Genes and (2011) 12, 663–666 & 2011 Macmillan Publishers Limited All rights reserved 1466-4879/11 www.nature.com/gene

SHORT COMMUNICATION A novel immunodeficiency disorder characterized by genetic amplification of 25

MR Green1,2, E Camilleri1, MK Gandhi2,3, J Peake4 and LR Griffiths1,2 1Genomics Research Centre, Griffith Institute for Health and Medical Research, Griffith University, Gold Coast, Queensland, Australia; 2Griffith Medical Research College, A Joint Program of Griffith University and the Queensland Institute of Medical Research, QIMR, Herston, Queensland, Australia; 3Clinical Immunohaematology Laboratory, Queensland Institute of Medical Research, Herston, Queensland, Australia and 4Queensland Pediatric and Service, Royal Children’s Hospital, Herston, Queensland, Australia

Many disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of . Here, we have used a whole- genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper bias in the immunodeficiency case provides a mechanistic explanation for recurrence of by met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses. Genes and Immunity (2011) 12, 663–666; doi:10.1038/gene.2011.50; published online 21 July 2011

Keywords: immunodeficiency; IL25; T-cell; Th1; Th2; copy number variation

Introduction interleukin (IL)-4mediated signaling as the drivers of Th1 and Th2 polarization, respectively. Signaling by these Primary immunodeficiency disorders affect approxi- causes helper T-cell polarization by invoking mately two people per 100 000, and predispose affected broad transcriptional changes. In Th1 cells, these individuals to recurrent infections and the development transcriptional changes are driven primarily by the of other disorders such as .1,2 Primary transcription factor TBX21 (T-bet5), whereas in Th2 cells, immunodeficiency disorders range in both severity they are primarily driven by the transcription factor and etiology, but the majority result in a deficiency in GATA3.6,7 These transcription factors induce the expres- production. Many of these disorders have been sion of cytokines by Th1- or Th2-polarized cells that well characterized and result from specific genetic function as a positive feedback loop to promote further defects that, in most cases, lead to the loss-of-function polarization of cells to the same subtype and inhibit of central components of immune maturation or signal- polarization to the opposing subtype. The culmination ing.3 The profound effect on immune function by genetic of these events is a dominance of one helper T-cell defects is testament to the importance of specific state over the other and dichotomization of the class of components in maintaining immune homeostasis. immune response mounted against invading pathogens. The establishment of a type 1 (Th1) or type 2 (Th2) In addition to the well-characterized interferon-g- and helper T-cell dominance is an important step in regulat- IL4-driven T-helper cell polarization mechanism, there is ing the class of immune response mounted against accumulating evidence for a role of other factors in pathogens (reviewed in Kidd4). Polarization of Th1 or regulating this axis. These include the Th1-associated Th2 subsets from non-committed naı¨ve T-cells is primar- IL12, the Th2-associated cytokine IL5, and more ily driven by cytokine signals that are directed by the recently, a newly defined member of the IL17 family, type of encountered by immune cells. The most IL25, has been shown to promote Th2 responses.8,9 definitive base of information regarding cytokine-driven