Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Vaseghi-Shanjani, Maryam
Doctor of Medicine / Doctor of Philosophy - MD/PhD
University of British Columbia
Inborn errors of immunity (IEIs) are a diverse group of genetic disorders that adversely affect the function and/or development of the immune system, resulting in increased susceptibility to infections, autoimmunity, allergy, and malignancies. Primary immune regulatory disorders and primary atopic disorders are two subcategories of IEIs. While IEIs are commonly associated with frequent, severe, and unconventional infections, primary immune regulatory disorders mainly present with immune dysregulation, and primary atopic disorders with severe, early-onset allergies. These manifestations can occur with or without the typical infectious symptoms expected with IEIs. Given the recently acknowledged diversity of symptoms associated with IEIs, there is a pressing need to sequence and investigate individuals presenting with unconventional and newly identified manifestations of IEIs. The overarching goal of this thesis was to use an established pipeline of studying single patients to discover and characterize novel monogenic causes of primary immune regulatory disorders and primary atopic disorders. The discovery of these novel classes of IEIs significantly enriches our understanding of fundamental human biology, presenting new pathways for therapeutic development and repurposing, which promise substantial enhancements in patient care. By employing a comprehensive approach that included the collection of phenotypic information from patients and the use of advanced genomic sequencing and bioinformatics analyses, we identified two novel diseases caused by germline variants in IKZF2 and ZBTB7B, which encode critical transcription factors – Helios and ThPOK – that regulate broad-ranging biological processes in the human body. The first study delineates the consequences of two dominant-negative variants in Helios, revealing an association with a complex syndrome in humans characterized by immune dysregulation and developmental anomalies, thus broadening our understanding of Helios’s role in human physiology and development. The second study describes the discovery of a multimorphic variant in ThPOK, outlining its critical involvement in CD4 T cell development and its unexpected association with tissue fibrosis in humans. These findings not only demonstrate the profound impact of transcriptional regulation on immune function and disease, but also expand the diagnostic and therapeutic approaches of IEIs, enriching the future landscape of IEI management.
CHARACTERIZING THE MOLECULAR AND CLINICAL IMPACT OF TWO NOVEL HUMAN INBORN ERRORS OF IMMUNITY CAUSED BY DEFECTS IN IKZF2 and ZBTB7B by Maryam Vaseghi-Shanjani B.Sc., McMaster University, 2017 M.Sc., McMaster University, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2024 © Maryam Vaseghi-Shanjani, 2024 ii The following individuals certify that they have read and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation titled: “CHARACTERIZING THE MOLECULAR AND CLINICAL IMPACT OF TWO NOVEL HUMAN INBORN ERRORS OF IMMUNITY CAUSED BY DEFECTS IN IKZF2 and ZBTB7B” submitted by Maryam Vaseghi-Shanjani in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine. Examining Committee: Dr. Stuart Turvey, Professor, Department of Pediatrics, UBC Supervisor Dr. Catherine Biggs, Clinical Assistant Professor, Department of Pediatrics, UBC Supervisory Committee Member Dr. Liam Brunham, Associate Professor, Department of General Internal Medicine, UBC Supervisory Committee Member Dr. Jan Friedman, Professor, Department of Medical Genetics, UBC Supervisory Committee Member Dr. Andrew Sandford, Professor, Respiratory Medicine Division, UBC Supervisory Committee Member Dr. Manish Sadarangani, Associate Professor, Department of Pediatrics, UBC University Examiner Dr. Marc Horwitz, Professor, Department of Microbiology and Immunology, UBC University Examiner Dr. Aleixo Muise, Professor, Department of Pediatrics, University of Toronto External Examiner iii Abstract Inborn errors of immunity (IEIs) are a diverse group of geneYc disorders that adversely affect the funcYon and/or development of the immune system, resulYng in increased suscepYbility to infecYons, autoimmunity, allergy, and malignancies. Primary immune regulatory disorders and primary atopic disorders are two subcategories of IEIs. While IEIs are commonly associated with frequent, severe, and unconvenYonal infecYons, primary immune regulatory disorders mainly present with immune dysregulaYon, and primary atopic disorders with severe, early-onset allergies. These manifestaYons can occur with or without the typical infecYous symptoms expected with IEIs. Given the recently acknowledged diversity of symptoms associated with IEIs, there is a pressing need to sequence and invesYgate individuals presenYng with unconvenYonal and newly idenYfied manifestaYons of IEIs. The overarching goal of this thesis was to use an established pipeline of studying single paYents to discover and characterize novel monogenic causes of primary immune regulatory disorders and primary atopic disorders. The discovery of these novel classes of IEIs significantly enriches our understanding of fundamental human biology, presenYng new pathways for therapeuYc development and repurposing, which promise substanYal enhancements in paYent care. By employing a comprehensive approach that included the collecYon of phenotypic informaYon from paYents and the use of advanced genomic sequencing and bioinformaYcs analyses, we idenYfied two novel diseases caused by germline variants in IKZF2 and ZBTB7B, which encode criYcal transcripYon factors – Helios and ThPOK – that regulate broad-ranging biological processes in the human body. The first study delineates the consequences of two dominant-negaYve variants in Helios, revealing an associaYon with a iv complex syndrome in humans characterized by immunodysregulaYon and developmental anomalies, thus broadening our understanding of Helios’s role in human physiology and development. The second study describes the discovery of a mulYmorphic variant in ThPOK, outlining its criYcal involvement in CD4 T cell development and its unexpected associaYon with Yssue fibrosis in humans. These findings not only demonstrate the profound impact of transcripYonal regulaYon on immune funcYon and disease, but also expand the diagnosYc and therapeuYc approaches of IEIs, enriching the future landscape of IEI management. v Lay Summary Our immune system protects us from diseases, but when malfuncYoning, it can lead to infecYons, allergies, autoimmune diseases, and cancer. Certain individuals are born with a rare group of geneYc disorders, known as inborn errors of immunity (IEIs), which means parts of their immune system are missing or do not funcYon well from birth. Understanding these geneYc disorders is important, as it can significantly impact the clinical care of the affected individuals and provide valuable insights for those similarly affected worldwide. With beeer knowledge, we can develop targeted treatments that directly address the problemaYc parts of their immune system, leading to improved paYent outcomes. In my thesis, I discovered two new human diseases that cause significant immune system defects. Besides helping to end the diagnosYc odyssey for these paYents, my studies have helped broaden our understanding of the molecular and geneYc causes of severe allergies and immune dysregulaYon in humans. vi Preface This thesis encompasses work undertaken at BC Children’s Hospital Research InsYtute and The University of BriYsh Columbia, in partnership with clinicians and scienYsts from the NaYonal InsYtutes of Health, University of Toronto, Baylor College of Medicine, Texas Children’s Hospital, and Care4Rare Canada. Research study protocols were approved by The University of BriYsh Columbia Clinical Research Ethics Board (H18-02853 and H15-00641). All study parYcipants, including paYents, healthy controls and/or their parents or legal guardians provided wrieen informed consent to parYcipate in our study. Parts of this thesis, notably chapter 4, is being prepared for submission. Majority of the data outlined in chapters 2 and 3 have already been successfully published, with the remaining data being prepared for submission or currently under review for publicaYon. The following secYon delineates the contribuYons of all collaborators and co-authors, underscoring the specifics of my involvement and the proporYon of research and wriYng executed by me. Chapter 2 Contributions Chapter 2 is comprised of three first-author review papers; all of which have been published: Vaseghi-Shanjani M, Smith KL, Sara RJ, Modi BP, Branch A, Sharma M, et al. Inborn errors of immunity manifesting as atopic disorders. J Allergy Clin Immunol. 2021;148(5):1130-9. The co-corresponding authors, Dr. Stuart E. Turvey and Dr. Catherine M. Biggs, supervised all aspects of this review arYcle, meYculously ediYng and refining the content to ensure its academic rigor. I took a lead role in defining its scope and focus areas and outlining the various vii secYons. As a co-first author, alongside Kelsey L. Smith, I contributed to approximately 60% of the manuscript's content, craoed Table 1, designed and executed the figures, and took the lead on revisions and addressing comments from peer reviewers and all co-authors. Kelsey L. Smith, sharing the co-first author disYncYon, was instrumental in composing the clinical secYons of the arYcle. Rahnuma J. Sara was tasked with assembling Table 2, which focused on the GWAS studies relaYng to PAD-causing genes and their links to common allergic diseases. Anna Branch's efforts were pivotal in the literature review, parYcularly concerning the red flags for primary atopic disorders as highlighted in our review. Dr. Bhavi P. Modi, Dr. Mehul Sharma, Dr. Henry Y. Lu, and Dr. Elliot L. James brought their specialized knowledge to the paper, ensuring its factual accuracy and enhancing its scholarly integrity with their revisions. Vaseghi-Shanjani M, Snow AL, Margolis DJ, Latrous M, Milner JD, Turvey SE, Biggs CM. Atopy as Immune Dysregulation: Offender Genes and Targets. J Allergy Clin Immunol Pract. 