Congenital erythropoietic porphyria: a single-observer clinical study of 29 cases.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an autosomal recessive cutaneous porphyria caused by decreased activity of uroporphyrinogen III synthase (UROS). Its predominant characteristics include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. Due to its rarity and genetic heterogeneity, clinical phenotypes are unclear and its impact on health-related quality of life (HRQoL) has not been previously assessed. OBJECTIVES: To define comprehensively CEP phenotypes and assess their impact on HRQoL, and to correlate these factors with laboratory parameters. METHODS: A single observer assessed patients with CEP from four European countries. RESULTS: Twenty-seven unrelated patients with CEP, aged between 7.6 and 65 years, participated in the study. The patients came from the U.K. (17), France (4), Switzerland (4) and Germany (2). Additional data were obtained for two deceased patients. Newly characterized features of CEP include acute-onset cutaneous and noncutaneous symptoms immediately following sunlight exposure, and pink erythematous facial papules. There was a lack of consistent genotype-phenotype correlation in CEP. The main poor prognostic factors in CEP are the early age of disease onset and haematological complications. CONCLUSIONS: CEP is a multisystem disease; cutaneous, ocular, oral and skeletal manifestations also contribute to disease severity and impact on HRQoL, in addition to the haematological complications. The rarity of the disease can lead to delayed diagnosis. The lack of consistent genotype-phenotype correlation in CEP suggests a contribution to phenotype from other factors, such as environment, patients' photoprotective behaviour and genes other than UROS. There is currently an unmet need for multidisciplinary management of patients with CEP.

A management algorithm for congenital erythropoietic porphyria derived from a study of 29 cases.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an autosomal recessive photomutilating porphyria with onset usually in childhood, where haematological complications determine prognosis. Due to its extreme rarity and clinical heterogeneity, management decisions in CEP are often difficult. OBJECTIVES: To develop a management algorithm for patients with CEP based on data from carefully characterized historical cases. METHODS: A single investigator collated data related to treatments and their outcomes in 29 patients with CEP from the U.K., France, Germany and Switzerland. RESULTS: Six children were treated with bone marrow transplantation (BMT); five have remained symptomatically cured up to 11.5 years post-transplantation. Treatments such as oral charcoal, splenectomy and chronic hypertransfusion were either of no benefit or were associated with complications and negative impact on health-related quality of life. Lack of consistent genotype-phenotype correlation meant that this could not be used to predict disease prognosis. The main poor prognostic factors were early age of disease onset and severity of haematological manifestations. CONCLUSIONS: A management algorithm is proposed where every patient, irrespective of disease severity at presentation, should receive a comprehensive, multidisciplinary clinical assessment and should then be reviewed at intervals based on their predicted prognosis, and the rate of onset of complications. A BMT should be considered in those with progressive, symptomatic haemolytic anaemia and/or thrombocytopenia. Uroporphyrinogen III synthase genotypes associated with poor prognosis would additionally justify consideration for a BMT. Rigorous photoprotection of the skin and eyes from visible light is essential in all patients.

Characterization of mutations in the CPO gene in British patients demonstrates absence of genotype-phenotype correlation and identifies relationship between hereditary coproporphyria and harderoporphyria.

Hereditary coproporphyria (HCP) is the least common of the autosomal dominant acute hepatic porphyrias. It results from mutations in the CPO gene that encodes the mitochondrial enzyme, coproporphyrinogen oxidase. A few patients have also been reported who are homoallellic or heteroallelic for CPO mutations and are clinically distinct from those with HCP. In such patients the presence of a specific mutation (K404E) on one or both alleles produces a neonatal hemolytic anemia that is known as "harderoporphyria"; mutations on both alleles elsewhere in the gene give rise to the "homozygous" variant of HCP. The molecular relationship between these disorders and HCP has not been defined. We describe the molecular investigation and clinical features of 17 unrelated British patients with HCP. Ten novel and four previously reported CPO mutations, together with three previously unrecognized single-nucleotide polymorphisms, were identified in 15 of the 17 patients. HCP is more heterogeneous than other acute porphyrias, with all but one mutation being restricted to a single family, with a predominance of missense mutations (10 missense, 2 nonsense, 1 frameshift, and 1 splice site). Of the four known mutations, one (R331W) has previously been reported to cause disease only in homozygotes. Heterologous expression of another mutation (R401W) demonstrated functional properties similar to those of the K404E harderoporphyria mutation. In all patients, clinical presentation was uniform, in spite of the wide range (1%-64%) of residual coproporphyrinogen oxidase activity, as determined by heterologous expression. Our findings add substantially to knowledge of the molecular epidemiology of HCP, show that single copies of CPO mutations that are known or predicted to cause "homozygous" HCP or harderoporphyria can produce typical HCP in adults, and demonstrate that the severity of the phenotype does not correlate with the degree of inactivation by mutation of coproporphyrinogen oxidase.

Comparison of complementary and genomic DNA sequencing for the detection of mutations in the HMBS gene in British patients with acute intermittent porphyria: identification of 25 novel mutations.

