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Brief Report IMMUNOBIOLOGY
A nonsense mutation in IKBKB causes combined immunodeficiency Talal Mousallem,1,2 Jialong Yang,2 Thomas J. Urban,3 Hongxia Wang,2,4 Mehdi Adeli,5 Roberta E. Parrott,2 Joseph L. Roberts,2 David B. Goldstein,3 Rebecca H. Buckley,2,4 and Xiao-Ping Zhong2,4 1
Department of Internal Medicine, Section of Pulmonary, Critical Care, Allergy and Immunological Diseases, Wake Forest University School of Medicine, Wake Forest Baptist Medical Center, Winston-Salem, NC; 2Department of Pediatrics, Division of Allergy and Immunology, 3Center for Human Genome Variation, and 4Department of Immunology, Duke University Medical Center, Durham, NC; 5Hamad Medical Corporation, Doha, Qatar; and 6Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
Key Points • A nonsense mutation in IKBKB caused the absence of IKKb and a lack of T- and B-cell activation through their antigen receptors. • IKKb is not necessary for development of T or B lymphocytes but is important for their activation and for the development/function of NK cells.
Identification of the molecular etiologies of primary immunodeficiencies has led to important insights into the development and function of the immune system. We report here the cause of combined immunodeficiency in 4 patients from 2 different consanguineous Qatari families with similar clinical and immunologic phenotypes. The patients presented at an early age with fungal, viral, and bacterial infections and hypogammaglobulinemia. Although their B- and T-cell numbers were normal, they had low regulatory T-cell and NK-cell numbers. Moreover, patients’ T cells were mostly CD45RA1-naive cells and were defective in activation after T-cell receptor stimulation. All patients contained the same homozygous nonsense mutation in IKBKB (R286X), revealed by whole-exome sequencing with undetectable IKKb and severely decreased NEMO proteins. Mutant IKKb(R286X) was unable to complex with IKKa/NEMO. Immortalized patient B cells displayed impaired IkBa phosphorylation and NFkB nuclear translocation. These data indicate that mutated IKBKB is the likely cause of immunodeficiency in these 4 patients. (Blood. 2014;124(13):2046-2050)
Introduction Mutations in genes important in T-cell or in both T- and B-cell development and function cause severe combined immunodeficiency (CID), with a majority of cases caused by mutations in IL2RG, IL7RA, ADA, JAK3, RAG1, RAG2, or DCLRE1C.1,2 In contrast to severe CID, T- and B-cell development is not as severely impaired in CID. CID has been associated with hypomorphic mutations in severe CID-causing genes as well as mutations in several other genes: ZAP70, MHC class II deficiencies, PNP, ORAI-1, STIM1, NEMO, CARD11, and MALT1.3-7 However, the etiology of many patients with CID remains unknown. The IkB kinase (IKK) complex contains 2 structurally related catalytic subunits, IKKa and IKKb, and a regulatory subunit, IKKg/ NEMO.8,9 Null alleles of the X-linked gene encoding NEMO (IKBKG) cause incontinentia pigmenti in heterozygous females and are lethal in hemizygous males.10 Hypomorphic alleles are compatible with life in males but cause immunodeficiency and accompanying developmental abnormalities of teeth, hair, or sweat glands in many patients.3,11,12 Hypermorphic mutations in the gene encoding IkBa cause autosomaldominant ectodermal dysplasia with T-cell immunodeficiency, undetectable memory T cells, and absence of response to CD3-T-cell receptor activation.13 The IKKa/b/NEMO complex, activated by antigen and other receptors, phosphorylates the IkB molecules, leading to subsequent NFkB nuclear translocation to activate transcription of
genes involved in immune responses.14 We report a homozygous nonsense mutation in IKBKB in 4 patients with CID from 2 unrelated families and compare and contrast our findings with those in 2 other recent reports of mutations in this gene.15,16
Submitted April 20, 2014; accepted August 4, 2014. Prepublished online as Blood First Edition paper, August 18, 2014; DOI 10.1182/blood-2014-04571265.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Patient, materials, and methods Abbreviated information is presented here. Details are provided in the supplemental Data, available on the Blood Web site. Patients All studies were performed with the approval of the Duke University Medical Center Institutional Review Board, and written informed consent of the patients’ parents was collected in accordance with the Declaration of Helsinki. The patients were members of 2 unrelated consanguineous families. Select clinical features are presented in Table 1, with further details available in the supplemental Data. Immunologic phenotype analysis Flow cytometry of peripheral blood leukocytes was performed with labeled antibodies. Lymphocyte proliferation was assessed as previously described.17
T.M. and J.Y. contributed equally to this study. The online version of this article contains a data supplement.
