ACTN1-related thrombocytopenia: Homozygosity for an ACTN1 variant results in a more severe phenotype.

ACTN1-related thrombocytopenia is a rare disorder caused by heterozygous variants in the ACTN1 gene characterized by macrothrombocytopenia and mild bleeding tendency. We describe for the first time two patients affected with ACTN1-RT caused by a homozygous variant in ACTN1 (c.982G>A) with mild heart valve defects unexplained by any other genetic variants investigated by WES. Within the reported family, the homozygous sisters have moderate thrombocytopenia and marked platelet macrocytosis with giant platelets, revealing a more severe haematological phenotype compared to their heterozygous relatives and highlighting a significant effect of allelic burden on platelet size. Moreover, we hypothesize that some ACTN1 variants, especially when present in the homozygous state, may also contribute to the cardiac abnormalities.

miR-129-5p inhibits anchorage-independent growth through silencing of ACTN1 and the ELK4/c-FOS axis in HPV-transformed keratinocytes.

A persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered microRNA (miRNA) expression, with miRNAs regulating anchorage-independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR-129-5p, a previously identified tumor suppressive miRNA involved in anchorage-independent growth and HPV-induced carcinogenesis. We predicted 26 potential miR-129-5p targets using online databases, followed by KEGG pathway enrichment analysis. RT-qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR-129-5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR-129-5p expression in HPV-transformed keratinocytes, and their silencing reduced anchorage-independent growth. Concordantly, miR-129-5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage-independent conditions. Additionally, c-FOS, a downstream target of ELK4, was downregulated upon miR-129-5p overexpression, suggesting regulation through the ELK4/c-FOS axis. ACTN1 and ELK4 expression was also upregulated in high-grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR-129-5p involved in the regulation of anchorage-independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR-129-5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR-129-5p-regulated ACTN1 and ELK4 expression contributes to HPV-induced carcinogenesis.

Metabotropic glutamate receptor 5 promotes blood-brain barrier recovery after traumatic brain injury.

Blood-brain barrier (BBB) impairment and glutamate release are two pathophysiological features of traumatic brain injury (TBI), contributing to secondary brain damage and neuroinflammation. However, our knowledge of BBB integrity damage and dysfunction are still limited due to the diverse and fluctuating expression of glutamate receptors after trauma. Here, we confirmed the downregulation of metabotropic glutamate receptor 5 (mGluR5) on microvascular endothelial cell within the acute phase of TBI, and the recovered mGluR5 levels on BBB was positively associated with blood perfusion and neurological recovery. In whole body mGluR5-knockout mice, BBB dysfunction and neurological deficiency were exacerbated after TBI compared with wild type mice. In terms of mechanism, the amino acid sequence 201-259 of cytoskeletal protein Alpha-actinin-1 (ACTN1) interacted with mGluR5, facilitating mGluR5 translocation from cytoplasmic compartment to plasma membrane in endothelial cells. Activation of plasma membrane mGluR5 triggers the PLC/PKCµ/c-Jun signaling pathway, leading to increased expression of the tight junction-actin cytoskeleton connecting protein zonula occludens-1 (ZO-1). Our findings uncover a novel mechanism mediated by membrane and cytoplasmic mGluR5 in endothelial cell integrity maintenance and repair, providing the potential therapeutic target for TBI treatment targeting at mGluR5 and mGluR5/ACTN1 complex in BBB.

