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Coutinho MF, Lacerda L, Macedo-Ribeiro S, Baptista E, Ribeiro H, Prata MJ, Alves S. Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients. The functional activity of lysosomal enzymes sialidase, β-galactosidase and N-acetylaminogalacto-6-sulfate-sulfatase in the cell depends on their association in a multienzyme complex with cathepsin A. Mutations in any of the components of this complex result in functional deficiency thereby causing severe lysosomal storage disorders. Here, we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in β-galactosidase; gene GLB1) in Portuguese patients. We performed molecular studies of the PPGB, NEU1 and GLB1 genes in biochemically diagnosed Portuguese patients. Gene expression was determined and the effect of each mutation predicted at protein levels. In the NEU1 gene, we found three novel missense mutations (p.P200L, p.D234N and p.Q282H) and one nonsense mutation (p.R341X). In the PPGB gene, we identified two missense mutations, one novel (p.G86V) and one already described (p.V104M), as well as two new deletions (c.230delC and c.991-992delT) that give rise to non-functional proteins. We also present the first molecular evidence of a causal missense mutation localized to the cathepsin A active site. Finally, in the GLB1 gene, we found six different mutations, all of them previously described (p.R59H, p.R201H, p.H281Y, p.W527X, c.1572-1577InsG and c.845-846delC). Seven novel mutations are reported here, contributing to our knowledge of the mutational spectrum of these diseases and to a better understanding of the genetics of the lysosomal multienzymatic complex. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling. [ABSTRACT FROM AUTHOR]

Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.

Introduction: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo., Methods: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models., Results: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish., Conclusion: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Niemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2 . The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants' associated transcripts.

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.

Lipids are essential for cellular function and are tightly controlled at the transcriptional and post-transcriptional levels. Dysregulation of these pathways is associated with vascular diseases, diabetes, cancer, and several inherited metabolic disorders. MicroRNAs (miRNAs), in particular, are a family of post-transcriptional gene repressors associated with the regulation of many genes that encode proteins involved in multiple lipid metabolism pathways, thereby influencing their homeostasis. Thus, this class of non-coding RNAs (ncRNAs) has emerged as a promising therapeutic target for the treatment of lipid-related metabolic alterations. Most of these miRNAs act at an intracellular level, but in the past few years, a role for miRNAs as intercellular signaling molecules has also been uncovered since they can be transported in bodily fluids and used as potential biomarkers of lipid metabolic alterations. In this review, we point out the current knowledge on the miRNA signature in a lysosomal storage disorder associated with lipid dysfunction, Niemann-Pick type C, and discuss the potential use of miRNAs as biomarkers and therapeutic targets for RNA-based therapies.

Mucopolysaccharidoses (MPSs) are rare inherited lysosomal storage diseases (LSDs) caused by deficient activity in one of the enzymes responsible for glycosaminoglycans lysosomal degradation. MPS II is caused by pathogenic mutations in the IDS gene, leading to deficient activity of the enzyme iduronate-2-sulfatase, which causes dermatan and heparan sulfate storage in the lysosomes. In MPS VI, there is dermatan sulfate lysosomal accumulation due to pathogenic mutations in the ARSB gene, leading to arylsulfatase B deficiency. Alterations in the immune system of MPS mouse models have already been described, but data concerning MPSs patients is still scarce. Herein, we study different leukocyte populations in MPS II and VI disease patients. MPS VI, but not MPS II patients, have a decrease percentage of natural killer (NK) cells and monocytes when compared with controls. No alterations were identified in the percentage of T, invariant NKT, and B cells in both groups of MPS disease patients. However, we discovered alterations in the naïve versus memory status of both helper and cytotoxic T cells in MPS VI disease patients compared to control group. Indeed, MPS VI disease patients have a higher frequency of naïve T cells and, consequently, lower memory T cell frequency than control subjects. Altogether, these results reveal MPS VI disease-specific alterations in some leukocyte populations, suggesting that the type of substrate accumulated and/or enzyme deficiency in the lysosome may have a particular effect on the normal cellular composition of the immune system.

