Functional associations of genetic variants involved in the clinical manifestation of erythropoietic protoporphyria in the Argentinean population.

BACKGROUND: Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP). OBJECTIVE: Identify the genetic variants in FECH gene and their associations in the expression of EPP in Argentina. Determine the allelic frequency of polymorphic variants, associations in cis and its linkage disequilibrium. METHODS: The FECH gene was PCR-amplified and sequenced. Allelic variants of intragenic polymorphisms were identified by PCR followed by sequencing or restriction digestion analysis. Residual FECH activity was determined by prokaryotic expression in Escherichia coli JM109. Data were analyzed using Haploview and Statistix 9. RESULTS: Ten mutations were identified: three novel (p.S222N; p.R298X and p.R367X) and seven already known (g.12490_18067del; p.R115X; p.I186T; c.580_584delTACAG; c.598 + 1 G>T; p.Y209X and p.W310X). The p.R115X mutation was found in two families. The p.S222N mutation expressed 5% of normal activity. Only individuals who inherited a mutation combined in trans to a low expression allele c.1-251G, c.68-23T, and c.315-48C, showed clinical symptoms. The absence of c.315-48C variant was sufficient for not triggering EPP. However, these variants showed high levels of cosegregation and GTC haplotype is over-represented in EPP patients. CONCLUSION: In the dominant inheritance form of EPP, c.315-48C variant in trans to the mutated allele is sufficient to trigger the disease. The presence of GTC haplotype in all patients with dominant EPP could be due to the high level of cosegregation of c.315-48C with c.1-251G and c.68-23T variants in our population.

Porfirias agudas en la unidad de cuidados intensivos / Acute porphyria in an intensive care unit

Las porfirias son enfermedades metabólicas hereditarias muy raras, causadas por la hipoactividad de determinadas enzimas implicadas en la síntesis del grupo hemo. Presentamos tres casos de pacientes jóvenes que debutaron con crisis de porfiria aguda, y en los que, como es frecuente, se retrasó el diagnóstico y llegaron a precisar ingreso en la unidad de cuidados intensivos (UCI) por encefalopatía grave. Tras realizar el tratamiento con hemina, la clínica mejoró rápidamente, pero en un paciente persistió una polineuropatía periférica grave como secuela durante meses. Además, comunicamos el primer caso de desencadenamiento de crisis porfírica por el uso de la "píldora del día después" (levonorgestrel) (AU)

Porphyrias are rare hereditary metabolic disorders caused by the inactivity of certain enzymes that participate in hemesynthesis. We report 3 cases in which porphyria debuted with acute episodes in young patients. As is often the case, diagnosis was delayed, and intensive care was required for severe encephalopathy. Symptoms improved rapidly after hemintherapy was started, but peripheral polyneuropathy persisted for several months in 1 patient. We report the first case of aporphyria-related seizure triggered by use of the morning-after pill (levonorgestrel) (AU)

Tumour VEGF/non tumour VEGF protein expression ratio as a biomarker for survival in colorectal cancer patients.

AIMS: Vascular endothelial growth factor (VEGF) over-expression is frequently considered as a marker of both, a poor prognosis and of an aggressive tumour phenotype. Colorectal carcinoma is still one of the most lethal malignancies. Thus, our purpose was to study the expression of VEGF in tumour tissue (VEGF(t)) and in the tissue surrounding tumours (VEGF(nt)) and analyse its correlation with clinico-pathological features and overall survival. METHODS: The study was designed to determine the concentration of vascular endothelial growth factor in tumour (n = 87) and non-tumour tissue (n = 230) obtained form the colorectal cancer patients. Accordingly, VEGF expression was studied in tissue homogenates by a quantitative sandwich ELISA method. RESULTS: The study was performed on 317 colorectal samples from 87 colorectal cancer patients. VEGF expression was higher in the tumour than in the non-tumour area (P < 0.0005). In areas of 5-10 cm around the tumours, VEGF expression was higher than the expression obtained in proximal or distal edge of the resection. VEGF(t) expression was lower in patients with stage I than in patients with stage II, III, or IV. However, a shorter overall survival time was evident when the ratio obtained between VEGF expression in the tumour and mean VEGF expression in the non-tumour areas of the same patient (VEGF(t)/VEGF(nt) ratio) was ≤2 (P = 0.019). CONCLUSIONS: VEGF expression in colorectal cancer tissue was higher in tumour than in non-tumour areas. VEGF(t) expression was lower in initial clinical stages. Indeed, patients who presented a VEGF(t)/VEGF(nt) ratio >2 survived longer. This is the first report showing that the clinical outcome could be related to the VEGF(nt) over-expression in colorectal cancer patients.

