A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle.

BACKGROUND: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. METHODS: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. RESULTS: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. CONCLUSIONS: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Lentiviral gene therapy with IGF2-tagged GAA normalizes the skeletal muscle proteome in murine Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa -/- mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa -/- mice. We found that muscle of Gaa -/- mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SIGNIFICANCE: Lysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease.

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.

Tagged IDS causes efficient and engraftment-independent prevention of brain pathology during lentiviral gene therapy for Mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (OMIM 309900) is a lysosomal storage disorder caused by iduronate 2-sulfatase (IDS) deficiency and accumulation of glycosaminoglycans, leading to progressive neurodegeneration. As intravenously infused enzyme replacement therapy cannot cross the blood-brain barrier (BBB), it fails to treat brain pathology, highlighting the unmet medical need to develop alternative therapies. Here, we test modified versions of hematopoietic stem and progenitor cell (HSPC)-mediated lentiviral gene therapy (LVGT) using IDS tagging in combination with the ubiquitous MND promoter to optimize efficacy in brain and to investigate its mechanism of action. We find that IDS tagging with IGF2 or ApoE2, but not RAP12x2, improves correction of brain heparan sulfate and neuroinflammation at clinically relevant vector copy numbers. HSPC-derived cells engrafted in brain show efficiencies highest in perivascular areas, lower in choroid plexus and meninges, and lowest in parenchyma. Importantly, the efficacy of correction was independent of the number of brain-engrafted cells. These results indicate that tagged versions of IDS can outperform untagged IDS in HSPC-LVGT for the correction of brain pathology in MPS II, and they imply both cell-mediated and tag-mediated correction mechanisms, including passage across the BBB and increased uptake, highlighting their potential for clinical translation.

Highly contractile 3D tissue engineered skeletal muscles from human iPSCs reveal similarities with primary myoblast-derived tissues.

Skeletal muscle research is transitioning toward 3D tissue engineered in vitro models reproducing muscle's native architecture and supporting measurement of functionality. Human induced pluripotent stem cells (hiPSCs) offer high yields of cells for differentiation. It has been difficult to differentiate high-quality, pure 3D muscle tissues from hiPSCs that show contractile properties comparable to primary myoblast-derived tissues. Here, we present a transgene-free method for the generation of purified, expandable myogenic progenitors (MPs) from hiPSCs grown under feeder-free conditions. We defined a protocol with optimal hydrogel and medium conditions that allowed production of highly contractile 3D tissue engineered skeletal muscles with forces similar to primary myoblast-derived tissues. Gene expression and proteomic analysis between hiPSC-derived and primary myoblast-derived 3D tissues revealed a similar expression profile of proteins involved in myogenic differentiation and sarcomere function. The protocol should be generally applicable for the study of personalized human skeletal muscle tissue in health and disease.

Lysosomal glycogen accumulation in Pompe disease results in disturbed cytoplasmic glycogen metabolism.

Pompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease onset and severity, although other factors, including dysregulation of cytoplasmic glycogen metabolism, are suspected to modulate the disease course. In this study, performed in mice and patient biopsies, we found elevated protein levels of enzymes involved in glucose uptake and cytoplasmic glycogen synthesis in skeletal muscle from mice with Pompe disease, including glycogenin (GYG1), glycogen synthase (GYS1), glucose transporter 4 (GLUT4), glycogen branching enzyme 1 (GBE1), and UDP-glucose pyrophosphorylase (UGP2). Expression levels were elevated before the loss of muscle mass and function. For first time, quantitative mass spectrometry in skeletal muscle biopsies from five adult patients with Pompe disease showed increased expression of GBE1 protein relative to healthy controls at the group level. Paired analysis of individual patients who responded well to treatment with enzyme replacement therapy (ERT) showed reduction of GYS1, GYG1, and GBE1 in all patients after start of ERT compared to baseline. These results indicate that metabolic changes precede muscle wasting in Pompe disease, and imply a positive feedforward loop in Pompe disease, in which lysosomal glycogen accumulation promotes cytoplasmic glycogen synthesis and glucose uptake, resulting in aggravation of the disease phenotype.

IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.

