Mature neurons from iPSCs unveil neurodegeneration-related pathways in mucopolysaccharidosis type II: GSK-3ß inhibition for therapeutic potential.

Mucopolysaccharidosis (MPS) type II is caused by a deficiency of iduronate-2-sulfatase and is characterized by the accumulation of glycosaminoglycans (GAGs). Without effective therapy, the severe form of MPS II causes progressive neurodegeneration and death. This study generated multiple clones of induced pluripotent stem cells (iPSCs) and their isogenic controls (ISO) from four patients with MPS II neurodegeneration. MPS II-iPSCs were successfully differentiated into cortical neurons with characteristic biochemical and cellular phenotypes, including axonal beadings positive for phosphorylated tau, and unique electrophysiological abnormalities, which were mostly rescued in ISO-iPSC-derived neurons. RNA sequencing analysis uncovered dysregulation in three major signaling pathways, including Wnt/ß-catenin, p38 MAP kinase, and calcium pathways, in mature MPS II neurons. Further mechanistic characterization indicated that the dysregulation in calcium signaling led to an elevated intracellular calcium level, which might be linked to compromised survival of neurons. Based on these dysregulated pathways, several related chemicals and drugs were tested using this mature MPS II neuron-based platform and a small-molecule glycogen synthase kinase-3ß inhibitor was found to significantly rescue neuronal survival, neurite morphology, and electrophysiological abnormalities in MPS II neurons. Our results underscore that the MPS II-iPSC-based platform significantly contributes to unraveling the mechanisms underlying the degeneration and death of MPS II neurons and assessing potential drug candidates. Furthermore, the study revealed that targeting the specific dysregulation of signaling pathways downstream of GAG accumulation in MPS II neurons with a well-characterized drug could potentially ameliorate neuronal degeneration.

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

A close-up view of the Hunter syndrome.

The Lysosomal Storage disease known as Mucopolysaccharidosis type II, is caused by mutations affecting the iduronate-2-sulfatase required for heparan and dermatan sulfate catabolism. The central nervous system (CNS) is mostly and severely affected by the accumulation of both substrates. The complexity of the CNS damage observed in MPS II patients has been limitedly explored. The use of mass spectrometry (MS)-based proteomics tools to identify protein profiles may yield valuable information about the pathological mechanisms of Hunter syndrome. In this further study, we provide a new comparative proteomic analysis of MPS II models by using a pipeline consisting of the identification of native protein complexes positioned selectively by using a specific antibody, coupled with mass spectrometry analysis, allowing us to identify changes involving in a significant number of new biological functions, including a specific brain antioxidant response, a down-regulated autophagic, the suppression of sulfur catabolic process, a prominent liver immune response and the stimulation of phagocytosis among others.

Lentiviral Gene Therapy for Mucopolysaccharidosis II with Tagged Iduronate 2-Sulfatase Prevents Life-Threatening Pathology in Peripheral Tissues But Fails to Correct Cartilage.

Deficiency of iduronate 2-sulfatase (IDS) causes Mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder characterized by systemic accumulation of glycosaminoglycans (GAGs), leading to a devastating cognitive decline and life-threatening respiratory and cardiac complications. We previously found that hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) employing tagged IDS with insulin-like growth factor 2 (IGF2) or ApoE2, but not receptor-associated protein minimal peptide (RAP12x2), efficiently prevented brain pathology in a murine model of MPS II. In this study, we report on the effects of HSPC-LVGT on peripheral pathology and we analyzed IDS biodistribution. We found that HSPC-LVGT with all vectors completely corrected GAG accumulation and lysosomal pathology in liver, spleen, kidney, tracheal mucosa, and heart valves. Full correction of tunica media of the great heart vessels was achieved only with IDS.IGF2co gene therapy, while the other vectors provided near complete (IDS.ApoE2co) or no (IDSco and IDS.RAP12x2co) correction. In contrast, tracheal, epiphyseal, and articular cartilage remained largely uncorrected by all vectors tested. These efficacies were closely matched by IDS protein levels following HSPC-LVGT. Our results demonstrate the capability of HSPC-LVGT to correct pathology in tissues of high clinical relevance, including those of the heart and respiratory system, while challenges remain for the correction of cartilage pathology.

Establishment of the Effectiveness of Early Versus Late Stem Cell Gene Therapy in Mucopolysaccharidosis II for Treating Central Versus Peripheral Disease.