Identification and characterization of HMBS gene mutations in Spanish patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant disorder with low penetrance that results from a partial deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. The disease is clinically characterized by acute neurovisceral attacks that are precipitated by several factors including certain drugs, steroid hormones, alcohol and fasting. Early diagnosis and counselling are essential to prevent attacks, being mutation analysis the most reliable method to identify asymptomatic carriers in AIP families. In this study we have investigated the molecular defect in 15 unrelated Spanish AIP patients. Mutation analysis of the HMBS gene revealed a total of fourteen mutations including six novel ones, two of them were on the same allele in one patient. The novel mutations were three missense (R26L, R173G and D178H), two frameshift (c.749_765dup and c.874insC) and one intronic deletion (IVS12+3_+11delAGGGCCTGT). RT-PCR and sequencing demonstrated that the intronic mutation caused abnormal splicing and exon 12 skipping. Prokaryotic expression of the novel missense mutations showed that only D178H had significant residual activity. These findings will facilitate the accurate identification of presymptomatic AIP carriers in these families and they further emphasize the molecular heterogeneity of AIP in Spain.

Pre-treatment serum lactate dehydrogenase level is an important prognostic factor in high-grade extremity osteosarcoma

BACKGROUND: Treatment of high-grade osteosarcoma remains a challenge. The prognostic significance of the pre-treatment serum lactate dehydrogenase (LDH) level is currently controversial. PATIENTS AND METHODS: We reviewed records from all patients diagnosed with conventional high-grade osteosarcoma at our institution over a 25-year period and analysed the prognostic significance of LDH in high-grade localised extremity osteosarcomas treated with chemotherapy. RESULTS: Between June 1977 and March 2003, 66 patients for whom follow-up was available were diagnosed with localised high-grade extremity osteosarcoma and treated with chemotherapy. The median age was 15 years, with only 3% older than 40 years, and the median follow-up was 100 months. The median progression-free survival (PFS) was 67 months and the median overall survival (OS) was 113 months. The absence of a response to chemotherapy was correlated with a trend toward lower PFS and OS. High serum pre-treatment LDH level was associated in multivariate analyses with a poorer prognosis for both PFS (HR=8.623, 95%CI: 1.71-43.37; p=0.009) and for OS (HR=9.38; 95%CI: 1.73-50.74; p=0.009). CONCLUSION: In this series, the pre-treatment serum LDH level had an independent prognostic value for both PFS and OS in patients with high-grade localised extremity osteosarcoma. This measurement should be included in a large prospective prognostic series (AU)

Epidermal growth factor receptor ligands in murine models for erythropoietic protoporphyria: potential novel players in the progression of liver injury.

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Induction of hepatic aminolevulinate acid synthetase activity by isoflurane in a genetic model for erythropoietic protoporphyria.

Erythropoietic Protoporphyria (EPP) is an inherited deficiency of ferrochelatase, the last enzyme of the heme pathway. Under general anaesthesia, some patients develop neurological dysfunction suggesting upregulation in heme biosynthesis similar to that described for acute porphyrias after xenobiotic administration. Our aim has been to evaluate whether Isoflurane induces alterations in the heme pathway in a mouse model for EPP. Administration of Isoflurane (a single dose of 2 ml/kg, i.p) to wild-type (+/+), heterozygous (+/Fechm1Pas) and homozygous (Fechm1Pas/Fechm1Pas) mice, was evaluated by measuring the activity of delta-aminolevulinic acid synthetase (ALA-S) and Porphobilinogen-deaminase (PBG-D) in different tissues, as well as Heme oxygenase (HO), cytochrome P-450, CYP2E1 and glutathione levels in liver. Porphyrin precursors were measured in 24 h-urine samples. Fechm1Pas/Fechm1Pas mice receiving anaesthesia show enhanced ALA-S and CYP2E1 activities in the liver and increased urinary excretion of porphyrin precursors. No alterations were found in either PBG-D or HO activities. Diminished glutathione levels suggest that anaesthesia may produce oxidative stress in these animals. In conclusion, Isoflurane induces ALA-S activity and increased excretion of porphyrin precursors in EPP mice. These findings appear to confirm our previous hypothesis and indicate that Isoflurane may be an unsafe anaesthetic not only for patients with acute porphyrias but also for individuals with non acute porphyrias.

