ACTN1 mutations cause congenital macrothrombocytopenia

Abstract

Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of rare platelet disorders characterized by a congenital reduction of platelet counts and abnormally large platelets, for which CMTP-causing mutations are only found in approximately half the cases. We herein performed whole-exome sequencing and targeted Sanger sequencing to identify mutations that cause CMTP, in which a dominant mode of transmission had been suspected but for which no known responsible mutations have been documented. In 13 Japanese CMTP-affected pedigrees, we identified six (46%) affected by ACTN1 variants cosegregating with CMTP. In the entire cohort, ACNT1 variants accounted for 5.5% of the dominant forms of CMTP cases and represented the fourth most common cause in Japanese individuals. Individuals with ACTN1 variants presented with moderate macrothrombocytopenia with anisocytosis but were either asymptomatic or had only a modest bleeding tendency. ACTN1 encodes α-actinin-1, a member of the actin-crosslinking protein superfamily that participates in the organization of the cytoskeleton. In vitro transfection experiments in Chinese hamster ovary cells demonstrated that altered α-actinin-1 disrupted the normal actin-based cytoskeletal structure. Moreover, transduction of mouse fetal liver-derived megakaryocytes with disease-associated ACTN1 variants caused a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number. Our findings provide an insight into the pathogenesis of CMTP.

Figure 1

A Flow Diagram of the Genomic Analysis (A) Detection of the candidate germline variants through Genomon-exome. (B) Screening of ACTN1 variants with high-throughput sequencing of pooled DNA.

Figure 2

Segregation of ACTN1 Variants, the ACTN1 Structure, α-Actinin-Actin Interaction, and the α-Actinin Domain Structure (A) The pedigrees for the six families affected by ACTN1 variants are shown together with the Sanger-sequencing electropherograms. Filled symbols represent individuals affected by macrothrombocytopenia. The identified ACTN1 variants are shown below the symbols. The arrows indicate nucleotide changes. The following abbreviations are used: WT, wild-type; and NA, not available. (B) Genomic organization of ACTN1, variants in which are identified in individuals with CMTP. (C) α-actinin cross-links actin filaments into actin-filament bundles. (D) α-actinin consists of an N-terminal actin-binding domain (ABD), composed of two calponin homology domains (CHD), four spectrin repeats (R1–R4), and a C-terminal calmodulin-like domain (CaM). Two molecules form an antiparallel dimer. The arrows indicate the positions of the identified ACTN1 variants.

Figure 3

The Platelet Morphology (A) Peripheral-blood smears were stained with May-Grünwald Giemsa for a normal control, the exome-1 proband, and the Sanger-3 proband. The affected individuals showed macrothrombocytopenia accompanied by anisocytosis. The number in each panel shows the mean platelet size (n = 200). Images were obtained with a BX50 microscope with a 100×/1.35 numeric aperture oil objective (Olympus, Tokyo, Japan). Images of the slides were acquired with a DP70 digital camera and DP manager software (Olympus). The original magnification is ×1,000. (B) The ultrastructure of platelets from a normal control, the exome-3 proband, and the Sanger-3 proband. Platelet-rich plasma was prepared from acid-citrate-dextrose-citrated whole blood that was fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide. The samples were then dehydrated in a graded ethanol series and n-butyl glycidyl ether and embedded in an epoxy resin (Epon 812; TAAB, Berkshire, UK). Ultrathin sections (0.1 μm) were doubly stained with 2% uranyl acetate and 1% lead citrate and were then observed with a transmission electron microscope (H-7650, Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV. The original magnification is ×1,500. The scale bar represents 2 μm.

Figure 4

The Subcellular Localization of Altered ACTN1 in CHO Cells CHO cells were transiently transfected with Myc-tagged wild-type or mutant ACTN1 cDNAs. After the cells were replated on fibronectin-coated coverslips, they were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. They were then stained with Myc-tagged antibody (Invitrogen), followed by Alexa-488-labeled goat anti-mouse IgG, Alexa-555-conjugated phalloidin (Invitrogen), and DAPI. Images were obtained with a BX50 fluorescence microscope with a 40×/1.00 numerical aperture oil objective lens. The cells shown are representative of eight independent experiments.

Figure 5

Abnormal Proplatelet Formation in Megakaryocytes Transduced with Altered ACTN1 (A) The organization of the actin filaments in megakaryocytes adhered to fibrinogen. The scale bar represents 50 μm. (B) The percentage of megakaryocytes extending proplatelets was evaluated under an IX71 fluorescence microscope with a 20×/0.40 objective lens (Olympus) 2–4 days after infection. For each specimen, at least 100 megakaryocytes were evaluated. (C) The number of proplatelet tips per megakaryocyte was decreased in megakaryocytes transduced with mutant ACTN1. (D) The size of proplatelet tips was increased in megakaryocytes transduced with altered ACTN1. (E) Representative megakaryocytes extending proplatelets 3 days after infection. Note that the size of the proplatelet tips was variable in the megakaryocytes transduced with altered ACTN1 (arrows indicate large tips, and arrowheads indicate small tips). The scale bar represents 50 μm. (F) The proplatelet morphology and α-tubulin localization of EGFP-positive megakaryocytes. Infection day 3 cultures were cytospun onto glass slides. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with GFP antibody (D153-3; Medical & Biological Laboratories, Nagoya, Japan) and α-tubulin antibody (RB-9281; Lab Vision, Fremont, CA), followed by Alexa-488-labeled goat anti-rat IgG and Alexa-555-labeled goat anti-rabbit IgG (Invitrogen). Images were obtained with a BX50 microscope with a 100×/1.35 numeric aperture oil objective lens (Olympus). The size of the proplatelet tips was variable in megakaryocytes transduced with altered ACTN1 (arrows indicate large tips, and arrowheads indicate small tips). The scale bar represents 20 μm. Representative images from four independent experiments are shown.