrin (1.7 +/- 1.4 nmol g-1) were the main metabolites in the tumour tissue. Erythrocytes also contained significant amounts of porphyrins (11.8 +/- 1.3 nmol g-1). The tumour and surrounding skin exhibited a different pattern of porphyrin metabolites. Unexpectedly, a single treatment of PDT with ALA-induced porphyrin resulted in only one complete remission out of six amelanotic melanomas when the final therapeutic outcome was assessed after 28 days. The therapeutic efficacy of PDT with ALA-induced porphyrins can be positively correlated with the fluorescence kinetics previously determined. The analysis of the porphyrin metabolites in amelanotic melanoma by HPLC indicates that the porphyrin accumulation is not due to a decreased activity of ferrochelatase. Moreover, the photodynamic effects may not be mediated solely by porphyrins localized in the tumour parenchyma, but also by significant amounts of porphyrins in the microvasculature. PDT with this endogenous photosensitizer failed to induce complete emission of the treated tumours despite irradiation at the time of maximum porphyrin concentration using the optimum therapeutic wavelength. Thus PDT with ALA-induced porphyrins is less effective in our model relative to that observed for the exogenous photosensitizer Photofrin or synthetic porphycenes after a single treatment.

Ex vivo application of delta-aminolevulinic acid induces high and specific porphyrin levels in human skin tumors: possible basis for selective photodynamic therapy.

In photodynamic therapy with topically applied delta-aminolevulinic acid porphyrins are acting as photosensitizers. The profile of porphyrin metabolites in normal or in neoplastic skin after administration of delta-aminolevulinic acid has not been determined in detail yet. Thus, to study porphyrin biosynthesis in human skin an organ culture model was developed. Explant pieces of normal skin, keratoacanthoma, and basal cell carcinoma were incubated with 1 mM delta-aminolevulinic acid for 36 h. Levels of delta-aminolevulinic acid, porphyrins and porphyrin metabolites were measured in tissues and supernatants. After incubation with delta-aminolevulinic acid, higher porphyrin levels were demonstrated in tumors as compared to normal skin. In supernatants, most of formed porphyrins, preferentially highly carboxylated porphyrin metabolites, were measured. The pattern of synthesized porphyrins differed between normal and neoplastic skin explants. In tissues of basal cell carcinomas protoporphyrin was preferentially shown and tissues of keratoacanthomas were characterized by a predominance of coproporphyrin as compared to normal skin. The results show that explant cultures offer an easy approach to examine the porphyrin biosynthesis of various tissues. The tumor-specific delta-aminolevulinic acid metabolism indicates additional porphyrin metabolites such as coproporphyrin apart from protoporphyrin as effective photosensitizers and may offer a novel approach to tumor-selective photodynamic damage.

Effects of clonidine in a primed rat model of acute hepatic porphyria.

Acute hepatic porphyrias can be induced by several drugs and acute attacks of porphyrias are often associated with severe hypertension. Therefore it is important to know if an antihypertensive drug used has porphyrogenic potency or not. As previously demonstrated in normal rats the alpha-receptor blocker clonidine (CAS 4205-90-7) has no significant influence on the porphyrin metabolism. Pretreatment of rats with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or allyl-isopropyl-acetamide (AIA) induces hepatic delta-aminolaevulinic acid synthase (ALA-S) and increases the urinary excretion of porphyrin precursors (ALA and PBG) comparable to the latent phase of acute hepatic porphyrias in humans. Clonidine did not induce hepatic ALA-S or urinary excretion of ALA or PBG in normal as well as in DDC or AIA pretreated rats. Moreover the induction of P4501A1 (7-ethoxyresorufin-O-deethylase) by DDC was abolished by simultaneous application of clonidine. From these findings one can probably conclude that clonidine is a safe drug in human acute hepatic porphyria.