2022;10(7):1737-56. The corresponding author, Dr. Catherine M. Biggs, supervised all aspects of this review arYcle, meYculously ediYng and refining the content to ensure its academic rigor. As the first author, I took the lead role in defining the scope and focus of this review paper under Dr. Catherine M. Biggs’ supervision and outlined the various secYons to ensure a comprehensive and coherent narraYve. I was responsible for authoring approximately 50% of the manuscript, in addiYon to creaYng Table 2. I also designed and executed all the figures that served as a visual anchor for the concepts discussed in the review. I spearheaded the revision process, which involved incorporaYon of feedback from peer reviewers and all co-authors. Dr. Andrew Snow, Dr. David Margolis, and Dr. Joshua Milner each provided invaluable insights by wriYng a secYon viii related to their respecYve areas of experYse, bringing depth and diverse perspecYves to the paper. Dr. Meriem Latrous was instrumental in draoing Table 1. Dr. Stuart E. Turvey performed a vital role in meYculously ediYng and refining the content, ensuring the accuracy of the scienYfic content presented in the paper. Vaseghi-Shanjani M, Yousefi P, Sharma M, Samra S, Sifuentes E, Turvey SE, Biggs CM. Transcription factor defects in inborn errors of immunity with atopy. Front Allergy. 2023;4:1237852. The corresponding author, Dr. Catherine M. Biggs, diligently oversaw every aspect of the review arYcle's preparaYon. She meYculously revised the content to ensure its scholarly integrity. As the lead author, I was tasked with defining the scope and direcYon of the review paper with Dr. Biggs' guidance, and I composed the enYre manuscript. Figures were developed by me and Pariya Yousefi. Following our preliminary drao, I led the revision phase, which entailed a detailed review and incorporaYon of feedback from peer reviewers and all co-authors. Dr. Mehul Sharma brought his experYse to the manuscript, assisYng in the refinement of concepts presented in the review and suggesYng valuable resources and scholarly work that contributed to the manuscript's compleYon. Simran Samra, Dr. Erika Sifuentes, and Dr. Stuart E. Turvey were instrumental in carefully reviewing and verifying the informaYon presented in the manuscript. Chapter 3 Contributions The work presented in Chapter 3 is a product of collaborative efforts between multiple research groups and clinicians that have significantly propelled our understanding of the spectrum of diseases that can be caused by variants in the DNA-binding domain of Helios. The data ix encompassed in this chapter has contributed to three distinct manuscripts, reflecting a spectrum of collaborative efforts. Firstly, I was the co-first author on a manuscript that is now published: Mohajeri A*,Vaseghi-Shanjani M*, Rosenfeld JA, Yang GX, Lu H, Sharma M, et al. Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay. J Med Genet. 2023;60(11):1092-104. This publication stands as a significant component of the research findings discussed in this chapter, where I contributed extensively to the conceptualization, execution of experiments, data analysis, and manuscript preparation. I planned, executed, and analyzed the data from all the laboratory experiments included in this manuscript. The genetic analysis and variant interpretation were done by co-first author, Arezoo Mohajeri. Writing the original draft was a joint effort between myself and Arezoo Mohajeri. The review and editing process was comprehensive, with invaluable contributions from all co-authors, ensuring a manuscript of the highest quality. This study was done under the supervision of Dr. Anna Lehman and Dr. Stuart E. Turvey. My role also extended to clinical data acquisition, working alongside Meriam Waqas and Areesha Salman to gather the necessary clinical data by meeting the patient family and reviewing the electronic medical records of the patient. The description of patient phenotype was a concerted effort, with Jill A. Rosenfeld, Dr. Mahshid Sababi Azamian, Dr. Frederick K. Kozak, Dr. Ronak Rahmanian, Dr. Catherine M. Biggs, Dr. Kyla J. Hildebrand, Dr. Seema R. Lalani, Dr. Sarah K. Nicholas, Dr. Daryl A. Scott, Dr. Erika Henkelman, Dr. Jessica Halparin, Dr. Connie L. Yang, Dr. Linlea Armstrong, Dr. Stuart E. Turvey, and Dr. Anna Lehman all providing valuable insights, which I helped to coordinate and integrate into our research. Members from the Undiagnosed Diseases Network and the Care4Rare Canada Consortium offered their multidisciplinary expertise. x Secondly, I am listed as a second author on a manuscript under review at a peer reviewed journal and posted on a preprint server. Lu HY, Vaseghi-Shanjani M, Lam AJ, Sharma M, Mohajeri A, Gillies J, et al. A germline heterozygous dominant negative IKZF2 variant causing syndromic primary immune regulatory disorder and ICHAD. medRxiv. 2023:2023.09.09.23295301. In this manuscript, I contributed to assessing the expression of the protein using immunoblotting and assessing the functional impact of the variant protein using luciferase reporter assay. I also extensively contributed to the clinical data acquisition, working alongside Dr. Henry Lu to gather the necessary clinical data by meeting the patient family and reviewing the electronic medical records of the patient. Lastly, I am listed as a second author on a manuscript currently in preparation: Velde H, Vaseghi-Shanjani M, Ramakrishnan G, Smits J, Oostrik J, Yntema H, Haer-Wigman L, Huynen M, Lehman A, Turvey S, Pennings R, Kremer H. Exome variant prioritization in a large cohort of hearing-impaired individuals indicates IKZF2 to be associated with non-syndromic hearing loss and guides future research for unsolved cases. This paper is being led by a research team at Radboud University under the supervision of Dr. Hannie Kremer. My contribution to this paper, while not in a lead capacity, involves the critical assessment of the expression of all variant proteins using immunoblotting techniques. Moreover, I have played a pivotal role in assessing the functional impact of the variants by employing a luciferase reporter assay which I developed and optimized. In addition, I contributed to the writing of the manuscript which is being prepared for submission. xi Chapter 4 Contributions Chapter 4 presents work from a collaborative effort involving research groups from the University of British Columbia (UBC), the National Institutes of Health (NIH), and the University of Toronto (UofT), as well as contributions from multiple clinicians at BC Children’s Hospital. This chapter reports the first case of a human with a genetic variant in ZBTB7B, leading to a complex syndrome that includes CD4 T cell deficiency, allergic disease, interstitial lung disease, hearing loss, developmental delay, and growth failure. The data in this chapter have been used to draft a manuscript that we plan to submit for publication soon. I am a co-first author on this manuscript. Vaseghi-Shanjani M*, Sharma M*, Yousefi P, Samra S, Laverty KU, Jolma A, Razavi R, Yang AHW, Albu M, Golding L, Lee AF, Tan R, Richmond PA, Bosticardo M, Rayment JH, Yang CL, Hildebrand KJ, Brager R, Demos MK, Lau YL, Notarangelo LD, Hughes TR, Bosselut R, Biggs CM, Turvey SE. A multimorphic variant in ThPOK causes a novel human disease characterized by T cell immune developmental abnormalities, immunodysregulation, allergy, and organ fibrosis. This study was conducted under the supervision of Dr. Stuart Turvey and Dr. Catherine Biggs, who oversaw the entire project. I had the privilege of being a co-first author, working closely under their guidance alongside Dr. Mehul Sharma, who is also a co-first author. I was principally responsible for all aspects of this study. This encompassed a wide range of tasks, from the initial study design and comprehensive literature review to the interpretation of the genetic variant. I designed and conducted the functional validation experiments, as well as in the detailed analysis of the majority of the data presented in the manuscript. I contributed to over 90% of the manuscript's planning, writing, and revising, under guidance from Dr. Stuart Turvey, Dr. Mehul Sharma, and Dr. Catherine Biggs. Dr. Mehul Sharma was instrumental in leading the single-cell RNA sequencing experiments, from planning and execution to analysis. He also conducted all the xii bioinformatic analysis of the bulk RNA sequencing data, while I was responsible for conducting the pre-processing step of the sequencing data. We had the support of collaborators from the University of Toronto in Dr. Timothy Hughes’ team, including Kaitlin U. Laverty, Arttu Jolma, Rozita Razavi, Ally H W Yang, and Mihai Albu, who provided valuable assistance with HT-SELEX and ChIP sequencing experiments. Other team members – Pariya Yousefi, Simran Samra, Ryan Tan, and Marita Bosticardo – were pivotal in assisting with various laboratory experiments. Dr. Phillip A. Richmond played a key role in the bioinformatic interpretation of the variant. The compilation of clinical information regarding the patient was a collaborative effort between Dr. Catherine Biggs, Dr. Stuart Turvey, Dr. Jonathan H. Rayment, Dr. Connie L. Yang, Dr. Kyla J. Hildebrand, and Dr. Anna Lee. Dr. Rémy Bosselut and Dr. Luigi D. Notarangelo, who are global experts in ThPOK and inborn errors of immunity affecting T cell development were instrumental in shaping the direction of our study, providing insights that greatly enhanced impact of our work. xiii Table of Contents Abstract ................................................................................................................................. iii Lay Summary .......................................................................................................................... v Preface .................................................................................................................................. vi Table of Contents ................................................................................................................. xiii List of Tables ....................................................................................................................... xviii List of Figures ....................................................................................................................... xix List of Abbreviations ............................................................................................................. xx Acknowledgements ............................................................................................................. xxiii Dedication ........................................................................................................................... xxv CHAPTER 1: Introduction ......................................................................................................... 