Pompe disease is caused by deficiency of acid α-glucosidase (GAA), resulting in glycogen accumulation in various tissues, including cardiac and skeletal muscles and the central nervous system (CNS). Enzyme replacement therapy (ERT) improves cardiac, motor, and respiratory functions but is limited by poor cellular uptake and its inability to cross the blood-brain barrier. Previously, we showed that hematopoietic stem cell (HSPC)-mediated lentiviral gene therapy (LVGT) with codon-optimized GAA (LV-GAAco) caused glycogen reduction in heart, skeletal muscles, and partially in the brain at high vector copy number (VCN). Here, we fused insulin-like growth factor 2 (IGF2) to a codon-optimized version of GAA (LV-IGF2.GAAco) to improve cellular uptake by the cation-independent mannose 6-phosphate/IGF2 (CI-M6P/IGF2) receptor. In contrast to LV-GAAco, LV-IGF2.GAAco was able to completely normalize glycogen levels, pathology, and impaired autophagy at a clinically relevant VCN of 3 in heart and skeletal muscles. LV-IGF2.GAAco was particularly effective in treating the CNS, as normalization of glycogen levels and neuroinflammation was achieved at a VCN between 0.5 and 3, doses at which LV-GAAco was largely ineffective. These results identify IGF2.GAA as a candidate transgene for future clinical development of HSPC-LVGT for Pompe disease.

Lentiviral gene therapy prevents anti-human acid α-glucosidase antibody formation in murine Pompe disease.

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT. Hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) provides a novel, potential lifelong therapy with a single intervention and may induce immune tolerance. Here, we investigated whether ERT can be safely applied as additional or alternative therapy following HSPC-LVGT in a murine model of Pompe disease. We found that lentiviral expression at subtherapeutic dose was sufficient to induce tolerance to the transgene product, as well as to subsequently administered ERT. Immune tolerance was established within 4-6 weeks after gene therapy. The mice tolerated ERT doses up to 100 mg/kg, allowing ERT to eliminate glycogen accumulation in cardiac and skeletal muscle and normalizing locomotor function. The presence of HSPC-derived cells expressing GAA in the thymus suggested the establishment of central immune tolerance. These findings demonstrate that lentiviral gene therapy in murine Pompe disease induced robust and long-term immune tolerance to GAA either expressed by a transgene or supplied as ERT.

Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy.

OBJECTIVE: To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II. STUDY DESIGN: Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue. RESULTS: Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10 years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity in vitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers. CONCLUSIONS: Patients with the neuronopathic form and lack of IDS protein expression were most at risk to develop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity in vitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels.

Antibodies against recombinant human alpha-glucosidase do not seem to affect clinical outcome in childhood onset Pompe disease.

BACKGROUND: Enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA, alglucosidase alfa) has improved survival, motor outcomes, daily life activity and quality of life in Pompe patients. However, ERT in Pompe disease often induces formation of antibodies, which may reduce the efficacy of treatment and can lead to adverse events. In this study antibody formation and their effect on clinical outcome in patients with childhood onset Pompe disease treated with enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA) are analyzed. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to determine anti-rhGAA antibody titers at predefined time points. The effect of antibodies on rhGAA activity (neutralizing effects) was measured in vitro. Clinical effects were evaluated by assessing muscle strength (MRC score) and function (QMFT-score), pulmonary function and infusion associated reactions (IARs). RESULTS: Twenty-two patients were included (age at start ERT 1.1-16.4 years, median treatment duration 12.4 years). Peak antibody titers were low (< 1:1250) in 9%, intermediate (1:1250-1:31,250) in 68% and high (≥ 1:31250) in 23% of patients; three patients (14%) had more than one titer of ≥ 1:31,250. Four patients (18%) experienced IARs; two patients from the high titer group had 86% of all IARs. Inhibition of intracellular GAA activity (58%) in vitro was found in one sample. The clinical course did not appear to be influenced by antibody titers. CONCLUSIONS: Ninety-one percent of childhood onset Pompe patients developed anti-rhGAA antibodies (above background level), a minority of whom had high antibody titers at repeated time points, which do not seem to interfere with clinical outcome. High antibody titers may be associated with the occurrence of IARs. Although the majority of patients does not develop high titers; antibody titers should be determined in case of clinical deterioration.

Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro.

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.

Outcome of Later-Onset Pompe Disease Identified Through Newborn Screening.

