Molecular Trojan Horses for treating lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are caused by monogenic mutations in genes encoding for proteins related to the lysosomal function. Lysosome plays critical roles in molecule degradation and cell signaling through interplay with many other cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes. Even though several strategies (i.e., protein replacement and gene therapy) have been attempted for LSDs with promising results, there are still some challenges when hard-to-treat tissues such as bone (i.e., cartilages, ligaments, meniscus, etc.), the central nervous system (mostly neurons), and the eye (i.e., cornea, retina) are affected. Consistently, searching for novel strategies to reach those tissues remains a priority. Molecular Trojan Horses have been well-recognized as a potential alternative in several pathological scenarios for drug delivery, including LSDs. Even though molecular Trojan Horses refer to genetically engineered proteins to overcome the blood-brain barrier, such strategy can be extended to strategies able to transport and deliver drugs to specific tissues or cells using cell-penetrating peptides, monoclonal antibodies, vesicles, extracellular vesicles, and patient-derived cells. Only some of those platforms have been attempted in LSDs. In this paper, we review the most recent efforts to develop molecular Trojan Horses and discuss how this strategy could be implemented to enhance the current efficacy of strategies such as protein replacement and gene therapy in the context of LSDs.

Identification of a novel fusion Iduronidase with improved activity in the cardiovascular system.

Background: Lysosomal diseases are a group of over 70 rare genetic conditions in which a protein deficiency (most often an enzyme deficiency) leads to multi-system disease. Current therapies for lysosomal diseases are limited in their ability to treat certain tissues that are major contributors to morbidity and mortality, such as the central nervous system (CNS) and cardiac valves. For this study, the lysosomal disease mucopolysaccharidosis type I (MPS I) was selected as the disease model. In MPS I, mutations in the IDUA gene cause a deficiency of the α-L-iduronidase (IDUA) enzyme activity, leading to disease pathology in tissues throughout the body, including the CNS and cardiac valves. Current therapies have been unable to prevent neurodevelopmental deficits and cardiac valvular disease in patients with MPS I. This study aimed to evaluate the delivery of IDUA enzyme, via a novel gene therapy construct, to target tissues. Methods: MPS I mice were hydrodynamically injected through the tail vein with plasmids containing either a codon-optimized cDNA encoding the wild-type IDUA protein or one of four modified IDUAs under the control of the liver-specific human α1-antitrypsin (hAAT) promoter. Two modified IDUAs contained a ligand for the CB1 receptor, which is a highly expressed receptor in the CNS. Iduronidase activity levels were measured in the tissues and plasma using an enzyme activity assay. Results: The modified IDUAs did not appear to have improved activity levels in the brain compared with the unmodified IDUA. However, one modified IDUA exhibited higher activity levels than the unmodified IDUA in the heart (p = 0.0211). This modified iduronidase (LT-IDUA) contained a sequence for a six amino acid peptide termed LT. LT-IDUA was further characterized using a noncompartmental pharmacokinetic approach that directly analyzed enzyme activity levels after gene delivery. LT-IDUA had a 2-fold higher area under the curve (AUC) than the unmodified IDUA (p = 0.0034) when AUC was estimated using enzyme activity levels in the plasma. Conclusion: The addition of a six amino acid peptide improved iduronidase's activity levels in the heart and plasma. The short length of this LT peptide facilitates its use as fusion enzymes encoded as gene therapy or administered as enzyme replacement therapy. More broadly, the LT peptide may aid in developing therapies for numerous lysosomal diseases.

Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson's Disease.

The association between Parkinson's disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD.

Transition from child to adult health care for patients with lysosomal storage diseases in France: current status and priorities-the TENALYS study, a patient perspective survey.