1 1.1. Overview of inborn errors of immunity (IEI) ........................................................................ 1 1.1.1 Disease prevalence ............................................................................................................................... 2 1.1.2 Molecular diagnosis of IEIs ................................................................................................................... 3 1.1.3 Challenges of using next-generation sequencing (NGS) in the diagnosis of IEIs ................................ 5 1.2. The expanding spectrum of clinical manifestations associated with IEIs .............................. 7 1.3. Primary immune regulatory disorders (PIRDs) ................................................................... 10 1.3.1. PIRDs manifesting as Tregopathies .................................................................................................... 11 1.3.1.1. Variants in IKZF2 as a novel cause of Tregopathy in humans .................................................. 12 xiv 1.4. Primary atopic disorders (PADs) ........................................................................................ 15 1.4.1. Variants in ZBTB7B as a novel cause of PADs in humans .................................................................. 16 1.5. Thesis rationale and objectives .......................................................................................... 18 CHAPTER 2: Constructing a framework for understanding the molecular and clinical underpinnings of primary atopic disorders ............................................................................ 21 2.1. Introduction and rationale ................................................................................................. 21 2.1.1 PADs informing our understanding of fundamental human biology ............................................... 26 2.1.2 Studying PADs reveals the genetic architecture of common allergies ............................................. 26 2.2. Clinical red flags for the diagnosis of PADs ......................................................................... 28 2.2.1 Atopy .................................................................................................................................................. 29 2.2.2 Autoimmunity .................................................................................................................................... 31 2.2.3 Malignancy ......................................................................................................................................... 32 2.2.4 Short stature ....................................................................................................................................... 33 2.2.5 Infections ............................................................................................................................................ 33 2.2.6 Connective tissue abnormalities ........................................................................................................ 35 2.3. Molecular mechanisms of allergic inflammation ................................................................ 36 2.3.1 Epithelial barrier disruption ............................................................................................................... 39 2.3.2 Cytokines and cytokine signaling defects .......................................................................................... 41 2.3.3 Actin cytoskeleton defects ................................................................................................................. 44 2.3.4 Antigen presentation defects ............................................................................................................ 46 2.3.5 Attenuated antigen receptor signaling .............................................................................................. 48 2.3.6 Altered cellular metabolism ............................................................................................................... 50 2.3.7 Granulocyte dysregulation ................................................................................................................. 52 xv 2.4. Transcription factor defects in IEIs with atopy ................................................................... 53 2.4.1 Transcription factors orchestrating allergic immune responses ....................................................... 54 2.4.2 Transcription factors implicated in the pathophysiology of PADs ................................................... 56 2.4.2.1 STAT family of transcription factors ......................................................................................... 57 2.4.2.2 T-box transcription factor TBX21 ............................................................................................. 62 2.4.2.3 Forkhead Box P3 ....................................................................................................................... 63 2.5. Conclusion ......................................................................................................................... 65 CHAPTER 3: Dominant-negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterized by immunodysregulation, craniofacial anomalies, hearing impairment, athelia, and developmental delay. ..................................................................................................... 66 3.1. INTRODUCTION AND RATIONALE ...................................................................................... 66 3.2. SUBJECTS AND METHODS .................................................................................................. 69 3.2.1. Sequencing, genomic data analysis, and variant validation ............................................................. 69 3.2.2. RT-PCR and sanger sequencing .......................................................................................................... 71 3.2.3. Protein 3D structure ........................................................................................................................... 71 3.2.4. Protein expression analysis ................................................................................................................ 71 3.2.5. Luciferase reporter assay ................................................................................................................... 73 3.2.6. Statistical analysis .............................................................................................................................. 73 3.3. RESULTS ............................................................................................................................ 74 3.3.1. Identification of two unrelated individuals with syndromic developmental and immune abnormalities .................................................................................................................................................... 74 3.3.2. Identification of two monoallelic de novo IKZF2 variants ................................................................. 80 xvi 3.3.3. HeliosGly136_Ser191dup protein expression is reduced while HeliosGly153Arg protein expression is comparable to WT Helios ................................................................................................................................. 86 3.3.4. HeliosGly136_Ser191dup and HeliosGly153Arg cannot effectively repress IL2 promoter activity and interfere with WT protein function in a dominant-negative manner ............................................................................ 89 3.4. DISCUSSION ....................................................................................................................... 91 CHAPTER 4: A multimorphic variant in ThPOK causes a novel inborn error of immunity characterized by T cell immune developmental abnormalities, immunodysregulation, atopy, and organ fibrosis. ................................................................................................................ 95 4.1 INTRODUCTION AND RATIONALE ...................................................................................... 95 4.2 SUBJECTS AND METHODS .................................................................................................. 97 4.2.1 Study design ....................................................................................................................................... 97 4.2.2 Study approval ................................................................................................................................... 98 4.2.3 Genomic analysis ................................................................................................................................ 98 4.2.4 Histopathology ................................................................................................................................... 98 4.2.5 Expression plasmid cloning ................................................................................................................ 99 4.2.6 Immunoblotting ................................................................................................................................. 99 4.2.7 Luciferase reporter assay ................................................................................................................. 