Despite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.

Over recent decades, the many functions of RNA have become more evident. This molecule has been recognized not only as a carrier of genetic information, but also as a specific and essential regulator of gene expression. Different RNA species have been identified and novel and exciting roles have been unveiled. Quite remarkably, this explosion of novel RNA classes has increased the possibility for new therapeutic strategies that tap into RNA biology. Most of these drugs use nucleic acid analogues and take advantage of complementary base pairing to either mimic or antagonize the function of RNAs. Among the most successful RNA-based drugs are those that act at the pre-mRNA level to modulate or correct aberrant splicing patterns, which are caused by specific pathogenic variants. This approach is particularly tempting for monogenic disorders with associated splicing defects, especially when they are highly frequent among affected patients worldwide or within a specific population. With more than 600 mutations that cause disease affecting the pre-mRNA splicing process, we consider lysosomal storage diseases (LSDs) to be perfect candidates for this type of approach. Here, we introduce the overall rationale and general mechanisms of splicing modulation approaches and highlight the currently marketed formulations, which have been developed for non-lysosomal genetic disorders. We also extensively reviewed the existing preclinical studies on the potential of this sort of therapeutic strategy to recover aberrant splicing and increase enzyme activity in our diseases of interest: the LSDs. Special attention was paid to a particular subgroup of LSDs: the mucopolysaccharidoses (MPSs). By doing this, we hoped to unveil the unique therapeutic potential of the use of this sort of approach for LSDs as a whole.

Mutations affecting constitutive splice donor sites (5'ss) are among the most frequent genetic defects that disrupt the normal splicing process. Pre-mRNA splicing requires the correct identification of a number of cis-acting elements in an ordered fashion. By disrupting the complementarity of the 5'ss with the endogenous small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, 5'ss mutations may result in exon skipping, intron retention or activation of cryptic splice sites. Engineered modification of the U1 snRNA seemed to be a logical method to overcome the effect of those mutations. In fact, over the last years, a number of in vitro studies on the use of those modified U1 snRNAs to correct a variety of splicing defects have demonstrated the feasibility of this approach. Furthermore, recent reports on its applicability in vivo are adding up to the principle that engineered modification of U1 snRNAs represents a valuable approach and prompting further studies to demonstrate the clinical translatability of this strategy.Here, we outline the design and generation of U1 snRNAs with different degrees of complementarity to mutated 5'ss. Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue., (© 2022. The Author(s).)

Background: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis., Methods: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels., Results: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER., Conclusion: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.

More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.

Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.

In recent years, the RNA molecule became one of the most promising targets for therapeutic intervention. Currently, a large number of RNA-based therapeutics are being investigated both at the basic research level and in late-stage clinical trials. Some of them are even already approved for treatment. RNA-based approaches can act at pre-mRNA level (by splicing modulation/correction using antisense oligonucleotides or U1snRNA vectors), at mRNA level (inhibiting gene expression by siRNAs and antisense oligonucleotides) or at DNA level (by editing mutated sequences through the use of CRISPR/Cas). Other RNA approaches include the delivery of in vitro transcribed (IVT) mRNA or the use of oligonucleotides aptamers. Here we review these approaches and their translation into clinics trying to give a brief overview also on the difficulties to its application as well as the research that is being done to overcome them.