ACTN1 promotes HNSCC tumorigenesis and cisplatin resistance by enhancing MYH9-dependent degradation of GSK-3ß and integrin ß1-mediated phosphorylation of FAK.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors globally. Understanding the molecular basis of tumor progression and drug resistance can offer innovative strategies to enhance clinical outcomes for HNSCC patients. METHODS: The cytoskeletal remodeling genes associated with cisplatin resistance were screened using a PCR array. The role of alpha-actinin 1 (ACTN1) in modulating cisplatin resistance and tumorigenesis in HNSCC was evaluated both in vitro and in vivo. Co-immunoprecipitation (Co-IP), IP-mass spectrometry (MS), western blotting, dual-luciferase assay, and bioinformatics analysis were performed to elucidate the underlying mechanisms involved. RESULTS: Our study identifies ACTN1 as a crucial contributor to cisplatin resistance and tumorigenesis in HNSCC, as evidenced across cellular, animal, and patient-derived xenograft models. From a clinical perspective, overexpression of ACTN1 significantly correlates with a suboptimal response to neoadjuvant chemotherapy and reduced overall survival in HNSCC patients. Mechanistically, ACTN1 predominantly activates ß-catenin-mediated signaling by promoting the interaction between myosin heavy chain 9 (MYH9) and GSK-3ß, leading to the ubiquitin-dependent degradation of GSK-3ß. ACTN1 also interacts with integrin ß1, subsequently activating the FAK/PI3K/AKT pathway, providing an additional avenue for the activation of ß-catenin signaling. Our study also unveils that the ß-catenin/c-Myc axis transcriptionally regulates ACTN1, thereby creating a positive feedback loop promoting HNSCC tumorigenesis and drug resistance. CONCLUSIONS: These insights underscore the novel mechanisms that highlight ACTN1's pivotal role in driving HNSCC progression and resistance to chemotherapy, suggesting ACTN1 as a promising therapeutic target in HNSCC management.

LLGL2 Inhibits Ovarian Cancer Metastasis by Regulating Cytoskeleton Remodeling via ACTN1.

Epithelial ovarian cancer is the most lethal gynecological malignant tumor. Although debulking surgery, chemotherapy, and PARP inhibitors have greatly improved survival, the prognosis for patients with advanced EOC without HRD is still poor. LLGL2, as a cell polarity factor, is involved in maintaining cell polarity and asymmetric cell division. In the study of zebrafish development, LLGL2 regulated the proliferation and migration of epidermal cells and the formation of cortical F-actin. However, the role of LLGL2 in ovarian cancer has not been described. Our study found, through bioinformatics analysis, that low expression of LLGL2 was significantly associated with a more advanced stage and a higher grade of EOC and a poorer survival of patients. Functional experiments that involved LLGL2 overexpression and knockdown showed that LLGL2 inhibited the migration and invasion abilities of ovarian cancer cells in vitro, without affecting their proliferation. LLGL2-overexpressing mice had fewer metastatic implant foci than the controls in vivo. Mechanistically, immunoprecipitation combined with mass spectrometry analysis suggested that LLGL2 regulated cytoskeletal remodeling by interacting with ACTN1. LLGL2 altered the intracellular localization and function of ACTN1 without changing its protein and mRNA levels. Collectively, we uncovered that LLGL2 impaired actin filament aggregation into bundles by interacting with ACTN1, which led to cytoskeleton remodeling and inhibition of the invasion and metastasis of ovarian cancer cells.

[Pedigree Analysis of ACTN1-Related Thrombocytopenia Attributed to A Novel Mutation].

