Aportaciones de Louis I. Woolf al Tratamiento y Diagnóstico Precoz de la Fenilcetonuria y otros Errores Congénitos del Metabolismo. Los comienzos de la Tría Neonatal en España, con referencia al Programa de Galicia. CENTENARIO DE LOUIS ISAAC WOOLF

3ª ediciónAportaciones de Louis I. Woolf al Tratamiento y Diagnóstico Precoz de la Fenilcetonuria y otros Errores Congénitos del Metabolismo. Los comienzos de la Tría Neonatal en España, con referencia al Programa de Galici

Newborn screening for Fabry disease in the north-west of Spain

Fabry disease is an X-linked lysosomal storage disorder caused by the impairment of α-galactosidase A. Enzyme replacement therapy is available to treat patients, who often experience delayed diagnosis. A newborn screening for Fabry disease was performed to study the prevalence of the pathology and to evaluate the possibility to implement the test in systematic screenings. We collected 14,600 dried blood spot samples (7575 males and 7025 females) and carried out a diagnostic study by fluorometric measurement of α-galactosidase A enzymatic activity and GLA gene sequencing. We detected one patient with a mutation in GLA associated with classical Fabry Disease (M290I), ten subjects carrying genetic variants of uncertain diagnosis (S126G, R118C, A143T), and a girl with the non-characterized variant F18Y, which was not previously described. Additional 25 samples presented nucleotide substitutions described as polymorphisms (D313Y, rs2071225, and rs2071397). The estimated prevalence for Fabry disease in north-western Spanish males is of 0.013%. Conclusion: These results confirm that the prevalence of Fabry disease is underestimated and systematic screening is feasible; however, further characterization of variants of uncertain clinical significance is necessary to establish protocols of patients’ managementThis studied was founded by the Spanish National Institute of Health-Instituto Carlos III/EU-FEDER, grant no. PI11-00842, to Ortolano S. and FEEL Foundation to Cristobal ColónS

V232D Mutation in Patients With Cystic Fibrosis: Not So Rare, Not So Mild

The frequency of some Cystic Fibrosis (CF) Transmembrane Conductance Regulator gene (CFTR) mutations varies between populations. Genetic testing during newborn screening (NBS) for CF can identify less common mutations with low clinical expression in childhood and previously considered mild but not fully characterized, such as the mutation p.Val232Asp (c.695T > A). The aim of this study was to describe CF patients with the V232D mutation. We identify CF children with the V232D mutation detected by NBS and compare them with CF adults with this mutation whose diagnosis was prompted by clinical symptoms in the same period. We studied clinical, biochemical, spirometric, and prognostic features in both populations. NBS program tested 276,523 children during a period of 14 years (2003-2017) and identified 54 cases of CF. Six children (11%) had the V232D mutation. Over the same period, 5 adults (age 37.6 ± 16.29 years old) with symptoms of CF and this mutation were also diagnosed. Follow-up duration was mean 10.1 years for adults and mean 6.5 years for children. In the adult group, lung function was impaired at diagnosis in all patients (Forced Expiratory Volume1-FEV1-67.12% ± 13.09) and worsened in children tested during evolution (FEV1first: 113%; FEV1last: 64%). Pancreatic insufficiency was present in adult group, with recurrent pancreatitis in 1 present. Although with less clinical expression in children, V232D is associated with pulmonary and pancreatic involvement during adulthood and CF cannot be considered mild. This mutation is present in 11% of all patients diagnosed with CF in our region. Its inclusion in some NBS programs should be taken into account in order to improve the prognosis of affected children.S

Characterization of New Proteomic Biomarker Candidates in Mucopolysaccharidosis Type IVA