BACKGROUND: Transition from childhood to adulthood (TCA) is usually difficult in rare, progressive and multisystemic diseases. New treatments and modalities of care for many lysosomal diseases (LD) can increase life expectancy, and a successful TCA can help patient who reach adulthood to avoid disruption to health care. In France, some TCA initiatives have been taken by referral centers but in view of the problems encountered by Vaincre les Maladies Lysosomales (VML), the LD patient association, they seem to be insufficient. The aim of this study is to determine the current state of the TCA process and to identify actions to improve it through interviews with patient families and physicians in LD referral centers. The study is based upon an observational, non-interventional, cross-sectional, national survey which used two anonymous questionnaires. These questionnaires, developed by a scientific committee including representatives from VML and medical specialists in LD, were sent to patients who were receiving care in pediatric departments at age 15 years or older. Questionnaires were also sent to their referral pediatricians. RESULTS: Fifty-four patients were included. Forty-two questionnaires were completed by patients and their corresponding physicians and 12 were completed by physicians only. The majority of the patients (80%) were informed that transfer to adult healthcare would occur, but 52% were informed after their eighteenth birthday. Forty-eight percent indicated that they were informed that a TCA coordinator would be appointed; for 39% the time frame for the transfer was communicated, and 31% were informed of the composition of the adult medical team. Among the actions that patients rated as "important/very important", and considered to be a priority in their comments, the most frequently cited were the provision of explanatory documents on the TCA (94%), the transmission of the medical file from the pediatric sector to the adult sector (94%) and a joint consultation with both pediatrician and adult unit physician (91%). Physicians were in agreement concerning the primary importance of the last two actions. CONCLUSION: This study provides a basis for the deployment, on the national level, of transition programs which include specific actions that patients view as priorities.

anexVis: A Transcriptome Tool to Visualize Organ/Tissue-Specific Glycosaminoglycan Biosynthetic and Catabolic Pathways in Human Health and Diseases.

Our lab has developed a new visualization tool, anexVis, for transcriptome analysis of glycosaminoglycan-related genes. This tool allows one to analyze a large number of genes that are related to biosynthetic and catabolic pathways of all glycosaminoglycans, such as heparan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid, in parallel across various human tissues/organs. Such visual analyses have not been accessible to the broad research community despite the accumulation of a large amount of RNA-seq data. We use publicly available data provided by the GTEx project with NIH permission to generate this new framework. Herein, we describe the use of anexVis in understanding the relationship among all biosynthetic and catabolic enzymes, core protein proteoglycans, and various transporters and understanding factors that control organ-specific GAG biosynthesis and catabolism in humans at the transcriptome level. This visualization tool may also assist us in understanding the impact of lysosomal diseases and rare glycan-related diseases on specific organs in humans.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy.

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

Lipofuscinosis ceroidea neuronal. Variante infantil tardía de tipo 6 en dos hermanos heterocigotos compuestos con mutaciones nuevas / Neuronal ceroid lipofuscinosis. Type 6 late infantile variant in two compound heterozygous siblings with novel mutations

Introducción: Existen 14 formas de lipofuscinosis. La de tipo 6, en su forma infantil tardía, comienza entre los 3 y 8 años con alteraciones motoras, mioclonos, disartria, ataxia, pérdida de la visión y las habilidades motoras, y muerte temprana. Ocurre por mutaciones en el gen CLN6. La mayoría de los pacientes presenta variantes en estado homocigoto, asociadas a consanguinidad o endogamia, y son poco frecuentes las variantes en estado heterocigoto compuesto. Casos clínicos: Hermanos con síntomas desde los 4 y 5 años, con marcha inestable, caídas frecuentes, posteriormente pérdida de la marcha, mioclonías, disfagia y alucinaciones visuales. En el examen físico presentaban atrofia del nervio óptico, Babinski y ataxia del tronco. El electroencefalograma mostraba brotes de ondas lentas generalizadas, sin respuesta fotoparoxística, y la resonancia magnética de cráneo, hiperintensidad de la sustancia blanca periventricular, y atrofia cerebelosa y cortical. El panel de lipofuscinosis reveló dos mutaciones nuevas en el gen CLN6, c.552del y c.244G>C (p.Gly82Arg), no descritas previamente. La madre resultó portadora de la deleción 552, y el padre y la abuela paterna, de la sustitución G>C (Gly82Arg). Conclusiones: El diagnóstico diferencial en los trastornos con neurorregresión se dificulta debido a que los signos clínicos son inespecíficos, similares a otras epilepsias mioclónicas progresivas. Presentamos los hallazgos clínicos en dos hermanos mexicanos con la variante infantil tardía de CLN6 por dos mutaciones heterocigotas nuevas que contribuyen al conocimiento de las mutaciones en la población mexicana y señalan la relevancia de realizar estudios genéticos aplicando la secuenciación de nueva generación para permitir un adecuado asesoramiento.(AU)

