Journal articles: 'Pompe disease; substrate localisation'

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Preisler, Nicolai, Pascal Laforêt, Karen Lindhardt Madsen, Edith Husu, Christoffer Rasmus Vissing, Gitte Hedermann, Henrik Galbo, Christopher Lindberg, and John Vissing. "Skeletal muscle metabolism during prolonged exercise in Pompe disease." Endocrine Connections 6, no. 6 (August 2017): 384–94. http://dx.doi.org/10.1530/ec-17-0042.

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Objective Pompe disease (glycogenosis type II) is caused by lysosomal alpha-glucosidase deficiency, which leads to a block in intra-lysosomal glycogen breakdown. In spite of enzyme replacement therapy, Pompe disease continues to be a progressive metabolic myopathy. Considering the health benefits of exercise, it is important in Pompe disease to acquire more information about muscle substrate use during exercise. Methods Seven adults with Pompe disease were matched to a healthy control group (1:1). We determined (1) peak oxidative capacity (VO2peak) and (2) carbohydrate and fatty acid metabolism during submaximal exercise (33 W) for 1 h, using cycle-ergometer exercise, indirect calorimetry and stable isotopes. Results In the patients, VO2peak was less than half of average control values; mean difference −1659 mL/min (CI: −2450 to −867, P = 0.001). However, the respiratory exchange ratio increased to >1.0 and lactate levels rose 5-fold in the patients, indicating significant glycolytic flux. In line with this, during submaximal exercise, the rates of oxidation (ROX) of carbohydrates and palmitate were similar between patients and controls (mean difference 0.226 g/min (CI: 0.611 to −0.078, P = 0.318) and mean difference 0.016 µmol/kg/min (CI: 1.287 to −1.255, P = 0.710), respectively). Conclusion Reflecting muscle weakness and wasting, Pompe disease is associated with markedly reduced maximal exercise capacity. However, glycogenolysis is not impaired in exercise. Unlike in other metabolic myopathies, skeletal muscle substrate use during exercise is normal in Pompe disease rendering exercise less complicated for e.g. medical or recreational purposes.

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Dajnoki, Angéla, Adolf Mühl, György Fekete, Joan Keutzer, Joe Orsini, Victor DeJesus, X. Kate Zhang, and Olaf A. Bodamer. "Newborn Screening for Pompe Disease by Measuring Acid α-Glucosidase Activity Using Tandem Mass Spectrometry." Clinical Chemistry 54, no. 10 (October 1, 2008): 1624–29. http://dx.doi.org/10.1373/clinchem.2008.107722.

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Abstract background: Pompe disease, caused by the deficiency of acid α-glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. methods: We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]- ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease–specific cutoff value using routine newborn screening samples. results: GAA activities in DBS from 29 known Pompe patients were <2 μmol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) μmol/h/L (n = 10 279, median 13.3, 95% CI 14.46–14.74 μmol/h/L) and in normal adult samples 9.3 (3.3) μmol/h/L (n = 229, median 9, 95% CI 8.88–9.72 μmol/h/L). GAA activity was stable for 28 days between 37 °C and −80 °C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 μmol/h/L and limit of quantification 0.35 μmol/h/L. conclusions: The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.

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Sista, Ramakrishna S., Allen E. Eckhardt, Tong Wang, Carrie Graham, Jeremy L. Rouse, Scott M. Norton, Vijay Srinivasan, et al. "Digital Microfluidic Platform for Multiplexing Enzyme Assays: Implications for Lysosomal Storage Disease Screening in Newborns." Clinical Chemistry 57, no. 10 (October 1, 2011): 1444–51. http://dx.doi.org/10.1373/clinchem.2011.163139.

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BACKGROUND Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods. METHODS We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h. RESULTS Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics. CONCLUSIONS A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

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Ullman, Julie C., Kevin T. Mellem, Yannan Xi, Terrence F. Satterfield, Tarunmeet Gujral, Rebeca Choy, Julian R. Homburger, et al. "Substrate reduction therapy for Pompe disease: Small molecule inhibition of glycogen synthase 1 in preclinical models." Molecular Genetics and Metabolism 135, no. 2 (February 2022): S122. http://dx.doi.org/10.1016/j.ymgme.2021.11.324.

