Importancia de la semiología y la bioquímica en el abordaje diagnóstico de un trastorno de la biogénesis peroxisómica / The importance of semiology and biochemistry in the diagnostic management of a peroxisomal biogenesis disorder

Introducción. Los trastornos de la biogénesis de los peroxisomas se deben a mutaciones en los genes PEX, que codifican peroxinas requeridas para la biogénesis peroxisómica. Clínicamente se expresan como un espectro del síndrome de Zellweger, y hay una amplia variedad fenotípica. Su diagnóstico se realiza bioquímicamente y la confirmación es molecular. El objetivo de este caso ilustrativo es resaltar la importancia de la clínica y de las pruebas bioquímicas en el abordaje de una enfermedad peroxisómica. Caso clínico. Niño de 3 años con hipotonía neonatal, retraso global del desarrollo y fallo de medro, con un patrón en resonancia cerebral de leucodistrofia hipomielinizante, en quien se había sospechado un trastorno de la biogénesis de los peroxisomas por encontrarse una variante de significado incierto en PEX5, pero su clínica, los estudios bioquímicos y el análisis crítico de las pruebas moleculares hacían improbable este diagnóstico. Se hace énfasis en el abordaje que debería tenerse cuando se sospecha un trastorno del espectro del síndrome de Zellweger. Conclusión. En el caso descrito se sospechó un trastorno de la biogénesis de los peroxisomas por una secuenciación exómica que, al analizarse críticamente junto con la clínica y los hallazgos bioquímicos, hacía muy poco probable una enfermedad peroxisómica. Cuando se tiene sospecha clínica y por neuroimágenes, el abordaje diagnóstico principal debe partir del análisis bioquímico. Aunque la confirmación es molecular, estas pruebas deben interpretarse con precaución

Introduction. Peroxisomal biogenesis disorders are due to mutations in the PEX genes, which code for peroxins that are required for peroxisomal biogenesis. Clinically, they are expressed as a Zellweger syndrome spectrum, and there is a wide phenotypic variety. They are diagnosed biochemically, and confirmation is molecular. The aim of this illustrative case is to highlight the importance of the clinical features and biochemical testing in the management of a peroxisomal disease. Case report. A 3-year-old boy with neonatal hypotonia, overall developmental delay and failure to thrive and a pattern of hypomyelinating leukodystrophy in brain resonance. The suspected diagnosis was a disorder affecting the biogenesis of the peroxisomes due to having found a variant with an uncertain meaning in PEX5. The clinical features, the biochemical studies and critical analysis, however, made this diagnosis unlikely. Emphasis is placed on the management that must be applied when a Zellweger syndrome spectrum is suspected. Conclusion. In the case reported here, a peroxisomal biogenesis disorder was suspected owing to an exome sequencing which, on being critically analysed together with the clinical features and the biochemical findings, made a peroxisomal disease very unlikely. In cases of clinical suspicion, backed up by neuroimaging, the main diagnostic management must be based on the biochemistry analysis. Although confirmation is molecular, these tests must be interpreted with caution

The important role of biochemical and functional studies in the diagnostics of peroxisomal disorders.

Peroxisomes are dynamic organelles that play an essential role in a variety of metabolic pathways. Peroxisomal dysfunction can lead to various biochemical abnormalities and result in abnormal metabolite levels, such as increased very long-chain fatty acid or reduced plasmalogen levels. The metabolite abnormalities in peroxisomal disorders are used in the diagnostics of these disorders. In this paper we discuss in detail the different diagnostic tests available for peroxisomal disorders and focus specifically on the important role of biochemical and functional studies in cultured skin fibroblasts in reaching the right diagnosis. Several examples are shown to underline the power of such studies.

Urine acylcarnitine analysis by ESI-MS/MS: a new tool for the diagnosis of peroxisomal biogenesis disorders.

BACKGROUND: Patients with peroxisomal biogenesis disorders (PBDs) have an abnormal profile of circulating acylcarnitines (i.e. elevated C16:0-DC-, C18:0-DC-, C24:0-, C26:0-carnitine). We developed an ESI-MS/MS method for quantification of urine acylcarnitines and tested its reliability for the diagnosis of PBDs. METHODS: Urine from 7 patients with PBDs (5 Zellweger syndrome, 2 infantile Refsum disease), from 2 patients with D-bifunctional protein (D-BP) deficiency, and from 130 healthy controls were analysed by ESI-MS/MS, using a multiple reactions monitoring (MRM) method, and quantified with labelled internal standards. Acylcarnitine levels between groups were analyzed by the STATA statistics data analysis and compared by the non parametric Mann-Whitney test. RESULTS: In PBDs, the urinary excretion of long-chain dicarboxylylcarnitines (C14:0-DC-, C16:0-DC-, and C18:0-DC-carnitine), and of very long-chain monocarboxylylcarnitines (C22:0-, C24:0-, C26:0-carnitine) were significantly elevated compared to controls (p<0.0001). Interestingly, among PBDs the most severe abnormalities of acylcarnitine profile were observed in patients with Zellweger syndrome. One patient with D-BP showed similar abnormalities to PBDs, while in the other only C16:0-DC-carnitine was markedly elevated. CONCLUSIONS: This study shows that MRM ESI-MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put.