100 4.2.8 Electrophoretic mobility shift assay (EMSA) ................................................................................... 101 4.2.9 High-Throughput Systematic Evolution of Ligands by EXponential enrichment (SELEX) ............... 103 4.2.10 Chromatin Immunoprecipitation Sequencing ............................................................................ 104 4.2.11 Isolation and culture of primary pulmonary fibroblasts ............................................................ 106 4.2.12 Lentiviral transduction of primary pulmonary fibroblasts ......................................................... 107 4.2.13 RNA sequencing ........................................................................................................................... 107 4.2.14 Single cell-RNA sequencing and TCR repertoire analysis ........................................................... 109 xvii 4.2.15 Flow cytometry ............................................................................................................................ 111 4.2.16 Treatment testing and qPCR of primary fibroblasts ................................................................... 113 4.2.17 Dose response assessment for pirfenidone and nintedanib ...................................................... 113 4.2.18 Lactate dehydrogenase release assay for assessing cytotoxicity ............................................... 114 4.2.19 Statistical Analysis ....................................................................................................................... 115 4.3 RESULTS .......................................................................................................................... 115 4.3.1 A novel de novo heterozygous ZBTB7B variant associated with symptoms of immunodeficiency, allergic disease, and interstitial lung disease ................................................................................................ 115 4.3.2 ThPOKK360N displays altered DNA binding specificity ...................................................................... 119 4.3.3 ThPOKK360N causes impaired T cell development and activation .................................................... 124 4.3.4 ThPOKK360N contributes to Th2 skewing in patient CD4 T cells ....................................................... 128 4.3.5 ThPOKK360N disrupts profibrotic gene suppression in pulmonary fibroblasts ................................. 131 4.3.6 Reversal of fibrotic gene signature in patient pulmonary fibroblasts is achieved by over-expression of WT ThPOK or Pirfenidone treatment ........................................................................................................ 135 4.4 DISCUSSION ........................................................................................................................... 138 CHAPTER 5: Conclusions and future directions ..................................................................... 144 Bibliography ....................................................................................................................... 152 xviii List of Tables Table 2.1. Monogenic allergic disorders (i.e. PADs) and their targeted treatments………………….22 Table 2.2. Number of published genetic association studies linking variation in known PAD genes to atopic disorders in the broader population……………………………………………………………….28 Table 2.3. Genes implicated in common allergic conditions with available targeted therapy.….43 Table 3.1. Clinical summary and variant description of Probands 1 and 2………………………………..74 Table 3.2. Representative laboratory tests of Proband 1 and 2………………………………………………..78 Table 3.3. Three somatic IKZF2 tandem duplications were reported previously in three cases of Peripheral T T-cell lymphoma Not Otherwise Specified (PTCL-NOS)………………………………..……….83 Table 4.1. List and sequence of IRDye labelled WT and variant probes used in EMSA………..….103 Table 4.2. List of antibodies used in the T cell phenotyping research flow cytometry panel…..112 xix List of Figures Figure 1.1. Beyond the tip of the iceberg: Unveiling the multifaceted clinical spectrum of IEIs.…9 Figure 2.1. Clinical red flags and warning signs that warrant assessment for PADs, and the benefits of timely diagnosis and treatment of these conditions…………………………………………………..............36 Figure 2.2. A roadmap for evaluating patients who present to the allergy and immunology clinic with symptoms of allergic disease…..............................................................................................38 Figure 2.3. Diverse mechanisms of gene expression regulation by transcription factors…….......56 Figure 2.4. Mechanisms by which variants in transcription factor-encoding genes cause inborn errors of immunity associated with atopy…..................................................................................61 Figure 3.1. Clinical phenotypes of Proband 1 and 2. ...................................................................77 Figure 3.2. Family pedigrees, RT-PCR, Sanger results, and schematic of the IKZF2 variants on the gene and protein…........................................................................................................................81 Figure 3.3. Sanger sequencing results from the bands corresponding to the RT-PCR products...83 Figure 3.4. Multiple alignments of ZFs 2 and 3 and the linker sequences in orthologs and paralogs of IKZF2.........................................................................................................................................84 Figure 3.5. Amino acid sequence alignment of human Helios (IKZF2) and human Aiolos (IKZF3) using BlastP...................................................................................................................................85 Figure 3.6. Assessment of variant protein expression in HEK293 cells……….................................87 Figure 3.7. Total protein staining using Revert 700 total protein stain….......................................88 Figure 3.8. Luciferase reporter assay to assess variants’ ability to repress IL2 activity………………90 Figure 4.1. Identification of a monoallelic missense variant in ZBTB7B in a pediatric patient with inborn error of immunity and fibroinflammatory lung disease….................................................118 Figure 4.2. Functional and molecular characterization of ThPOKK360N……………………..…..............123 Figure 4.3. ThPOK's impact on T cell development and activation……………………………………........127 Figure 4.4. Enhanced Th2 Response in Patient CD4+ T Cells. ......................................................130 Figure 4.5. ThPOKK360N disruption of ThPOK-mediated fibrosis suppression in fibroblasts.........134 Figure 4.6. Evaluating the efficacy and toxicity of pirfenidone and nintedanib in TGF-β-treated patient primary pulmonary fibroblasts……………………………………………………………………………………137 xx List of Abbreviations ABR: Auditory Brainstem Response ACMG: American College of Medical GeneYcs and Genomics AD-HIES: autosomal dominant hyper IgE syndrome AD: Atopic dermatitis ALPS: Autoimmune Lymphoproliferative Syndrome AP-1: Activator protein 1 Arp2/3: Actin Related Protein 2/3 ARPC1B: Actin Related Protein 2/3 Complex Subunit 1B ASCT2: Alanine, serine, cysteine-preferring transporter 2 BCL10: B-cell CLL/lymphoma 10 cAMP: Cyclic adenosine monophosphate C2H2: Cys2His2 CADINS: CARD11-associated atopy with dominant interference of NF-κB signaling CARD: Caspase recruitment domain CBM: CARD11–BCL10–MALT1 CDC42: Cell division cycle 42 ChIP-seq: Chromatin immunoprecipitation sequencing CID: Combined immunodeficiency COL1A1: Collagen type I alpha 1 chain COL1A2: Collagen type I alpha 2 chain CTLA4: Cytotoxic T lymphocyte-associated antigen 4 DBD: DNA binding domain DHRs: Drug hypersensitivity reactions DLBCL: Diffuse large B cell lymphoma DMEM: Dulbecco modified Eagle medium DN: Dominant-negative DOCK8: Dedicator of Cytokinesis 8 DP: Double positive EMSA: Electrophoretic Mobility Shift Assay ERBIN: ERBB2-interacting protein EV: Empty vector ExAC: Exome AggregaYon ConsorYum FISH: Fluorescence in situ hybridizaYon FKH: forkhead FLG: Filaggrin gene FOXP3: forkhead box P3 GAS: Genetic association studies GATA-3: GATA Binding Protein 3 GH: Growth hormone GlcNAc: N-aceytlglucosamine GLUT1: Glucose transporter 1 xxi gnomAD: Genome AggregaYon Database GOF: Gain-of-function GWAS: Genome-wide association studies HD: Helper-deficient HEK293: Human Embryonic Kidney 293 HIES: Hyper IgE syndrome HLA: Human leukocyte antigen HPV: Human Papilloma Virus HSCT: Hematopoietic stem cell transplantation HT-SELEX: High-Throughput Systematic Evolution of Ligands by EXponential enrichment IBD: Inflammatory bowel diseases IEI: Inborn errors of immunity IFN-α: Interferon alpha IFN-γ: Interferon gamma IgE: Immunoglobulin E IGF-1: Insulin-like growth factor IgG: Immunoglobulin G IgM: Immunoglobulin M IKZF: IKAROS Family Zinc Finger IL2RG: Interleukin 2 receptor subunit gamma IL-13RA1: IL-13 receptor α1 chain IL-1R: Interleukin-1 receptor IL-31RA: IL-31 receptor α chain IL-4Ra: IL-4 receptor a chain IL: Interleukin IL2: Interleukin 2 Indels: Insertions/deletions IPEX: immune dysregulation, polyendocrinopathy, enteropathy, X-linked IRAK4: Interleukin-1 receptor associated kinase-4 IUIS: International Union of Immunological Societies JAK: Janus kinase JNK: c-Jun N-terminal kinase KIR: Killer Ig-like receptors LOF: loss-of-function LRBA: LPS-responsive and beige-like anchor protein LZ: leucine-zipper MALT1: Mucosa associated lymphoid tissue lymphoma translocation gene 1 MHC: Major histocompatibility complex mTOR: mammalian target of rapamycin mTORC1: Mammalian target of rapamycin complex 1 MyD88: Myeloid differentiation primary response 88 NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells xxii NFAT: nuclear factor of activated T cells NGS: Next-generation sequencing NK: Natural killer PAD: Primary atopic disorder PBMC: Peripheral blood mononuclear cells PCA: Principal Component Analysis PGM3: Phosphoglucomutase 3 PID: Primary immunodeficiency disease PIRD: Primary immune