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van Diggelen, O. P., L. F. Oemardien, N. A. M. E. van der Beek, M. A. Kroos, H. K. Wind, Y. V. Voznyi, D. Burke, M. Jackson, B. G. Winchester, and A. J. J. Reuser. "Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates." Journal of Inherited Metabolic Disease 32, no. 3 (April 19, 2009): 416–23. http://dx.doi.org/10.1007/s10545-009-1082-3.

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Njeim, Mario, and Frank Bogun. "Selecting the Appropriate Ablation Strategy: the Role of Endocardial and/or Epicardial Access." Arrhythmia & Electrophysiology Review 4, no. 3 (2015): 184. http://dx.doi.org/10.15420/aer.2015.4.3.184.

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Percutaneous catheter ablation has emerged as an effective treatment modality for the management of ventricular tachycardia. Despite years of progress in this field, the role of epicardial mapping and ablation needs to be further refined. In this review, we discuss the relationship between the type of underlying heart disease and the location of the arrythmogenic substrate as it pertains to a procedural approach. We describe the contribution of preprocedural and intraprocedural diagnostic tools for the localisation of the arrhythmogenic substrate, with a special emphasis on cardiac MRI and electrophysiological mapping. In our opinion, the preferred approach to target ventricular tachycardia should depend on the patient’s underlying heart disease and the location of scar tissue, which can be best visualised using cardiac MRI.

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Clayton, Nicholas P., Carol A. Nelson, Timothy Weeden, Kristin M. Taylor, Rodney J. Moreland, Ronald K. Scheule, Lucy Phillips, Andrew J. Leger, Seng H. Cheng, and Bruce M. Wentworth. "Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease." Molecular Therapy - Nucleic Acids 3 (January 2014): e206. http://dx.doi.org/10.1038/mtna.2014.57.

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Clayton, Nicholas P., Carol A. Nelson, Timothy Weeden, Kristin M. Taylor, Rodney J. Moreland, Ronald K. Scheule, Andrew J. Leger, Lucy Phillips, Seng H. Cheng, and Bruce M. Wentworth. "Antisense oligonucleotide-mediated suppression of muscle glycogen synthase 1 synthesis as an approach for substrate reduction therapy of Pompe disease." Molecular Genetics and Metabolism 114, no. 2 (February 2015): S32. http://dx.doi.org/10.1016/j.ymgme.2014.12.055.

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Tortorelli, Silvia, Coleman T. Turgeon, Dimitar K. Gavrilov, Devin Oglesbee, Kimiyo M. Raymond, Piero Rinaldo, and Dietrich Matern. "Simultaneous Testing for 6 Lysosomal Storage Disorders and X-Adrenoleukodystrophy in Dried Blood Spots by Tandem Mass Spectrometry." Clinical Chemistry 62, no. 9 (September 1, 2016): 1248–54. http://dx.doi.org/10.1373/clinchem.2016.256255.

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Abstract BACKGROUND Newborn screening for lysosomal storage disorders (LSD) has revealed that late-onset variants of these conditions are unexpectedly frequent and therefore may evade diagnosis. We developed an efficient and cost-effective multiplex assay to diagnose six LSDs and several peroxisomal disorders in patients presenting with diverse phenotypes at any age. METHODS Three 3-mm dried blood spot (DBS) punches were placed into individual microtiter plates. One disc was treated with a cocktail containing acid sphingomyelinase-specific substrate and internal standard (IS). To the second DBS we added a cocktail containing substrate and IS for β-glucosidase, acid α-glucosidase, α-galactosidase A, galactocerebrosidase, and α-L-iduronidase. The third DBS was extracted with methanol containing d4-C26 lysophosphatidylcholine as IS and stored until the enzyme plates were combined and purified by liquid–liquid and solid-phase extraction. The extracts were evaporated, reconstituted with the extract from the lysophosphatidylcholine plate, and analyzed by flow injection tandem mass spectrometry. RESULTS Reference intervals were determined by analysis of 550 samples from healthy controls. DBS from confirmed patients with 1 of the 6 LSDs (n = 33), X-adrenoleukodystrophy (n = 9), or a peroxisomal biogenesis disorder (n = 5), as well as carriers for Fabry disease (n = 17) and X-adrenoleukodystrophy (n = 5), were analyzed for assay validation. Prospective clinical testing of 578 samples revealed 25 patients affected with 1 of the detectable conditions. CONCLUSIONS Our flow injection tandem mass spectrometry approach is amenable to high-throughput population screening for Hurler disease, Gaucher disease, Niemann–Pick A/B disease, Pompe disease, Krabbe disease, Fabry disease, X-adrenoleukodystrophy, and peroxisomal biogenesis disorder in DBS.