Introduction: There are 14 forms of lipofuscinosis, among them type 6 in its late childhood form is found, it starts between three and eight years with epilepsy, motor disorders, myoclonus, dysarthria, ataxia and neurological regression associated with vision loss and motor skills, and early death. It occurs from mutations in the CLN6 gene, most patients have homozygote variants associated with consanguinity, and rarely, with compound heterozygote variants. Case report: Siblings, started at 4 and 5 years each, with unstable gear, frequent falls and difficult running. Subsequently, loss of gait, myoclonus, dysphagia, and hallucinations. On physical examination, present optic nerve atrophy, Babinski and trunk ataxia. Electroencephalogram with widespread slow wave bursts during non-REM sleep, non photoparoxystic response, MRI with periventricular white substance hyperintensity, cerebellar atrophy and cortical. Panel of lipofuscinosis report two mutations, c.552del and c.244G>C, not described previously, in both patients. The mother was the carrier of the 552 deletion and the father and paternal grandmother of the G>C substitution (Gly82Arg). Conclusions: Differential diagnosis in neuroregression disorders is difficult because clinical signs are nonspecific, like many other neurodegenerative disorders with progressive myoclonic epilepsy. We report the clinical findings in two Mexican siblings with the late childhood variant of CLN6 with two new heterozygote mutations that contribute to the knowledge of mutations in the Mexican population and point out the relevance of performing next-generation genetic sequencing studies which will allow a better genetic counseling practice.(AU)

Clinical Trials for Gene Therapy in Lysosomal Diseases With CNS Involvement.

There are over 70 known lysosomal storage disorders (LSDs), most caused by mutations in genes encoding lysosomal hydrolases. Central nervous system involvement is a hallmark of the majority of LSDs and, if present, generally determines the prognosis of the disease. Nonetheless, brain disease is currently poorly targeted by available therapies, including systemic enzyme replacement therapy, mostly (but not only) due to the presence of the blood-brain barrier that restricts the access of orally or parenterally administered large molecules into the brain. Thus, one of the greatest and most exciting challenges over coming years will be to succeed in developing effective therapies for the treatment of central nervous system manifestations in LSDs. Over recent years, gene therapy (GT) has emerged as a promising therapeutic strategy for a variety of inherited neurodegenerative diseases. In LSDs, the ability of genetically corrected cells to cross-correct adjacent lysosomal enzyme-deficient cells in the brain after gene transfer might enhance the diffusion of the recombinant enzyme, making this group of diseases a strong candidate for such an approach. Both in vivo (using the administration of recombinant adeno-associated viral vectors) and ex vivo (auto-transplantation of lentiviral vector-modified hematopoietic stem cells-HSCs) strategies are feasible. Promising results have been obtained in an ever-increasing number of preclinical studies in rodents and large animal models of LSDs, and these give great hope of GT successfully correcting neurological defects, once translated to clinical practice. We are now at the stage of treating patients, and various clinical trials are underway, to assess the safety and efficacy of in vivo and ex vivo GT in several neuropathic LSDs. In this review, we summarize different approaches being developed and review the current clinical trials related to neuropathic LSDs, their results (if any), and their limitations. We will also discuss the pitfalls and the remaining challenges.

Chitotriosidase as a biomarker for gangliosidoses.