regulatory disorders PRR: proline-rich region RAG: RecombinaYon acYvaYng gene SCID: Severe combined immune deficiency SCORAD: Scoring Atopic Dermatitis scRNA-seq: Single cell RNA sequencing SH2: Src homology 2 SLE: Systemic lupus erythematosus SLEA: Sample level enrichment analyses SNPs: Single nucleotide polymorphisms SNV: Single nucleotide variants SPINK5: Serine Peptidase Inhibitor Kazal Type 5 STAT: signal transducer and activator of transcription TCR: T cell receptor TF: transcription factor TFH: T follicular helper TGF-b: Transforming growth factor beta Th: T helper Th2: T helper type 2, Type 2 immune responses ThPOK: T-helper-inducing POZ/Kruppel-like factor Treg: Regulatory T cells TSLP: Thymic stromal lymphopoietin TYK2: tyrosine kinase 2 UMAP: Uniform manifold approximation and projection VUS: Variant of unknown significance WAS: Wiskott-Aldrich syndrome WASP: Wiskott–Aldrich Syndrome protein WES: Whole exome sequencing WGS: Whole genome sequencing WIP: WASp-interacting protein WT: Wild-type ZAP70: Zeta Chain Of T Cell Receptor Associated Protein Kinase 70 ZBTB: Zinc finger and BTB/POZ domain-containing ZF: Zinc finger xxiii Acknowledgements First and foremost, I am deeply thankful to my supervisor, Dr. Stuart Turvey for bringing me into his research team. His generosity in providing countless learning opportunities and the chance to engage with patients and their families has been invaluable. Dr. Turvey's impactful work in improving patients' lives through science has constantly inspired me, and his belief in my abilities has encouraged me to aim higher. I also I extend my deepest gratitude to the patients and their families for entrusting us with their care and stories. Their trust and cooperation have been the cornerstone of this research, and for that, I am profoundly thankful. I extend my sincere gratitude to my supervisory committee – Dr. Jan Friedman, Dr. Liam Brunham, Dr. Andrew Sandford, and Dr. Catherine Biggs – as well as the MD-PhD program directors and administrative staff – Dr. Liam Brunham, Dr. Torsten Nielsen, Jane, and Naureen. Your ongoing support throughout this program, your insightful advice, and constant encouragement have been invaluable to my success in this long journey. I am also immensely grateful to my mentor, Dr. Catherine Biggs, for her support that went beyond academic advice. Dr. Biggs was always there to listen when I faced challenges, both in my studies and personal life, demonstrating what it means to be a strong and successful woman in science and medicine – a true "superwoman," and an inspiration to us all. To my lab mates, Mehul, Pariya, Simran, Courtney, Josh, and Liam, your support has been the foundation of my research experience. The many late nights of doing science together, sneaking free food, along with the fun times, have made this journey unforgettable. xxiv A special thank you to my many friends, especially Pariya, Mahtab, Kyle, Mahdi, Nami, Sami, Katie, Dij, Leilynaz, and Mohammad. Your unwavering support and encouragement have been crucial in helping me navigate this challenging process. To Parpar goli, your presence in both the lab and my personal life has been invaluable. You have been my constant companion, my confidante, and my life saver, embodying the true definition of friendship. Special thank you to my partner, Sam. Every single day, you have supported me, inspired me, motivated me to be a better me and to keep going when things got tough. You inspired me to be as good of a scientist as you are, to pursue immunology in the first place, and to master all the skills necessary for success, from creating polished PowerPoint presentations to excelling in wet lab experiments. Despite the distance between us, you never failed to pick up my phone calls and guide me through my experiments, serving as my “pocket immunologist”. I am indebted to you for your unwavering support, now, then, and forever. To my family, particularly my father, Mahdi, and my sister, Marjan, you have been my rock. Your encouragement, pride in my achievements, and unconditional love and support have been the source of my strength during the challenging times of being miles away from home. You have been there every step of the way, embodying all that a sister and father can be. Lastly, I want to acknowledge the broader scientific community and all those who have indirectly contributed to my path. The discussions, conferences, and collaborations with you have deepened my understanding and appreciation of science. To everyone mentioned and those unmentioned, I extend my heartfelt thanks. This thesis reflects not just my work, but the collective support, guidance, and inspiration I have received from all of you. xxv Dedication To my family and friends, for their unconditional love and support; to my mentors, for their invaluable guidance and wisdom; to the patients I have had the privilege to learn from, who remind me daily of the real-world impact of our scientific endeavors; and to those I shall have the privilege of serving in the future, may the wisdom gained through this journey enable me to provide care with insight, expertise, and above all, compassion. 1 CHAPTER 1: Introduction 1.1. Overview of inborn errors of immunity (IEI) Inborn errors of immunity (IEI), also known as primary immunodeficiency diseases (PID), encompass a heterogenous group of ~485 genetic disorders that adversely affect the immune system’s development and function1. IEIs commonly occur due to variants in genes that encode components of the immune system, such as cytokines, receptors, structural proteins, and enzymes, or genes that regulate immune system development, activation, and differentiation such as transcription factors and signalling molecules1,2. The mechanisms of pathogenesis in IEIs depend on the nature of the variant as well as the mode of inheritance. Vast majority of the IEI-causing variants are monogenic germline variants and in rare cases somatic variants that result in the loss of expression (null), loss-of-function (LOF; amorphic/hypomorphic), or gain-of-function (GOF; hypermorphic, neomorphic) of the protein encoded by the affected gene. Monoallelic (heterozygous) variants may cause autosomal dominant traits by GOF, haploinsufficiency, or negative dominance. Biallelic (homozygous, compound heterozygous) variants mainly cause autosomal recessive traits by LOF or loss of expression of the encoded protein, while X-linked recessive traits occur due to LOF of genes on the X chromosome. In rare cases, X-linked dominant traits can be caused by LOF or GOF variants1. Often referred to as ‘experiments of nature’, IEIs serve as a unique model for linking monogenic defects with their associated clinical outcomes in humans3. Historically, IEIs have demonstrated the importance of individual genes and their products in immune responses. They 2 have also contributed to the development of targeted therapeutics for diseases with immunological underpinnings4-7, laid the groundwork for fundamental, clinical, and applied immunological research, and offered valuable insights into the molecular pathogenesis of common diseases4,8,9. 1.1.1 Disease prevalence IEIs were historically considered as extremely rare disorders, affecting only 1 in 10,000 to 50,000 live births8. In the recent years, advancements in next-generation sequencing (NGS) technologies, heightened awareness among healthcare professionals, more precise clinical characterization, expanded newborn screening programs, active patient advocacy, collaborative research efforts, and the emergence of precision medicine, all have contributed to more rapid and accurate diagnoses of IEIs9,10. The prevalence of IEIs is now estimated to be between 1 in 1,000 to 1 in 5,000 individuals8. Yet, these frequencies are very likely to be an underestimate, with the true prevalence of these disorders speculated to be closer to 1 in 500 affected individuals. The underestimation of the prevalence of IEIs can be attributed to several factors, including the limited use of NGS in the clinic, the precocious mortality of patients before they get diagnosed, the phenomenon of incomplete penetrance where some variant gene carriers show no or partial disease symptoms, the emergence of IEI patients with symptoms in distal organ systems which leads to their management outside of immunology and genetics, and the significant yet underexplored role of somatic variants which are likely the cause of adult-onset IEIs9,11. Despite recent advances in diagnosing IEIs, it is esYmated that ~70 to 90% of individuals affected by IEIs remain undiagnosed12. Among children who are undiagnosed, the mortality rate 3 can be as high as 50%, underscoring the criYcal nature of early detecYon and intervenYon11,13. These disorders, while potenYally severe, can ooen be managed effecYvely, and in some cases be cured, with prompt diagnosis and appropriate treatment. Thus, improving the idenYficaYon, understanding, and treatment of these disorders is urgently needed. 