Background: Mucopolysaccharidoses type III (MPS III) are a group of autosomal recessive lysosomal storage diseases, caused by mutations in genes that code for enzymes involved in the lysosomal degradation of heparan sulphate: heparan sulfate sulfamidase (SGSH), α-Nacetylglucosaminidase (NAGLU), heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT), and N-acetylglucosamine-6-sulfatase (GNS)., Methods: In this study, we have performed the molecular analysis of the SGSH, NAGLU and HGSNAT genes in 10 patients from 6 different MPS III Tunisian families., Results: In the SGSH gene, two mutations were identified: one novel (p.D477N) and one already described (p.Q365X). In the NAGLU gene, two novel mutations were discovered (p.L550P and p.E153X). For the novel missense mutations found in these two genes we performed an in silico structural analysis and the results were consistent with the clinical course of the patients harboring those mutations. Finally, in HGSNAT gene, we found the splicesite mutation c.234+1G>A that had already been reported as relatively frequent in MPS IIIC patients from countries surrounding the basin of the Mediterranean sea. Its presence in two Tunisian MPS IIIC families points to the hypothesis of its peri Mediterranean origin. With the exception of the c.234+1G>A mutation, that was identified in two unrelated MPS IIIC families, the other identified mutations were family-specific and were always found in homozygosity in the patients studied, thus reflecting the existence of consanguinity in MPS III Tunisian families., Conclusions: Three novel mutations are reported here, further contributing to the knowledge of the molecular basis of these diseases. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to an improvement in genetic counseling.

Mucopolysaccharidosis (MPS) and Mucolipidosis (ML) share common phenotypes (coarse facial features, organomegaly, dysostosis multiplex) despite having different molecular basis. Thus, they pose great diagnostic challenge to treating clinicians. Differentiating between the two conditions requires a battery of tests from screening to molecular diagnosis. Besides discussing differential diagnosis of MPS like features with negative urinary Glycosaminoglycans (GAG), the authors also discuss the utility of p-nitrocatechol sulphate based chemical test as an important screening tool, besides establishing molecular basis in index case.

Lysosomal storage diseases are a group of rare genetic disorders characterized by the accumulation of storage molecules in late endosomes/lysosomes. Most of them result from mutations in genes encoding for the catabolic enzymes that ensure intralysosomal digestion. Conventional therapeutic options include enzyme replacement therapy, an approach targeting the functional loss of the enzyme by injection of a recombinant one. Even though this is successful for some diseases, it is mostly effective for peripheral manifestations and has no impact on neuropathology. The development of alternative therapeutic approaches is, therefore, mandatory, and striking innovations including the clinical development of pharmacological chaperones and gene therapy are currently under evaluation. Most of them, however, have the same underlying rationale: an attempt to provide or enhance the activity of the missing enzyme to re-establish substrate metabolism to a level that is consistent with a lack of progression and/or return to health. Here, we will focus on the one approach which has a different underlying principle: substrate reduction therapy (SRT), whose uniqueness relies on the fact that it acts upstream of the enzymatic defect, decreasing storage by downregulating its biosynthetic pathway. Special attention will be given to the most recent advances in the field, introducing the concept of genetic SRT (gSRT), which is based on the use of RNA-degrading technologies (RNA interference and single stranded antisense oligonucleotides) to promote efficient substrate reduction by decreasing its synthesis rate.

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests.

Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT), whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.

Sixty years after its discovery, the lysosome is no longer considered as cell's waste bin but as an organelle playing a central role in cell metabolism. Besides its well known association with lysosomal storage disorders (mostly rare and life-threatening diseases), recent data have shown that the lysosome is also a player in some of the most common conditions of our time; and, perhaps even most important, it is not only a target for orphan drugs (rare disease therapeutic approaches) but also a putative target to treat patients suffering from common complex diseases worldwide. Here we review the striking associations linking rare lysosomal storage disorders such as the well-known Gaucher disease, or even the recently discovered, extremely rare Neuronal Ceroid Lipofuscinosis-11 and some of the most frequent, multifaceted and complex disorders of modern society such as cancer, Parkinson's disease and frontotemporal lobar degeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of β-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We report a neonate who was diagnosed as a case of skeletal dysplasia during pregnancy, and was subsequently diagnosed as a case of MLII alpha/beta on the basis of clinical and radiological findings and molecular testing of the parents. A novel GNPTAB mutation c.1701delC [p.F566LfsX5] was identified in the father. The case reiterates the severe prenatal phenotype of MLII alpha/beta which mimics skeletal dysplasia and illustrates the utility of molecular genetic analysis in confirmation of diagnosis and subsequent genetic counselling., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/β-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients., (© 2013 WILEY PERIODICALS, INC.)

