Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

Kunishima, Shinji; Hamaguchi, Motohiro; Saito, Hidehiko

doi:10.1182/blood-2007-10-116194

Cited by 42 publications

(48 citation statements)

References 36 publications

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“…11,12 The activation state of ␣IIb␤3 was evaluated by the binding of the ligand-mimetic antibody PAC-1 (BD Biosciences) and FITC-labeled fibrinogen. 13 Cloning, mutagenesis, and retroviral transduction ITGA2B and ITGB3 sequences were amplified from the patient's platelet cDNA and cloned into pcDNA3.1 (Invitrogen).…”

Section: Platelet Glycoprotein Analysismentioning

confidence: 99%

Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia

Kunishima

Kashiwagi

Otsu

et al. 2011

Blood

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Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. ␣IIb␤3 has not been implicated in these conditions. We identified a novel, conserved heterozygous ITGA2B R995W mutation in 4 unrelated families. The surface expression of platelet ␣IIb␤3 was decreased to 50% to 70% of control. There was spontaneous PAC-1 and fibrinogen binding to resting platelets without CD62p expression. The activation state of ␣IIb␤3 in 293T cells was higher for ␣IIb-W995 than for ␤3-H723 but was weaker than for ␤3-

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Section: Platelet Glycoprotein Analysismentioning

confidence: 99%

Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia

Kunishima

Kashiwagi

Otsu

et al. 2011

Blood

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“…22 In brief, peripheral blood smears were fixed in methanol, permeabilized with acetone and blocked with normal goat serum. The slides were concomitantly incubated with anti-FLNA (MAB1678, Millipore) and anti-β1-tubulin antibodies, 23 and then reacted with Alexa Fluor 555-labeled antimouse IgG and Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen).…”

Section: Immunofluorescence Analysismentioning

confidence: 99%

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function

et al. 2015

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Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), cardiovascular complications, thrombocytopenia and EhlersDanlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-function mutation in FLNA. They presented with multiple complications, including valvulopathy, intestinal malrotation and chronic intestinal pseudo-obstruction (CIPO). However, these siblings had atypical clinical courses, such as a lack of PVNH and a spontaneous improvement of CIPO. Trio-based whole-exome sequencing revealed a 4-bp deletion in exon 40 that was predicted to cause a lethal premature protein truncation. However, molecular investigations revealed that the mutation induced in-frame skipping of the mutated exon, which led to the translation of a mutant FLNA missing an internal region of 41 amino acids. Functional analyses of the mutant protein suggested that its binding affinity to integrin, as well as its capacity to induce focal adhesions, were comparable to those of the wild-type protein. These results suggested that exon skipping of FLNA partially restored its protein function, which could contribute to amelioration of the siblings' clinical courses. This study expands the diversity of the phenotypes associated with loss-of-function mutations in FLNA.

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“…10 Cytospin preparations of differentiated megakaryocytes were analyzed by immunofluorescence staining. 10 …”

Section: Analysis Of Cultured Megakaryocytesmentioning

confidence: 99%

Mutation of the β1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly

Kunishima

Kobayashi

Itoh

et al. 2009

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Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet ␤1-tubulin localization/marginal band organization was observed, the level of ␤1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patient's peripheral blood CD34 ؉ cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant ␤1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant ␤1-tubulin may not be transported from the megakaryocytes into platelets. W318 ␤1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia. IntroductionCongenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. [1][2][3] The most frequent forms include MYH9 disorders, such as May-Hegglin anomaly, and Bernard-Soulier syndrome. In approximately half of the cases the pathogenesis remains unknown; thus, a definite diagnosis is not possible. The linkage between the membrane skeleton and cytoskeletal actin filaments as well as the marginal microtubule band maintains normal platelet morphology. 4,5 Defects in these systems may result in macrothrombocytopenia. The microtubules are assembled from ␣-and ␤-tubulin heterodimers. ␤1-Tubulin expression is restricted in the megakaryocyte/platelet lineage. 6 Tubb1 knockout mice show thrombocytopenia and spherical platelets. 7 TUBB1 Q43P functional polymorphism has been reported. However, it may not be relevant to macrothrombocytopenia. 8 We identified the first TUBB1 mutation affecting microtubule assembly in congenital macrothrombocytopenia. Methods PatientThe patient was a 7-year-old boy who was incidentally found to have thrombocytopenia (platelets, 40-60 ϫ 10 9 /L). He was diagnosed with immune thrombocytopenic purpura. Peripheral blood smears showed the prominent appearance of giant platelets. Electron microscopy showed no other abnormalities ( Figure 1A,B). There were no leukocyte inclusion bodies, confirmed by myosin IIA localization. 9 The platelets aggregated normally with adenosine diphosphate (ADP), collagen, and ristocetin. Flow cytometry showed normal expression of platelet GPIb/IX. An initial bone marrow examination revealed normal megakaryocyte number and morphology. The mother of the patient also had macrothrombocytopenia. Peripheral blood samples were obtained after the mother gave informed consent in accordance with the Declaration of Helsinki for the study, which was approved by the...

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Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders

Cited by 42 publications

References 36 publications

Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia

Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function

Mutation of the β1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly

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