1.1.2 Molecular diagnosis of IEIs Methods used in the diagnosis of IEIs have evolved with advances in medical technology and geneYc research. In the pre-genomic era between 1984 and 2009, various geneYc methods including familial aggregaYon, linkage analysis, convenYonal Sanger sequencing, and cytogeneYcs were uYlized to uncover the molecular geneYc causes of IEIs14. IniYally, cytogeneYc tools, including fluorescence in situ hybridizaYon (FISH), were employed to idenYfy chromosomal abnormaliYes. For example, FISH played a crucial role in idenYfying small deleYons of chromosome 22q11.2 in individuals with Di George syndrome as a cause for severe combined immunodeficiency (SCID)15. Before the emergence of NGS, another commonly employed approach was familial aggregaYon and linkage analysis. In families exhibiYng recurrent infecYons and immune dysregulaYon, linkage analysis and mapping was conducted. The aim of linkage mapping is to establish the posiYon of the disease-causing gene in relaYon to a known DNA sequence, such as a microsatellite marker or single-nucleoYde polymorphism (SNP). This is based on the principle of linkage which states that genes and geneYc markers that are located close to each other on a chromosome have the tendency to be inherited together. Through the analysis of the inheritance paeerns of these microsatellite markers/polymorphic loci in the affected individuals and their 4 relaYves, the general chromosomal areas where genes linked to the disease phenotype were likely located were idenYfied. Following the idenYficaYon of these chromosomal regions, posiYonal cloning and Sanger sequencing was used to precisely locate and idenYfy the specific genes responsible14. This method played an important role in idenYfying genes associated with various IEIs. Namely, linkage mapping was used to pinpoint the locaYon of X-linked SCID to the proximal long arm of the X chromosome, leading to the subsequent idenYficaYon of the Interleukin 2 receptor subunit gamma (IL2RG) gene as the causaYve gene for X-linked SCID16. Variants associated with IEIs were historically, and conYnue to be, idenYfied through a candidate gene strategy subsequent to the cloning of homologous genes, typically in mice. The deficiencies of RecombinaYon acYvaYng gene-1 (RAG1) and RAG2, linked to autosomal recessive SCID in humans, were discovered using this method, relying on similar characterisYcs observed in Rag1- and Rag2-knockout mice14. Despite the use of these tradiYonal methods leading to the idenYficaYon of over 180 genes associated with IEIs, many paYents remained undiagnosed. Since 2010, NGS has revoluYonized the diagnosis of IEIs17,18. GeneYc tests, including targeted gene sequencing, whole-exome sequencing (WES), and whole-genome sequencing (WGS), have become essenYal tools in idenYfying the underlying geneYc defects responsible for IEIs. These technologies allow for the detecYon of pathogenic variants in known IEI-associated genes, as well as the discovery of novel gene defects. AddiYonally, funcYonal assays and immunological studies are ooen employed to complement geneYc tesYng and provide a comprehensive assessment of immune funcYon17,18. 5 1.1.3 Challenges of using next-generation sequencing (NGS) in the diagnosis of IEIs Although NGS has significantly improved the diagnosis of IEIs by enabling the simultaneous analysis of mulYple genes, it is not without limitaYons. One of the challenges is the interpretaYon and validaYon of the vast list of geneYc variants that is generated by the analysis of NGS data. To interpret geneYc variants idenYfied through geneYc tesYng, clinicians and scienYsts ooen use the 5-Yer classificaYon system and the set of guidelines that have been established by the American College of Medical GeneYcs and Genomics (ACMG)19. The guidelines are based on the integraYon of mulYple lines of evidence and require careful consideraYon of various factors to place the variant in one of the five categories: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign. These include the examinaYon of variant frequency within healthy control populaYons, leveraging populaYon databases like the Genome AggregaYon Database (gnomAD) or Exome AggregaYon ConsorYum (ExAC). ComputaYonal and predicYve analyses uYlizing tools such as PolyPhen, SIFT, and MutaYonTaster, are employed to assess the potenYal impact on gene or protein funcYon. Furthermore, funcYonal data derived from laboratory experiments, segregaYon analysis demonstraYng the co-occurrence of the variant with the disease in family studies, and evaluaYon of de novo variants in affected individuals, substanYally inform this classificaYon process. AddiYonally, the consideraYon of allelic data, specifically other known pathogenic variants in the same gene, and a broad spectrum of other relevant informaYon, including literature from databases like PubMed, ClinVar, and the Human Gene MutaYon Database (HGMD), further strengthens the assessment19. 6 To classify a variant as ‘pathogenic’, several stringent criteria must be met. These include the presence of null variants (such as nonsense or frameshio variants) in a gene where loss of funcYon is a known disease mechanism, de novo occurrence of the variant (with proven paternity and maternity) in a paYent with the disease but not in healthy individuals, and segregaYon of the variant with the disease in mulYple affected family members. AddiYonally, the variant must be absent or extremely rare in healthy controls, show a staYsYcally significant associaYon with the disease in quesYon, and exhibit funcYonal studies supporYve of the damaging effect. For a variant to be considered ‘likely pathogenic’, it must meet a subset of these criteria with a moderate level of evidence, indicaYng a strong suspicion of pathogenicity, though not as conclusive as for the ‘pathogenic’ classificaYon19. If a geneYc variant does not meet the criteria to be classified as ‘pathogenic’ or ‘likely pathogenic’, and there is also insufficient evidence to classify it as ‘benign’ or ‘likely benign’, it is typically categorized as a ‘variant of uncertain significance’ (VUS). It is esYmated that VUSs consYtute ~40% of all variants idenYfied19. The VUS designaYon indicates that the current understanding and available evidence are not adequate to make a definiYve classificaYon regarding the variant's impact on protein funcYon and paYent health, making the VUS not clinically acYonable. Over Yme, as more research is conducted and more data becomes available, the classificaYon of a VUS may be revised. This dynamic aspect of variant classificaYon underscores the significant need for ongoing research, functional validation, and data sharing to better understand the implications of VUSs, to reclassify them accurately over time, and to 7 elucidate the complete genetic basis of IEIs. This will ultimately improve the accuracy of diagnoses and guide more effective treatments. In addressing VUSs within the realm of rare IEIs, Dr. Jean-Laurent Casanova has proposed a methodological framework that emphasizes the importance of individual case studies for the interpretation and resolution of VUSs20. Casanova advocates for the use of stringent criteria to establish a definitive link between a genotype and its clinical phenotype, a process especially critical when the evidence is based on single-case data. Firstly, the genotype under consideration should not be found in individuals lacking the associated clinical phenotype (although the possibility of having a disease with variable penetrance should always be considered). Secondly, experimental validation is required to demonstrate that the VUS adversely affects, modifies, or alters the gene product's expression or function. Lastly, the relationship between the genotype and the clinical phenotype must be corroborated through relevant cellular models, or, when this is not achievable, through analogous animal phenotypes20. This approach underlines the necessity of rigorous and comprehensive experimental and clinical validation when resolving VUSs, especially in the context of rare diseases where cohort studies may not be feasible. 1.2. The expanding spectrum of clinical manifestations associated with IEIs Historically, the image of children with IEIs living in sterile 'bubbles' due to their compromised immune systems has dominated the public perception. Clinical practice often mirrors this notion, with diagnostic criteria heavily weighted towards increased susceptibility to infections, as listed in the Jeffrey Modell Foundations' 10 warning signs of primary immune deficiency used by many clinicians21. However, this perception only scratches the surface of a much broader spectrum22. 8 We now recognize that IEIs may manifest in diverse ways, including malignancies, autoimmune disorders, and allergic conditions, with or without the hallmark of enhanced susceptibility to infections. These manifestations can occur in isolation or in combination, showcasing the immune system’s critical role in a wide array of pathological processes, and reflecting the complexity of these diseases. In fact, several subgroups of IEIs have been described that redefine our understanding and approach to treatment. These subgroups include primary immunodeficiency diseases (PIDDs), primary immune regulatory disorders (PIRDs), and the most recently recognized primary atopic disorders (PADs)23-26. PIDDs consist of conditions where the predominant feature is recurrent and/or severe infections23. PIRDs are characterized by a dysregulation of the immune system, leading to a range of clinical presentations that may include autoimmune phenomena, lymphoproliferation, and at times, a paradoxical predisposition to infections26. Conversely, PADs typically present with severe, early-onset allergic symptoms that can overshadow the traditional infectious complications expected in IEIs4,22,24,25,27. This revelation indicates that the current clinical guidelines and warning signs may be insufficient, as they fail to encompass the full breadth of IEI presentations. Expanding our diagnostic criteria to include these additional clinical features is crucial to uncovering the vast 'iceberg' of undiagnosed cases and developing a more nuanced approach to treatment and management of IEIs. Recognizing the distinct clinical patterns of PIRDs and PADs allows clinicians to move beyond the outdated 'bubble' concept and adopt more individualized diagnostic and therapeutic strategies, tailored to the intricate presentations of each patient's immune dysregulation and/or deficiency. 9 Beyond the immune system, IEIs can also be associated with systemic effects due to pleiotropy and the interconnectedness of immune function with other bodily systems. Genetic variants implicated in IEIs often influence multiple pathways, leading to pleiotropic effects that can manifest in the central nervous, digestive, renal, or pulmonary systems. Chronic inflammation, immune dysregulation, and susceptibility to infections further contribute to the multisystem involvement seen in these disorders1,9. Consequently, clinicians must be vigilant for a spectrum of warning signs that extend beyond typical immunological symptoms; these may encompass anomalies in other body systems leading to developmental delays, atypical growth patterns, and organ-specific dysfunctions, all of which could indicate the systemic reach of IEIs. Figure 1.1. Beyond the tip of the iceberg: Unveiling the multifaceted clinical spectrum of IEIs. Schematic iceberg model illustrating that while increased infection susceptibility is the most recognized sign, it represents just a small visible portion of the broad spectrum of IEI manifestations, which include malignancy, autoimmunity, and allergy —elements that form the substantial, yet often overlooked mass below the surface. 