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder caused by mutations in the hydroxymethylbilane synthase (HMBS) gene. Direct detection of mutations is becoming the method of choice for the accurate identification of asymptomatic affected individuals within AIP families so that they can be advised to avoid drugs and other compounds that provoke the life-threatening acute neurovisceral crises that characterise the condition. We describe a prospective comparison of direct automated sequencing of cDNA (29 patients) or genomic DNA (28 patients) to identify HMBS mutations in 57 patients referred consecutively for mutational analysis; 39 different mutations were identified in 54 patients. The sensitivity of the cDNA and genomic DNA methods was 69% and 95%, respectively, indicating that analysis of genomic DNA provides a higher mutation detection rate. Thirty mutations were restricted to a single family; only one (R173W) occurred in more than three families. Of the mutations (6 missense, 8 splice defects, 10 frameshifts, 1 nonsense), 25 have not been reported previously. One novel mutation (344+33G-->T) was located in a putative intron splice enhancer in intron7. Our results define the extent of allelic heterogeneity and the types (41% missense; 59% truncating) and distribution (35% in exons 10, 12, 14) of HMBS mutations, for AIP in the United Kingdom.

Alterations in heart failure of cyclic AMP-dependent inotropic and lusitropic properties of cardiac and skeletal muscle.

A central working hypothesis in our laboratory is that deficient cellular cyclic AMP concentrations may be responsible, at least in part, for striated muscle dysfunction, both cardiac and skeletal, in heart failure. These results suggest that therapy aimed at restoring cyclic AMP to normal levels may be effective with regard to improving systolic and diastolic function in the heart and may decrease the development of fatigue in skeletal muscle of patients with failure. The use of cyclic AMP-dependent drugs in clinical practice has been limited by side effects associated with raising total cellular content of this cyclic nucleotide. However, evidence suggesting that separate pools of cyclic AMP may exist within the cell raises the possibility that those pools associated with excitation/contraction coupling could serve as more specific therapeutic targets.

Negative inotropic effect of methylecgonidine, a major product of cocaine base pyrolysis, on ferret and human myocardium.

This study examined the physiological effects and potential mechanisms of action of methylecgonidine (MEG), the major pyrolysis product from smoking "crack cocaine," on cardiac function. Ferret right ventricular papillary muscles and human ventricular trabeculae were isolated and placed in a physiological solution at 30 degrees C containing 2.5 mM Ca2+ and stimulated at 0.33 Hz. MEG decreased peak tension and peak intracellular Ca2+ transients in a concentration-dependent manner (10 microM-1 mM). The negative inotropic effect (NIE) of MEG was reversible by atropine (1 microM). Atropine shifted the concentration-response curve of MEG rightward (pA2 = 9.17) similar to that of carbachol (pA2 = 8.70). With prior addition of histamine (1 microM) and Ca2+ (4.5 mM) in equiinotropic concentrations, MEG and carbachol decreased contractility to a greater extent in the histamine-stimulated muscles. To clarify whether the treatments altered responsiveness of the contractile elements to Ca2+, the effect of 2,3-butanedione monoxime (BDM), an agent that interferes with the interaction of actin and myosin, was tested after prior addition of histamine or increased Ca2+. No differential effect occurred. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME; 0.1 mM), lessened the NIE of MEG compared with prior (pre-L-NAME) values. Furthermore, in human ventricular trabeculae (n = 7), MEG exhibited an NIE that was also reversible by atropine. We concluded that the NIE of MEG is caused by decreased calcium availability; the effect is not the result of a local anesthetic action but is mediated by stimulation of cholinergic receptors. This effect is potentiated by the nitric oxide pathway.

Effect of cocaine and methylecgonidine on intracellular Ca2+ and myocardial contraction in cardiac myocytes.

We evaluated the cardiac effects of the principle pyrolysis product of crack cocaine smoking, methylecgonidine (MEG), in comparison with cocaine. Peak cell shortening and intracellular Ca2+, as detected by the Ca2+ indicator indo 1, were recorded in enzymatically isolated ferret myocytes. Both cocaine and MEG decreased peak cell shortening and peak intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner (10(-8)-10(-4) M). MEG shifted the peak [Ca2+]i-to-peak shortening relationship downward and was more potent than cocaine. Atropine (10(-6) M) upwardly shifted the dose-response curves of MEG, cocaine, and carbachol but not of procaine. The negative inotropic effects of MEG were inhibited by methoctramine, a selective M2 receptor blocker but not by M1 (pirenzepine) or M3 (4-diphenylacetoxy-N-methylpiperidine methiodide) blocking agents. In contrast to cocaine, the effects of large doses of MEG were irreversible over the time course of our experiments, raising the possibility of structural damage. We conclude that MEG acts primarily on M2 cholinergic receptors in the heart to produce acute cardiac intoxication and, in contrast to cocaine, may decrease the myofilament Ca2+ responseness and cause structural damage to myocytes by a direct toxic effect.

Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.

Deficient cellular cyclic AMP may cause both cardiac and skeletal muscle dysfunction in heart failure.