Here, we describe the molecular characterization of a polarization of helper T-cells highlights interferon-g- and novel immunodeficiency disorder. This case shows profound combined immunodeficiency that manifested from an early age with recurrent Varicella infections, Correspondence: Professor LR Griffiths, Genomics Research Centre, bronchitis, skin infections, upper respiratory tract infec- Griffith Institute for Health and Medical Research, Griffith tions and otitis media (For detailed case history, see University, Gold Coast Campus, Queensland 4215, Australia. E-mail: [email protected] Supplementary Information). No molecular alterations Received 16 March 2011; accepted 13 June 2011; published online characteristic of currently defined immunodeficiency 21 July 2011 disorders were found, and the case tested negative for Primary immunodeficiency disorders MR Green et al 664 human immunodeficiency . We therefore utilized (Conley et al.11 and Supplementary Information), we genome-wide DNA and transcriptional profiling, to undertook a comprehensive screen for potentially etio- define the etiology of the disorder. logic DNA copy number alterations in the case genome with reference to 100 HapMap controls. This highlighted small, previously defined, naturally occurring copy number variations, as well as a larger tetraploid region Results and discussion at 14q11.2 (chr11:19,272,965-23,891,430; Figure 1a), which To define the molecular etiology of a novel immunode- did not align to copy number variations previously ficiency disorder, we utilized whole-genome high resolu- identified in a large population of healthy control tion DNA copy number analysis to elucidate potentially subjects.12 The presence of this alteration was confirmed causative genetic alterations. Using this approach, we by matrix assisted laser desorption/ionisation time of identified a tetraploid region at 14q11.2 that mapped flight (MALDI-TOF) copy number analysis (Figure 1b) over the IL25 gene, which has a role in promoting and encompassed the gene encoding IL25, a cytokine Th2 responses and thereby suppressing Th1 responses. known to induce Th2 immune responses.8,9 In vitro By pairing genetic analysis with transcriptional profiling, analysis of IL25 gene expression in purified we identified a large number of genes associated following T-cell activation found that this cytokine was with Th1/Th2 profiles to be differentially expressed expressed at levels 2.49-fold higher in case lymphocytes, in peripheral blood lymphocytes of the case, including a compared with lymphocytes from control subjects (t-test significant enrichment of a GATA3 transcriptional P-value ¼ 0.001, Figure 1c). We therefore hypothesized signature, indicative of a Th2 bias that was also that genetic amplification and overexpression of IL25 in confirmed in the case by flow cytometric analysis. this case may skew immunity towards Th2 responses Single-nucleotide polymorphism (SNP) microarray- and thereby result in insufficient Th1 immune responses. based platforms are an effective tool for gaining To provide further evidence towards this hypothesis, high resolution DNA copy number information, with we utilized the genome-wide transcriptional profiling. extensive genome coverage.10 Because of the inability to Comparison of gene expression profiles from peripheral define the etiology of this immunodeficiency syndrome blood lymphocytes of the case and an age-, gender-, based on molecular tests for known disorders ethnicity-matched healthy control subject identified a