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© 2014 by The American Society of Hematology
BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
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IKBKB MUTATION CAUSES COMBINED IMMUNODEFICIENCY
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Table 1. Clinical features, immunoglobulins, and lymphocyte subsets Family 1 Parameter
Family 2
Patient 1
Patient 2
Patient 3
Patient 4
Normal range
Clinical features Sex
Female
Male
Female
Male
5 months
11 months
7 months
6 months
Infections
Candida, BCG,
Candida, rotavirus, BCG
Candida
Candida, Klebsiella,
Treatment
Ablated, HLA-identical
Ablated, unrelated donor
Ablated, T-cell depleted
Ablated, T-cell depleted
paternal marrow
cord blood hematopoietic
paternal marrow
maternal marrow
hematopoietic stem cell
stem cell transplantation
hematopoietic stem cell
hematopoietic stem cell
transplantation
transplantation
Alive and well
Died 2 months later
Died 1 year later
Died 13 months later
Age at presentation
cytomegalovirus
transplantaion Outcome Immunoglobulins (Igs)* IgG
82
712†
367
173
192-515
IgA
Low
0
0
0
12-31
IgM
Low
0
0
22
39-92
Flow cytometry‡ T and NK cells CD3
11 550 (96)
14 792 (96)
6861 (81)
9 118 (82)
2940-4560 (49-76){
CD4
8 656 (72)
12 380 (81)
5946 (70)
7 498 (67)
1860-3360 (31-56){
CD8
2 881 (24)
2 458 (16)
1262 (15)
1 888 (17)
720-1440 (12-24){
CD28
11 118 (93)
14 792 (96)
6852 (81)
9 062 (81)
3000-4680 (50-78)§
Ti a/b
11 502 (96)
14 561 (95)
3480-5040 (58-84)§
6683 (79)
9 040 (81)
Ti g/d
180 (2)
15 (0)
42 (1)
939 (8)
CD16
120 (1)
154 (1)
644 (8)
335 (3)
CD4,CD25bright
240 (2)
ND
158 (1.6)
CD19
384 (3)
461 (3)
881 (10)
894 (8)
840-2220 (14-37){
CD20
312 (3)
476 (3)
813 (10)
950 (9)
300-1020 (5-17)§
ND
0-540 (0-9)§ 180-1080 (3-18)§ 24-336 (0.4-5.6)§
B cells
Switched
2 (13.3)
ND
2 (9.2)
ND
1-224 (22.4-79.4)§
memory B Activation CD45
11 994 (100)
CD45RO,CD3
1 063 (9)
CD45RA,CD3
9 783 (85)
CD45RA,CD3,
8 697 (75.3)
15 345 (100) 59 (0) 14 274 (97) ND
8428 (100) 521 (8) 6360 (93) 6237 (90.9)
10 526 (94) 27 (0) 8 589 (94) ND
5520-6000 (92-100)§ 177-1140 (6-25){ 441-2006 (15-44)§ 338-2513 (11.5-55.1)§
CD62L BCG, Bacillus Calmette-Guerin; ND, not done. *Values are expressed as milligrams per deciliter. †The patient was receiving immunoglobulin replacement. ‡Values are expressed as cells per cubic millimeter or (percentage of lymphocytes). {Normal values are the 95% confidence intervals for ninety 6- to 12-month-old healthy control subjects.21 §Normal values are the 95% confidence intervals for 2338 healthy adult control subjects from the authors’ laboratory.
Exome sequencing, alignment, and variant calling Exome sequencing was performed in the Genomic Analysis Facility in the Center for Human Genome Variation at Duke University. Sequencing libraries were prepared from DNA extracted from patients’ leukocytes using the Illumina TruSeq library preparation kit, following the manufacturer’s protocol. Establishment of Epstein-Barr virus cell lines Epstein-Barr virus (EBV)-transformed B-cell lines were established from peripheral blood leukocytes, as previously described.18 Cloning of full-length and mutant HuIKKb, transfection, and retroviral transduction Wild-type full-length (FL-hIKKb) or R286X mutant IKKb cDNA, amplified from a wild-type human IKKb template (Addgene) with a forward primer 59-GTGAACCGTCAGAATTGATCT-39 and a reverse primer 59-gagtGtttaaacACATCATGAGGCCTGCTCCA-39 or 59CggaattcTCAGGGGTGCCACATCAGCATCA-39, was cloned into
the MIGR1 retroviral vector. Retroviral vectors were transfected into the Phoenix-Ampho cells to generate amphotropic retroviruses. Cell stimulation, isolation of nuclear and cytoplasmic fractions, and immunoblot analysis EBV-transformed B cells, which were rested in Dulbecco’s phosphate buffered saline at 37°C for 30 minutes, were stimulated with phorbol myristate acetate (PMA) (20 ng/mL) or anti-human CD40 (10 mg/mL, Biolegend) at 37°C for 10 or 20 minutes. Nuclear extracts and cytosolic or total cell lysates were subjected to standard immunoblotting analysis. Real-time quantitative polymerase chain reaction Total RNA from peripheral blood mononuclear cells and EBV-transformed B cells was used for real-time quantitative polymerase chain reaction analysis. Statistical analysis Two-tail Student t test was performed (*P , .05; **P , .01; ***P , .001; Figure 1).
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MOUSALLEM et al
BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
Figure 1. Contribution of IKKb(R286X) mutation to CID. (A-C) Patients’ blood lymphocytes responded normally when stimulated with phytohemagglutinin (PHA) but had variably low responses to concanavalin A (Con A) and pokeweed mitogen (A). However, they failed to respond when stimulated with candida and tetanus antigens (B) or soluble or immobilized anti-CD3 (C). (D) Impaired upregulation of T-cell activation markers in patients’ CD4 T cells after overnight stimulation with plate-bound anti-CD3 or phytohemagglutinin. (E-F) Neither full-length nor truncated mutant IKKb(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKKb antibodies. (G) mRNA levels of IKKa, IKKb, and NEMO in PMCs detected by real-time quantitative polymerase chain reaction. (H) IKKb(R286X) is not able to form a complex with IKKa/NEMO. Cell lysates from Phoenix-Eco cells transfected with Flag-tagged-FL-IKKb, Flag-tagged-IKKb(R286X), or vector control were subjected to immunoblotting analysis directly (left) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads (right). (I) Defective IkBa/NFkB signaling in patient-derived B cells can be reverted by full-length (long) but not mutant IKKb (R286X). EBV-transformed B cells of a sibling and patients stably infected with retrovirus expressing WT IKKb or with control vector were rested in phosphate-buffered saline at 37°C for 30 minutes, followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of cytosolic fractions (top) or nuclear extracts (bottom) with indicated antibodies. (J) Decreased expansion of patient-derived B cells can be corrected by full-length but not mutant IKKb. *P , .05; **P , .01; ***P , .001 determined by Student t test.