10 1.3. Primary immune regulatory disorders (PIRDs) PIRD are a growing subset of IEIs that are characterized by immune dysregulation. Out of the ~485 genes associated with IEIs listed by the International Union of Immunological Societies (IUIS) Expert Committee on inborn errors of immunity, approximately 30% of them have clinical manifestations predominantly resulting from immune-mediated pathology26,28. Unlike classical IEIs that often present with severe, recurrent, or unusual infections, PIRDs present with a diverse range of clinical manifestations including autoimmunity, autoinflammation, hyperinflammation, lymphoproliferation and malignancy that arise from defects in diverse immune regulatory pathways. Based on the immune pathways involved, PIRDs can be further be subcategorized into subsets including Tregopathies which are characterized by defective regulatory T cells (Tregs) leading to conditions such as Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome (IPEX) and IPEX-like disorders. PIRDs can also stem from exaggerated innate and adaptive inflammatory responses, as observed in autoinflammatory syndromes and hyperinflammatory disorders. Furthermore, PIRDs may develop from abnormal inflammatory signals due to improper sensing and clearance of cell debris (debris defects), and can manifest as non-malignant lymphoproliferation, such as in Autoimmune Lymphoproliferative Syndrome (ALPS) or ALPS-like disorders. Additionally, certain PIRD genes may contribute to hematopoietic malignancies. Other PIRD categories include monogenic inflammatory bowel diseases (IBD) resulting from dysfunction in intestinal immune regulation, and rheumatologic diseases arising from a breakdown in self-tolerance. It is also possible for PIRD patients to exhibit a combination 11 of these phenotypes if the gene defect affects multiple immune regulatory pathways26. In the following section, I will discuss the subcategories that are relevant to the chapters of this thesis. 1.3.1. PIRDs manifesting as Tregopathies Tregopathies are a subset of PIRDs that result in regulatory T cell (Treg) deficiency or dysfunction, which can lead to multiorgan autoimmunity. Tregs play a crucial role in maintaining peripheral tolerance and immune homeostasis and preventing autoimmune diseases29,30. The main function of Tregs is to suppress the activity of other immune cells and they achieve this through various mechanisms, including the release of inhibitory cytokines, inducing cytolysis, metabolic disruption of target cells, modulation of dendritic cell function, and direct cell-to-cell contact with the target cells31. Tregs release cytokines such as Interleukin-10 (IL-10) and Transforming growth factor-beta (TGF-β) to inhibit the activity of other immune cells. They can also directly kill other immune cells through the release of cytotoxic molecules like Granzyme A and B. Tregs can induce apoptosis in other immune cells by depriving them of cytokines like IL-2, or by generating immunosuppressive molecules like Cyclic AMP (cAMP) and adenosine. Additionally, Tregs can suppress the activation and maturation of dendritic cells, which are antigen-presenting cells that play a crucial role in initiating immune responses. It is important to note that Tregs likely use a combination of these mechanisms to achieve their suppressive effects, and the relative importance of each mechanism may vary depending on the context and target cell type29-32. They are also involved in preventing excessive immune responses to harmless environmental antigens like allergens. Dysfunction or deficiency of Tregs can lead to autoimmune diseases, chronic inflammation, and allergies25,26. 12 The most extensively studied Tregopathies are IPEX syndrome and IPEX-like disorders which result from germline variants that result in Treg deficiency or dysfunction33. These disorders are primarily associated with non-infectious gut pathology, cytopenias, Type I Diabetes, and inflammation in the lungs and skin. IPEX syndrome is caused by variants in the FOXP3 gene, which encodes for an essential transcription factor that regulates the development and function of Tregs34. Additionally, several other genes encoding proteins that regulate Treg function have been associated with Treg cell defects leading to Tregopathies33. These genes include LOF variants in CD25, which encodes the high-affinity Interleukin-2 (IL-2) receptor on Tregs that acts as an IL-2 sink by binding and sequestering IL-2, a cytokine that promotes the growth, differentiation, and survival of T cells35. Additionally, LOF variants in Cytotoxic T lymphocyte-associated antigen 4 (CTLA4), encoded by CTLA4, have been identified, as CTLA4 serves as an inhibitory immune regulator necessary for Treg function36. Furthermore, LOF variants in LPS-responsive and beige-like anchor protein (LRBA), essential for CTLA4 trafficking to the cell surface, have been associated with Tregopathies37,38. 1.3.1.1. Variants in IKZF2 as a novel cause of Tregopathy in humans Helios, encoded by IKAROS Family Zinc Finger 2 (IKZF2), is a transcription factor that plays a crucial role in immune cell development and function. As Tregs are among the highest IKZF2 expressors, most studies have focused on the role of Helios in Treg development and function39-49. In particular, Helios has been shown to play an important role in maintaining the phenotype, stability, and function of Tregs,50-54. Tregs express CD25 that help clear IL-2 from the environment, depriving other T cells from gaining access to IL-2, which is an essential cytokine 13 for their activation, proliferation, and differentiation.55 In addition to acting as IL-2 ‘sponges’, Tregs do not produce or secrete IL-2. Helios has been previously shown to stabilize Treg phenotype and function by suppressing Il2 transcription in murine models.52 Interestingly, loss of Helios in Tregs leads to defective CD25 expression and abrogates the suppressive function of these cells by de-depressing the Il2 locus and allowing the production of IL-2 and other effector cytokines such as Interferon gamma (IFN-γ), Interleukin-17 (IL-17) and Tumor necrosis factor alpha (TNF-α).51,52 This has been linked to delayed onset autoimmunity in mice51,53. Both Ikzf2-/- and Treg-specific Ikzf2-/- mice develop progressive autoimmune disease associated with activated CD4/CD8 T cells, increased T follicular helper (TFH) and germinal center B cell numbers, autoantibody production, and increased proinflammatory cytokine production41,42,56. These findings highlight the importance of IKZF2 in maintaining immune homeostasis and preventing autoinflammatory responses in mice. Recent studies have implicated IKZF2 variants as a novel cause of Tregopathies in humans57-59. Hetemäki et al. described two related individuals presenting with combined immunodeficiency resulting from a heterozygous truncating LOF variant in IKZF2. This variant resulted in chronic T cell activation and increased production of proinflammatory cytokines both by effector T cells and Tregs.57 Patient Tregs exhibited a more activated and mature phenotype, as evidenced by increased IL-2 production and higher expression levels of immunosuppressive proteins CTLA-4 and CD39. Additionally, these Tregs retained their suppressive capacity while producing more IL-2, indicating an enhanced proinflammatory phenotype. In another study, Shahin et al. described an individual with immunodeficiency resulting from a homozygous 14 missense variant in IKZF2. Functionally, this variant resulted in defective Tregs with widespread transcriptional disturbances that led to an increase in the production of proinflammatory cytokines such as IL-2 and IFN-γ upon T cell stimulation, confirming the proinflammatory and dysregulated state of the patient.58 While the patient did not exhibit any autoimmune symptoms, the variant resulted in a dysregulated and proinflammatory state, which could potentially contribute to the development of autoimmune diseases in the future. In a second study, Shahin et al. idenYfied heterozygous IKZF2 variants in a cohort of paYents with diverse autoimmune manifestaYons, immune dysregulaYon, and lymphoproliferaYon, providing compelling evidence for the contribuYon of IKZF2 to the spectrum of Tregopathies. In a study conducted in our group, Lu et al. further elucidated the impact of a novel dominant-negaYve heterozygous IKZF2 variant on Treg cell funcYon and immune tolerance60. This patient exhibited several immunodysregulatory autoimmune features, including autoimmune hemolytic anemia (AIHA) and atopic dermatitis. Additionally, the patient's CD4+ T cells were hyperactive, producing elevated levels of nearly all T helper (TH) cytokines and readily proliferating in response to stimulation. Furthermore, the patient's Tregs developed normally but aberrantly produced high levels of many TH cytokines and had impaired suppressive function60. These studies collecYvely highlight the emerging role of IKZF2 as a geneYc determinant of Tregopathies in humans and underscore the significance of IKZF2 in the maintenance of immune homeostasis and the prevenYon of autoimmunity. In chapter 3 of this thesis, I will introduce and describe a newly idenYfied disorder in two unrelated individuals characterized by immunodysregulaYon, craniofacial anomalies, hearing impairment, athelia and developmental delay (designated as 15 ICHAD) caused by two novel de novo heterozygous variants in IKZF2, further elucidaYng the role of Helios in immune regulaYon and the pathogenesis of autoimmunity in humans. 