Figure 1 Increased DNA copy number of IL25 results in overexpression following T-cell activation. (a) Genome-wide DNA copy number analysis was performed using Sty 250 K SNP arrays (Affymetrix, Santa Clara, CA, USA), on peripheral blood DNA from the case and three control subjects. Copy number was calculated as in Supplementary Materials, and identified a quadraploid region at 14q11.2 that mapped over the IL25 locus. (b) DNA copy number increase was confirmed by matrix assisted laser desorption/ionization time of flight (MALDI- TOF) mass spectrometry-based analysis of two markers within the hyperploid region (rs4562981 and rs6489721), compared with a control marker within the glyceraldehydes 3-phosphate dehydrogenase gene (rs6489721). The significantly larger area under the curve of the homozygous SNP rs4562981 and heterozygous SNP rs6489721 compared with the heterozygous control marker confirm hyperploidy of the candidate region. (c) Expression of IL25 was measured by quantitative PCR in purified peripheral blood cultures following T-cell activation, using anti-CD3 T-cell activation plates. Expression of IL25 in the case sample was found to be significantly (P ¼ 0.001) higher than that in an age-, gender-, ethnicity-matched healthy control subject. For detailed methods, refer to Supplementary Material. *Po0.05.

Genes and Immunity Primary immunodeficiency disorders MR Green et al 665 large number of differentially expressed genes. Among case subject, there was a profound absence of lympho- these were a large number of genes associated with Th1 cytes with a Th1 phenotype, but a clearly defined or Th2 transcriptional profiles, which supported a Th2 population (Figure 2c). This provided additional decreased Th1 signature and increased Th2 signature evidence towards a skewed Th2 dominance in the (Figure 2a). This included 4.17-fold decreased expression immunodeficiency case, which we posit is a direct result of the Th1-associated gene TBX21, a central transcription of genetic amplification of the IL25 locus. factor driving Th1 polarization. Although the transcrip- Several factors of the case history further substantiate a tion factor responsible for driving Th2 polarization Th2 bias. Patients with profound immunodeficiency (GATA3) was not found to be significantly differentially commonly suffer from recurrent infections by pathogens expressed between case and control subjects by gene that are cleared by Th2-mediated responses, such as expression microarray analysis, the activity of this Pneumocystis carinii or acid-fast bacteria,16,17 transcription factor may be modified by post-transla- whereas there is no evidence of these infections within tional modifications that would not be detected at the the case history of this novel disorder. In contrast, this transcript level.13 We therefore investigated GATA3 patient suffered from recurrent infections by Varicella, activity by gene set enrichment analysis (for detailed Staphylococcus aureus and , which methods, refer to Supplementary Information) of the are more effectively cleared by Th1 responses.18–20 previously defined GATA3PATHWAY signature14,15 and An overactive Th2 response is also further supported found evidence that GATA3 activity is significantly by other features of the case history, including enriched in case lymphocytes (enrichment score ¼ 0.512, (i) occurrence idiopathic purpura P-value o0.001; Figure 2b). Additionally, the previously at age 3 without relapse, which is consistent with a defined IL4PATHWAY and IL5PATHWAY signatures Th2 imbalance,21 (ii) the normal levels of IgE despite (http://www.broadinstitute.org/gsea/msigdb/index.jsp), lymphopenia and deficiencies of other immunoglobu- which may also be considered suggestive of a Th2 bias, lins, as an IgE switch is supported by a Th2 also showed enrichment in the immunodeficiency case response,22 and (iii) the normal proliferative response of (enrichment scores ¼ 0.549 and 0.620, respectively), lymphocytes to pokeweed, which elicits a Th2 re- but did not obtain statistical significance with robust sponse,23 but poor proliferative responses to all other multiple hypothesis testing correction. mitogens. Together, these support our hypothesis that Finally, evidence for a Th2 bias within the immuno- genetic amplification and resulting overexpression of deficiency case was gained by flow cytometric analysis IL25 drives a Th2 bias in this case and renders the subject of lymphocyte populations, using the CD4 helper susceptible to infections cleared by Th1 responses, in T-cell marker and intracellular staining for the Th1- or addition to other pathologies associated with overactive Th2-associated cytokines, interferon-g and IL4. Impor- Th2 responses. tantly, T-cell activation of healthy control subjects by In conclusion, this study not only shows the utility of anti-CD3 stimulation resulted in quantifiable popula- whole-genome screens in defining the molecular basis tions of Th1 cells (data not shown). However, in the for novel disorders, but also provides insight into a novel

Figure 2 IL25 amplification and overexpression is associated with a Th2 bias in the case. (a) Total RNA from peripheral blood of the case and matched control were profiled using HG U133 Plus2.0 microarrays (Affymetrix). A large number of genes associated with Th1/Th2 profiles were found to be differentially expressed in the case. Genes associated with a Th1 profile, including the Th1-associated transcription factor TBX21, were found to be downregulated, and genes associated with a Th2 profile were found to be upregulated. (b) Although GATA3 was not found to be differentially expressed between case and control subjects, gene set enrichment analysis of the GATA3PATHWAY signature found it to be significantly enriched in the case (enrichment score ¼ 0.512, Po0.001). (c) A Th2 skew was confirmed by cytoflow analysis, using the CD4 helper T-cell marker and intracellular staining for the Th1-associated cytokine, interferon-gg, or the Th2-associated cytokine, IL4. This showed that, following in vitro T-cell activation with anti-CD3, there were no detectable cells of a Th1 phenotype in the case, and the majority of CD4 þ cells showed intracellular expression of IL4, indicating they were of a Th2 phenotype and confirming a Th2 bias in the case. For detailed methods, see Supplementary Information.

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Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)

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