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Results and discussion All 4 infants were hypogammaglobulinemic (Table 1). Three demonstrated normal or elevated numbers of T cells and normal numbers of B cells but low numbers of switched memory B cells and NK cells. Most T cells were CD45RA1. There were abnormally low numbers of CD45RO1 T cells and CD41CD25bright or CD41 FOXP31 T-regulatory cells (Treg) (supplemental Figure 1). All patient T cells displayed normal responses to phytohemagglutinin, but low responses to pokeweed mitogen or concanavalin A were seen in 3 of them (Figure 1A). Strikingly, all patients’ T cells failed to respond to candida or tetanus toxoid antigens or anti-CD3 stimulation (Figure 1B-C). Moreover, patients’ CD4 T cells were impaired for T-cell receptor-induced CD25 and CD69 upregulation (Figure 1D). NK-cell function, tested in 1 patient from each family, was impaired in both patients (data not shown). Whole exome sequencing on patients 1 and 2 representing the 2 unrelated families identified a single candidate nonsense homozygous variant in IKBKB (R286X). The other 2 patients and the parents carried the same homozygous and heterozygous variant, respectively (supplemental Figure 1C-D), which was absent in the 998 sequenced controls and in the exome variant server public database (Exome Variant Server, National Heart, Lung and Blood Institute [NHLBI] Grand Oppportunity [GO] Exome Sequencing Project, Seattle, WA [URL: http://evs.gs.washington.edu/EVS/]). Contrary to in healthy controls and a heterozygous sibling, antiN- or -C-terminal IKKb antibodies failed to detect any full-length or truncated IKKb in patients’ peripheral blood mononuclear cells and EBV-transformed B-cell lines (Figure 1E-F) that was not caused by possible low quality of the antibody (supplemental Figure 1E). Interestingly, NEMO was virtually undetectable, but IKKa was not obviously altered in patient samples (Figure 1E-F). Both IkKb and NEMO mRNA levels were considerably increased in patients’ peripheral blood mononuclear cells (Figure 1G). Contrary to wildtype IKKb, IKKb(R286X) could not associate with either IKKa or NEMO when overexpressed in Phoenix-eco cells (Figure 1H). Patients’ EBV-transformed B cells displayed reduced IkBa phosphorylation and degradation and impaired nuclear accumulation of NFkB after stimulation with PMA, which activates the PKC-IKKNFkB pathway (Figure 1I).19 Such defects were corrected after reconstitution with full-length but not IKKb(R286X), suggesting that IKKb(R286X) caused defective IkBa/NFkB activation in patient-derived B cells (Figure 1J and supplemental Figure 1F). Concordantly, expansion of patient’s EBV-transformed B cells, which was slower in vitro than their sibling’s or parents’ counterparts, could be accelerated to wild-type levels when reconstituted with full-length, but not mutant, IKKb (Figure 1J). Interestingly, patient B cells expressing IKKb(R286X) expanded slower than cells expressing green fluorescence protein alone, suggesting that IKKb(R286X) exerted a dominant-negative function. At this time, it is unclear whether IKKb(R286X) may interfere with the residual IKKa/NFkB signaling in these cells. Together, these observations indicate that IKKb protein is absent in the patients and that the C terminus of IKKb mediates its association with IKKa/NEMO, which may be important for the stability of itself and of NEMO, supporting that the nonsense mutation in IKBKB caused CID in these patients. Our results indicate that IKKb is not required for the development of T and B cells and show a novel cause of CID related to defects in the NFkB pathway. These findings highlight the diversity of phenotypes associated with mutations in
IKBKB MUTATION CAUSES COMBINED IMMUNODEFICIENCY
2049
different components of the NFkB pathway. For example, most patients with mutations in IKBKG had ectodermal dysplasia,20 which was absent in our IKKb(R286X) patients even though they have almost undetectable NEMO. Interestingly, while this manuscript was in preparation/review, 2 other IKBKB mutations in CID patients were reported: one a duplication of exon 13 (IKBKB13Dup),15 and the other Y107X nonsense mutation of IKBKB.16 The clinical features of our patients were similar to those in the other 2 reports, with early infections with candida, gram-negative bacteria, viruses, and mycobacteria. The immunological features were also similar: They all have elevated numbers of naive T cells that were poorly activated by antigens and anti-CD3, low numbers of B cells that were also naive, and low numbers and function of NK cells. There was a paucity of CD45RO-positive T cells, Tregs, and g/d T cells. Similar to our study, mutant IKKb protein was undetectable and NEMO was decreased in IKBKB13Dup patients.15 However, IKKa was not obviously decreased in our IKKb(R286X) patients but was severely decreased in the IKBKB13Dup patients.15 The effect of IKKb(Y107X) mutation on IKKa and NEMO expression was not reported.16
Acknowledgments The authors thank the Cancer Center flow cytometry core facility at Duke University for cell sorting. We acknowledge the following individuals for the contributions of control samples: Dr Gianpiero Cavalleri, Dr Norman Delanty, Dr Chantal Depondt, Dr Sanjay Sisodiya, Dr William B. Gallentine, Dr Erin L. Heinzen, Dr Aatif M. Husain, Kristen N. Linney, Dr Rodney A. Radtke, Dr Saurabh R. Sinha, Nicole M. Walley, Dr Julie Hoover-Fong, Dr Nara L. Sobreira, Dr David Valle, Dr William L. Lowe, Dr Scott M. Palmer, Dr Zvi Farfel, Dr Doron Lancet, Dr Elon Pras, Arthur Holden, Dr Elijah Behr, Dr Annapurna Poduri, Dr Patricia Lugar, Dr Rasheed Gbadegesin, Dr Michelle Winn, Dr Robert Brown, Dr Gianpiero Cavalleri, Dr Norman Delanty, Dr Chantal Depondt, Dr Yong-Hui Jiang, Dr Vandana Shashi, Kelly Schoch, Dr Eli J. Holtzman, Dr Sarah Kerns, Dr Harriet Oster, Dr Doug Marchuk, Dr Demetre Daskalakis, Dr Nicole Calakos, Dr Francis J. McMahon, Nirmala Akula, and Dr M. Chiara Manzini. The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the Women’s Health Initiative Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010); the National Institute of Allergy and Infectious Diseases (AI076357, AI079088, and AI101206 to X.-P.Z.); and an award from Clinical Immunology Society/Grifols (CIS/Grifols Senior Fellowship Award to T.M.). The collection of control samples and the production of sequence data were funded in part by the Epilepsy Phenome/Genome Project U01NS053998; Epi4K Project 1 – Epileptic Encephalopathies U01NS077364; Epi4K Sequencing, Biostatistics and Bioinformatics Core U01NS077303; National Institute of Allergy and Infectious Diseases Grant UO1AIO67854 (Center for HIV/AIDS Vaccine Immunology), and an award from Biogen Idec.