Molecular heterogeneity of familial porphyria cutanea tarda in Spain: characterization of 10 novel mutations in the UROD gene.

BACKGROUND: Porphyria cutanea tarda (PCT) results from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. In the majority of patients, the disease is sporadic (S-PCT or type I) and the enzyme deficiency is limited to the liver. Familial PCT (F-PCT or type II) is observed in 20-30% of patients in whom mutations on one allele of the UROD gene reduce UROD activity by approximately 50% in all tissues. Another variant of PCT (type III) is characterized by family history of the disease although it is biochemically indistinguishable from S-PCT. OBJECTIVES: To investigate the molecular basis of PCT in Spain and to compare enzymatic and molecular analysis for the identification of patients with F-PCT. METHODS: Erythrocyte UROD activity measurement and mutation analysis of the UROD gene were carried out in a cohort of 61 unrelated Spanish patients with PCT and 50 control individuals. Furthermore, each novel missense mutation identified was characterized by prokaryotic expression studies. RESULTS: Of these 61 patients, 40 (66%) were classified as having S-PCT, 16 (26%) as having F-PCT and five (8%) as having type III PCT. Discordant results between enzymatic and molecular analysis were observed in two patients with F-PCT. In total, 14 distinct mutations were found, including 10 novel mutations: five missense, one nonsense, three deletions and an insertion. Prokaryotic expression of the novel missense mutations demonstrated that each results in decreased enzyme activity or stability. CONCLUSIONS: These results confirm the high degree of molecular heterogeneity of F-PCT in Spain and emphasize the usefulness of molecular genetic analysis to distinguish between F-PCT and S-PCT.

Photodynamic and light independent action of 8 to 2 carboxylic free porphyrins on some haem-enzymes.

BACKGROUNDS AND AIMS: skin lesions in cutaneous porphyrias appear to be determined by the structural properties of the porphyrins accumulated. To better understand the relationship between the structure and physicochemical properties of porphyrins and their specific effect on protein configuration, the action of a whole range of 8 to 2 carboxylic porphyrins has been studied. MATERIALS AMD METHODS: delta-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) partially purified from bovine liver, were exposed to 10 microM uroporphyrin (Uro), phyriaporphyrin (Phyria), hexaporphyrin (Hexa), pentaporphyrin (Penta), coproporphyrin (Copro) or protoporphyrin (Proto), either in the dark or under UV light. All experiments were performed in the enzyme solutions after removing the porphyrins. RESULTS: under both illuminating conditions, all porphyrins inactivated the enzymes (20-70% under control values), indicating photodynamic action mediated by oxidative reactions and conformational changes due to direct binding of porphyrins to the protein. Total thiol content in ALA-D was not significantly changed by most porphyrins under UV light, while all porphyrins increase total sulfhydryl groups in PBG-D (23-52% over the control values) indicating changes in the redox status of SH residues. Free amino groups were reduced by all porphyrins in ALA-D (23-56% under controls), instead they were enhanced in PBG-D (23-51% over controls), suggesting protein fragmentation. The formation of molecular aggregates would be the consequence of cross-links between oxidation products, while fragmentation can be attributed to either rupture of disulphur bridges and/or enhancement of free amino groups on the protein enzyme. CONCLUSIONS: the effect of the porphyrins on enzyme activity, total SH groups and free amino groups content, was different for ALA-D and PBG-D, even under the same illuminating conditions. On the basis of these results, no correlation between enzyme alterations and the physico-chemical properties of porphyrins could be established.

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells.