Plasma low-density lipoprotein cholesterol (LDL-C) levels are a key determinant of the risk of cardiovascular disease, which is why many studies have attempted to elucidate the pathways that regulate its metabolism. Novel latest-generation sequencing techniques have identified a strong association between the 1p13 locus and the risk of cardiovascular disease caused by changes in plasma LDL-C levels. As expected for a complex phenotype, the effects of variation in this locus are only moderate. Even so, knowledge of the association is of major importance, since it has unveiled a new metabolic pathway regulating plasma cholesterol levels. Crucial to this discovery was the work of three independent teams seeking to clarify the biological basis of this association, who succeeded in proving that SORT1, encoding sortilin, was the gene in the 1p13 locus involved in LDL metabolism. SORT1 was the first gene identified as determining plasma LDL levels to be mechanistically evaluated and, although the three teams used different, though appropriate, experimental methods, their results were in some ways contradictory. Here we review all the experiments that led to the identification of the new pathway connecting sortilin with plasma LDL levels and risk of myocardial infarction. The regulatory mechanism underlying this association remains unclear, but its discovery has paved the way for considering previously unsuspected therapeutic targets and approaches., (Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.)

Lysosomal hydrolases have long been known to be responsible for the degradation of different substrates in the cell. These acid hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network (TGN). From there, they are delivered to endosomal/lysosomal compartments, where they finally become active due to the acidic pH characteristic of the lysosomal compartment. The majority of the enzymes leave the TGN after modification with mannose-6-phosphate (M6P) residues, which are specifically recognized by M6P receptors (MPRs), ensuring their transport to the endosomal/lysosomal system. Although M6P receptors play a major role in the intracellular transport of newly synthesized lysosomal enzymes in mammalian cells, several lines of evidence suggest the existence of alternative processes of lysosomal targeting. Among them, the two that are mediated by the M6P alternative receptors, lysosomal integral membrane protein (LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes, whereas sortilin has been suggested to be a multifunctional receptor capable of binding several different ligands, including neurotensin and receptor-associated protein (RAP), and of targeting several proteins to the lysosome, including sphingolipid activator proteins (prosaposin and GM2 activator protein), acid sphingomyelinase and cathepsins D and H. Here, we review the current knowledge on these two proteins: their discovery, study, structural features and cellular function, with special attention to their role as alternative receptors to lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to disease are also extensively reviewed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lysosomal hydrolases are synthesized in the rough endoplasmic reticulum and specifically transported through the Golgi apparatus to the trans-Golgi network, from which transport vesicles bud to deliver them to the endosomal/lysosomal compartment. The explanation of how are the lysosomal enzymes accurately recognized and selected over many other proteins in the trans-Golgi network relies on being tagged with a unique marker: the mannose-6-phosphate (M6P) group, which is added exclusively to the N-linked oligosaccharides of lysosomal soluble hydrolases, as they pass through the cis-Golgi network. Generation of the M6P recognition marker depends on a reaction involving two different enzymes: UDP-N-acetylglucosamine 1-phosphotransferase and α-N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase. The M6P groups are then recognized by two independent transmembrane M6P receptors, present in the trans-Golgi network: the cation-independent M6P receptor and/or the cation-dependent M6P receptor. These proteins bind to lysosomal hydrolases on the lumenal side of the membrane and to adaptins in assembling clathrin coats on the cytosolic side. In this way, the M6P receptors help package the hydrolases into vesicles that bud from the trans-Golgi network to deliver their contents to endosomes that ultimately will develop into mature lysosomes, where recently-delivered hydrolases may start digesting the endocyted material. The above described process is known as the M6P-dependent pathway and is responsible for transporting most lysosomal enzymes. This review synthesizes the current knowledge on each of the major proteins involved in the M6P-dependent pathway. Impairments in this pathway will also be addressed, highlighting the lysosomal storage disorders associated to GlcNAc-1-phosphotransferase loss of function: mucolipidosis type II and III., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Impaired degradation of glycosaminoglycans (GAGs) with consequent intralysosomal accumulation of undegraded products causes a group of lysosomal storage disorders known as mucopolysaccharidoses (MPSs). Characteristically, MPSs are recognized by increased excretion in urine of partially degraded GAGs which ultimately result in progressive cell, tissue, and organ dysfunction. There are eleven different enzymes involved in the stepwise degradation of GAGs. Deficiencies in each of those enzymes result in seven different MPSs, all sharing a series of clinical features, though in variable degrees. Usually MPS are characterized by a chronic and progressive course, with different degrees of severity. Typical symptoms include organomegaly, dysostosis multiplex, and coarse facies. Central nervous system, hearing, vision, and cardiovascular function may also be affected. Here, we provide an overview of the molecular basis, enzymatic defects, clinical manifestations, and diagnosis of each MPS, focusing also on the available animal models and describing potential perspectives of therapy for each one.