Elevated serum chitotriosidase (CHITO) is an indication of macrophage activation, and its capacity have been explored as a marker of inflammation in a number of disease states. For over a decade, CHITO plasma levels have been used by clinicians as a biomarker of inflammation in the lysosomal disease, Gaucher disease, including monitoring response to therapies in patients with Gaucher disease type I. Although it is becoming increasingly recognized that inflammation is a prominent component of many lysosomal diseases, the relation of CHITO levels to disease burden has not been well-characterized in the large majority of lysosomal diseases. Moreover, the role of CHITO in lysosomal diseases that affect the central nervous system (CNS) has not been systematically studied. In this study, one hundred and thirty-four specimens of CSF and serum were collected from 34 patients with lysosomal diseases affecting the CNS. This study included patients with GM1-gangliosidosis, GM2-gangliosidosis, mucopolysaccharidoses (MPS), multiple sulfatase deficiency and Gaucher disease. CHITO levels in the CSF were significantly higher in patients with more rapidly progressing severe neurological impairment: GM1-gangliosidosis vs MPS (p < 0.0001); GM2-gangliosidosis vs MPS (p < 0.0001). CHITO levels were higher in patients with the more severe phenotypes compared to milder phenotypes in GM1-gangliosidosis and GM2-gangliosidosis (serum CHITO in GM1-gangliosidosis infantile vs juvenile p = 0.025; CSF CHITO in Tay-Sachs infantile vs Tay-Sachs late-onset p < 0.0001). Moreover, higher CHITO levels in the CSF were significantly associated with lower cognitive test scores in patients with GM1-gangliosidosis, GM2-gangliosidosis, and MPS (p = 1.12*10-5, R2 = 0.72). Patients with infantile GM1-gangliosidosis showed increasing CSF CHITO over time, suggesting that CSF CHITO reflects disease progression and a possible surrogate endpoint for future clinical trials with infantile GM1-gangliosidosis. In summary, these results support the use of CSF CHITO to diagnose between different disease phenotypes and as a valuable tool for monitoring disease progression in patients. These results necessitate the inclusion of CHITO as an exploratory biomarker for clinical trials.

TDP-43 proteinopathy occurs independently of autophagic substrate accumulation and underlies nuclear defects in Niemann-Pick C disease.

AIMS: Neuronal cytoplasmic inclusions of TAR-DNA binding protein of 43 kDa (TDP-43) are a pathological hallmark of diverse neurodegenerative disorders, yet the processes that mediate their formation and their functional significance remain incompletely understood. Both dysfunction in autophagy and neuroinflammation have been linked to TDP-43 mislocalisation. Here, we investigate TDP-43 proteinopathy in Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disease (LSD) distinguished by the accumulation of unesterified cholesterol within late endosomes and lysosomes. NPC is characterised by neurodegeneration, neuroinflammation and multifocal disruption of the autophagy pathway. METHODS: We utilised immunohistochemistry, confocal microscopy, electron microscopy and biochemical and gene expression studies to characterise TDP-43 pathology and autophagic substrate accumulation in Npc1-deficient mice. RESULTS: In the NPC brain, cytoplasmic TDP-43 mislocalisation was independent of autophagic substrate accumulation. These pathologies occurred in distinct neuronal subtypes, as brainstem cholinergic neurons were more susceptible to TDP-43 mislocalisation, whereas glutamatergic neurons exhibited hallmarks of autophagic dysfunction. Furthermore, TDP-43 mislocalisation did not co-localise with markers of stress granules or progress to ubiquitinated aggregates over months in vivo, indicating a stable, early stage in the aggregation process. Neither microgliosis nor neuroinflammation were sufficient to drive TDP-43 proteinopathy in the NPC brain. Notably, cytoplasmic TDP-43 co-localised with the nuclear import factor importin α, and TDP-43 mislocalised neurons demonstrated nuclear membrane abnormalities and disruption of nucleocytoplasmic transport. CONCLUSION: Our findings highlight the relationship between LSDs and TDP-43 proteinopathy, define its functional importance in NPC by triggering nuclear dysfunction, and expand the spectrum of TDP-43 pathology in the diseased brain.

Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.

Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years.

Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation.