AbstractObjective: To investigate the clinical phenotype and genotype of an ACTN1-associated thrombocytopenic family and explore its molecular pathogenesis. METHODS: All the family members' peripheral blood was collected for routine blood tests, blood smear, coagulation function, and platelet aggregation test. Flow cytometry was used to detect the expression of platelet CD41 and CD61. The proband and her father were tested bone marrow cytomorphology. Whole-exome sequencing techniques were performed to detect and uncover mutant loci of suspected pathogenic genes. Bioinformatics was used to assess the conserved nature of the mutated loci and to analyze the effect of the mutated genes leading to the function of the corresponding amino acid sequences. RESULTS: The platelet count of the proband was 88×109/L, and the blood smear showed dumbbell-shaped platelets, snake-shaped platelets and platelets of various sizes. Her bone marrow cytomorphology revealed normal megakaryocyte morphology with a count of 270. The platelet count of the proband's father was 74×109/L, with large platelets and platelets of various sizes observed in the blood smear, and the morphology of megakaryocytes was normal in bone marrow with a megakaryocyte count of 239. Her grandfather had a platelet count of 83×109/L, with snake-shaped platelets and platelets of various sizes on blood smears. Other family members were normal in all tests. The missense mutation c.2396G > A in exon 20 of the ACTN1 gene in the proband resulted in the mutation of 799 amino acids of the encoded protein, i.e., Arg, to His. The sequencing results of her father and grandfather at this locus were found to be consistent with her. Furthermore, bioinformatics analysis indicated that the locus was highly conserved across species and that variation in this locus might lead to functional impairment of the protein. The protein model analysis demonstrated that α-actin-1 at position 799 Arg and Glu at position 811 could form a critical salt bridge which stabilizes the conformation of the Ca2+ binding loop within the calmodulin-like motif. the mutation of R799H lost this critical salt bridge and destabilized this structural domain. CONCLUSION: In the present study, the newly uncovered missense mutation c.2396G＞A in exon 20 of the ACTN1 gene is potentially the molecular mechanism for the thrombocytopenia.

The Importance of Alpha-Actinin Proteins in Platelet Formation and Function, and Their Causative Role in Congenital Macrothrombocytopenia.

The actin cytoskeleton plays a central role in platelet formation and function. Alpha-actinins (actinins) are actin filament crosslinking proteins that are prominently expressed in platelets and have been studied in relation to their role in platelet activation since the 1970s. However, within the past decade, several groups have described mutations in ACTN1/actinin-1 that cause congenital macrothrombocytopenia (CMTP)-accounting for approximately 5% of all cases of this condition. These findings are suggestive of potentially novel functions for actinins in platelet formation from megakaryocytes in the bone marrow and/or platelet maturation in circulation. Here, we review some recent insights into the well-known functions of actinins in platelet activation before considering possible roles for actinins in platelet formation that could explain their association with CMTP. We describe what is known about the consequences of CMTP-linked mutations on actinin-1 function at a molecular and cellular level and speculate how these changes might lead to the alterations in platelet count and morphology observed in CMTP patients. Finally, we outline some unanswered questions in this area and how they might be addressed in future studies.

Case Report: Exome Sequencing Identified a Novel Frameshift Mutation of α-Actin 1 in a Chinese Family With Macrothrombocytopenia and Mild Bleeding.

Inherited macrothrombocytopenia (IMTP) is a rare disorder characterized by a reduced platelet count and abnormally large platelets. The main clinical symptom of IMTP is mild bleeding in some patients. At present, more than 30 genes have been identified in patients with syndromic and non-syndromic IMTP. In this study, a 3-year-old boy and his mother who presented with mild epistaxis and/or gingival bleeding were diagnosed as having IMTP. Wen then selected whole sequencing to explore the genetic lesion of the patients. After data filtering and mutation validation, a novel frameshift mutation (NM_001130004: c.398_399insTGCG, p.F134AfsX60) of α-actin 1 (ACTN1) was identified in the proband and his mother but absent in other unaffected individuals. Previous studies have proven that mutations in ACTN1 may lead to IMTP with mild to absent bleeding phenotype. The novel mutation, resulting in a truncated protein in exon 4 of the ACTN1 gene, was absent in the public database, such as 1000G and genomAD. Further Western blot revealed that the expression of α-actin 1 in the proband was decreased overtly, which indicated that the novel frameshift mutation may induce non-sense-mediated mRNA decay. In summary, this study not only broadened the variants spectrum of ACTN1 gene, which may contribute to the genetic counseling of IMTP, but also confirmed the diagnosis of IMTP, which may help the management and prognosis for the family members.

ACTN1 supports tumor growth by inhibiting Hippo signaling in hepatocellular carcinoma.