Mucolipidosis type II α/β is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII α/β patients, but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602 + 343del897) encompassing exon 19, identified in a ML II α/β patient. Long-range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21 bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5' and 3' breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18.This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.

Mucolipidosis II (ML II alpha/beta), or I-cell disease, is a rare genetic disease in which activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes, GNPTAB and GNPTG. A spectrum of mutations in GNPTAB has been recently reported to cause ML II alpha/beta. Most of these mutations were found to be private or rare. However, the mutation c.3503_3504delTC has been detected among Israeli and Palestinian Arab-Muslim, Turkish, Canadian, Italian, Portuguese, Irish traveller and US patients. We analysed 44 patients who were either homozygous or compound heterozygous for this deletion (22 Italians, 8 Arab-Muslims, 1 Turk, 3 Argentineans, 3 Brazilians, 2 Irish travellers and 5 Portuguese) and 16 carriers (15 Canadians and 1 Italian) for three intragenic polymorphisms: c.-41_-39delGGC, c.18G>A and c.1932A>G as well as two microsatellite markers flanking the GNPTAB gene (D12S1607 and D12S1727). We identified a common haplotype in all chromosomes bearing the c.3503_3504delTC mutation. In summary, we showed that patients carrying the c.3503_3504delTC deletion presented with a common haplotype, which implies a common origin of this mutation. Additionally, the level of diversity observed at the most distant locus indicates that the mutation is relatively ancient (around 2063 years old), and the geographical distribution further suggests that it probably arose in a peri-Mediterranean region., (2010 John Wiley & Sons A/S.)

This paper discusses the theme therapeutic assistance (TA), understood as homecare-based mental health intervention. We emphasize the importance of community interventions for dealing with psychic suffering, either through reading the symptoms based on visibility, or through a psychoanalytic approach mainly concerned with listening. Lacking an independent theoretical background to support this practice, therapeutic assistance makes use of theories coming from other related fields of knowledge. Therefore, we discuss the influence of psychoanalysis and its role among broad spectrum mental health practice through clinical interventions belonging to the field of TA, focusing on two long-range operative concepts: Lacan's subject supposed to know and Winnicott's care (or caring process). Both concepts guide the clinical action and provide answers to theoretical problems within the TA field. We conclude that TA meets some requirements of the classical management of transference by means of a complex care process developed in the daily life and environment of the patient, in which desire and subjectivity are necessarily recognized although no psychotherapic setting is intentionally settled. Therapeutic assistance performs the role of an advanced clinical sentinel in the field of community psychiatry and public health.

