A germline heterozygous dominant negative IKZF2 variant causing syndromic primary immune regulatory disorder and ICHAD

Monogenic defects that impair the control of inflammation and tolerance lead to profound immune dysregulation, including autoimmunity and atopy. Studying these disorders reveals important molecular and cellular factors that regulate human immune homeostasis and identifies potential precision medicine targets. Here, we provide a detailed immunological assessment of a pediatric patient with a recently discovered syndrome causing Immunodysregulation, Craniofacial anomalies, Hearing impairment, Athelia, and Developmental delay (or ICHAD syndrome). The immunodysregulation resulted in autoimmune hemolytic anemia (AIHA) and atopic dermatitis. The patient carried a de novo germline heterozygous c.406+540\_574+13477dup;p.Gly136\_Ser191dup variant in IKAROS family zinc finger 2 ( IKZF2 ), which encodes Helios. This variant led to reduced Helios protein expression and dominant interference of wild-type Helios-mediated repression of the IL2 promoter. Multi-parameter flow cytometric analyses of patient peripheral blood mononuclear cells revealed strongly impaired natural killer cell differentiation and function, and increased CD8+ T cell activation and cytokine secretion. Strikingly, patient CD4+ T cells were hyperactive, produced elevated levels of nearly all T helper (TH) cytokines, and readily proliferated in response to stimulation. Patient regulatory T cells (Tregs) developed normally but aberrantly produced high levels of many TH cytokines. Single-cell RNA sequencing revealed largely normal Tregs (albeit mostly memory), but naïve CD4+ T cells that were more enriched in genes related to activation, proliferation, metabolism, and TH differentiation. This work describes the immunological phenotype of one of the first reported cases of germline dominant negative Helios deficiency, expands our understanding of the pathogenesis of AIHA on a single cell level, and provides valuable insights into Helios function in a variety of lymphocyte subsets. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported in part by grants from the Canadian Institutes of Health Research (PJT 178054 to S.E.T.; FDN-154304 to M.K.L.), Genome British Columbia (SIP007) (S.E.T.), and BC Children's Hospital Foundation. S.E.T. holds a Tier 1 Canada Research Chair in Pediatric Precision Health and the Aubrey J. Tingle Professor of Pediatric Immunology. M.K.L. receives a BCCHR salary award and holds a Tier 1 Canada Research Chair in Engineered Immune Tolerance. H.Y.L. is supported by a CIHR Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award (CGS-D), University of British Columbia Four Year Doctoral Fellowship (4YF), Killam Doctoral Scholarship, Friedman Award for Scholars in Health, and a BC Children's Hospital Research Institute Graduate Studentship. M.V.S. is supported by a Vanier Canada Graduate Scholarship and 4YF. A.J.L. is supported by a CGS-D and 4YF. M.S. is supported by a CGS-D and 4YF. We would like to acknowledge the Flow Cytometry and Imaging cores at BC Children's Hospital Research Institute for providing the equipment for microscopy, flow cytometry, and cell sorting, the Biomedical Research Centre Sequencing Core (BRC-Seq) for their assistance with single-cell RNA sequencing and data processing, The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada for their assistance with whole genome sequencing, and the BC Children's Hospital BioBank for providing access to age-matched and sex-matched control peripheral blood mononuclear cells. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All study participants and/or their parents/guardians provided written informed consent. Research study protocols (H18-02853, H18-02912, H15-00092) were approved by The University of British Columbia Clinical Research Ethics Board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.