Authorship Contribution: T.M. designed the study, analyzed sequencing data, and wrote the paper; J.Y. designed and performed functional studies and contributed to manuscript preparation; T.J.U. analyzed the
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BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
MOUSALLEM et al
sequencing data; H.W. participated in functional studies; M.A. referred the patients and conducted immunologic investigations; R.E.P. performed extensive immunologic studies and contributed to manuscript preparation; J.L.R. contributed to data analysis and manuscript composition; D.B.G. oversaw the whole-genome sequencing and data analysis and contributed to the manuscript; R.H.B. designed the study, wrote the paper, and performed
extensive immunologic evaluations; X.-P.Z. designed functional studies, analyzed data, and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Rebecca H. Buckley, Box 2898 or 362 Jones Building, Duke University Medical Center, Durham, NC 27710, e-mail: [email protected].
References 1. Fischer A, Le Deist F, Hacein-Bey-Abina S, et al. Severe combined immunodeficiency. A model disease for molecular immunology and therapy. Immunol Rev. 2005;203:98-109. 2. Buckley RH. Transplantation of hematopoietic stem cells in human severe combined immunodeficiency: longterm outcomes. Immunol Res. 2011;49(1-3):25-43. 3. Orange JS, Brodeur SR, Jain A, et al. Deficient natural killer cell cytotoxicity in patients with IKKgamma/NEMO mutations. J Clin Invest. 2002; 109(11):1501-1509. 4. Feske S, Gwack Y, Prakriya M, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441(7090): 179-185. 5. Picard C, McCarl CA, Papolos A, et al. STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N Engl J Med. 2009;360(19):1971-1980. 6. Jabara HH, Ohsumi T, Chou J, et al. A homozygous mucosa-associated lymphoid tissue 1 (MALT1) mutation in a family with combined immunodeficiency. J Allergy Clin Immunol. 2013; 132(1):151-158. 7. Stepensky P, Keller B, Buchta M, et al. Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects. J Allergy Clin Immunol. 2013;131(2):477-485, e1. 8. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IkappaB kinase complex (IKK)
contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell. 1997;91(2): 243-252. 9. Courtois G, Israel ¨ A. IKK regulation and human genetics. Curr Top Microbiol Immunol. 2011;349:73-95. 10. Smahi A, Courtois G, Vabres P, et al; The International Incontinentia Pigmenti (IP) Consortium. Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. Nature. 2000; 405(6785):466-472. 11. Niehues T, Reichenbach J, Neubert J, et al. Nuclear factor kappaB essential modulatordeficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(6):1456-1462. 12. Orange JS, Levy O, Brodeur SR, et al. Human nuclear factor k B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(3):650-656. 13. Courtois G, Smahi A, Reichenbach J, et al. A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J Clin Invest. 2003;112(7): 1108-1115. 14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[k]B activity. Annu Rev Immunol. 2000;18:621-663.
15. Pannicke U, Baumann B, Fuchs S, et al. Deficiency of innate and acquired immunity caused by an IKBKB mutation. N Engl J Med. 2013;369(26):2504-2514. 16. Burns SO, Plagnol V, Gutierrez BM, et al. Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKb. J Allergy Clin Immunol. 2014;134(1):215-218. 17. Buckley RH, Schiff SE, Sampson HA, et al. Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation. J Immunol. 1986;136(7):2398-2407. 18. Sugden B, Mark W. Clonal transformation of adult human leukocytes by Epstein-Barr virus. J Virol. 1977;23(3):503-508. 19. Krishna S, Zhong X. Role of diacylglycerol kinases in T cell development and function. Crit Rev Immunol. 2013;33(2):97-118. 20. Hanson EP, Monaco-Shawver L, Solt LA, et al. Hypomorphic nuclear factor-kappaB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity. J Allergy Clin Immunol. 2008;122(6): 1169-1177, e16. 21. Shearer WT, Rosenblatt HM, Gelman RS, et al; Pediatric AIDS Clinical Trials Group. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol. 2003;112(5):973-980.