Cognitive dysfunction is common in multiple sclerosis (MS), yet few studies have examined effects of treatment on neuropsychological (NP) performance. To evaluate the effects of interferon beta-1a (IFNbeta-1a, 30 microg administered intramuscularly once weekly [Avonex]) on cognitive function, a Comprehensive NP Battery was administered at baseline and week 104 to relapsing MS patients in the phase III study, 166 of whom completed both assessments. A Brief NP Battery was also administered at 6-month intervals. The primary NP outcome measure was 2-year change on the Comprehensive NP Battery, grouped into domains of information processing and learning/memory (set A), visuospatial abilities and problem solving (set B), and verbal abilities and attention span (set C). NP effects were most pronounced in cognitive domains vulnerable to MS: IFNbeta-1a had a significant beneficial effect on the set A composite, with a favorable trend evident on set B. Secondary outcome analyses revealed significant between-group differences in slopes for Brief NP Battery performance and time to sustained deterioration in a Paced Auditory Serial Addition Test processing rate, favoring the IFNbeta-1a group. These results support and extend previous observations of significant beneficial effects of IFNbeta-1a for relapsing MS.

Histological and immunohistochemical analyses were made of biopsy specimens from 50 consecutive patients who experienced putative graft rejection. The mean age of the patients was 44.5 years (range, 17-69 years) and 26 were men. There were 67 evaluable allograft specimens, which were grouped according to the histological diagnosis: group 1, acute tubulointerstitial rejection (n = 42); group 2, acute vascular rejection (n = 18); and group 3, diffuse thrombosis (n = 7). Over a follow-up period of 21-57 months, the mean number of rejection episodes was 1.7, 2.8, and 3.3 in groups 1, 2, and 3, respectively. Allograft loss occurred in 7 out of 30, 10 out of 16, and 4 out of 4 patients in groups 1, 2, and 3, respectively. The following histological parameters differed significantly (P < 0.05) among the groups: interstitial edema, congestion of peritubular capillaries, glomerular thrombosis, and glomerular ischemia (group 3 > group 2 > group 1). Interstitial bleeding was seen more often in group 2 and 3 tissues than in group 1 specimens (P < 0.01). Immunohistochemical analyses showed that vascular rejection was associated with WT14 staining for monocytes and macrophages around the tubuli and with interstitial deposition of complement factor 3. With regard to serology, positive anti-endothelial cell antibody-dependent cellular cytotoxicity was associated with vascular rejection and thrombosis of the graft in all patients tested, and with graft loss in 75%. Pre-existent positive anti-IgG immunofluorescence on peritubular capillaries in pretransplant biopsy specimens incubated with patient serum was found in only 3 of the 50 patients, but was associated with graft loss in 2 of the 3. Cytomegalovirus infection was associated with a higher percentage of graft loss. There were significant intergroup differences in panel reactive antibodies before transplantation (P < 0.001), with higher titers in groups 2 and 3. The findings in relation to interstitial rejection are compatible with cellular rejection, while the data on vascular rejection support a humorally mediated pathogenesis.

This study aimed to determine whether dietary supplementation with fish oil has a beneficial effect on graft function and the incidence of rejection in renal allograft recipients treated with cyclosporin A (CsA). Renal function, blood pressure, the incidence of acute rejection episodes, graft survival, and renal histology and immunochemistry were investigated. In a randomized, placebo-controlled, double-blind trial, groups of 25 recipients of primary cadaveric renal allografts who had been treated with CsA took fish oil (30% C20:5 omega-3 and 20% C22:6 omega-3) or coconut oil (63% C8:0 and 36% C10:0) at 6 g/day for 3 months. There were no differences between the two patient groups with regard to HLA matching, panel-reactive antibody titers, or the demographic characteristics of donors or recipients. The GFR and effective RPF were determined at 1, 3, and 12 months after transplantation by simultaneous measurement of (125I-)iothalamate and (131I-)hippuran clearances. At 1 yr after transplantation, patients treated with fish oil showed better renal function than did the control patients, but this difference was not statistically significant. Blood pressure and antihypertensive drug use were similar in both groups. The number of rejection episodes was also similar, and renal histopathological and immunohistochemical studies showed no significant differences between the fish-oil group and the control patients. It is concluded that fish oil, at a dose of 6 g/day, has no beneficial effect after renal transplantation within the time scale of the study.

