The Journal of Immunology Disruption of Thrombocyte and T Lymphocyte Development by a Mutation in ARPC1B Raz Somech,*,† Atar Lev,*,† Yu Nee Lee,*,† Amos J. Simon,*,†,‡ Ortal Barel,†,x Ginette Schiby,{ Camila Avivi,{ Iris Barshack,{ Michele Rhodes,‖ Jiejing Yin,‖ Minshi Wang,‖ Yibin Yang,‖ Jennifer Rhodes,‖ Nufar Marcus,# Ben-Zion Garty,# Jerry Stein,** Ninette Amariglio,†,‡,x,†† Gideon Rechavi,†,x David L. Wiest,‖,1 and Yong Zhang‖,1 Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott–Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott–Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed. The Journal of Immunology, 2017, 199: 4036–4045. he actin cytoskeleton is a network of actin filaments that widespread immunological defects observed upon disruption of are polymerized from actin monomers. A key regulator of this regulation (3). Consequently, it is perhaps not surprising that T this process, in hematopoietic cells, is the Wiskott–Aldrich the molecules regulating this critical process have been linked to syndrome protein (WASP). WASP regulates actin polymerization the etiology of immunodeficiency. The first described and most by activating the Arp2/3 complex, allowing nucleation of new extensively studied actin-related protein causing primary immu- actin filaments and cross-linking them from end to side branch (1). nodeficiency is WASP, resulting in Wiskott–Aldrich syndrome The Arp2/3 complex consists of seven subunits: Arp2, Arp3, Arc-p16, (WAS) (4). WAS is an X-linked immunodeficiency disease Arc-p20, Arc-p21, Arc-p34, and Arc-p41 (2). Among these sub- with a characteristic clinical phenotype that includes micro- units, Arc-p41, also known as ARPC1B, is proposed to have a thrombocytopenia, eczema, combined T and B cell immunodefi- regulatory role, facilitating assembly and maintenance of the whole ciency, and an increased incidence of autoimmune manifestations complex. and malignancies (5). Mutations in WIP, a known stabilizer of The precise regulation of actin cytoskeleton dynamics is critical WASP, were also reported to cause a similar clinical phenotype (6). to nearly every stage of the immune response, as evidenced by the Mutations in the Arp2/3 complex or its activators, have long been *Pediatric Department A and Immunology Service, Jeffrey Modell Foundation Center, Received for publication March 30, 2017. Accepted for publication October 6, 2017. Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, This work was supported by National Institutes of Health Grants P30 CA006927 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 52621, Israel; R21 AI111208 (to D.L.W.), an appropriation from the Commonwealth of Pennsyl- †The Wohl Institute for Translational Medicine, Sheba Medical Center, Tel Hashomer, vania (to D.L.W.), and also by the Bishop Fund (to D.L.W.). Y.Z. was supported by Tel Aviv 52621, Israel; ‡Hematology Laboratory, Sheba Medical Center, Tel Hashomer, x Leukemia Specialized Program of Research Excellence Grant CA100632 from MD Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 52621, Israel; Sheba Cancer Anderson Cancer Center and Institutional Postdoctoral Training Grant T32 Research Center, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, { CA009035 from Fox Chase Cancer Center. Tel Aviv University, Tel Aviv 52621, Israel; Department of Pathology, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv Address correspondence and reprint requests to Dr. Yong Zhang and Dr. David 52621, Israel; ‖Blood Cell Development and Function Program, Fox Chase Cancer L. Wiest, Fox Chase Cancer Center, 333 Cottman Avenue, Room 364, Reimann Center, Philadelphia, PA 19111; #Allergy and Immunology Unit, Schneider Children’s Building, Philadelphia, PA 19111. E-mail addresses: [email protected] (Y.Z.) Medical Center of Israel, Felsenstein Medical Research Center, Kipper Institute of and [email protected] (D.L.W.) Immunology, Petach Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv The online version of this article contains supplemental material. 