Mucopolysaccharidosis type II (MPSII) is a rare pediatric X-linked lysosomal storage disease, caused by heterogeneous mutations in the iduronate-2-sulfatase (IDS) gene, which result in accumulation of heparan sulfate (HS) and dermatan sulfate within cells. This leads to severe skeletal abnormalities, hepatosplenomegaly, and cognitive deterioration. The progressive nature of the disease is a huge obstacle to achieve full neurological correction. Although current therapies can only treat somatic symptoms, a lentivirus-based hematopoietic stem cell gene therapy (HSCGT) approach has recently achieved improved central nervous system (CNS) neuropathology in the MPSII mouse model following transplant at 2 months of age. In this study, we evaluate neuropathology progression in 2-, 4- and 9-month-old MPSII mice, and using the same HSCGT strategy, we investigated somatic and neurological disease attenuation following treatment at 4 months of age. Our results showed gradual accumulation of HS between 2 and 4 months of age, but full manifestation of microgliosis/astrogliosis as early as 2 months. Late HSCGT fully reversed the somatic symptoms, thus achieving the same degree of peripheral correction as early therapy. However, late treatment resulted in slightly decreased efficacy in the CNS, with poorer brain enzymatic activity, together with reduced normalization of HS oversulfation. Overall, our findings confirm significant lysosomal burden and neuropathology in 2-month-old MPSII mice. Peripheral disease is readily reversible by LV.IDS-HSCGT regardless of age of transplant, suggesting a viable treatment for somatic disease. However, in the brain, higher IDS enzyme levels are achievable with early HSCGT treatment, and later transplant seems to be less effective, supporting the view that the earlier patients are diagnosed and treated, the better the therapy outcome.

Fusion of Rabies Virus Glycoprotein or gh625 to Iduronate-2-Sulfatase for the Treatment of Mucopolysaccharidosis Type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.

Cognitive and adaptive behaviors associated with disease severity and genotype in patients with mucopolysaccharidosis II.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is a rare, X-linked lysosomal storage disease caused by pathogenic variants of the iduronate-2-sulfatase gene (IDS) and is characterized by a highly variable disease spectrum. MPS II severity is difficult to predict based on IDS variants alone; while some genotypes are associated with specific phenotypes, the disease course of most genotypes remains unknown. This study aims to refine the genotype-phenotype categorization by combining information from the scientific literature with data from two clinical studies in MPS II. METHODS: Genotype, cognitive, and behavioral data from 88 patients in two clinical studies (NCT01822184, NCT02055118) in MPS II were analyzed post hoc in combination with published information on IDS variants from the biomedical literature through a semi-automated multi-stage review process. The Differential Ability Scales, second edition (DAS-II) and the Vineland Adaptive Behavior Scales™, second edition (VABS-II) were used to measure cognitive function and adaptive behavior. RESULTS: The most common category of IDS variant was missense (47/88, 53.4% of total variants). The mean (standard deviation [SD]) baseline DAS-II General Conceptual Ability (GCA) and VABS-II Adaptive Behavior Composite (ABC) scores were 74.0 (16.4) and 82.6 (14.7), respectively. All identified IDS complete deletions/large rearrangements (n = 7) and large deletions (n = 1) were associated with a published 'severe' or 'predicted severe' progressive neuronopathic phenotype, characterized by central nervous system involvement. In categories comprising more than one participant, mean baseline DAS-II GCA scores (SD) were lowest among individuals with complete deletions/large rearrangements 64.0 (9.1, n = 4) and highest among those with splice site variants 83.8 (14.2, n = 4). Mean baseline VABS-II ABC scores (SD) were lowest among patients with unclassifiable variants 79.3 (4.9, n = 3) and highest among those with a splice site variant 87.2 (16.1, n = 5), in variant categories with more than one participant. CONCLUSIONS: Most patients in the studies had an MPS II phenotype categorized as 'severe' or 'predicted severe' according to classifications, as reported in the literature. Patients with IDS complete deletion/large rearrangement variants had lower mean DAS-II GCA scores than those with other variants, as well as low VABS-II ABC, confirming an association with the early progressive 'severe' (neuronopathic) disease. These data provide a starting point to improve the classification of MPS II phenotypes and the characterization of the genotype-phenotype relationship.

A novel CRISPR/Cas9-based iduronate-2-sulfatase (IDS) knockout human neuronal cell line reveals earliest pathological changes.

Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment.