OBJECTIVE: To determine the outcomes of patients with later-onset Pompe disease (LOPD) identified through newborn screening (NBS). STUDY DESIGN: A prospective observational cohort study was conducted from the initiation of Pompe disease NBS by following subjects every 3-12 months for motor development and biochemical markers. RESULTS: Between 2005 and 2018, 39 of 994 975 newborns evaluated were classified as having LOPD based on low acid α-glucosidase (GAA) activity but no cardiac involvement at the time of screening. As of December 2020, 8 of these 39 infants (21%) were treated with enzyme replacement therapy owing to persistent elevation of creatine kinase (CK), cardiac involvement, or developmental delay. All subjects' physical performance and endurance improved after treatment. Subjects carrying c.[752C>T;761C>T] and c.[546+5G>T; 1726G>A] presented a phenotype of nonprogressive hypotonia, muscle weakness, and impairment in physical fitness tests, but they have not received treatment. CONCLUSIONS: One-fifth of subjects identified through NBS as having LOPD developed symptoms after a follow-up of up to 15 years. NBS was found to facilitate the early detection and early treatment of those subjects. GAA variants c.[752C>T;761C>T] and c.[546+5G>T; 1726G>A] might not cause Pompe disease but still may affect skeletal muscle function.

Broad variation in phenotypes for common GAA genotypes in Pompe disease.

Patients with the common c.-32-13T > G/null GAA genotype have a broad variation in age at symptom onset, ranging from early childhood to late adulthood. Phenotypic variation for other common GAA genotypes remains largely unexplored. Here, we analyzed variation in age at symptom onset for the most common GAA genotypes using the updated and extended Pompe GAA variant database. Patients with the c.2647-7G > A/null genotype invariably presented symptoms at adulthood, while the c.-32-13T > G/null, c.546G > T/null, c.1076-22T > G/null, c.2238G > C/null, and c.2173C > T/null genotypes led to presentations from early childhood up to late adulthood. The c.1309C > T/null genotype was associated with onset at early to late childhood. Symptom onset shifted toward higher ages in homozygous patients. These findings indicate that a broad variation in symptom onset occurs for various common GAA genotypes, suggesting the presence of modifying factors. We identified three new compound heterozygous c.-32-13T > G/null patients who carried the genetic modifier c.510C > T and who showed symptom onset at childhood. While c.510C > T acted by lowering GAA enzyme activity, other putative genetic modifiers did not at the group level, suggesting that these act in trans on processes downstream of GAA enzyme activity.

An in vitro assay to quantify satellite cell activation using isolated mouse myofibers.

Isolated myofibers offer the possibility of in vitro study of satellite cells in their niche. We describe a mouse myofiber isolation assay to assess satellite cell activation by quantifying myofiber-derived satellite cell progeny. The assay allows isolation of myofibers from a mouse using standard equipment and reagents. It can be used to compare satellite cells across different mouse models or to evaluate their response to treatments, offering a valuable complementary tool for in vitro experimentation.

Update of the Pompe variant database for the prediction of clinical phenotypes: Novel disease-associated variants, common sequence variants, and results from newborn screening.

Pompe disease is an inherited disorder caused by disease-associated variants in the acid α-glucosidase gene (GAA). The Pompe disease GAA variant database (http://www.pompevariantdatabase.nl) is a curated, open-source, disease-specific database, and lists disease-associated GAA variants, in silico predictions, and clinical phenotypes reported until 2016. Here, we provide an update to include 226 disease-associated variants that were published until 2020. We also listed 148 common GAA sequence variants that do not cause Pompe disease. GAA variants with unknown severity that were identified only in newborn screening programs were listed as a new feature to indicate the reason why phenotypes were still unknown. Expression studies were performed for common missense variants to predict their severity. The updated Pompe disease GAA variant database now includes 648 disease-associated variants, 26 variants from newborn screening, and 237 variants with unknown severity. Regular updates of the Pompe disease GAA variant database will be required to improve genetic counseling and the study of genotype-phenotype relationships.

Hip disease in Mucopolysaccharidoses and Mucolipidoses: A review of mechanisms, interventions and future perspectives.

The hips are frequently involved in inheritable diseases which affect the bones. The clinical and radiological presentation of these diseases may be very similar to common hip disorders as developmental dysplasia of the hip, osteoarthritis and avascular necrosis, so the diagnosis may be easily overlooked and treatment may be suboptimal. Mucopolysaccharidosis (MPS) and Mucolipidosis (ML II and III) are lysosomal storage disorders with multisystemic involvement. Characteristic skeletal abnormalities, known as dysostosis multiplex, are common in MPS and ML and originate from intra-lysosomal storage of glycosaminoglycans in cells of the cartilage, bones and ligaments. The hip joint is severely affected in MPS and ML. Hip pathology results in limitations in mobility and pain from young age, and negatively affects quality of life. In order to better understand the underlying process that causes hip disease in MPS and ML, this review first describes the normal physiological (embryonic) hip joint development, including the interplay between the acetabulum and the femoral head. In the second part the factors contributing to altered hip morphology and function in MPS and ML are discussed, such as abnormal development of the pelvic- and femoral bones (which results in altered biomechanical forces) and inflammation. In the last part of this review therapeutic options and future perspectives are addressed.