BACKGROUND: Alpha actinins (ACTNs) are major cytoskeletal proteins and exhibit many non-muscle functions. Emerging evidence have uncovered the regulatory role of ACTNs in tumorigenesis, however, the expression pattern, biological functions, and underlying mechanism of ACTN1 in hepatocellular carcinoma (HCC) remain largely unexplored. METHODS: Immunohistochemical analysis of a HCC tissue microarray (n = 157) was performed to determine the expression pattern and prognostic value of ACTN1 in HCC. In vitro loss-of-function study in HCC cells were carried out to investigate ACTN1 knockdown on cell proliferation. In vivo subcutaneous xenograft model and intrahepatic transplantation model were generated to decipher the contribution of ACTN1 in the tumor growth of HCC. Gene set enrichment analysis, quantitative real-time PCR, Co-immunoprecipitation, immunofluorescence and western blotting were performed to identify the underlying molecular mechanism. RESULTS: It was found that ACTN1 was significantly upregulated in HCC tissues and closely related to llpha-fetoprotein level, tumor thrombus, tumor size, TNM stage and patient prognoses. Knockdown of ACTN1 suppressed in vitro cell proliferation and in vivo tumor growth of HCC cells. Mechanistically, knockdown of ACTN1 increased Hippo signaling pathway activity and decreased Rho GTPases activities. Mechanistically, ACTN1 could competitively interact with MOB1 and decrease the phosphorylation of LATS1 and YAP. The growth-promoting effect induced by ACTN1 was significantly abrogated by pharmacological inhibition of YAP with verteporfin or super-TDU. CONCLUSIONS: ACTN1 is highly expressed in HCC tissues and acts as a tumor promoter by suppressing Hippo signaling via physical interaction with MOB1. ACTN1 may serve as a potential prognostic marker and therapeutic target for HCC.

Oroxylin A suppresses ACTN1 expression to inactivate cancer-associated fibroblasts and restrain breast cancer metastasis.

Tumor initiation and progression are not only ascribed to the behavior of cancer cells, but also profoundly influenced by the tumor microenvironment. Inside, cancer-associated fibroblasts (CAFs) have become key factors to accelerate growth and metastasis for the abundance in most solid tumors. Our group previously reported that Oroxylin A (OA), a flavone from Scutellaria Baicalensis Georgi, possess the ability to suppress growth and invasion of several tumor cells. However, the regulatory effect of OA on stromal microenvironment is poorly understood. In this study, breast cancer-induced fibroblasts and primary breast CAFs from MMTV-PyMT mice were used to evaluate the influence of OA on the activation of fibroblasts. Results showed that OA could decrease the expression of α-SMA, fibronectin, vimentin and matrix metalloproteinases (MMPs). Thus, OA-deactivated CAFs did not further promote the proliferation and invasion in breast cancer cells. In vivo experiments, OA could also impede tumor metastasis through exhausting progressive CAFs. Mechanically, OA could specifically bind ACTN1 and significantly inhibit its expression to prevent CAF activation. As a consequence, OA could decrease the phosphorylation of FAK and STAT3, and reduce the secretion of CCL2 in CAFs. Altogether, OA could remodel stromal microenvironment and it is a potential therapeutic agent in breast cancer.

Investigation of calmodulin-like and rod domain mutations suggests common molecular mechanism for α-actinin-1-linked congenital macrothrombocytopenia.

Actinin-1 mutations cause dominantly inherited congenital macrothrombocytopenia (CMTP), with mutations in the actin-binding domain increasing actinin's affinity for F-actin. In this study, we examined nine CMTP-causing mutations in the calmodulin-like and rod domains of actinin-1. These mutations increase, to varying degrees, actinin's ability to bundle actin filaments in vitro. Mutations within the calmodulin-like domain decrease its thermal stability slightly but do not dramatically affect calcium binding, with mutant proteins retaining calcium-dependent regulation of filament bundling in vitro. The G764S and E769K mutations increase cytoskeletal association of actinin in cells, and all mutant proteins colocalize with F-actin in cultured HeLa cells. Thus, CMTP-causing actinin-1 mutations outside the actin-binding domain also increase actin association, suggesting a common molecular mechanism underlying actinin-1 related CMTP.

Alternative splicing events during adipogenesis from hMSCs.

