Confirmation of ACTN1 Variants as the Cause of Macrothrombocytopenia Using an in Vitrocellular Model
ISTH Academy. Takagi Y. Jul 10, 2019; 273840; OC 72.4 Topic: Platelet Disorders, Hereditary
Yuki Takagi
Yuki Takagi
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OC 72.4

Confirmation of ACTN1 Variants as the Cause of Macrothrombocytopenia Using an in Vitrocellular Model

Y. Takagi1, Q. Chen1,2, J. Mackay3, I. Morison4, D. Rabbolini1,2, Y. Zhu5, J. Joseph6, P. Crispin7, A. Bennett8, W. Stevenson1,2, C. Ward1,2, M.-C. Morel-Kopp1,2
1The University of Sydney, Northern Blood Research Centre, Kolling Institute, Sydney, Australia, 2Royal North Shore Hospital, Department of Haematology and Transfusion Medicine, Sydney, Australia, 3The University of Sydney, School of Molecular Biosciences, Sydney, Australia, 4University of Otago, Department of Pathology, Dunedin School of Medicine, Dunedin, New Zealand, 5The University of Sydney, Kolling Institute, Sydney, Australia, 6St Vincent Hospital, Department of Haematology, Sydney, Australia, 7Canberra Hospital, Department of Haematology, Canberra, Australia, 8Monash Pathology, Melbourne, Australia

Main Topic: Platelet Disorders
Category: Platelet Disorders, Hereditary

Background: Genetic variants of alpha-actinin-1 (ACTN1) have been reported to cause autosomal dominant macrothrombocytopenia, however, confirmation of pathogenicity requires proof of altered cellular structure.
Aims: To assess the pathogenicity of ACTN1 variants of uncertain significance (VUS) in macrothrombocytopenic patients using an in vitro cellular model.
Methods: To evaluate the effect of ACTN1 VUS on the cytoskeleton, CHO cells were transfected with Myc-tagged wild-type (WT) or mutated ACTN1. Cytoskeletal and cytosol fractions were prepared for Western blotting. Myc-tagged ACTN1 was semi-quantitated in both fractions. Transfected cells also were spread on fibronectin, and stained with anti-cMyc antibody and phalloidin. Previously reported pathogenic variants Val105Ile and Leu395Gln were used as abnormal controls.
Results: Four heterozygous missense ACTN1 variants were identified by targeted sequencing of macrothrombocytopenic subjects and confirmed by Sanger sequencing. Ser43Thr (c.127T>A) and Trp128Cys (c.384G>T) are located within the actin-binding domain (ABD) while Leu547Pro (c.1640T>C) and Asn703Ser (c.2108A>G) are in the rod domain.
By Western blotting, cytoskeletal Myc-ACTN1 was significantly increased for ABD variants S43T, W128C and V105I compared to WT, while cytosolic Myc-ACTN1 was reduced suggesting increased actin affinity. Redistribution of Myc-ACTN1 to the cytoskeleton was not seen with L547P, N703S and L395Q . Immunofluorescence on spread cells showed diffuse staining and finely dispersed ACTN1 filaments colocalizing with actin for WT and N703S transfected cells, whereas cells transfected with S43T, W128C, V105I and L395Q showed less diffuse staining and coarse, irregular fibers suggesting a less fluid actin network. Results for L547P were inconclusive with mixed patterns.
Conclusions: Using an in vitro cellular model, the two novel ABD variants of ACTN1 may increase affinity for actin, leading to a more rigid cytoskeleton that may explain the macrothrombocytopenia. Our model has proven useful for confirming the pathogenicity of ABD variants, but will need refining to assess the impact of rod domain variants.

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