Study of Bernard–Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells

Mekchay, Ponthip; Ingrungruanglert, Praewphan; Suphapeetiporn, Kanya; Sosothikul, Darintr; Ji-au, Wilawan; Grand, Supang Maneesri le; Israsena, Nipan; Rojnuckarin, Ponlapat

doi:10.1055/s-0039-1693409

Cited by 7 publications

(7 citation statements)

References 21 publications

(23 reference statements)

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“…In addition, GpIbα expression was also low which may explain the reduced ristocetin responses and dilated membrane system by EM in this case similar to Bernard Soulier syndrome platelets. [ 28 ]…”

Section: Discussionmentioning

confidence: 99%

Whole exome sequencing for diagnosis of hereditary thrombocytopenia

Mekchay

Ittiwut

et al. 2020

Medicine

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Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management. To evaluate the role of whole exome sequencing (WES) in these Pts. Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM). Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9. This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

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Section: Discussionmentioning

confidence: 99%

Whole exome sequencing for diagnosis of hereditary thrombocytopenia

Mekchay

Ittiwut

et al. 2020

Medicine

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“…generated induced pluripotent stem cells (iPSCs) from BSS type A1 and type B patients. 26 Platelets derived from BSS-iPSCs were larger than their wild-type (WT) counterparts. Importantly, lentiviral correction restored GPIb-V-IX expression, improved platelet production, and reduced platelet size in both BSS-iPSC models.…”

Section: Introductionmentioning

confidence: 99%

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Martinez-Navajas,

Ceron-Hernandez,

Simon

et al. 2023

Molecular Therapy - Nucleic Acids

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“…The underlying causes are mutations in one of the GPIb-IX-V complex encoding genes. BSS is characterized by macrothrombocytopenia, bleeding tendency, reduced ristocetin-induced platelet agglutination (RIPA), and reduced the GPIb-IX expression on platelet surface (15,34,38). Clinical manifestations of BSS are epistaxis, ecchymosis, gingival and cutaneous bleeding, purpura, hemorrhage, and menorrhagia (34,37,39).…”

Section: Gp1ba Mutationsmentioning

confidence: 99%

“…Wild-type hiPSCs (the gift from Assoc. Prof. Nipan Israsena, Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University) were derived from skin fibroblasts and the GP1BA-null (BSS-A) hiPSCs derived from a Bernard Soulier syndrome patient were constructed in our group as previously reported (38). This experimental study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (The certificate number 010/2020) and complied with the Declaration of Helsinki and ICH-GCP.…”

Section: Generation Of the Gp1ba-mutated Hipscs By Crispr-cas9mentioning

confidence: 99%

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Study of platelet production from megakaryocyte by using induced pluripotent stem cell

Pawinwongchai¹

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Thrombopoiesis is the process of platelet production from hematopoietic stem cells (HSCs). Glycoproteins (GP) Ib-IX-V that is expressed on the surface of megakaryocytes and platelets binds von Willebrand factor (VWF) plays roles in platelet production. Either the GPIb deficiency or hyper-function can cause macrothrombocytopenia, the molecular mechanisms remain unclear. In this study, the pathogenesis investigations were performed in the human induced pluripotent stem cell (hiPSC) model. CRISPR-Cas9 was used to generate the hiPSCs carrying a gain-of-function GP1BA p.M255V mutation which was described in platelet-type von Willebrand disease (PT-VWD). The GPIb deficiency hiPSCs were previously derived from a Bernard Soulier syndrome (BSS) patient. After megakaryocyte differentiation, both hiPSC mutations showed large proplatelet tips and yielded fewer but larger platelets compared with normal hiPSCs. The Capillary Western analyses revealed the lower ERK1/2 activation and higher MLC2 (Myosin light chain 2) phosphorylation in megakaryocytes with mutated GPIb. Adding a mitogen-activated protein kinase (MAPK) pathway inhibitor to normal hiPSCs recapitulated the phenotypes of GPIb mutations and increased MLC2 phosphorylation. Notably, a ROCK inhibitor which could inhibit MLC2 phosphorylation rescued the macrothrombocytopenia phenotypes of both GPIb alterations and normal hiPSCs with a MAPK inhibitor. In conclusion, the genetically-modified hiPSCs can be used to model disorders of proplatelet formation. Both loss- and gain-of-function GPIb reduced MAPK/ERK activation but enhanced ROCK/MLC2 phosphorylation resulting in dysregulated platelet generation.

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Study of Bernard–Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells

Cited by 7 publications

References 21 publications

Whole exome sequencing for diagnosis of hereditary thrombocytopenia

Whole exome sequencing for diagnosis of hereditary thrombocytopenia

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Study of platelet production from megakaryocyte by using induced pluripotent stem cell

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