Advances and Challenges in Classical Galactosemia. Pathophysiology and Treatment

Abstract Classical galactosemia is caused by the genetic deficiency of galactose-1-phosphate-urydyl-transferase resulting in clinical symptoms development during the first weeks of life including jaundice, hypotonia, lethargy, emesis, hepatomegaly, among others. Currently, dietary restriction of galactose is considered the standard for classical galactosemia management. For several years, severe dietary galactose restriction was considered necessary, implying restriction not only of dairy products, but also fruits, vegetables, legumes, and viscera. Such management failed to improve or prevent the appearance of long-term complications, by contrast, such restrictive approach may lead to nutritional deficiencies development. Thus, the last consensus suggests guidelines that are more flexible. In addition, the lack of knowledge regarding the physiopathology of the disease, and the toxicity threshold of the metabolites accumulated, make even more difficult to propose novel and more effective therapeutic approaches. This review summarizes the current state of knowledge regarding classical galactosemia in terms of physiopathology, long-term complications, newborn screening and genetic variants and their implications on galactosemia treatment, summed to the challenges that researchers working on this disease must address in future studies including the analysis of galactose content in foods, galactose tolerance threshold and search for novel therapeutic targets.

1H-Nuclear Magnetic Resonance Analysis of Urine as Diagnostic Tool for Organic Acidemias and Aminoacidopathies.

The utility of low-resolution 1H-NMR analysis for the identification of biomarkers provided evidence for rapid biochemical diagnoses of organic acidemia and aminoacidopathy. 1H-NMR, with a sensitivity expected for a field strength of 400 MHz at 64 scans was used to establish the metabolomic urine sample profiles of an infant population diagnosed with small molecule Inborn Errors of Metabolism (smIEM) compared to unaffected individuals. A qualitative differentiation of the 1H-NMR spectral profiles of urine samples obtained from individuals affected by different organic acidemias and aminoacidopathies was achieved in combination with GC-MS. The smIEM disorders investigated in this study included phenylalanine metabolism; isovaleric, propionic, 3-methylglutaconicm and glutaric type I acidemia; and deficiencies in medium chain acyl-coenzyme and holocarboxylase synthase. The observed metabolites were comparable and similar to those reported in the literature, as well as to those detected with higher-resolution NMR. In this study, diagnostic marker metabolites were identified for the smIEM disorders. In some cases, changes in metabolite profiles differentiated post-treatments and follow-ups while allowing for the establishment of different clinical states of a biochemical disorder. In addition, for the first time, a 1H-NMR-based biomarker profile was established for holocarboxylase synthase deficiency spectrum.

Use of a neuron-glia genome-scale metabolic reconstruction to model the metabolic consequences of the Arylsulphatase a deficiency through a systems biology approach.

Metachromatic leukodystrophy (MLD) is a human neurodegenerative disorder characterized by progressive damage on the myelin band in the nervous system. MLD is caused by the impaired function of the lysosomal enzyme Arylsulphatase A (ARSA). The physiopathology mechanisms and the biochemical consequences in the brain of ARSA deficiency are not entirely understood. In recent years, the use of genome-scale metabolic (GEM) models has been explored as a tool for the study of the biochemical alterations in MLD. Previously, we modeled the metabolic consequences of different lysosomal storage diseases using single GEMs. In the case of MLD, using a glia GEM, we previously predicted that the metabolism of glycosphingolipids and neurotransmitters was altered. The results also suggested that mitochondrial metabolism and amino acid transport were the main reactions affected. In this study, we extended the modeling of the metabolic consequences of ARSA deficiency through the integration of neuron and glial cell metabolic models. Cell-specific models were generated from Recon2, and these were used to create a neuron-glial bi-cellular model. We propose a workflow for the integration of this type of model and its subsequent study. The results predicted the impairment pathways involved in the transport of amino acids, lipids metabolism, and catabolism of purines and pyrimidines. The use of this neuron-glial GEM metabolic reconstruction allowed to improve the prediction capacity of the metabolic consequences of ARSA deficiency, which might pave the way for the modeling of the biochemical alterations of other inborn errors of metabolism with central nervous system involvement.

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

Low-scale expression and purification of an active putative iduronate 2-sulfate sulfatase-Like enzyme from Escherichia coli K12.

The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.