A novel mucopolysaccharidosis type II mouse model with an iduronate-2-sulfatase-P88L mutation.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder characterized by an accumulation of glycosaminoglycans (GAGs), including heparan sulfate, in the body. Major manifestations involve the central nerve system (CNS), skeletal deformation, and visceral manifestations. About 30% of MPS II is linked with an attenuated type of disease subtype with visceral involvement. In contrast, 70% of MPS II is associated with a severe type of disease subtype with CNS manifestations that are caused by the human iduronate-2-sulfatase (IDS)-Pro86Leu (P86L) mutation, a common missense mutation in MPS II. In this study, we reported a novel Ids-P88L MPS II mouse model, an analogous mutation to human IDS-P86L. In this mouse model, a significant impairment of IDS enzyme activity in the blood with a short lifespan was observed. Consistently, the IDS enzyme activity of the body, as assessed in the liver, kidney, spleen, lung, and heart, was significantly impaired. Conversely, the level of GAG was elevated in the body. A putative biomarker with unestablished nature termed UA-HNAc(1S) (late retention time), one of two UA-HNAc(1S) species with late retention time on reversed-phase separation,is a recently reported MPS II-specific biomarker derived from heparan sulfate with uncharacterized mechanism. Thus, we asked whether this biomarker might be elevated in our mouse model. We found a significant accumulation of this biomarker in the liver, suggesting that hepatic formation could be predominant. Finally, to examine whether gene therapy could enhance IDS enzyme activity in this model, the efficacy of the nuclease-mediated genome correction system was tested. We found a marginal elevation of IDS enzyme activity in the treated group, raising the possibility that the effect of gene correction could be assessed in this mouse model. In conclusion, we established a novel Ids-P88L MPS II mouse model that consistently recapitulates the previously reported phenotype in several mouse models.

Clinical characteristics and genotypes of 201 patients with mucopolysaccharidosis type II in China: A retrospective, observational study.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a disease-associated variant in the IDS gene, which encodes iduronate 2-sulfatase (IDS). We aimed to characterize the clinical characteristics and genotypes of the largest cohort of Chinese patients with MPS II and so gain a deeper understanding of natural disease progression. Patients with confirmed MPS II and without treatment were included. The disease was classified as severe in patients with neurological impairment, and as attenuated in patients aged >6 years without neurological impairment. Of the 201 male patients, 78.1% had severe MPS II. Cognitive regression occurred before age 6 years in 94.3% of patients. Of 122 IDS variants identified, 37 were novel. Among the large gene alteration types identified, only the frequency of IDS-IDS2 recombination was significantly higher in severe versus attenuated MPS II (P = 0.032). Some identified point variants could inform the understanding of genotype-phenotype correlations. In conclusion, this study showed that classification of the disease as attenuated should only be made in patients aged >6 years. Our findings expand the understanding of the genotype-phenotype relationship, inform the diagnostic process, and provide an indication of the likely prognosis.

Newborn screening for mucopolysaccharidosis type II: Lessons learned.

We describe our experience with population-based newborn screening for mucopolysaccharidosis type II (MPS II) in 586,323 infants by measurement of iduronate-2-sulfatase activity in dried blood spots between December 12, 2017 and April 30, 2022. A total of 76 infants were referred for diagnostic testing, 0.01% of the screened population. Of these, eight cases of MPS II were diagnosed for an incidence of 1 in 73,290. At least four of the eight cases detected had an attenuated phenotype. In addition, cascade testing revealed a diagnosis in four extended family members. Fifty-three cases of pseudodeficiency were also identified, for an incidence of 1 in 11,062. Our data suggest that MPS II may be more common than previously recognized with a higher prevalence of attenuated cases.

A Case of Mucopolysaccharidosis II Caused by a Novel Variant with Skin Linear Hyperpigmented Streaks along Blaschko's Lines.

We report a case of an eight-year-old boy with mucopolysaccharidosis (MPS) II with atypical skin lesions of hyperpigmented streaks along Blaschko's lines. This case presented with mild symptoms of MPS such as hepatosplenomegaly, joint stiffness, and quite mild bone deformity, which was the reason for the delay in diagnosis until the age of seven years. However, he showed an intellectual disability that did not meet the diagnostic criteria for an attenuated form of MPS II. Iduronate 2-sulfatase activity was reduced. Clinical exome sequencing of DNA from peripheral blood revealed a novel pathogenic missense variant (NM_000202.8(IDS_v001):c.703C>A, p.(Pro235Thr)) in the IDS gene, which was confirmed in the mother with a heterozygous state. His brownish skin lesions differed from the Mongolian blue spots or "pebbling" of the skin that are observed in MPS II.

Evidence and recommendation for mucopolysaccharidosis type II newborn screening in the United States.