Genetic, epidemiological and experimental evidence implicate lysosomal dysfunction in Parkinson's disease (PD) and related synucleinopathies. Investigate several mouse models of lysosomal storage diseases (LSDs) and evaluate pathologies reminiscent of synucleinopathies. We obtained brain tissue from symptomatic mouse models of Gaucher, Fabry, Sandhoff, Niemann-Pick A (NPA), Hurler, Pompe and Niemann-Pick C (NPC) diseases and assessed for the presence of Lewy body-like pathology (proteinase K-resistant α-synuclein and tau aggregates) and neuroinflammation (microglial Iba1 and astrocytic GFAP) by immunofluorescence. All seven LSD models exhibited evidence of proteinopathy and/or inflammation in the central nervous system (CNS). However, these phenotypes were divergent. Gaucher and Fabry mouse models displayed proteinase K-resistant α-synuclein and tau aggregates but no neuroinflammation; whereas Sandhoff, NPA and NPC showed marked neuroinflammation and no overt proteinopathy. Pompe disease animals uniquely displayed widespread distribution of tau aggregates accompanied by moderate microglial activation. Hurler mice also demonstrated proteinopathy and microglial activation. The present study demonstrated additional links between LSDs and pathogenic phenotypes that are hallmarks of synucleinopathies. The data suggest that lysosomal dysregulation can contribute to brain region-specific protein aggregation and induce widespread neuroinflammation in the brain. However, only a few LSD models examined exhibited phenotypes consistent with synucleinopathies. While no model can recapitulate the complexity of PD, they can enable the study of specific pathways and mechanisms contributing to disease pathophysiology. The present study provides evidence that there are existing, previously unutilized mouse models that can be employed to study pathogenic mechanisms and gain insights into potential PD subtypes, helping to determine if they are amenable to pathway-specific therapeutic interventions.

Elevated Dipeptidyl Peptidase IV (DPP-IV) Activity in Plasma from Patients with Various Lysosomal Diseases.

Increased activity of dipeptidyl peptidase IV (DPP-IV) was reported earlier in patients with different types of mucopolysaccharidoses. DPP-IV (also known as CD26 lymphocyte T surface antigen) is a transmembrane protein showing protease activity. This enzyme displays various functions in the organism and plays an important role in multiple processes like glucose metabolism, nociception, cell-adhesion, psychoneuroendocrine regulation, immune response and cardiovascular adaptation. In order to evaluate DPP-IV in lysosomal storage diseases (LSD), we examined its activity in plasma samples from 307 patients affected with 24 different LSDs and in 75 control persons. Our results revealed elevated DPP-IV activity especially in individuals affected with mucolipidosis II/III, alpha-mannosidosis, and mucopolysaccharidoses types III, II, and I (p < 0.05). In other LSDs the DPP-IV activity was still significantly increased, but to a lesser extent. In patients with Gaucher disease, ceroid lipofuscinosis type 1 (CLN1), Niemann-Pick disease type C and A, Krabbe and Pompe diseases, gangliosidosis GM2 and metachromatic leukodystrophy discreet or no changes in DPP-IV activity were observed. DPP-IV may serve as a first-tier diagnostic procedure or additional biochemical analysis in recognizing patients with some LSDs. DPP-IV may become an object of basic research for a better understanding of LSDs.

Enzymatic diagnosis of neuronal lipofuscinoses in dried blood spots using substrates for concomitant tandem mass spectrometry and fluorimetry.

Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM-MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM-MS determination of multiple enzymes were synthesized by chemical coupling of alkyl-umbelliferone building blocks with the corresponding peptidyl-substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM-MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl-(4-alkyl-umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins-D, -F, and -B), to provide a differentiation of proteolytic specificities.

[Impact of the inclusion of second-tier tests in the newborn screening program of Catalonia and in other international programs.] / Impacto de la inclusión de pruebas de segundo nivel en el programa de cribado neonatal de Cataluña y en otros programas internacionales.

OBJECTIVE: Newborn screening programmes (NBSP) have experienced a qualitative breakthrough due to the implementation of tandem mass spectrometry. However, the tests used give rise to false positives (FP) generating an excessive request for second samples with the consequent anxiety of the families. In order to avoid this problem several programmes have developed second-tier tests (2TT). METHODS: This article presents our experience in the implementation of 2TT in the NBSP of Catalonia, as well as in other international programmes. RESULTS: From 2004 to the present, 2TT tests have been developed for more than 30 diseases. The use of 2TT helps to decrease the FP rate and increase the positive predictive value (PPV). In the NBSP of Catalonia, the implementation of 2TT for the detection of methylmalonic and propionic acidemias, homocystinurias, maple syrup disease and citrulinaemia, has managed to increase the PPV to 95% and decrease the PF rate to less than 0.01%. In cystic fibrosis, the application of 2TT slightly increases PPV but with a significant decrease in the request for second samples and in the number of cases referred to clinical units. CONCLUSIONS: The introduction of 2TT in the NBSP allows to reduce considerably the FP, decreases the number of requested samples, as well as both anxiety and stress of the families, at the same time that the hospital costs are reduced and the PPV is increased, improving notably the efficiency of the NBSP.

