Subject(s)
B-Lymphocytes/physiology , Leukocyte-Adhesion Deficiency Syndrome/genetics , Membrane Proteins/genetics , Neoplasm Proteins/genetics , Pneumonia, Bacterial/genetics , Adaptive Immunity/genetics , Antibodies/blood , Cell Differentiation/genetics , Child, Preschool , Female , Humans , Immunoglobulin Class Switching/genetics , Infant , Leukocyte-Adhesion Deficiency Syndrome/diagnosis , Mutation/genetics , Pedigree , Pneumonia, Bacterial/diagnosisABSTRACT
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet productionin vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.
Subject(s)
Blood Platelets/metabolism , Cytoskeleton/metabolism , Induced Pluripotent Stem Cells/metabolism , Megakaryocyte Progenitor Cells/metabolism , Megakaryocytes/metabolism , Thrombopoiesis , Wiskott-Aldrich Syndrome/metabolism , Actins/metabolism , Antigens, CD34/metabolism , Blood Platelets/ultrastructure , Cell Lineage , Cell Shape , Cell Size , Coculture Techniques , Cytoskeleton/ultrastructure , Feeder Cells , Genetic Predisposition to Disease , Humans , Induced Pluripotent Stem Cells/ultrastructure , Megakaryocyte Progenitor Cells/ultrastructure , Megakaryocytes/ultrastructure , Mutation , Phenotype , Thrombopoiesis/genetics , Transfection , Wiskott-Aldrich Syndrome/blood , Wiskott-Aldrich Syndrome/diagnosis , Wiskott-Aldrich Syndrome/genetics , Wiskott-Aldrich Syndrome Protein/genetics , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
AIM: to describe genetic features of five unrelated Thai families with infantile-onset Pompe disease caused by mutations in the acid α-glucosidase (GAA) gene. METHODS: total RNA and genomic DNA were extracted from peripheral blood leukocytes, and mutation analysis of the entire coding regions of the GAA gene was performed in our first patient. Polymerase chain reaction-restriction fragment length polymorphism analysis was also used for a particular mutation in subsequent patients. RESULTS: the mutation analysis revealed that all patients harbored the same mutation, c.1935C > A (p.D645E), with three being homozygotes. The p.D645E, therefore, accounted for 80% (8 out of 10 alleles) of the mutations. CONCLUSIONS: we identified five unrelated Thai patients with infantile-onset Pompe disease with no history of consanguinity. Finding of the most common mutation, p.D645E, in this study will help facilitate prenatal diagnosis of their family members and molecular diagnosis of future suspected patients. Analysis of common mutations could be the most effective strategy in identifying GAA mutations responsible for Pompe disease in the Thai population.