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2014 124: 2046-2050 doi:10.1182/blood-2014-04-571265 originally published online August 18, 2014
A nonsense mutation in IKBKB causes combined immunodeficiency Talal Mousallem, Jialong Yang, Thomas J. Urban, Hongxia Wang, Mehdi Adeli, Roberta E. Parrott, Joseph L. Roberts, David B. Goldstein, Rebecca H. Buckley and Xiao-Ping Zhong
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
Brief Report IMMUNOBIOLOGY
A nonsense mutation in IKBKB causes combined immunodeficiency Talal Mousallem,1,2 Jialong Yang,2 Thomas J. Urban,3 Hongxia Wang,2,4 Mehdi Adeli,5 Roberta E. Parrott,2 Joseph L. Roberts,2 David B. Goldstein,3 Rebecca H. Buckley,2,4 and Xiao-Ping Zhong2,4 1
Department of Internal Medicine, Section of Pulmonary, Critical Care, Allergy and Immunological Diseases, Wake Forest University School of Medicine, Wake Forest Baptist Medical Center, Winston-Salem, NC; 2Department of Pediatrics, Division of Allergy and Immunology, 3Center for Human Genome Variation, and 4Department of Immunology, Duke University Medical Center, Durham, NC; 5Hamad Medical Corporation, Doha, Qatar; and 6Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
Key Points • A nonsense mutation in IKBKB caused the absence of IKKb and a lack of T- and B-cell activation through their antigen receptors. • IKKb is not necessary for development of T or B lymphocytes but is important for their activation and for the development/function of NK cells.
Identification of the molecular etiologies of primary immunodeficiencies has led to important insights into the development and function of the immune system. We report here the cause of combined immunodeficiency in 4 patients from 2 different consanguineous Qatari families with similar clinical and immunologic phenotypes. The patients presented at an early age with fungal, viral, and bacterial infections and hypogammaglobulinemia. Although their B- and T-cell numbers were normal, they had low regulatory T-cell and NK-cell numbers. Moreover, patients’ T cells were mostly CD45RA1-naive cells and were defective in activation after T-cell receptor stimulation. All patients contained the same homozygous nonsense mutation in IKBKB (R286X), revealed by whole-exome sequencing with undetectable IKKb and severely decreased NEMO proteins. Mutant IKKb(R286X) was unable to complex with IKKa/NEMO. Immortalized patient B cells displayed impaired IkBa phosphorylation and NFkB nuclear translocation. These data indicate that mutated IKBKB is the likely cause of immunodeficiency in these 4 patients. (Blood. 2014;124(13):2046-2050)
Introduction Mutations in genes important in T-cell or in both T- and B-cell development and function cause severe combined immunodeficiency (CID), with a majority of cases caused by mutations in IL2RG, IL7RA, ADA, JAK3, RAG1, RAG2, or DCLRE1C.1,2 In contrast to severe CID, T- and B-cell development is not as severely impaired in CID. CID has been associated with hypomorphic mutations in severe CID-causing genes as well as mutations in several other genes: ZAP70, MHC class II deficiencies, PNP, ORAI-1, STIM1, NEMO, CARD11, and MALT1.3-7 However, the etiology of many patients with CID remains unknown. The IkB kinase (IKK) complex contains 2 structurally related catalytic subunits, IKKa and IKKb, and a regulatory subunit, IKKg/ NEMO.8,9 Null alleles of the X-linked gene encoding NEMO (IKBKG) cause incontinentia pigmenti in heterozygous females and are lethal in hemizygous males.10 Hypomorphic alleles are compatible with life in males but cause immunodeficiency and accompanying developmental abnormalities of teeth, hair, or sweat glands in many patients.3,11,12 Hypermorphic mutations in the gene encoding IkBa cause autosomaldominant ectodermal dysplasia with T-cell immunodeficiency, undetectable memory T cells, and absence of response to CD3-T-cell receptor activation.13 The IKKa/b/NEMO complex, activated by antigen and other receptors, phosphorylates the IkB molecules, leading to subsequent NFkB nuclear translocation to activate transcription of
genes involved in immune responses.14 We report a homozygous nonsense mutation in IKBKB in 4 patients with CID from 2 unrelated families and compare and contrast our findings with those in 2 other recent reports of mutations in this gene.15,16
Submitted April 20, 2014; accepted August 4, 2014. Prepublished online as Blood First Edition paper, August 18, 2014; DOI 10.1182/blood-2014-04571265.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
Patient, materials, and methods Abbreviated information is presented here. Details are provided in the supplemental Data, available on the Blood Web site. Patients All studies were performed with the approval of the Duke University Medical Center Institutional Review Board, and written informed consent of the patients’ parents was collected in accordance with the Declaration of Helsinki. The patients were members of 2 unrelated consanguineous families. Select clinical features are presented in Table 1, with further details available in the supplemental Data. Immunologic phenotype analysis Flow cytometry of peripheral blood leukocytes was performed with labeled antibodies. Lymphocyte proliferation was assessed as previously described.17
T.M. and J.Y. contributed equally to this study. The online version of this article contains a data supplement.