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is an X-linked condition caused by pathogenic variants in the iduronate-2-sulfatase gene. The resulting reduced activity of the enzyme iduronate-2-sulfatase leads to accumulation of glycosaminoglycans that can progressively affect multiple organ systems and impair neurologic development. In 2006, the US Food and Drug Administration approved idursulfase for intravenous enzyme replacement therapy for MPS II. After the data suggesting that early treatment is beneficial became available, 2 states, Illinois and Missouri, implemented MPS II newborn screening. Following a recommendation of the Advisory Committee on Heritable Disorders in Newborns and Children in February 2022, in August 2022, the US Secretary of Health and Human Services added MPS II to the Recommended Uniform Screening Panel, a list of conditions recommended for newborn screening. MPS II was added to the Recommended Uniform Screening Panel after a systematic evidence review reported the accuracy of screening, the benefit of presymptomatic treatment compared with usual case detection, and the feasibility of implementing MPS II newborn screening. This manuscript summarizes the findings of the evidence review that informed the Advisory Committee's decision.

A novel preclinical model of mucopolysaccharidosis type II for developing human hematopoietic stem cell gene therapy.

A hematopoietic stem cell (HSC) gene therapy (GT) using lentiviral vectors has attracted interest as a promising treatment approach for neuropathic lysosomal storage diseases. To proceed with the clinical development of HSC-GT, evaluation of the therapeutic potential of gene-transduced human CD34+ (hCD34+) cells in vivo is one of the key issues before human trials. Here, we established an immunodeficient murine model of mucopolysaccharidosis type II (MPS II), which are transplantable human cells, and demonstrated the application of those mice in evaluating the therapeutic efficacy of gene-modified hCD34+ cells. NOG/MPS II mice, which were generated using CRISPR/Cas9, exhibited a reduction of disease-causing enzyme iduronate-2-sulfatatase (IDS) activity and the accumulation of glycosaminoglycans in their tissues. When we transplanted hCD34+ cells transduced with a lentiviral vector carrying the IDS gene into NOG/MPS II mice, a significant amelioration of biochemical pathophenotypes was observed in the visceral and neuronal tissues of those mice. In addition, grafted cells in the NOG/MPS II mice showed the oligoclonal integration pattern of the vector, but no obvious clonal dominance was detected in the mice. Our findings indicate the promising application of NOG/MPS II mice to preclinical study of HSC-GT for MPS II using human cells.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.

Phenotypic Correction of Murine Mucopolysaccharidosis Type II by Engraftment of Ex Vivo Lentiviral Vector-Transduced Hematopoietic Stem and Progenitor Cells.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.

Diagnosis of patients with mucopolysaccharidosis type II via RNA sequencing.

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder caused by various variants in the IDS gene. It is known that genomic recombinants between IDS and its homologous pseudogene IDSP1 account for a small number of patients, for whom genetic diagnosis usually relies on restriction enzyme digestion at specific loci. Nevertheless, such approach cannot reveal the impact of rearrangements on IDS transcription, which is crucial for the interpretation of the pathogenicity of rearrangement variants. METHODS: RNA sequencing (RNA-seq) was explored to analyze transcriptional alterations in four male MPS II patients who were negative for Sanger sequencing of the IDS gene. Reverse transcription-polymerase chain reaction and TA clone sequencing were used to validate RNA-seq analysis results. The IDS-IDSP1 recombinant was determined by sequencing the indicated loci in genome. RESULTS: Differential expression analysis showed the expression levels of IDS gene in patients were largely reduced compared to the healthy individuals. Differential splicing analysis revealed skipping of exons 8 and 9 of IDS, without any splice-junction defects at the genomic level. In addition, two types of fusion transcripts, IDS_EOLA1 and IDS_EOLA1-DT_EOLA1 were identified by gene fusion analysis. Sequencing of the known rearrangement alleles showed these four patients have the same type of IDS-IDSP1 recombinant. CONCLUSION: We establish an RNA-seq workflow to analyze transcriptional characteristics of IDS gene from multiple perspectives. Our study validates the diagnostic value of RNA-seq in MPS II, including the discovery of transcriptional alterations and the potential to suggest genome-level rearrangements in IDS.

Intrathecal idursulfase-IT in patients with neuronopathic mucopolysaccharidosis II: Results from a phase 2/3 randomized study.