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Skeletal dysplasia and the related clinical features of MPS IVA are caused by disruption of the cartilage and its extracellular matrix, leading to a growth imbalance. Enzyme replacement therapy (ERT) with recombinant human GALNS has yielded positive results in activity of daily living and endurance tests. However, no data have demonstrated improvements in bone lesions and bone grow thin MPS IVA after ERT, and there is no correlation between therapeutic efficacy and urine levels of keratan sulfate, which accumulates in MPS IVA patients. Using qualitative and quantitative proteomics approaches, we analyzed leukocyte samples from healthy controls (n = 6) and from untreated (n = 5) and ERT-treated (n = 8, sampled before and after treatment) MPS IVA patients to identify potential biomarkers of disease. Out of 690 proteins identified in leukocytes, we selected a group of proteins that were dysregulated in MPS IVA patients with ERT. From these, we identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: lactotransferrin, coronin 1A, neutral alpha-glucosidase AB, and vitronectin. Further studies of cartilage and bone alterations in MPS IVA will be required to verify the validity of these proteins as potential biomarkers of MPS IVAS

Proteomic analysis in morquio a cells treated with immobilized enzymatic replacement therapy on nanostructured lipid systems

[ENG]Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERTS

Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems

Mucopolysaccharidosis IVA (Morquio A) is a rare inherited metabolic disease caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). Until now, treatments employed included hematopoietic stem cell transplantation and enzyme replacement therapy (ERT); the latter being the most commonly used to treat mucopolysaccharidoses, but with serious disadvantages due to rapid degradation and clearance. The purpose of this study was to develop and evaluate the potential of nanostructured lipid carriers (NLCs) by encapsulating elosulfase alfa and preserving its enzyme activity, leading to enhancement of its biological effect in chondrocyte cells. A pegylated elosulfase alfa-loaded NLC was characterized in terms of size, ζ potential, structural lipid composition (DSC and XRD), morphology (TEM microscopy), and stability in human plasma. The final formulation was freeze-dried by selecting the appropriate cryoprotective agent. Viability assays confirmed that NLCs were non-cytotoxic to human fibroblasts. Imaging techniques (confocal and TEM) were used to assess the cellular uptake of NLCs loaded with elosulfase alfa. This study provides evidence that the encapsulated drug exhibits enzyme activity inside the cells. Overall, this study provides a new approach regarding NLCs as a promising delivery system for the encapsulation of elosulfase alfa or other enzymes and the preservation of its activity and stability to be used in enzymatic replacement therapy (ERT).This research was funded by Xunta de Galicia, grant number GRC2013/015 and GPC2017/015S

Diagnóstico precoz de los errores congénitos del metabolismo

Los avances científicos recientes han permitido identificar un número importante de enfermedades consideradas como raras, aquellas que tienen una baja prevalencia o aparecen ocasionalmente en la población –en Europa, menos de 1 caso cada 2.000 ciudadanos–, entre las que se encuentran las enfermedades metabólicas. En España, se estima que hay, aproximadamente, tres millones de afectados por estas dolencias, cuya atención está siendo objeto de interés por las administraciones sanitarias. Las enfermedades metabólicas, o errores congénitos del metabolismo (ECM), son un grupo numeroso de dolencias hereditarias, cada una producida por el bloqueo de alguna vía metabólica en el organismo. La mayoría de ellas se heredan de forma autosómica recesiva, y su frecuencia se estima entre 1/1.000-3.000 recién nacidos vivos. Esta memoria recoge las investigaciones y trabajos llevados a cabo en la Unidad de Diagnóstico y Tratamiento de los Errores Congénitos del Metabolismo (UDTECM), del Departamento de Pediatría del Hospital Clínico Universitario de Santiago de Compostela, dedicado a evitar enfermedades que supongan un riesgo para el pleno desarrollo del recién nacido. En ella se presentan los resultados globales del programa de diagnóstico precoz neonatal desarrollado durante más de 30 años, del que se han beneficiado cerca de 700.000 niños. La unidad, formada por un equipo multiprofesional, ha recibido el Premio Reina Sofía 2008, de Prevención de la Discapacidad