2046
© 2014 by The American Society of Hematology
BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
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IKBKB MUTATION CAUSES COMBINED IMMUNODEFICIENCY
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Table 1. Clinical features, immunoglobulins, and lymphocyte subsets Family 1 Parameter
Family 2
Patient 1
Patient 2
Patient 3
Patient 4
Normal range
Clinical features Sex
Female
Male
Female
Male
5 months
11 months
7 months
6 months
Infections
Candida, BCG,
Candida, rotavirus, BCG
Candida
Candida, Klebsiella,
Treatment
Ablated, HLA-identical
Ablated, unrelated donor
Ablated, T-cell depleted
Ablated, T-cell depleted
paternal marrow
cord blood hematopoietic
paternal marrow
maternal marrow
hematopoietic stem cell
stem cell transplantation
hematopoietic stem cell
hematopoietic stem cell
transplantation
transplantation
Alive and well
Died 2 months later
Died 1 year later
Died 13 months later
Age at presentation
cytomegalovirus
transplantaion Outcome Immunoglobulins (Igs)* IgG
82
712†
367
173
192-515
IgA
Low
0
0
0
12-31
IgM
Low
0
0
22
39-92
Flow cytometry‡ T and NK cells CD3
11 550 (96)
14 792 (96)
6861 (81)
9 118 (82)
2940-4560 (49-76){
CD4
8 656 (72)
12 380 (81)
5946 (70)
7 498 (67)
1860-3360 (31-56){
CD8
2 881 (24)
2 458 (16)
1262 (15)
1 888 (17)
720-1440 (12-24){
CD28
11 118 (93)
14 792 (96)
6852 (81)
9 062 (81)
3000-4680 (50-78)§
Ti a/b
11 502 (96)
14 561 (95)
3480-5040 (58-84)§
6683 (79)
9 040 (81)
Ti g/d
180 (2)
15 (0)
42 (1)
939 (8)
CD16
120 (1)
154 (1)
644 (8)
335 (3)
CD4,CD25bright
240 (2)
ND
158 (1.6)
CD19
384 (3)
461 (3)
881 (10)
894 (8)
840-2220 (14-37){
CD20
312 (3)
476 (3)
813 (10)
950 (9)
300-1020 (5-17)§
ND
0-540 (0-9)§ 180-1080 (3-18)§ 24-336 (0.4-5.6)§
B cells
Switched
2 (13.3)
ND
2 (9.2)
ND
1-224 (22.4-79.4)§
memory B Activation CD45
11 994 (100)
CD45RO,CD3
1 063 (9)
CD45RA,CD3
9 783 (85)
CD45RA,CD3,
8 697 (75.3)
15 345 (100) 59 (0) 14 274 (97) ND
8428 (100) 521 (8) 6360 (93) 6237 (90.9)
10 526 (94) 27 (0) 8 589 (94) ND
5520-6000 (92-100)§ 177-1140 (6-25){ 441-2006 (15-44)§ 338-2513 (11.5-55.1)§
CD62L BCG, Bacillus Calmette-Guerin; ND, not done. *Values are expressed as milligrams per deciliter. †The patient was receiving immunoglobulin replacement. ‡Values are expressed as cells per cubic millimeter or (percentage of lymphocytes). {Normal values are the 95% confidence intervals for ninety 6- to 12-month-old healthy control subjects.21 §Normal values are the 95% confidence intervals for 2338 healthy adult control subjects from the authors’ laboratory.
Exome sequencing, alignment, and variant calling Exome sequencing was performed in the Genomic Analysis Facility in the Center for Human Genome Variation at Duke University. Sequencing libraries were prepared from DNA extracted from patients’ leukocytes using the Illumina TruSeq library preparation kit, following the manufacturer’s protocol. Establishment of Epstein-Barr virus cell lines Epstein-Barr virus (EBV)-transformed B-cell lines were established from peripheral blood leukocytes, as previously described.18 Cloning of full-length and mutant HuIKKb, transfection, and retroviral transduction Wild-type full-length (FL-hIKKb) or R286X mutant IKKb cDNA, amplified from a wild-type human IKKb template (Addgene) with a forward primer 59-GTGAACCGTCAGAATTGATCT-39 and a reverse primer 59-gagtGtttaaacACATCATGAGGCCTGCTCCA-39 or 59CggaattcTCAGGGGTGCCACATCAGCATCA-39, was cloned into
the MIGR1 retroviral vector. Retroviral vectors were transfected into the Phoenix-Ampho cells to generate amphotropic retroviruses. Cell stimulation, isolation of nuclear and cytoplasmic fractions, and immunoblot analysis EBV-transformed B cells, which were rested in Dulbecco’s phosphate buffered saline at 37°C for 30 minutes, were stimulated with phorbol myristate acetate (PMA) (20 ng/mL) or anti-human CD40 (10 mg/mL, Biolegend) at 37°C for 10 or 20 minutes. Nuclear extracts and cytosolic or total cell lysates were subjected to standard immunoblotting analysis. Real-time quantitative polymerase chain reaction Total RNA from peripheral blood mononuclear cells and EBV-transformed B cells was used for real-time quantitative polymerase chain reaction analysis. Statistical analysis Two-tail Student t test was performed (*P , .05; **P , .01; ***P , .001; Figure 1).