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fech(m1Pas)/Fech(m1Pas) murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Aproximación de laboratorio al diagnósticoy tipificación de una porfiria / Laboratory approach to diagnosis and classification of porphyrias

El diagnóstico y tipificación de las porfirias se consigue mediante una adecuada interrelación entre la sintomatología clínica y la detección de anomalías específicas del metabolismo del hemo. Las crisis de las porfirias se caracterizan por un frecuentemente confuso cuadro de síntomas abdomino-psiquico-neurológicos. El rasgo patognomónico de las porfirias agudas es un incremento en la eliminación urinaria de porfobilinógeno, que es detectado rápidamente mediante el sencillo test de Hoesch. Las lesiones dérmicas características de las porfirias cutáneas están asociadas a un acúmulo plasmático de porfirinas. Su presencia es fácil y rápidamente detectada mediante un barrido fluorimétrico de la muestra diluida en PBS (buffer fosfato salino). La caracterización de los defectos moleculares de los genes que codifican las enzimas implicadas en la síntesis del hemo es un complemento para el análisis bioquímico que permite clarificar los casos bioquímicamente inclasificables y facilita el diagnóstico antenatal en los casos homocigotos más severos (AU)

Serum levels of coenzyme Q10 in patients with Parkinson's disease.

We compared serum levels of coenzyme Q10 and the coenzyme Q10/cholesterol ratio in 33 patients with Parkinson's disease (PD) and 31 matched controls. The mean serum coenzyme Q10 levels did not differ significantly between the 2 study groups. Coenzyme Q10 levels were not correlated with age, age at onset, duration of the disease, scores of the Unified Parkinson Disease Rating Scale (UPDRS) or the Hoehn and Yahr staging in the PD group. The coenzyme Q10/cholesterol ratio had a significant correlation (although low) with duration of the disease (r = -0.46), total UPDRS score (r = -0.39), motor examination of the UPDRS (r = 0.45). These values were not influenced significantly by therapy with levodopa or dopamine agonists. The normality of serum coenzyme Q10 and coenzyme Q10/cholesterol ratio suggest that these values are not related with the risk for PD.

Serum levels of coenzyme Q10 in patients with Alzheimer's disease.

We compared serum levels of coenzyme Q10 and the coenzyme Q10/cholesterol ratio in 44 patients with Alzheimer's disease (AD), 17 patients with vascular dementia (VD), and 21 matched controls. The mean serum coenzyme Q10 and cholesterol levels and the coenzyme Q10/cholesterol ratio of patients with AD or VD did not differ significantly from those of controls. Coenzyme Q10 levels and coenzyme Q10/cholesterol ratio of AD or VD patients were not correlated with age, age at onset, duration of the disease or scores of the MiniMental State Examination. These results suggest that these values are not related with the risk for AD or VD.

Lipoprotein abnormalities in patients with asymptomatic acute porphyria.

There have been discrepancies in reports of total cholesterol and low density lipoprotein (LDL)-cholesterol levels in patients with acute porphyria. Some studies have found that acute porphyria patients have increased levels while others do not. The aim of this study has been to evaluate the lipid profile in a series of patients with acute porphyria, in order to help clarify these differences. Serum lipoprotein levels were studied in 30 patients (25 women and five men; age:38+/-10 years) with asymptomatic acute porphyria. Controls were 30 healthy volunteers matched for age and gender. For 13 patients and 15 controls, lipoprotein lipase and hepatic lipase activities were determined. Patients exhibited increased levels of total-cholesterol, LDL-cholesterol, high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo)-A1 compared with controls (P4 mmol/l in 15 patients (50%). Levels of total triglycerides, very low density lipoprotein (VLDL)-triglycerides, VLDL-cholesterol, apo-B and lipoprotein(a) were similar in patients and controls. The hepatic lipase activity tended to be lower in patients than controls (33.8+/-17.7 vs. 50.4+/-23.0 pkat/ml; P=0.05). In conclusion, in patients with asymptomatic acute porphyria an increase of total and LDL-cholesterol was found. The cardiovascular risk conferred by this factor may be attenuated by increased HDL-cholesterol and apo-A1.

[Laboratory approximation to the diagnosis and typification of porphyria]. / Aproximación de laboratorio al diagnóstico y tipificación de una porfiria.

The relationship between clinical features and specific alterations of heme metabolism allows the accurate diagnosis and classification of porphyrias. The acute porphyric attack is characterized by frequently confusing clinical pattern of abdominal-psiquic- and neurological symptoms. An increased urinary excretion of porphobilinogen, which can be quickly detected by the Hoesch test, confirms the diagnosis of this acute attack. Increased plasma porphyrin levels are associated with skin lesions, which are the characteristic features of the cutaneous porphyrias. Their presence is easily and rapidly detected by a fluorimetric scanning of PBS (phosphate buffer saline) diluted samples. Characterization of the molecular defects in genes coding for the enzymes involved in the heme synthetic pathway is complementary to the biochemical methods. Molecular analysis permits an accurate classification of those biochemically unclassified patients and allows prenatal diagnosis in those homozygotic cases where a severe prognosis is suspected.

