Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in alpha‐glucosidase (GAA) ‐ an enzyme responsible of hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Skeletal muscle, motor neuron pathology and airway smooth muscle cells are known to contribute to respiratory insufficiency in Pompe disease, but the role of distal airway cells including alveolar type 1 and type 2 cells (AT1 and AT2, respectively) has not been evaluated. AT1 cells depend on lysosomes for cellular homeostasis to maintain a thin barrier for gas exchange whereas AT2 cells depend on lysosomal structures for surfactant production. Using an established mouse model of Pompe disease, the Gaa−/− mouse, we compared lung histology using Periodic acid‐Schiff staining, quantification of immunohistochemistry staining and electron microscopy, and pulmonary mechanics using forced oscillometry FlexiVent system, between Gaa−/− and age‐matched wild‐type (WT) mice. We also perfomed single cell‐RNA seq (scRNA‐seq) on the distal airways of Gaa‐/‐ mice. Lysosomal glycogen accumulation and engorged lamellar bodies were observed in the AT2 cells in Gaa−/− but not WT mice. Furthermore, AT2 positive surfactant protein‐C (SPC) staining and lysosomal positive lysosomal associated membrane protein 1 (LAMP1) staining is more evident with increased colocalization of LAMP1 with the type 2 cell marker after quantification of each marker in Gaa−/− airway cells. The Gaa−/− mice exhibited a significant decrease in total and central airway resistance (Rrs and Rn, respectively), as well as a significant increase in lung compliance (Crs) versus WT mice. Moreover, in the dimensionless and volume‐independent shape constant k that describes the curvature of the upper portion of the deflation limb of the Pressure‐Volume curves, a statistically significant change was observed in Gaa−/− mice. This indicates a change in the intrinsic elastic properties of the respiratory system in Pompe disease. Finally, a robust transcriptomic dysregulation in AT1 and AT2 cells was detected scRNA‐seq in Gaa‐/‐mice relative to WT mice. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the distal airway stem cells that may contribute to respiratory impairments in Pompe disease. Our findings will inform the clinical care of patients with Pompe disease and will provide essential information for the development of novel therapies in Pompe disease that will address this airway pathology.
Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in the gene that encodes acid alpha-glucosidase (GAA) - an enzyme responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Glycogen accumulation in skeletal muscle, motor neurons, and airway smooth muscle cells are known to contribute to respiratory insufficiency in Pompe disease. However, the impact of GAA deficiency on the distal alveolar type 1 and type 2 cells (AT1 and AT2) has not been evaluated. AT1 cells rely on lysosomes for cellular homeostasis so that they can maintain a thin barrier for gas exchange, while AT2 cells depend on lysosome-like structures (lamellar bodies) for surfactant production. Using a mouse model of Pompe disease, the Gaa−/− mouse, we investigated the consequences of GAA deficiency on AT1 and AT2 cells using histology, pulmonary function and mechanics, and transcriptional analysis. Histological analysis revealed increased accumulation of lysosomal associated membrane protein 1 (LAMP1) in the Gaa-/- mice lungs. Further, ultrastructural examination showed extensive intracytoplasmic vacuoles enlargement and lamellar body engorgement. Respiratory dysfunction was confirmed using whole body plethysmography and forced oscillometry. Finally, transcriptomic analysis demonstrated dysregulation of surfactant proteins in AT2 cells, specifically reduced levels of surfactant protein D in the Gaa-/- mice. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the distal airway cells that disrupts surfactant homeostasis and contributes to respiratory impairments in Pompe disease.
IntroductionBladder cancer (BC) is a highly prevalent malignancy worldwide and particularly in Lebanon. Several studies have reported genetic risk factors in GSTM1 and NAT2, as well as many modifiable and non-modifiable risk factors, associated with BC.MethodsThis is a retrospective study that includes 51 Lebanese patients with BC. Peripheral blood samples were collected from patients and DNA extraction was performed using the standard salting-out method. Whole exome sequencing (WES) was performed and was followed by a thorough analysis of GSTM1, NAT2 and a panel of 127 genes known to be implicated in different hereditary cancers, for the detection of pathogenic germline mutations in the studied individuals.ResultsOur cohort included 42 men (82.4%) and 9 women (17.6%), with a mean age at diagnosis equal to 67 years. Of all participants, 10.8% had a family member diagnosed with BC and 17.3% with other cancer types. Our WES analysis allowed the detection of a high-risk variant found in APC (rs1801155) and a variant in BRIP1 (rs28903098) that are frequent in our cohort and more prevalent in our Lebanese in-house database compared to other international databases. Furthermore, two actionable variants, one in PALB2 (rs864622498) and another in ERCC2 (rs121913016) were detected in 2 different individuals from our cohort, paving the way to personalized therapies in these patients. A variant in NAT2 (rs1799931), shown to be associated with BC in other populations, was detected in 4 patients from our cohort.ConclusionThis is the first study in Lebanon and one of very few international studies carried out by NGS in patients with BC. Further studies in larger cohorts are required to confirm our findings in the Lebanese population.
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