Renal biopsies were performed 1 week following renal transplantation at a time without clinical evidence of rejection in 43 patients (13 females, mean age 48 years range 18-60 and 30 males, mean age 43 years range 17-59 years). Thirty-six biopsies were available for histological or immunohistochemical analysis. Immunohistochemical analyses were performed with monoclonal antibodies against leukocytes (CD45), monocytes (WT14), complement factor 3 (C3), T-cells (Leu4), T-cell receptor alpha beta and gamma delta, tumour necrosis factor alpha (TNF alpha), IL-2 receptor (IL2-R, TAC), intercellular adhesion molecule-1 (ICAM1) and HLA-DR. The slides were scored semiquantitatively with the observers having no knowledge of clinical or patient data. TNF alpha and IL-2R were also measured by quantative PCR. None of the studied parameters correlated to delayed graft function or graft loss. Histological analysis showed that both focal interstitial infiltrate (18/35) and tubular basement membrane disruption (11/35) were followed by a higher incidence of subsequent rejection (P = 0.03 and 0.02 respectively). Also positivity for WT14 around tubuli (P = 0.02) was associated with subsequent occurrence of rejection. The intensity of staining of ICAM-1 on PTC as well as TAC on proximal tubular cells was associated with the number of subsequent rejection episodes. The association between the IL-2 receptor and subsequent rejection was also found applying PCR to the tissue specimens. We conclude that the presence of focal interstitial infiltrates and tubulitis in 1-week biopsies from well-functioning grafts carries an increased risk of subsequent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors review the visual prognosis of 44 patients with Behçet's disease referred to the Ophthalmology and Rheumatology Departments (Hospital S. João-one of the two major referral centers in Northern Portugal), due to ocular complaints or for routine examination, in the last ten years. All fulfilled the clinical criteria for diagnosis of Behçet's disease. Twenty-six were male and 18 female, with mean age 37 years (range 23-66). The mean evolution time since the first clinical manifestations was ten years (range 1-21). Aphthous stomatitis (100%) and genital ulcers (77.3%) were the initial manifestations preceding eye involvement. HLA-B(51)(5) was present in 27 of 36 typed patients (75%). Ocular manifestations were present in 33 patients (75%)-22 patients with panuveitis, eight with hypopyon, four with chronic anterior uveitis and three with episcleritis. Mean age of onset of ocular complications was 32 years (range 20-54). Retinal vaso-occlusive vasculitis was diagnosed in 26 patients (22 with panuveitis and four with posterior uveitis). To prevent ocular relapses, all needed immunosuppression with corticosteroids (drops, depo or systemic), combined with cyclosporine A (CsA) (5 mg/kg/day) in 13 patients and with chlorambucil or cyclophosphamide in six patients, when sight threatening uveitis had previously been refractory to treatment with systemic steroids. The 13 patients under CsA were observed for a period ranging from eight months to five years. Lowdose CsA was found to abrogate the intraocular inflammation, was well tolerated and had no major adverse effects. Regardless of the type of treatment, 21% (14/66) of the eyes lost useful vision five to ten years after initial diagnosis.

Idiopathic uveitis is the most common form of chronic uveitis seen in our area. Characteristically it is a heterogeneous entity in its clinical severity, evolution and therapeutic response, and immune mechanisms are thought to be involved. The phenotype frequencies of HLA antigens were studied in 62 patients with idiopathic uveitis and compared with their frequencies in northern Portugal. The authors also included two groups with a well known immunogenetic background-Behçet's syndrome (BS: n=20) and ankylosing spondylitis (AS: n=18) which are, respectively, the most severe and the most frequent of the rheumatic forms in the area. An increased frequency of HLA B5(B51) in Behçet uveitis (55%; RR=8) was confirmed in the authors' population. They also found in idiopathic uveitis an increased risk for HLA B27 (RR=14). HLA B27 positive idiopathic uveitis was predominantly unilateral (80%) and anterior (97%), whereas B27 negative forms showed a higher prevalence of severe panuveitis. In conclusion, the authors confirmed in their population the association of HLA B27 with uveitis, and HLA B51 with Behçet uveitis. In this last entity an intermediate frequency between northern Europe and the Mediterranean area was observed.