Adipogenesis, the developmental process of progenitor-cell differentiating into adipocytes, leads to fat metabolic disorders. Alternative splicing (AS), a ubiquitous regulatory mechanism of gene expression, allows the generation of more than one unique messenger RNA (mRNA) species from a single gene. Till now, alternative splicing events during adipogenesis from human mesenchymal stem cells (hMSCs) are not yet fully elucidated. We performed RNA-Seq coupled with bioinformatics analysis to identify the differentially expressed AS genes and events during adipogenesis from hMSCs. A global survey separately identified 1262, 1181, 1167, and 1227 ASE involved in the most common types of AS including cassette exon, alt3, and alt5, especially with cassette exon the most prevalent, at 7, 14, 21, and 28 days during adipogenesis. Interestingly, 122 differentially expressed ASE referred to 118 genes, and the three genes including ACTN1 (alt3 and cassette), LRP1 (alt3 and alt5), and LTBP4 (cassette, cassette_multi, and unknown), appeared in multiple AS types of ASE during adipogenesis. Except for all the identified ASE of LRP1 occurred in the extracellular topological domain, alt3 (84) in transmembrane domain significantly differentially expressed was the potential key event during adipogenesis. Overall, we have, for the first time, conducted the global transcriptional profiling during adipogenesis of hMSCs to identify differentially expressed ASE and ASE-related genes. This finding would provide extensive ASE as the regulator of adipogenesis and the potential targets for future molecular research into adipogenesis-related metabolic disorders.

High Expression Levels of ACTN1 and ACTN3 Indicate Unfavorable Prognosis in Acute Myeloid Leukemia.

Background: Actinins are major cytoskeletal proteins that mediate sarcomere function, and they also have important non-muscle functions such as regulating cytokinesis, cell adhesion and migration. There are four isoforms of actinins in mammals (ACTN1-4). Recently, the relationship between actinins and cancer has been discovered in many types of malignancy, yet their prognostic significance in acute myeloid leukemia (AML) remains unclear. Methods: We collected data of 155 de novo AML patients from The Cancer Genome Atlas (TCGA) database; 85 patients received chemotherapy only and 70 patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We divided each treatment groups into sub-groups based on the median expression levels of ACTN1-4. Results: Survival analysis showed that in the chemotherapy-only group, high ACTN1 and ACTN3 expression were associated with shorter event-free survival (EFS) and overall survival (OS) (p<0.01). Multivariate analysis suggested that high expression of ACTN1 and ACTN3 (p<0.05) were independent poor prognostic factors. In the allo-HSCT group, ACTN1-4 expression had no impact on survival. Conclusions: Our study suggested that high expression levels of ACTN1 and ACTN3 adversely affected the survival of AML patients, but their harmful impact could be overcome by allo-HSCT.

Novel ACTN1 variants in cases of thrombocytopenia.

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.

ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia.

The inherited thrombocytopenias (IT) are a heterogeneous group of diseases resulting from mutations in more than 30 different genes. Among them, ACTN1-related thrombocytopenia (ACTN1-RT; Online Mendelian Inheritance in Man: 615193) is one of the most recently identified forms. It has been described as a mild autosomal dominant macrothrombocytopenia caused by mutations in ACTN1, a gene encoding for one of the two non-muscle isoforms of α-actinin. We recently identified seven new unrelated families with ACTN1-RT caused by different mutations. Two of them are novel missense variants (p.Trp128Cys and p.Pro233Leu), whose pathogenic role has been confirmed by in vitro studies. Together with the 10 families we have previously described, our cohort of ACTN1-RT now consists of 49 individuals carrying ACTN1 mutations. This is the largest case series ever collected and enabled a critical evaluation of the clinical aspects of the disease. We concluded that ACTN1-RT is the fourth most frequent form of IT worldwide and it is characterized by platelet macrocytosis in all affected subjects and mild thrombocytopenia in less than 80% of cases. The risk of bleeding, either spontaneous or upon haemostatic challenge, is negligible and there are no other associated defects, either congenital or acquired. Therefore, ACTN1-RT is a benign form of IT, whose diagnosis provides affected individuals and their families with a good prognosis.

