Novel ACTN1 variants in cases of thrombocytopenia

Abstract

The ACTN1 gene has been implicated in inherited macrothrombocytopenia. To decipher the spectrum of variants and phenotype of ACTN1-related thrombocytopenia, we sequenced the ACTN1 gene in 272 cases of unexplained chronic or familial thrombocytopenia. We identified 15 rare, monoallelic, nonsynonymous and likely pathogenic ACTN1 variants in 20 index cases from 20 unrelated families. Thirty-one family members exhibited thrombocytopenia. Targeted sequencing was carried out on 12 affected relatives, which confirmed presence of the variant. Twenty-eight of 32 cases with monoallelic ACTN1 variants had mild to no bleeding complications. Eleven cases harbored 11 different unreported ACTN1 variants that were monoallelic and likely pathogenic. Nine variants were located in the α-actinin-1 (ACTN1) rod domain and were predicted to hinder dimer formation. These variants displayed a smaller increase in platelet size compared with variants located outside the rod domain. In vitro expression of the new ACTN1 variants induced actin network disorganization and led to increased thickness of actin fibers. These findings expand the repertoire of ACTN1 variants associated with thrombocytopenia and highlight the high frequency of ACTN1-related thrombocytopenia cases. The rod domain, like other ACTN1 functional domains, may be mutated resulting in actin disorganization in vitro and thrombocytopenia with normal platelet size in most cases.

Keywords: ACTN1; actin; actinin; constitutional; platelet; rod domain; thrombocytopenia.

Conflict of interest statement

The authors declare that there are no conflict of interests.

© 2019 The Authors. Human Mutation Published by Wiley Periodicals, Inc.

Figures

Figure 1

Identification and characterization of novel ACTN1 variants. (a) Pedigrees of the affected families Squares denote males, and circles denote females. Black filled, and empty symbols represent thrombocytopenic and nonthrombocytopenic family members, respectively. Dotted‐line symbols were used when platelet count was unavailable. ATCN1 gene constitutional variant status is indicated at the top of symbols representing family members. All identified variants were monoallelic. (b) Structural domains of ACTN1 and localization of ACTN1 variants: (i) previously described with proven functional impact (dark), (ii) previously described but untested for functional impact (gray), (iii) previously described with proven functional impact and reported here (green), and (iv) previously undescribed and reported here (red). The four SRs form the rod domain of the protein. CaM, calmodulin‐like domain; CH, calponin homology domain; SR, spectrin repeat

Figure 2

Bleeding, platelet counts and platelet diameters in ACTN1 variant carriers. (a) Characterization of bleeding phenotype in ACTN1 variant carriers. Bleeding episodes of the 30 affected patients were recorded. The cases are annotated with the identifiers described in Figure 1a. The number of patients suffering from a bleeding phenotype is reported on the left. The number of bleeding symptoms for each patient is indicated at the bottom. The type of bleeding disorder is indicated on the right. Exposure to surgery is indicated in the last line. (b,c) Platelet phenotype of ACTN1 variant carriers according to variant location (rod domain vs.nonrod domain variant): platelet count (×109 L−1) (b) and platelet diameter (µm) (c). Among nonrod domain ACTN1 variant carriers, those with variants in the AB domain or the AB/rod domain neck are depicted in gray and those with variants in the CaM domain or the rod domain/CaM domain neck are depicted in black. The mean ± standard error of the mean is shown for each group. MPD values represent the mean of 200 platelet diameter values. The intergroup difference was evaluated using an unpaired t test. *p < .05. The lower limit of the reference range for platelet counts (155 × 109 L−1) and the upper limit of the reference range for platelet diameters (2.9 µm) are represented with a dotted line. ACTN1, α‐actinin‐1; N, no; NA, not available; ns, nonsignificant; Y, yes

Figure 3

Visualization of the ACTN1 variant protein structures. The dimeric structure of ACTN2 was assembled from two halves of the ACTN2 protomer (AB domain‐neck‐SR1‐SR2‐SR3‐SR4‐CaM1‐CaM2) through a crystallographic twofold axis. One monomer is represented as a blue surface (bottom view) and the other is shown as a yellow ribbon (top view). The two views are rotated 90° around the horizontal axis. Residues corresponding to the variants in ACTN1 are indicated in red when located at the dimer interface and in purple when located elsewhere. The upper panels correspond to close‐up views of the p.(Lys324Glu), p.(Gln329Arg), p.(Val328Met), p.(Lys398Thr), p.(Arg450Cys), and p.(Ala432Val) variants. The monomer colored in blue is shown in stick representation with the residues corresponding to the variants colored in red. The other monomer is represented as a yellow surface. ACTN1, α‐actinin‐1; CaM, calmodulin

Figure 4

Novel ACTN1 variants alter ACTN1 localization and actin network organization (a) Representative immunofluorescence microscopy images of CHO cells transiently transfected with wild‐type or variant ACTN1 constructs using the standard procedure. The images were visualized after ACTN1 and actin staining, as previously described (Kunishima et al., 2013). Magnification: ×100. (b) Quantification of actin fiber diameter, perimeter and area. For each variant, six 100‐pixel square areas with the greatest density of actin fibers were analyzed to measure the feret diameter, perimeter and area of actin fibers within two or three cells using the “analyze particle” plugin of the ImageJ program. Low levels of fluorescent signal attributed to low‐level autofluorescence and background noise were corrected using a 9.75% threshold value. The intergroup difference was evaluated using one‐way ANOVA with Dunnett's posthoc test. The data are shown as the mean ± standard error of the mean. ref: reference (wild‐type). **p ≤ .01, ***p ≤ .001, ****p ≤ .0001. ACTN1, α‐actinin‐1