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Lin, Na, Jingyu Huang, Sara Violante, Joseph J. Orsini, Michele Caggana, Erin E. Hughes, Colleen Stevens, et al. "Liquid Chromatography–Tandem Mass Spectrometry Assay of Leukocyte Acid α-Glucosidase for Post-Newborn Screening Evaluation of Pompe Disease." Clinical Chemistry 63, no. 4 (April 1, 2017): 842–51. http://dx.doi.org/10.1373/clinchem.2016.259036.

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Abstract BACKGROUND Pompe disease (PD) is the first lysosomal storage disorder to be added to the Recommended Uniform Screening Panel for newborn screening. This condition has a broad phenotypic spectrum, ranging from an infantile form (IOPD), with severe morbidity and mortality in infancy, to a late-onset form (LOPD) with variable onset and progressive weakness and respiratory failure. Because the prognosis and treatment options are different for IOPD and LOPD, it is important to accurately determine an individual's phenotype. To date, no enzyme assay of acid α-glucosidase (GAA) has been described that can differentiate IOPD vs LOPD using blood samples. METHODS We incubated 10 μL leukocyte lysate and 25 μL GAA substrate and internal standard (IS) assay cocktail for 1 h. The reaction was purified by a liquid–liquid extraction. The extracts were evaporated and reconstituted in 200 μL methanol and analyzed by LC-MS/MS for GAA activity. RESULTS A 700-fold higher analytical range was observed with the LC-MS/MS assay compared to the fluorometric method. When GAA-null and GAA-containing fibroblast lysates were mixed, GAA activity could be measured accurately even in the range of 0%–1% of normal. The leukocyte GAA activity in IOPD (n = 4) and LOPD (n = 19) was 0.44–1.75 nmol · h−1 · mg−1 and 2.0–6.5 nmol · h−1 · mg−1, respectively, with no overlap. The GAA activity of pseudodeficiency patients ranged from 3.0–28.1 nmol · h−1 · mg−1, showing substantial but incomplete separation from the LOPD group. CONCLUSIONS This assay allows determination of low residual GAA activity in leukocytes. IOPD, LOPD, and pseudodeficiency patients can be partially differentiated by measuring GAA using blood samples.

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Zhang, X. Kate, Carole S. Elbin, Wei-Lien Chuang, Samantha K. Cooper, Carla A. Marashio, Christa Beauregard, and Joan M. Keutzer. "Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry." Clinical Chemistry 54, no. 10 (October 1, 2008): 1725–28. http://dx.doi.org/10.1373/clinchem.2008.104711.

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Abstract Background: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories. Methods: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay. Results: In our study, the median enzyme activity measured in adults was generally increased 2–3–fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples. Conclusions: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

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Metz, Thomas F., Thomas P. Mechtler, Joseph J. Orsini, Monica Martin, Bori Shushan, Joseph L. Herman, Rene Ratschmann, et al. "Simplified Newborn Screening Protocol for Lysosomal Storage Disorders." Clinical Chemistry 57, no. 9 (September 1, 2011): 1286–94. http://dx.doi.org/10.1373/clinchem.2011.164640.

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BACKGROUND Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays. METHODS After overnight incubation (16–20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC–tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid–liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate. RESULTS Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory. CONCLUSIONS Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.

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Lun, Yi, Su Xu, Rebecca Soska, Anju Nair, Michelle Frascella, Anadina Garcia, Russell Gotschall, Hung Do, Kenneth J. Valenzano, and Richie Khanna. "A novel recombinant human acid alpha-glucosidase, ATB200, leads to greater substrate reduction and improvement in Pompe disease-relevant markers compared to alglucosidase alfa in GAA KO mice." Molecular Genetics and Metabolism 120, no. 1-2 (January 2017): S88. http://dx.doi.org/10.1016/j.ymgme.2016.11.216.

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Soussi-Yanicostas, N., C. Faivre-Sarrailh, J. P. Hardelin, J. Levilliers, G. Rougon, and C. Petit. "Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner." Journal of Cell Science 111, no. 19 (October 1, 1998): 2953–65. http://dx.doi.org/10.1242/jcs.111.19.2953.

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Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.