Two-thirds of patients with mucopolysaccharidosis II (MPS II; Hunter syndrome) have cognitive impairment. This phase 2/3, randomized, controlled, open-label, multicenter study (NCT02055118) investigated the effects of intrathecally administered idursulfase-IT on cognitive function in patients with MPS II. Children older than 3 years with MPS II and mild-to-moderate cognitive impairment (assessed by Differential Ability Scales-II [DAS-II], General Conceptual Ability [GCA] score) who had tolerated intravenous idursulfase for at least 4 months were randomly assigned (2:1) to monthly idursulfase-IT 10 mg (n = 34) via an intrathecal drug delivery device (IDDD; or by lumbar puncture) or no idursulfase-IT treatment (n = 15) for 52 weeks. All patients continued to receive weekly intravenous idursulfase 0.5 mg/kg as standard of care. Of 49 randomized patients, 47 completed the study (two patients receiving idursulfase-IT discontinued). The primary endpoint (change from baseline in DAS-II GCA score at week 52 in a linear mixed-effects model for repeated measures analysis) was not met: although there was a smaller decrease in DAS-II GCA scores with idursulfase-IT than with no idursulfase-IT at week 52, this was not significant (least-squares mean treatment difference [95% confidence interval], 3.0 [-7.3, 13.3]; p = 0.5669). Changes from baseline in Vineland Adaptive Behavioral Scales-II Adaptive Behavior Composite scores at week 52 (key secondary endpoint) were similar in the idursulfase-IT (n = 31) and no idursulfase-IT (n = 14) groups. There were trends towards a potential positive effect of idursulfase-IT across DAS-II composite, cluster, and subtest scores, notably in patients younger than 6 years at baseline. In a post hoc analysis, there was a significant (p = 0.0174), clinically meaningful difference in change from baseline in DAS-II GCA scores at week 52 with idursulfase-IT (n = 13) versus no idursulfase-IT (n = 6) among those younger than 6 years with missense iduronate-2-sulfatase gene variants. Overall, idursulfase-IT reduced cerebrospinal glycosaminoglycan levels from baseline by 72.0% at week 52. Idursulfase-IT was generally well tolerated. These data suggest potential benefits of idursulfase-IT in the treatment of cognitive impairment in some patients with neuronopathic MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Mutational spectrum of the iduronate-2-sulfatase gene in Mexican patients with Hunter syndrome.

OBJECTIVE: Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which is responsible for degrading heparan and dermatan sulfate. The IDS gene is located on chromosome Xq28; pathological variants in this gene mostly consist of missense mutations and small and larger deletions, which produce different phenotypes. However, there is only one record in our population concerning the molecular mechanism of this disease; a genotype-phenotype description is not available. PATIENTS AND METHODS: There were included 24 unrelated male patients; clinical features were recorded at a database, fluorometric IDS enzyme activity testing was done for each individual, followed by Sanger sequencing to identify mutations. RESULTS: The mutational spectrum was found in 16 out of 24 Mexican patients with MPS II, and its range of phenotypes was described. The most frequent variants were of the missense type. The most affected exons were exon 3 (c.275T>G, c.284_287del, c.325T>C), exon 8 (c.1035G>C, c.550G>A), exon 9 (c.1403G>C, c.1229_1229del), and exon 7 (c.979A>C; this variant has not been previously reported). Exon 5 (c.438C>T, a non-pathogenic variant) was the least frequent. It was also found that the most severely affected patients were those with large deletions (2 out of 24) [rsaIDS: IDSP1 (P164)x0, FMR1, AFF2 (P164)x2] involving genes and pseudogenes. We found 2 patients with a synonymous mutation in exon 4. CONCLUSIONS: Our results confirmed reports in the literature, since the most frequent variants were reported in exons 3 and 8. However, this result varies from one previous report in our population, which mentions large deletions and rearrangements as the most frequent alterations, since complex rearrangements were not found. According to what has been previously found, the most severely affected patients are those in which a whole gene has been deleted.

[Genetic analysis and prenatal diagnosis of a Chinese pedigree affected with mucopolysaccharidosis type II due to deletion of exon 2 of IDS gene].

OBJECTIVE: To explore the genetic etiology of a patient with mucopolysaccharidosis type II (MPSII). METHODS: The IDS gene of the proband and his mother was detected by Sanger sequencing, agarose gel electrophoresis, real-time PCR and multiple ligation-dependent probe amplification (MLPA). Prenatal diagnosis was performed on amniotic fluid sample. RESULTS: Agarose gel electrophoresis, real-time PCR, and MLPA all showed that exon 2 of IDS gene of the proband was deleted, for which his mother was normal. Prenatal diagnosis showed that the fetus was a normal male. CONCLUSION: The de novo deletion of exon 2 of the IDS gene probably underlay the MPSII in this patient. Above finding has broadened the mutation spectrum of the IDS gene. The combined methods for the detection of IDS gene mutations could make accurate prenatal diagnosis for MPSII.