OBJETIVO: Los programas de cribado neonatal (PCN) han experimentado un gran avance cualitativo debido a la implementación de la espectrometría de masas en tándem. Sin embargo, las pruebas utilizadas dan lugar a falsos positivos (FP) generando una excesiva solicitud de segundas muestras con la consiguiente ansiedad de las familias. Con el fin de evitar este problema diversos programas han desarrollado pruebas de segundo nivel (2TT). METODOS: En este artículo se presenta nuestra experiencia en la implementación de 2TT en el PCN de Cataluña, así como en otros programas internacionales. RESULTADOS: Desde el año 2004 hasta la actualidad se han desarrollado pruebas de 2TT para más de 30 enfermedades. La utilización de 2TT ayuda a disminuir la tasa de FP y aumentar el valor predictivo positivo (VPP). En el PCN de Cataluña, la implementación de 2TT para la detección de acidemias metilmalónicas y propiónica, homocistinurias, jarabe de arce y citrulinemia, ha conseguido aumentar el VPP a un 95% y disminuir la tasa de FP a menos del 0,01%. En la fibrosis quística la aplicación de 2TT aumenta ligeramente el VPP pero con disminución significativa de la solicitud de segundas muestras y de los casos referidos a las unidades clínicas. CONCLUSIONES: La introducción de los 2TT en los PCN permite reducir considerablemente los FP, disminuye el número de muestras solicitadas, así como la ansiedad y el estrés de las familias, a la vez que se reducen los costes hospitalarios y se aumenta el VPP, mejorando notablemente la eficiencia de los PCN.

Lysosomal Diseases and Neuropsychiatry: Opportunities to Rebalance the Mind.

The brain is the physical organ of the mind but efforts to understand mental illness within a neurobiological context have hitherto been unavailing. Mental disorders (anxiety, depression, bipolar disorder, and schizophrenia) affect about one fifth of the population and present an almost endless societal challenge at the frontier of human sciences. Prodigious technological advances in functional neuroimaging and large-scale genetics have not yet delivered the prospect of refined molecular understanding of mental illness beyond early anatomical descriptions of brain metabolism. However, intensive clinical phenotyping and quantitative metabolic studies using sophisticated radio-ligands in positron-emission tomography, persistently favor the neurobiological approach. This Perspective pursues a familiar maxim in Medicine, aptly summarized in the words of Arthur Koestler: "Nature is generous in her senseless experiments on mankind." Hitherto, studies in neuropsychiatry have largely ignored rare genetic disorders but derangements of specific components within the cerebral laboratory offer rich opportunities for mechanistic exploration. Aberrant psychic behavior is characteristic of many inborn errors of metabolism and although each disorder represents a universe of its own, we are at a threshold for understanding, since contemporary genetics and cell biology furnish abundant materials to take on the perturbing enigma of mental derangement. A further development relates to orphan drugs with actions on defined molecular targets: these represent new ways to study the pathogenesis of psychiatric phenomena associated with rare diseases and in a manner not formerly possible. Here we introduce the frontier of schizophrenia and its strong association with late-onset Tay-Sachs disease as a paradigm to explore.

The Lysosomal Diseases Testing Laboratory: A review of the past 47 years.