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MOUSALLEM et al
BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
Figure 1. Contribution of IKKb(R286X) mutation to CID. (A-C) Patients’ blood lymphocytes responded normally when stimulated with phytohemagglutinin (PHA) but had variably low responses to concanavalin A (Con A) and pokeweed mitogen (A). However, they failed to respond when stimulated with candida and tetanus antigens (B) or soluble or immobilized anti-CD3 (C). (D) Impaired upregulation of T-cell activation markers in patients’ CD4 T cells after overnight stimulation with plate-bound anti-CD3 or phytohemagglutinin. (E-F) Neither full-length nor truncated mutant IKKb(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKKb antibodies. (G) mRNA levels of IKKa, IKKb, and NEMO in PMCs detected by real-time quantitative polymerase chain reaction. (H) IKKb(R286X) is not able to form a complex with IKKa/NEMO. Cell lysates from Phoenix-Eco cells transfected with Flag-tagged-FL-IKKb, Flag-tagged-IKKb(R286X), or vector control were subjected to immunoblotting analysis directly (left) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads (right). (I) Defective IkBa/NFkB signaling in patient-derived B cells can be reverted by full-length (long) but not mutant IKKb (R286X). EBV-transformed B cells of a sibling and patients stably infected with retrovirus expressing WT IKKb or with control vector were rested in phosphate-buffered saline at 37°C for 30 minutes, followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of cytosolic fractions (top) or nuclear extracts (bottom) with indicated antibodies. (J) Decreased expansion of patient-derived B cells can be corrected by full-length but not mutant IKKb. *P , .05; **P , .01; ***P , .001 determined by Student t test.
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Results and discussion All 4 infants were hypogammaglobulinemic (Table 1). Three demonstrated normal or elevated numbers of T cells and normal numbers of B cells but low numbers of switched memory B cells and NK cells. Most T cells were CD45RA1. There were abnormally low numbers of CD45RO1 T cells and CD41CD25bright or CD41 FOXP31 T-regulatory cells (Treg) (supplemental Figure 1). All patient T cells displayed normal responses to phytohemagglutinin, but low responses to pokeweed mitogen or concanavalin A were seen in 3 of them (Figure 1A). Strikingly, all patients’ T cells failed to respond to candida or tetanus toxoid antigens or anti-CD3 stimulation (Figure 1B-C). Moreover, patients’ CD4 T cells were impaired for T-cell receptor-induced CD25 and CD69 upregulation (Figure 1D). NK-cell function, tested in 1 patient from each family, was impaired in both patients (data not shown). Whole exome sequencing on patients 1 and 2 representing the 2 unrelated families identified a single candidate nonsense homozygous variant in IKBKB (R286X). The other 2 patients and the parents carried the same homozygous and heterozygous variant, respectively (supplemental Figure 1C-D), which was absent in the 998 sequenced controls and in the exome variant server public database (Exome Variant Server, National Heart, Lung and Blood Institute [NHLBI] Grand Oppportunity [GO] Exome Sequencing Project, Seattle, WA [URL: http://evs.gs.washington.edu/EVS/]). Contrary to in healthy controls and a heterozygous sibling, antiN- or -C-terminal IKKb antibodies failed to detect any full-length or truncated IKKb in patients’ peripheral blood mononuclear cells and EBV-transformed B-cell lines (Figure 1E-F) that was not caused by possible low quality of the antibody (supplemental Figure 1E). Interestingly, NEMO was virtually undetectable, but IKKa was not obviously altered in patient samples (Figure 1E-F). Both IkKb and NEMO mRNA levels were considerably increased in patients’ peripheral blood mononuclear cells (Figure 1G). Contrary to wildtype IKKb, IKKb(R286X) could not associate with either IKKa or NEMO when overexpressed in Phoenix-eco cells (Figure 1H). Patients’ EBV-transformed B cells displayed reduced IkBa phosphorylation and degradation and impaired nuclear accumulation of NFkB after stimulation with PMA, which activates the PKC-IKKNFkB pathway (Figure 1I).19 Such defects were corrected after reconstitution with full-length but not IKKb(R286X), suggesting that IKKb(R286X) caused defective IkBa/NFkB activation in patient-derived B cells (Figure 1J and supplemental Figure 1F). Concordantly, expansion of patient’s EBV-transformed B cells, which was slower in vitro than their sibling’s or parents’ counterparts, could be accelerated to wild-type levels when reconstituted with full-length, but not mutant, IKKb (Figure 1J). Interestingly, patient B cells expressing IKKb(R286X) expanded slower than cells expressing green fluorescence protein alone, suggesting that IKKb(R286X) exerted a dominant-negative function. At this time, it is unclear whether IKKb(R286X) may interfere with the residual IKKa/NFkB signaling in these cells. Together, these observations indicate that IKKb protein is absent in the patients and that the C terminus of IKKb mediates its association with IKKa/NEMO, which may be important for the stability of itself and of NEMO, supporting that the nonsense mutation in IKBKB caused CID in these patients. Our results indicate that IKKb is not required for the development of T and B cells and show a novel cause of CID related to defects in the NFkB pathway. These findings highlight the diversity of phenotypes associated with mutations in
IKBKB MUTATION CAUSES COMBINED IMMUNODEFICIENCY
2049