Diagnóstico precoz de los errores congénitos del metabolismo

Los avances científicos recientes han permitido identificar un número importante de enfermedades consideradas como raras, aquellas que tienen una baja prevalencia o aparecen ocasionalmente en la población –en Europa, menos de 1 caso cada 2.000 ciudadanos–, entre las que se encuentran las enfermedades metabólicas. En España, se estima que hay, aproximadamente, tres millones de afectados por estas dolencias, cuya atención está siendo objeto de interés por las administraciones sanitarias. Las enfermedades metabólicas, o errores congénitos del metabolismo (ECM), son un grupo numeroso de dolencias hereditarias, cada una producida por el bloqueo de alguna vía metabólica en el organismo. La mayoría de ellas se heredan de forma autosómica recesiva, y su frecuencia se estima entre 1/1.000-3.000 recién nacidos vivos. Esta memoria recoge las investigaciones y trabajos llevados a cabo en la Unidad de Diagnóstico y Tratamiento de los Errores Congénitos del Metabolismo (UDTECM), del Departamento de Pediatría del Hospital Clínico Universitario de Santiago de Compostela, dedicado a evitar enfermedades que supongan un riesgo para el pleno desarrollo del recién nacido. En ella se presentan los resultados globales del programa de diagnóstico precoz neonatal desarrollado durante más de 30 años, del que se han beneficiado cerca de 700.000 niños. La unidad, formada por un equipo multiprofesional, ha recibido el Premio Reina Sofía 2008, de Prevención de la Discapacidad

Evaluation and perspective of 20 years of neonatal screening in Galicia: Program results

El Programa Gallego para la Detección Precoz de Enfermedades Endocrinas y Metabólicas se inició en 1978 y fue pionero en España en el cribado neonatal ampliado con la incorporación de la espectrometría de masas en julio de 2000. Como objetivo primario se criban veintiocho enfermedades, incluyendo las de la cartera básica del Servicio Nacional de Salud excepto la anemia de células falciformes, que está en fase de inclusión. En sus veinte años de trayectoria se analizaron 404.616 recién nacidos (RN), identificando 547 casos afectos de las enfermedades incluidas, con una incidencia global de 1:739 RN vivos y de 1:1.237 RN de las enfermedades metabólicas congénitas (EMC) cribadas (1:1.580 RN excluyendo la hiperfenilalaninemia benigna-HPA), con una participación media del 99,35%, progresivamente creciente durante el período analizado. Entre las patologías cribadas destacan por su incidencia el hipotirodismo congénito (1:2.211 RN), la cistinuria (1:4.129 RN) y la HPA (1:5.699 RN), seguida de fenilcetonuria y fibrosis quística (1:10.936 RN). Se identificaron sesenta y seis casos de falsos positivos (diecisiete de los mismos en relación con patología materna) y cinco falsos negativos, siendo el VPP (valor predictivo positivo) y el VPN (valor predictivo negativo) global del programa del 89,2% y 99,99%, respectivamente, con una sensibilidad de 99,09% y una especificidad del 99,98%. La tasa de mortalidad de los pacientes con EMC diagnosticados fue del 1,52%, presentando once casos sintomatología previa al resultado del cribado (2%). El cociente intelectual de los pacientes con EMC y riesgo de afectación neurológica es normal en más del 95% de los casosGalician newborn screening program for early detection of endocrine and metabolic diseases began in 1978 and was a pioneer in expanded newborn screening in Spain with the incorporation of mass spectrometry in July 2000. As a primary objective, 28 diseases are screened, including those recommended SNS except sickle cell anemia which is in the inclusion phase. In its 20-year history, 404,616 newborns (nb) have been analyzed, identifying 547 cases affected by the diseases included, with a global incidence of 1: 739 newborns and 1: 1.237 of the screened inborn errors of metabolism (IEM) (1:1.580 nb if excluding benign hyperphenylalaninemia-HPA), with an average participation of 99.35%, progressively higher during the analyzed period. Among the pathologies screened, congenital hypothyroidism (1:2.211 nb), cystinuria (1:4.129 nb) and HPA (1:5.699 nb), followed by phenylketonuria and cystic fibrosis (1:10,936 nb) stand out for their incidence. Sixty-six cases of false positives were identified (seventeen of them in relation to maternal pathology) and five false negatives, being the overall PPV and NPV of the program respectively of 89.2% and 99.99%, with a sensitivity of 99.09% and a specificity of 99.98%. The mortality rate of diagnosed CME patients is 1.52%, with eleven cases presenting symptoms prior to the screening result (2%). The intelligence quotient of IEM patients at risk of neurological involvement is normal in more than 95% of casesS