Deficient myocardial cyclic AMP concentrations contribute to abnormal Ca2+ handling and systolic and diastolic dysfunction in chronic heart failure (CHF). We tested the hypothesis that decreased cyclic AMP in skeletal muscle of animals with failure may contribute to the weakness and easy fatiguability also common in patients with CHF. We compared intracellular Ca2+ signaling and contractility in skeletal muscle preparations from rats 6 weeks after myocardial infarction-induced CHF versus sham-operated controls. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control and CHF rats. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches. Treatment with 2mM dibutyryl cyclic AMP returned tension and Ca2+ towards normal levels. There was no evidence of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling that could be reversed with cyclic AMP supplementation as previously reported for the heart. This suggests that deficient cyclic AMP levels may contribute to both cardiac and skeletal muscle dysfunction in CHF.

A robotics-based liquid chromatographic assay for the measurement of atovaquone in plasma.

A precise and specific robotics-based liquid chromatographic (LC) method for measuring atovaquone concentrations in plasma was developed and validated, and the method was compared with an existing manual LC method. The compound was isolated from plasma by liquid-liquid extraction, separated by reversed-phase LC, and quantitated against an internal standard with UV detection. Least-squares linear regression with 1/concentration2 weighting was used as the calibration model. The range of the calibration curve for the assay under routine conditions was 0.25-50 micrograms ml-1. No endogenous interferences with the compound or the internal standard were noted in either untreated human plasma or in plasma from patients enrolled in Phase III clinical trials of atovaquone. The accuracy of the assay (determined as the percent bias) ranged from -4.8% to -9.4% in the validation runs. The intra- and interassay precisions (determined as the relative standard deviation) were less than 6.8% and 6.4%, respectively. The contribution of an internal standard on assay accuracy and precision also was examined. Interassay variability was marginally improved by the incorporation of an internal standard to the assay; accuracy and intra-assay precision were essentially unchanged. A paired t-test between estimates of atovaquone concentrations in healthy volunteer and HIV + patient human plasma samples assayed by the automated and manual methods demonstrated no significant difference (p = 0.31) between the values determined by each method.

Detection of latent variegate porphyria by fluorescence emission spectroscopy of plasma.

The plasma of patients with overt variegate porphyria contains porphyrin with a fluorescence emission maximum at about 626 nm, which is diagnostic for the condition. We have evaluated qualitative fluorescence emission scanning of saline-diluted plasma as a method for the identification of asymptomatic carriers of the gene for variegate porphyria. Plasma from 36 unrelated patients with variegate porphyria, 136 of their asymptomatic first- and second-degree relatives aged 15 years or over, and 322 normal subjects was scanned. An emission maximum between 621 and 627 nm was observed in the 36 patients with variegate porphyria and 54 of their relatives, but not in any normal subject, nor in 56 patients with other types of porphyria. For the detection of asymptomatic adult carriers of the gene for variegate porphyria, fluorescence emission scanning of plasma appears to be 100% specific, with a sensitivity of 86% (95% confidence interval 71-98%). In contrast, the sensitivity of faecal porphyrin analysis as a test for adult gene carriers was 36%. These results suggest that fluorescence emission scanning of plasma should replace faecal porphyrin analysis as the test of first choice for this purpose.

Differences in the metabolism of the aromatic amino acid hydroxylase cofactor, tetrahydrobiopterin, in mutant mice with neurological and immunological defects.

Tetrahydrobiopterin (BH4) levels and GTP cyclohydrolase activity (GTP-CH) were measured in tissues from mutants and controls of 24 different mouse strains to identify mutants that might be suitable models for diseases which are characterized by a deficiency of the biopterin cofactor, such as parkinsonism and atypical phenylketonuria. BH4 levels and GTP-CH activity were determined in brain, liver, and spleen obtained from 24 mutants with neurological or immunological defects. BH4 levels in brain were slightly but significantly decreased in only two mutants, spastic (spa) and jittery (ji), while GTP-CH activity in brain was not significantly lower than controls in any of the strains examined. GTP-CH levels in liver were significantly decreased in four mutant strains (jittery, ji; leaner, tgla; reeler, rl; and anorexia, anx); however, BH4 levels were significantly lower only in the mutant anorexia (anx). The most significant and widespread changes in both BH4 levels and GTP-CH activity were observed in spleen. In those mutants which were most affected, BH4 levels and GTP-CH activity were decreased 85-90%.

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate.

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.

Urinary biopterin and neopterin excretion and pituitary-adrenal activity in psychiatric patients.

The urinary excretion of biopterin and neopterin, pterin compounds related to tetrahydrobiopterin, the cofactor for the initial steps in monoamine synthesis, was serially measured in a heterogeneous group of psychiatric patients and compared to excretion in control subjects, to state of illness, and to the results of the dexamethasone suppression test. Patients with major depression had increased excretion of biopterin compared to normal subjects. There was no relationship between biopterin or neopterin excretion and postdexamethasone cortisol levels. Pterin excretion did not significantly change with improvement in mood or with conversion from nonsuppressor to suppressor status. The meaning of increased urinary biopterin is presently unclear, although its relation to hormonal state and sympathoadrenal tone deserves further study.