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Hulme, Benjamin J., Kathrin K. Geyer, Josephine E. Forde-Thomas, Gilda Padalino, Dylan W. Phillips, Wannaporn Ittiprasert, Shannon E. Karinshak, et al. "Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production." PLOS Pathogens 18, no. 1 (January 13, 2022): e1009828. http://dx.doi.org/10.1371/journal.ppat.1009828.

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α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

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Niño, Monica Y., Mark Wijgerde, Douglas Oliveira Soares de Faria, Marianne Hoogeveen-Westerveld, Atze J. Bergsma, Mike Broeders, Nadine A. M. E. van der Beek, et al. "Enzymatic diagnosis of Pompe disease: lessons from 28 years of experience." European Journal of Human Genetics, November 8, 2020. http://dx.doi.org/10.1038/s41431-020-00752-2.

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Abstract 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.

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Stevens, David, Shadi Milani-Nejad, and Tahseen Mozaffar. "Pompe Disease: a Clinical, Diagnostic, and Therapeutic Overview." Current Treatment Options in Neurology, August 4, 2022. http://dx.doi.org/10.1007/s11940-022-00736-1.

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Abstract Purpose of Review This review summarizes the clinical presentation and provides an update on the current strategies for diagnosis of Pompe disease. We will review the available treatment options. We examine newly approved treatments as well as upcoming therapies in this condition. We also provide commentary on the unmet needs in clinical management and research for this disease. Recent Findings In March 2015, Pompe disease was added to the Recommended Uniform Screening Panel (RUSP) and since then a number of states have added Pompe disease to their slate of diseases for their Newborn Screening (NBS) program. Data emerging from these programs is revising our knowledge of incidence of Pompe disease. In 2021, two randomized controlled trials involving new forms of enzyme replacement therapy (ERT) were completed and one new product is already FDA-approved and on the market, whereas the other product will come up for FDA review in the fall. Neither of the new ERT were shown to be superior to the standard of care product, alglucosidase. The long-term effectiveness of these newer forms of ERT is unclear. Newer versions of the ERT are in development in addition to multiple different strategies of gene therapy to deliver GAA, the gene responsible for producing acid alpha-glucosidase, the defective protein in Pompe Disease. Glycogen substrate reduction is also in development in Pompe disease and other glycogen storage disorders. Summary There are significant unmet needs as it relates to clinical care and therapeutics in Pompe disease as well as in research. The currently available treatments lose effectiveness over the long run and do not have penetration into neuronal tissues and inconsistent penetration in certain muscles. More definitive gene therapy and enzyme replacement strategies are currently in development and testing.

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Zhang, Xiao, Huiying Liu, Naresh Meena, Chao Li, Guanghui Zong, Nina Raben, Rosa Puertollano, and Lai-Xi Wang. "Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game." Chemical Science, 2021. http://dx.doi.org/10.1039/d1sc03188k.

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An efficient one-pot M6P glycan remodeling of a multiply glycosylated lysosomal enzyme (Lumizyme) affords a new glycoengineered protein that shows greatly improved receptor binding, cellular uptake, and degradation of lysosomal glycogen in an in vitro model of Pompe disease.

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Main, Alice, Andri Boguslavskyi, Jacqueline Howie, Chien-Wen Kuo, Aileen Rankin, Francis L. Burton, Godfrey L. Smith, et al. "Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies." Frontiers in Physiology 13 (October 5, 2022). http://dx.doi.org/10.3389/fphys.2022.1023237.

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S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

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Hanson, Filipe, Rachel E. Hodgson, Madalena I. Ribeiro de Oliveira, Elizabeth Allen, and Susan Gerarda Campbell. "Regulation and function of elF2B in neurological and metabolic disorders." Bioscience Reports, May 17, 2022. http://dx.doi.org/10.1042/bsr20211699.

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Eukaryotic initiation factor 2B, eIF2B is a guanine nucleotide exchange, factor with a central role in coordinating the initiation of translation. During stress and disease, the activity of eIF2B is inhibited via the phosphorylation of its substrate eIF2 (p-eIF2α). A number of different kinases respond to various stresses leading to the phosphorylation of the alpha subunit of eIF2 and collectively this regulation is known as the Integrated Stress Response, ISR. This targeting of eIF2B allows the cell to regulate protein synthesis and reprogramme gene expression to restore homeostasis. Advances within structural biology have furthered our understanding of how eIF2B interacts with eIF2 in both the productive GEF active form and the non-productive eIF2ɑ phosphorylated form. Here, current knowledge of the role of eIF2B in the ISR is discussed within the context of normal and disease states focussing particularly on diseases such as Vanishing White Matter Disease (VWMD) and Permanent Neonatal Diabetes Mellitus (PNDM), which are directly linked to mutations in eIF2B. The role of eIF2B in synaptic plasticity and memory formation is also discussed. In addition, the cellular localisation of eIF2B is reviewed and considered along with the role of additional in vivo eIF2B binding factors and protein modifications that may play a role in modulating eIF2B activity during health and disease.