A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease.

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.

Opportunities and challenges for antisense oligonucleotide therapies.

Antisense oligonucleotide (AON) therapies involve short strands of modified nucleotides that target RNA in a sequence-specific manner, inducing targeted protein knockdown or restoration. Currently, 10 AON therapies have been approved in the United States and Europe. Nucleotides are chemically modified to protect AONs from degradation, enhance bioavailability and increase RNA affinity. Whereas single stranded AONs can efficiently be delivered systemically, delivery of double stranded AONs requires capsulation in lipid nanoparticles or binding to a conjugate as the uptake enhancing backbone is hidden in this conformation. With improved chemistry, delivery vehicles and conjugates, doses can be lowered, thereby reducing the risk and occurrence of side effects. AONs can be used to knockdown or restore levels of protein. Knockdown can be achieved by single stranded or double stranded AONs binding the RNA transcript and activating RNaseH-mediated and RISC-mediated degradation respectively. Transcript binding by AONs can also prevent translation, hence reducing protein levels. For protein restoration, single stranded AONs are used to modulate pre-mRNA splicing and either include or skip an exon to restore protein production. Intervening at a genetic level, AONs provide therapeutic options for inherited metabolic diseases as well. This review provides an overview of the different AON approaches, with a focus on AONs developed for inborn errors of metabolism.

Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience.

Pompe disease is a lysosomal and neuromuscular disorder caused by deficiency of acid alpha-glucosidase (GAA), and causes classic infantile, childhood onset, or adulthood onset phenotypes. The biochemical diagnosis is based on GAA activity assays in dried blood spots, leukocytes, or fibroblasts. Diagnosis can be complicated by the existence of pseudodeficiencies, i.e., GAA variants that lower GAA activity but do not cause Pompe disease. A large-scale comparison between these assays for patient samples, including exceptions and borderline cases, along with clinical diagnoses has not been reported so far. Here we analyzed GAA activity in a total of 1709 diagnostic cases over the past 28 years using a total of 2591 analyses and we confirmed the clinical diagnosis in 174 patients. We compared the following assays: leukocytes using glycogen or 4MUG as substrate, fibroblasts using 4MUG as substrate, and dried blood spots using 4MUG as substrate. In 794 individuals, two or more assays were performed. We found that phenotypes could only be distinguished using fibroblasts with 4MUG as substrate. Pseudodeficiencies caused by the GAA2 allele could be ruled out using 4MUG rather than glycogen as substrate in leukocytes or fibroblasts. The Asian pseudodeficiency could only be ruled out in fibroblasts using 4MUG as substrate. We conclude that fibroblasts using 4MUG as substrate provides the most reliable assay for biochemical diagnosis and can serve to validate results from leukocytes or dried blood spots.

A Generic Assay to Detect Aberrant ARSB Splicing and mRNA Degradation for the Molecular Diagnosis of MPS VI.

Identification and characterization of disease-associated variants in monogenic disorders is an important aspect of diagnosis, genetic counseling, prediction of disease severity, and development of therapy. However, the effects of disease-associated variants on pre-mRNA splicing and mRNA degradation are difficult to predict and often missed. Here we present a generic assay for unbiased identification and quantification of arylsulfatase B (ARSB) mRNA for molecular diagnosis of patients with mucopolysaccharidosis VI (MPS VI). We found that healthy control individuals have inefficient ARSB splicing because of natural skipping of exon 5 and inclusion of two pseudoexons in introns 5 and 6. Analyses of 12 MPS VI patients with 10 different genotypes resulted in identification of a 151-bp intron inclusion caused by the c.1142+2T>C variant and detection of low ARSB expression from alleles with the c.629A>G variant. A special case showed skipping of exon 4 and low ARSB expression. Although no disease-associated DNA variant could be identified in this patient, the molecular diagnosis could be made based on RNA. These results highlight the relevance of RNA-based analyses to establish a molecular diagnosis of MPS VI. We speculate that inefficient natural splicing of ARSB may be a target for therapy based on promotion of canonical splicing.