Familial macrothrombocytopenia due to a double mutation in cis in the alpha-actinin 1 gene (ACTN1), previously considered to be chronic immune thrombocytopenic purpura.

Congenital thrombocytopenia can easily be misdiagnosed as immune thrombocytopenic purpura, as is illustrated by this case of a woman and her two children. Doubts arose when steroid/IVIG therapy failed in the mother and the thrombocytopenia in the children persisted. By means of next-generation sequencing, two missense variants in cis in the ACTN1 gene of the affected family members were identified, both of unknown significance. We conclude, after further analysis of these mutations with, among others, in silico prediction tools, that the thrombocytopenia has a genetic cause, in particular the ACTN1 mutations, and is not immune mediated.

EFA6 proteins regulate lumen formation through α-actinin 1.

A key step of epithelial morphogenesis is the creation of the lumen. Luminogenesis by hollowing proceeds through the fusion of apical vesicles at cell-cell contacts. The small nascent lumens grow through extension, coalescence and enlargement, coordinated with cell division, to give rise to a single central lumen. Here, by using MDCK cells grown in 3D-culture, we show that EFA6A (also known as PSD) participates in luminogenesis. EFA6A recruits α-actinin 1 (ACTN1) through direct binding. In polarized cells, ACTN1 was found to be enriched at the tight junction where it acts as a primary effector of EFA6A for normal luminogenesis. Both proteins are essential for the lumen extension and enlargement, where they mediate their effect by regulating the cortical acto-myosin contractility. Finally, ACTN1 was also found to act as an effector for the isoform EFA6B (also known as PSD4) in the human mammary tumoral MCF7 cell line. EFA6B restored the glandular morphology of this tumoral cell line in an ACTN1-dependent manner. Thus, we identified new regulators of cyst luminogenesis essential for the proper maturation of a newly-formed lumen into a single central lumen.

Congenital macrothrombocytopenia-linked mutations in the actin-binding domain of α-actinin-1 enhance F-actin association.

Mutations in the actin cross-linking protein actinin-1 were recently linked to dominantly inherited congenital macrothrombocytopenia. Here, we report that several disease-associated mutations that are located within the actinin-1 actin-binding domain cause increased binding of actinin-1 to actin filaments and enhance filament bundling in vitro. These actinin-1 mutants are also more stably associated with the cytoskeleton in cultured cells, as assessed by biochemical fractionation and fluorescence recovery after photobleaching experiments. For two mutations the disruption of contacts between the calponin homology domains within the actinin actin-binding domain may explain increased filament binding--providing mechanistic and structural insights into the basis of actinin-1 dysfunction in congenital macrothrombocytopenia.

ACTN1 rod domain mutation associated with congenital macrothrombocytopenia.

Mutations in ACTN1, the gene encoding the actin-crosslinking protein α-actinin-1, cause autosomal dominant macrothrombocytopenia. α-Actinin-1 exists as antiparallel dimers, composed of an N-terminal actin-binding domain (ABD), four spectrin-like repeats (SLRs), which form the spacer rod, and a C-terminal calmodulin-like (CaM) domain. All of the previously reported ACTN1 mutations associated with macrothrombocytopenia reside within the ABD and the CaM domain and not within the SLR domain. In this report, we describe a mutation in SLR2 of α-actinin-1 (p.Leu395Gln) associated with familial macrothrombocytopenia. A 3-year-old boy and his mother both had this mutation. They showed a mild form of thrombocytopenia without severe bleeding, accompanied by an elevated mean platelet volume. Consistent with the previous reports of mutations that reside in the ABD or the CaM domain, immunofluorescence examination revealed disorganization of the actin cytoskeleton in Gln395 mutant-transduced Chinese hamster ovary cells. Our findings suggest a novel mechanism for the pathogenesis of ACTN1-related macrothrombocytopenia that does not involve functional domain mutations.