4920235, Israel; **Bone Marrow Transplantation Unit, Schneider Children’s Medical Center of Israel, Petach Tikva, Sackler Faculty of Medicine, Tel Aviv University, Tel Abbreviations used in this article: dpf, day postfertilization; hpf, hour postfertiliza- Aviv 4920235, Israel; and ††The Mina and Everard Goodman Faculty of Life Sciences, tion; MO, morpholino oligonucleotide; pt1, patient 1; pt2, patient 2; TREC, TCR Bar-Ilan University, Ramat Gan 52900, Israel excision circle; WAS, Wiskott–Aldrich syndrome; WASP, Wiskott–Aldrich syndrome protein; WISH, whole mount in situ hybridization; WT, wild-type. 1D.L.W. and Y.Z. contributed equally to this work. ORCIDs: 0000-0001-8648-5253 (Y.N.L.); 0000-0002-0225-0586 (M.W.); 0000-0001- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 7298-1213 (J.R.); 0000-0001-6352-6956 (J.S.); 0000-0002-0792-3188 (D.L.W.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700460 The Journal of Immunology 4037 sought as a cause of immunodeficiency syndromes with WAS-like turing was done using the Illumina Nextera DNA sample preparation pathologies; however, such mutations have not been observed, kit. Overall, ∼50 M sequence reads were produced for each sample. perhaps because many of these genes are essential for normal de- BWA-mem algorithm (11) for alignment versus the hg19 version of the human genome was applied. Around 40 M reads were properly velopment and so the loss of their function would likely result in aligned. The median coverage was around 40 reads per base. GATK early lethality (7). In support, the loss of Arp2/3 function in Arc- (12) version 2.4.7 with the UnifiedGenotyper algorithm was applied p21–deficient mice is embryonic lethal (8). Nevertheless, here we for variant calling including all steps mentioned in the best practice report the finding of two brothers with a WAS-like clinical phe- pipeline. KGG-sEquation (13) was used for annotation of detected variants and for comparing with allele frequency population data- notype that harbor a novel mutation in the ARPC1B gene. A distinct bases. In-house scripts were applied for filtering, based on family mutation in this gene was also recently reported in a patient with pedigrees and intersections. The ARPC1B mutation was validated by defects in platelets and neutrophils (9). We describe here the clinical dideoxy Sanger sequencing in the patients and carriers. PCR ampli- phenotype of the patients and definitively link their developmental fication products were directly sequenced using BigDye 3.1 Termi- defects in platelets and T cells to the ARPC1B mutation by reca- nator chemistry (Applied Biosystems) and separated on an ABI 3500 genetic analyzer (Applied Biosystems). Data were evaluated using pitulating the disease in a zebrafish model. Sequencer v5.0 software (Gene Codes, Ann Arbor, MI). The resulting data were deposited as a batch deposit of “Human; version 1.0” Materials and Methods samples in the National Center for Biotechnology Information BioSample database (http://www.ncbi.nlm.nih.gov/biosample/; Submis- Patients sion SUB2910482; Accession STUDY; PRJNA397356, SRS2412383 and The patients were diagnosed at the Edmond and Lily Safra Children’s SRX3067724). Hospital. The Institutional Review Board (Sheba Medical Center, Tel Identification of zebrafish arpc1b ortholog Hashomer) approved this study and a written informed consent was obtained from their parents. The zebrafish ortholog (NM_213156) of human ARPC1B was identified using the reciprocal “best-hits” basic local alignment search tool Immunological evaluation search strategy (14). Then multiple alignments of amino acid se- Cell surface markers of PBMCs, lymphocyte proliferative responses to quences for human, mouse, and zebrafish ARPC1B were obtained mitogens, and the amount of signal joint TCR excision circles (TRECs) by using the Clustal V algorithm (http://www.ebi.ac.uk/Tools/msa/ were determined as previously described (10). Serum concentration of Igs clustalo/). Synteny analysis