Evidence for increased oxidative stress in peroxisomal D-bifunctional protein deficiency.

Peroxisome biogenesis disorders (PBDs) and D-bifunctional protein (D-BP) deficiency are two types of inherited peroxisomal disorders. Patients with a PBD lack functional peroxisomes and patients with D-BP deficiency lack the enzyme, which is responsible for the second and third step of the peroxisomal beta-oxidation. The clinical presentation of these peroxisomal disorders is severe and includes several neurological abnormalities. The pathological mechanisms underlying these disorders are not understood and no therapies are available. Because peroxisomes have been associated with oxidative stress, as oxygen radicals are both produced and scavenged in peroxisomes, we have investigated whether oxidative stress is involved in the pathogenesis of PBDs and D-BP deficiency. We found in D-BP-deficient patients increased levels of thiobarbituric acid-reactive substances (TBARS) and 8-hydroxydeoxyguanosine (8-OHdG), which are markers for lipid peroxidation and oxidative DNA damage, respectively, whereas the levels of the lipophilic antioxidants alpha-tocopherol and coenzyme Q(10) were decreased. In addition, we found in skin fibroblasts from D-BP-deficient patients an imbalance between the activities of the peroxisomal H(2)O(2)-generating straight-chain acyl-CoA oxidase (SCOX) and the peroxisomal H(2)O(2)-degrading enzyme catalase. In conclusion, we have found clear evidence for the presence of increased oxidative stress in patients with D-BP deficiency, but not in patients with a PBD.

Urinary bile acid profile in children with inborn errors of bile acid metabolism and chronic cholestasis; screening technique using electrospray tandem mass-spectrometry (ES/MS/MS).

BACKGROUND: It is well known that urine becomes the major route for bile acid excretion in liver diseases and thus we examined bile acid profile in urine obtained from normal children and children having chronic liver diseases using electrospray tandem mass spectrometry (ES/MS/MS). MATERIAL/METHODS: Bile acid were extracted from 5 ml of urine obtained from five healthy children or from twenty patients with various liver diseases including patients with unknown chronic liver diseases, Zellweger syndrome, peroxisomal bifunctional protein deficiency disease, tyrosinema type 1, biliary atresia, and patients with progressive familial intrahepatic cholestasis (PFIC) of undetermined type. Identification and quantification of bile acids were achieved in 5 minutes using electrospray tandem mass spectrometry (ES/MS/MS). RESULTS: Urinary bile acid excretion increased in liver diseases an average of 100 times as compared to control values. There was a specific profile for different liver disease which confirms the pathology of the disease and could be used for its diagnosis. The results also show that the ions used for the diagnosis of oxo-steroid reductase deficiency disease were present in other chronic liver diseases suggesting that these atypical bile acids may not be a result of an inborn error of bile acid metabolism. CONCLUSIONS: The urinary bile acid profile obtained in this study by ES/MS/MS can be of use for the diagnosis of certain chronic liver diseases.

Urinary organic acids in peroxisomal disorders: a simple screening method.

Using GC-MS, we studied urinary organic acids in 20 Japanese patients with peroxisomal disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and single deficiency of peroxisomal beta-oxidation enzymes. Non-ketotic dicarboxylic aciduria with elevated sebacate/adipate molar ratio was observed in 19 of the 20 patients. Elevation of 2-hydroxysebacate and epoxydicarboxylic acids were seen in 13 and 18, respectively. Tyrosyluria was remarkable in all patients. In two ZS patients, we tracked the time course from birth to infancy, and all the above stated findings were detected, except for one sample. Urinary organic acid analysis is indeed useful for screening subjects with peroxisomal disorders.

The identification of unusual bile acid metabolites by tandem mass spectrometry: use of low-energy collision-induced dissociation to produce informative spectra.