Lysosomal disorders are diseases that involve mutations in genes responsible for the coding of lysosomal enzymes, transport proteins, activator proteins and protein processing enzymes. These defects lead to the storage of specific metabolites within lysosomes resulting in a great variety of clinical features depending on the tissues with the storage, the storage products and the extent of the storage. The methods for rapidly diagnosing patients started in the late 1960's when the enzyme defects were identified eliminating the need for tissue biopsies. The first requests for diagnostic help in this laboratory came in 1973. In that year, patients with Krabbe disease and Niemann-Pick type A were diagnosed. Since that time samples from about 62 000 individuals have been received for diagnostic studies, and 4900 diagnoses have been made. The largest number of diagnosed individuals had metachromatic leukodystrophy and Krabbe disease because of our research interest in leukodystrophies. A number of new disorders were identified and the primary defects in other disorders were clarified. With new methods for diagnosis, including newborn screening, molecular analysis, microarrays, there is still a need for biochemical confirmation before treatment is considered. With new treatments, including gene therapy, stem cell transplantation, enzyme replacement used alone or in combination becoming more available, the need for rapid, accurate diagnosis is critical.

Precision Medicine for Lysosomal Disorders.

Precision medicine (PM) is an emerging approach for disease treatment and prevention that accounts for the individual variability in the genes, environment, and lifestyle of each person. Lysosomal diseases (LDs) are a group of genetic metabolic disorders that include approximately 70 monogenic conditions caused by a defect in lysosomal function. LDs may result from primary lysosomal enzyme deficiencies or impairments in membrane-associated proteins, lysosomal enzyme activators, or modifiers that affect lysosomal function. LDs are heterogeneous disorders, and the phenotype of the affected individual depends on the type of substrate and where it accumulates, which may be impacted by the type of genetic change and residual enzymatic activity. LDs are individually rare, with a combined incidence of approximately 1:4000 individuals. Specific therapies are already available for several LDs, and many more are in development. Early identification may enable disease course prediction and a specific intervention, which is very important for clinical outcome. Driven by advances in omics technology, PM aims to provide the most appropriate management for each patient based on the disease susceptibility or treatment response predictions for specific subgroups. In this review, we focused on the emerging diagnostic technologies that may help to optimize the management of each LD patient and the therapeutic options available, as well as in clinical developments that enable customized approaches to be selected for each subject, according to the principles of PM.

Pre-clinical Mouse Models of Neurodegenerative Lysosomal Storage Diseases.

There are over 50 lysosomal hydrolase deficiencies, many of which cause neurodegeneration, cognitive decline and death. In recent years, a number of broad innovative therapies have been proposed and investigated for lysosomal storage diseases (LSDs), such as enzyme replacement, substrate reduction, pharmacologic chaperones, stem cell transplantation, and various forms of gene therapy. Murine models that accurately reflect the phenotypes observed in human LSDs are critical for the development, assessment and implementation of novel translational therapies. The goal of this review is to summarize the neurodegenerative murine LSD models available that recapitulate human disease, and the pre-clinical studies previously conducted. We also describe some limitations and difficulties in working with mouse models of neurodegenerative LSDs.

[Mechanism of action of cell therapy in hereditary diseases]. / Mecanismo de acción de la terapia celular en enfermedades hereditarias.

Inherited metabolism disorders are serious childhood diseases that lead to significant cognitive impairment and regression of psychomotor development. The pathophysiology of the neural progressive deterioration is usually associated with severe neuroinflammation and demyelination, and as a consequence, neurodegeneration. At the moment they have no adequate treatment and require early and aggressive therapeutic approaches, which entail high mortality rates and, very frequently, low degrees of functional improvement and survival. Bone marrow transplantation and bone marrow mesenchymal cells grafts are therapeutic and experimental therapies that improve the course of these diseases through different mechanisms of action: enzyme replacement, membrane exchange and regulation of the inflammatory process.

Los trastornos heredados del metabolismo son enfermedades graves de la infancia que cursan con un gran deterioro cognitivo y del desarrollo psicomotor. La fisiopatología del progresivo deterioro del sistema nervioso suele estar asociada a una severa neuroinflamación y desmielinización, y como consecuencia, neurodegeneración. Por el momento no tienen cura y precisan de actitudes terapéuticas precoces y agresivas, que conllevan altas tasas de mortalidad y, muy frecuentemente, escasos grados de mejoría funcional y supervivencia. El trasplante de médula ósea y de células mesenquimales de médula ósea son terapias de elección y experimentales que consiguen mejorar el curso de estas enfermedades mediante diferentes mecanismos de acción: remplazo de enzima deficiente, intercambio de membranas y regulación del proceso inflamatorio.