Survival of patients with colorectal carcinoma: possible prognostic value of tissular carbohydrate antigen 19.9 determination.

BACKGROUND: An important goal of serum tumor marker research is to provide a test for detecting cancer in early stages. Expression of carbohydrate antigen (CA) 19.9 has been described in various malignancies. METHODS: The possible prognostic value measuring cytosol CA 19.9 expression in tumors was evaluated in a study of 63 colorectal carcinoma (CRC) patients who were followed for at least 2 years. CA 19.9 expression in cytosol was determined by enzyme-linked immunoadsorbant assay, and measurement of this protein achieved by quantitative assay. RESULTS: Mean levels of cytosol CA 19.9 found in tumor samples were significantly higher than those in nontumoral areas in CRC patients (P<0.0005). Patients with more than 3 positive lymph nodes had a higher expression of the marker (P<0.05). Univariate and multivariate analyses revealed that cytosol CA 19.9 concentration was an independent prognostic variable for relapse. Furthermore, the probability of relapse increased 4.2 times for every increase in cytosol tumor marker of 5000 U/mg, and tumors located in the rectum had a probability of relapse 9.5 times greater. CONCLUSIONS: Cystol CA 19.9 expression in CRC can be an independent prognostic factor for relapse. Patients with high levels of CA 19.9 have worse prognosis than those with lower values. Therefore, this group of patients should receive special management with regard to their follow-up and treatment. Moreover, quantitative cytosol tumor marker measurement is an easy and highly effective method for determining the prognoses of CRC patients.

[Serum lipoproteins in patients with porphyria cutanea tarda]. / Lipoproteínas séricas en pacientes con porfiria cutánea tarda.

BACKGROUND: To know the lipid profile in patients with porphyria cutanea tarda (PCT). PATIENTS AND METHODS: Serum lipoproteins have been studied in 64 males with PCT: 42 with chronic hepatitis C and 22 seronegatives for hepatitis B and C virus. Thirteen patients without porphyria, but with chronic hepatitis C, and 13 healthy subjects were also studied. RESULTS: Patients with chronic hepatitis C, with or without PCT, had a decrease of total and VLDL cholesterol (TC and VLDLc) and apolipoprotein (apo) B in comparison with healthy controls and seronegative for hepatitis C virus patients with PCT. CONCLUSIONS: Lipid abnormalities found in PCT are not related with this disease, but with the presence of chronic hepatitis C often associated to PCT.

Delta aminolevulinate dehydratase (ALA-D) activity in human and experimental diabetes mellitus.

The haem pathway is impaired in porphyrias and a frequent coexistence of diabetes mellitus and porphyria disease has been reported. We have therefore decided to investigate delta-aminolevulinate dehydratase, one of the more sensitive enzymes in the haem pathway, in both human diabetic patients and diabetic rats. We have studied 131 diabetes mellitus patients, 32 insulin dependent and 99 non-insulin dependent. The latter group was further subdivided according to treatment: diet alone (n = 24), diet plus oral hypoglycemic agents (n = 28) and diet plus insulin (n = 47). We have also performed similar studies in the rat model of diabetes mellitus, induced in 11 Wistar rats by streptozotocin. Control groups of both humans and animals were used. Erythrocytic aminolevulinate dehydratase activity was reduced in both insulin dependent and non-insulin dependent diabetic patients as compared to their controls (p < 0.001). This activity was only partially restored by addition of zinc and thiols to the incubation media. In insulin-dependent diabetes mellitus, reduction of enzyme activity was related to the glycosilated hemoglobin concentration (p < 0.05) and in non-insulin dependent diabetes mellitus to the glycemia (p < 0.01). In the diabetic rat, aminolevulinate dehydratase activity was diminished on both erythrocytes (p < 0.01) and hepatic tissue (p < 0.01) when compared to the control group. The decrease in activity of erythrocyte aminolevulinate dehydratase observed in diabetic patients, may represent an additional and useful parameter for the assessment of the severity of carbohydrate metabolism impairment.