The use of collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) has been shown to produce fragmentation that is useful for the structural analysis of bile acids and their conjugates. Low-energy CID using a triple quadrupole has been used to help characterise bile acid identity but the majority of work has been conducted using high-energy CID on specialised instrumentation. This paper describes the use of low-energy CID as a rapid method for identification of urinary bile acids and presents some examples of its use in the diagnosis of liver disease in infants. These include the differential diagnosis of peroxisomal disorders, identification of compounds (e.g. 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulphate) indicative of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase deficiency and the confirmation of the identity of an unusual bile acid series consisting of different conjugates of lithocholic acid. The use of lithium cationisation and derivatisation with aminosulfonic acids for the analysis of unconjugated and glycine-conjugated bile acids has also been evaluated.

Urinary bile acids and peroxisomal bifunctional enzyme deficiency.

The biosynthesis of normal bile acids involves beta-oxidation of the 8-carbon side-chain of cholesterol, in addition to numerous modifications of the sterol nucleus. Because beta-oxidation of the sterol side-chain has been localized to the peroxisome, bile acid analysis has been suggested to be useful in the diagnostic evaluation of individuals suspected of having peroxisomal disorders. Although data from subjects with generalized peroxisomal disorders support this, few data exist regarding the bile acids in individuals having single peroxisomal beta-oxidation enzyme disorders. In this study, we analyzed the urinary bile acids from 12 patients with peroxisomal bifunctional protein deficiency using continuous flow fast atom bombardment mass spectrometry. All 12 patients had abnormal spectra, although their ion profiles and rank order of intensity of ions varied considerably. Ten of 12 individuals had abnormal spectra with presence of taurine-conjugated tetrahydroxycholestenoates, allowing a definite diagnosis of a peroxisomal beta-oxidation defect and a presumptive diagnosis of bifunctional protein deficiency; the other two cases were nondiagnostically abnormal. The strengths and limitations of urinary bile acid analysis for the diagnosis of peroxisomal beta-oxidation disorders are discussed.

Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders.

Urine extracts from children diagnosed with generalized peroxisomal disorders were screened by continuous flow-negative ion fast atom bombardment-mass spectrometry. In 45 of 60 children with generalized peroxisomal disorders, we observed one or more intense ions (m/z 489, 505, 461, and others) that are infrequently found in children with cholestatic liver disease or normal children. Compounds giving rise to these ions were isolated using reverse phase and anion exchange chromatography. After appropriate derivatization and/or methanolysis the compounds were analyzed using capillary gas chromatography-mass spectrometry. The major compounds were found to be 12,13-dihydroxy-9-octadecenoic acid and 9,10-dihydroxy-12-octadecenoic acid, with one of the hydroxyl groups in glycosidic linkage with glucuronic acid. Minor compounds were glucuronic acid conjugates of 9,10-dihydroxy-octadecanoic acid, and 12,13-dihydroxy-6,9-, 15,16-dihydroxy-9,12-, and 9, 10-dihydroxy-12,15-octadecadienoic acids. A series of hexadecanoic, hexadecenoic, and hexadecadienoic acid glucuronides which appear to be beta-oxidation products of the C18 fatty acids were also observed, with the major species being 10, 11-dihydroxy-7-hexadecenoic acid glucuronide. In all, 16 C16 and C18 dihydroxy fatty acids were identified by gas chromatography-mass spectrometry. A series of at least 11 trihydroxy fatty acids was also observed but not fully characterized. Measurement of these compounds may prove to be useful in the diagnosis of some peroxisomal disorders.

12- and 15-hydroxyeicosatetraenoic acid are excreted in the urine of peroxisome-deficient patients: evidence for peroxisomal metabolism in vivo.

To determine the importance of peroxisomes and mitochondria in hydroxyeicosatetraenoic acid (HETE) oxidation in vivo, urinary excretion of 12- and 15-HETE was measured in eight patients with a peroxisome deficiency disorder (Zellweger syndrome) showing normal mitochondrial beta-oxidation capacity, in three patients with a defect of mitochondrial long-chain fatty acid oxidation (long-chain acyl-CoA dehydrogenase deficiency), and in eight healthy subjects. 12- and 15-HETE were identified and quantified by gas chromatography/negative ion chemical ionization-mass spectrometry and specific RIA. The free compounds were found exclusively in the urine of peroxisome-deficient subjects (12-HETE: median 26 pg/mL, range 17-36 pg/mL; 15-HETE: median 40 pg/mL, range 29-61 pg/mL), whereas both compounds were below the detection limit (< 0.5 pg/mL) in the urine of patients with defective mitochondrial long-chain fatty acid oxidation and normal subjects (p < 0.002). These results implicate that peroxisomes are the main cellular organelle responsible for HETE oxidation in vivo. Analysis of HETE excretion in urine represents an additional new specific diagnostic tool in patients with Zellweger syndrome.