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Cappabianca, Lucia, Antonietta Rosella Farina, Lucia Di Marcotullio, Paola Infante, Daniele De Simone, Michela Sebastiano, and Andrew Reay Mackay. "Discovery, characterization and potential roles of a novel NF-YAx splice variant in human neuroblastoma." Journal of Experimental & Clinical Cancer Research 38, no. 1 (December 2019). http://dx.doi.org/10.1186/s13046-019-1481-8.

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Abstract Background Identification of novel cancer-associated splice variants is of potential diagnostic, prognostic and therapeutic importance. NF-Y transcription factor is comprised of NF-YA, NF-YB and NF-YC subunits, binds inverted CCAAT-boxes in ≈70% of gene promoters, regulates > 1000 cancer-associated genes and proteins involved in proliferation, staminality, differentiation, apoptosis, metabolism and is subject to component alternative splicing. RT-PCR evaluation of alternative NF-YA splicing in primary human neuroblastomas (NBs), led to discovery of a novel NF-YAx splice variant, also expressed during mouse embryo development and induced by doxorubicin in NB cells. Here, we report the discovery and characterisation of NF-YAx and discus its potential roles in NB. Methods NF-YAx cDNA was RT-PCR-cloned from a stage 3 NB (provided by the Italian Association of Haematology and Paediatric Oncology, Genova, IT), sequenced and expressed as a protein using standard methods and compared to known fully-spliced NF-YAl and exon B-skipped NF-YAs isoforms in: EMSAs for capacity to form NF-Y complexes; by co-transfection, co-immunoprecipitation and Western blotting for capacity to bind Sp1; by IF for localisation; in AO/EtBr cell-death and colony formation assays for relative cytotoxicity, and by siRNA knockdown, use of inhibitors and Western blotting for potential mechanisms of action. Stable SH-SY5Y transfectants of all three NF-YA isoforms were also propagated and compared by RT-PCR and Western blotting for differences in cell-death and stem cell (SC)-associated gene expression, in cell-death assays for sensitivity to doxorubicin and in in vitro proliferation, substrate-independent growth and in vivo tumour xenograft assays for differences in growth and tumourigenic capacity. Results NF-YAx was characterized as a novel variant with NF-YA exons B, D and partial F skipping, detected in 20% of NF-YA positive NBs, was the exclusive isoform in a stage 3 NB, expressed in mouse stage E11.5–14 embryos and induced by doxorubicin in SH-SY5Y NB cells. The NF-YAx protein exhibited nuclear localisation, competed with other isoforms in CCAAT box-binding NF-Y complexes but, in contrast to other isoforms, did not bind Sp1. NF-YAx expression in neural-related progenitor and NB cells repressed Bmi1 expression, induced KIF1Bβ expression and promoted KIF1Bβ-dependent necroptosis but in NB cells also selected tumourigenic, doxorubicin-resistant, CSC-like sub-populations, resistant to NF-YAx cytotoxicity. Conclusions The discovery of NF-YAx in NBs, its expression in mouse embryos and induction by doxorubicin in NB cells, unveils a novel NF-YA splice mechanism and variant, regulated by and involved in development, genotoxic-stress and NB. NF-YAx substitution of other isoforms in NF-Y complexes and loss of capacity to bind Sp1, characterises this novel isoform as a functional modifier of NF-Y and its promotion of KIF1Bβ-dependent neural-lineage progenitor and NB cell necroptosis, association with doxorubicin-induced necroptosis and expression in mouse embryos coinciding with KIF1Bβ-dependent sympathetic neuroblast-culling, confirm a cytotoxic function and potential role in suppressing NB initiation. On the other hand, the in vitro selection of CSC-like NB subpopulations resistant to NF-YAx cytotoxicity not only helps to explain high-level exclusive NF-YAx expression in a stage 3 NB but also supports a role for NF-YAx in disease progression and identifies a potential doxorubicin-inducible mechanism for post-therapeutic relapse.

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Journal articles on the topic 'Pompe disease; substrate localisation'

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