different components of the NFkB pathway. For example, most patients with mutations in IKBKG had ectodermal dysplasia,20 which was absent in our IKKb(R286X) patients even though they have almost undetectable NEMO. Interestingly, while this manuscript was in preparation/review, 2 other IKBKB mutations in CID patients were reported: one a duplication of exon 13 (IKBKB13Dup),15 and the other Y107X nonsense mutation of IKBKB.16 The clinical features of our patients were similar to those in the other 2 reports, with early infections with candida, gram-negative bacteria, viruses, and mycobacteria. The immunological features were also similar: They all have elevated numbers of naive T cells that were poorly activated by antigens and anti-CD3, low numbers of B cells that were also naive, and low numbers and function of NK cells. There was a paucity of CD45RO-positive T cells, Tregs, and g/d T cells. Similar to our study, mutant IKKb protein was undetectable and NEMO was decreased in IKBKB13Dup patients.15 However, IKKa was not obviously decreased in our IKKb(R286X) patients but was severely decreased in the IKBKB13Dup patients.15 The effect of IKKb(Y107X) mutation on IKKa and NEMO expression was not reported.16
Acknowledgments The authors thank the Cancer Center flow cytometry core facility at Duke University for cell sorting. We acknowledge the following individuals for the contributions of control samples: Dr Gianpiero Cavalleri, Dr Norman Delanty, Dr Chantal Depondt, Dr Sanjay Sisodiya, Dr William B. Gallentine, Dr Erin L. Heinzen, Dr Aatif M. Husain, Kristen N. Linney, Dr Rodney A. Radtke, Dr Saurabh R. Sinha, Nicole M. Walley, Dr Julie Hoover-Fong, Dr Nara L. Sobreira, Dr David Valle, Dr William L. Lowe, Dr Scott M. Palmer, Dr Zvi Farfel, Dr Doron Lancet, Dr Elon Pras, Arthur Holden, Dr Elijah Behr, Dr Annapurna Poduri, Dr Patricia Lugar, Dr Rasheed Gbadegesin, Dr Michelle Winn, Dr Robert Brown, Dr Gianpiero Cavalleri, Dr Norman Delanty, Dr Chantal Depondt, Dr Yong-Hui Jiang, Dr Vandana Shashi, Kelly Schoch, Dr Eli J. Holtzman, Dr Sarah Kerns, Dr Harriet Oster, Dr Doug Marchuk, Dr Demetre Daskalakis, Dr Nicole Calakos, Dr Francis J. McMahon, Nirmala Akula, and Dr M. Chiara Manzini. The authors would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the Women’s Health Initiative Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010); the National Institute of Allergy and Infectious Diseases (AI076357, AI079088, and AI101206 to X.-P.Z.); and an award from Clinical Immunology Society/Grifols (CIS/Grifols Senior Fellowship Award to T.M.). The collection of control samples and the production of sequence data were funded in part by the Epilepsy Phenome/Genome Project U01NS053998; Epi4K Project 1 – Epileptic Encephalopathies U01NS077364; Epi4K Sequencing, Biostatistics and Bioinformatics Core U01NS077303; National Institute of Allergy and Infectious Diseases Grant UO1AIO67854 (Center for HIV/AIDS Vaccine Immunology), and an award from Biogen Idec.
Authorship Contribution: T.M. designed the study, analyzed sequencing data, and wrote the paper; J.Y. designed and performed functional studies and contributed to manuscript preparation; T.J.U. analyzed the
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BLOOD, 25 SEPTEMBER 2014 x VOLUME 124, NUMBER 13
MOUSALLEM et al
sequencing data; H.W. participated in functional studies; M.A. referred the patients and conducted immunologic investigations; R.E.P. performed extensive immunologic studies and contributed to manuscript preparation; J.L.R. contributed to data analysis and manuscript composition; D.B.G. oversaw the whole-genome sequencing and data analysis and contributed to the manuscript; R.H.B. designed the study, wrote the paper, and performed
extensive immunologic evaluations; X.-P.Z. designed functional studies, analyzed data, and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Rebecca H. Buckley, Box 2898 or 362 Jones Building, Duke University Medical Center, Durham, NC 27710, e-mail: [email protected].
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contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Cell. 1997;91(2): 243-252. 9. Courtois G, Israel ¨ A. IKK regulation and human genetics. Curr Top Microbiol Immunol. 2011;349:73-95. 10. Smahi A, Courtois G, Vabres P, et al; The International Incontinentia Pigmenti (IP) Consortium. Genomic rearrangement in NEMO impairs NF-kappaB activation and is a cause of incontinentia pigmenti. Nature. 2000; 405(6785):466-472. 11. Niehues T, Reichenbach J, Neubert J, et al. Nuclear factor kappaB essential modulatordeficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(6):1456-1462. 12. Orange JS, Levy O, Brodeur SR, et al. Human nuclear factor k B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(3):650-656. 13. Courtois G, Smahi A, Reichenbach J, et al. A hypermorphic IkappaBalpha mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J Clin Invest. 2003;112(7): 1108-1115. 14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[k]B activity. Annu Rev Immunol. 2000;18:621-663.
15. Pannicke U, Baumann B, Fuchs S, et al. Deficiency of innate and acquired immunity caused by an IKBKB mutation. N Engl J Med. 2013;369(26):2504-2514. 16. Burns SO, Plagnol V, Gutierrez BM, et al. Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKb. J Allergy Clin Immunol. 2014;134(1):215-218. 17. Buckley RH, Schiff SE, Sampson HA, et al. Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation. J Immunol. 1986;136(7):2398-2407. 18. Sugden B, Mark W. Clonal transformation of adult human leukocytes by Epstein-Barr virus. J Virol. 1977;23(3):503-508. 19. Krishna S, Zhong X. Role of diacylglycerol kinases in T cell development and function. Crit Rev Immunol. 2013;33(2):97-118. 20. Hanson EP, Monaco-Shawver L, Solt LA, et al. Hypomorphic nuclear factor-kappaB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity. J Allergy Clin Immunol. 2008;122(6): 1169-1177, e16. 21. Shearer WT, Rosenblatt HM, Gelman RS, et al; Pediatric AIDS Clinical Trials Group. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol. 2003;112(5):973-980.
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2014 124: 2046-2050 doi:10.1182/blood-2014-04-571265 originally published online August 18, 2014
A nonsense mutation in IKBKB causes combined immunodeficiency Talal Mousallem, Jialong Yang, Thomas J. Urban, Hongxia Wang, Mehdi Adeli, Roberta E. Parrott, Joseph L. Roberts, David B. Goldstein, Rebecca H. Buckley and Xiao-Ping Zhong
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