1.4. Primary atopic disorders (PADs) It is now appreciated that some IEIs can predominantly cause severe and early onset allergic disease, such as asthma, food allergy, elevated eosinophil counts and atopic dermatitis. The term PADs was recently coined to describe this group of heritable diseases which result from single-gene defects that affect the development, function, and regulation of the immune system and the skin epithelial barrier, leading to dysregulated pathogenic allergic effector responses that occur irrespective of initial sensitization 24,25,27. These disorders are clinically and genetically heterogeneous with more than 46 known monogenic allergic diseases identified to date 4,22,61. Some monogenic allergic diseases result from variants in genes that encode components of the immune system and the skin epithelial barrier, such as cytokines, receptors, structural proteins, and enzymes, while others are caused by variants in genes that regulate immune system development, activation, or differentiation such as transcription factors and signalling molecules 4. PADs can be categorized mechanistically based on the pathways identified as critical in the development of atopy. These pathways range from focal defects in immune cells and epithelial barrier function to global changes in metabolism. Some of the subgroups and mechanisms include impaired T cell receptor (TCR) signaling and cytoskeletal remodeling, altered cytokine signaling, T cell repertoire restriction, tolerance failure, metabolic disturbance, skin barrier disruption, and mast cell deregulation2,4,22,24,25,27. The clinical and molecular heterogeneity of PADs underscores their complexity and emphasizes the need to understand the underlying 16 molecular and immunological mechanisms involved in their pathogenesis to develop targeted therapeutics that can effectively address the dysregulated physiological pathways. Chapter 2 of this thesis offers a comprehensive discussion of PADs, drawing upon extensive research presented in three of my detailed review papers. Each paper concentrates on a different facet of PADs: the first provides an exhaustive compilation of the clinical manifestations of known PADs, thereby aiding in establishing a set of diagnostic red flags for these disorders22. The second review delves into the molecular mechanisms underlying PADs, with a particular emphasis on their genetic underpinnings and the potential for targeted precision therapies4. The third review examines PADs that are attributed to variants in transcription factor genes, shedding light on a more nuanced understanding of their pathogenesis2. Building upon this foundation, in Chapter 4, we describe a novel human PAD caused by a de novo heterozygous variant in ZBTB7B in an individual with early-onset, severe allergic inflammation accompanied by multiorgan fibrosis. 1.4.1. Variants in ZBTB7B as a novel cause of PADs in humans T-helper-inducing POZ/Kruppel-like factor (ThPOK), encoded by ZBTB7B, is a transcription factor that belongs to the zinc finger and BTB/POZ domain-containing (ZBTB) family of proteins62. In recent years, ThPOK has garnered significant attention in immunological research, primarily through insights gained from murine models. ThPOK plays a crucial role in the immune system, particularly in the regulation of T cell development, CD4/CD8 lineage decision, differentiation, and function63. The most studied role of ThPOK is its function in directing the differentiation of T cell lineages, particularly in promoting the development of CD4+ T cells and inhibiting the 17 development of CD8+ T cells in the thymus64. This discovery was initially made based on a spontaneous mutant mouse strain with biallelic missense variant in the second zinc finger domain of ThPOK, predicted to directly interact with DNA, leading to the inability of ThPOK to bind important DNA targets62. Helper-deficient (HD) mice showed a selective absence of mature CD4+ T cells and increased frequency of CD8+ cells65. The absence of functional CD4+ T cells in HD mice was linked to impaired humoral immune responses, altered cytokine production, impaired immune regulation, and altered immune cell populations. HD mice were noted to have reduced levels of serum immunoglobulins and showed impaired antibody responses to T-dependent antigens. They also had altered cytokine production profiles. Moreover, these mice had reduced numbers of Tregs and exhibited impaired immune regulation, as evidenced by increased susceptibility to autoimmune diseases and reduced ability to suppress immune responses to allogeneic grafts. Additionally, HD mice have altered immune cell populations, including increased numbers of CD8+ T cells and NK cells, and reduced numbers of CD4-CD8- double negative T cells62,65. As such, the HD mouse model and other conditional knock-out mouse models of ThPOK deficiency or LOF have been instrumental in elucidating the gene's role in immune system regulation. While the primary focus on ZBTB7B has been its immune functions, its role extends to other biological processes, although these are less well-understood. ThPOK is highly expressed in the skin and it is shown to be a negative regulator of type I collagen gene expression by binding specifically to the regulator regions of the type I collagen genes (COL1A1 and COL1A2) in mice66. 18 Despite advances in our understanding of the role of ThPOK in immunity, the direct implications of ThPOK variants in human health have remained largely unexplored. To date, there have been no documented cases of ThPOK variants reported in humans, nor has ThPOK been previously implicated as a cause of IEI or disease in humans. In Chapter 4 of this thesis, I describe the first human with de novo heterozygous variant in ZBTB7B presenting with severe, early-onset allergic diseases, multi-organ fibrosis, and growth failure. Our findings not only challenge our current understanding of ThPOK's role in the human immune system, but also opens new avenues for exploring its broader implications in other tissues and body systems in humans. 1.5. Thesis rationale and objectives Recent advances in genomics and bioinformatics have revolutionized our understanding of IEIs. Studying IEIs provides unique insights into the intricate workings of the immune system and offers opportunities for developing targeted therapeutic interventions. Related to this theme, the overarching goals of this thesis are as follows: 1. Novel candidate gene discovery and validation: The primary objective of this thesis is to identify and validate new genes associated with IEIs. This involves isolating DNA from patients with severe, early-onset allergic diseases and/or immunodysregulatory features, followed by genomic sequencing and bioinformatics analysis with the help of our collaborators. The goal is to uncover novel genetic variants that could be potential causative factors for these immune disorders. 19 2. Detailed functional characterization of genetic variants: Upon identifying candidate variants, the next step is to comprehensively characterize these variants at a molecular and cellular level. This includes studying the impact of these variants on gene expression (using assays such as immunoblotting and flow cytometry), protein function (using assays such as luciferase reporter experiments, electrophoretic mobility shift assay, single-cell RNA sequencing, and flow cytometry), and cellular signaling pathways (using experiments to assess the transcriptome of transfected or stably transduced cells). Understanding the functional consequences of these variants is key to elucidating their role in the pathophysiology of IEIs. 3. Discovery of novel immune pathways and molecules: This objective focuses on uncovering new immune pathways and molecules that are affected by the identified variants. We aim gain insights into previously unrecognized mechanisms underlying immune dysregulation and atopy. 4. Application to precision therapies and patient management: The final goal of this research is to translate our findings from functional studies into practical clinical applications. This will involve a thorough investigation to determine if there are existing precision medicine therapies that can be effectively repurposed to treat patients with the newly identified genetic variants associated with IEIs. In cases where current precision therapies are not applicable, the focus will shift to informing patients and their clinical 20 teams about our findings. This information will be crucial in guiding the management of these patients, aiding in the prevention of future complications. The insights gained will be grounded in our research findings and supported by existing literature, aiming to provide a comprehensive understanding of the disease implications. Ultimately, this approach seeks to enhance patient care by aligning treatment strategies with the unique genetic and clinical profiles revealed through our studies. The pivotal data chapters of this thesis collectively advance our understanding of IEIs, with a particular focus on PADs and PIRDs. Chapter 2 lays the foundational clinical and molecular insights into PADs, setting the stage for the exploration of novel genetic underpinnings of PADs. Chapter 3 introduces ICHAD syndrome as a new disorder with a distinctive clinical picture, driven by dominant-negative variants in IKZF2, thereby expanding the catalog of immunodysregulatory diseases. Chapter 4 describes a new PAD caused by a novel dominant-interfering heterozygous variant in ZBTB7B, which is implicated in severe allergy and multiorgan fibrotic disease in a single patient. Together, these chapters offer new perspectives on the diagnosis and management IEIs and highlight the profound impact of precise genomic insights on patient care. 21 CHAPTER 2: Constructing a framework for understanding the molecular and clinical underpinnings of primary atopic disorders 2.1. Introduction and rationale We have begun to appreciate that severe allergic inflammation may be the sole or the earliest manifestation of a monogenic immune defect67,68. In fact, some monogenic inborn errors of immunity (IEIs) predominantly cause severe allergic disease, such as asthma, food allergy, elevated eosinophil counts and/or atopic dermatitis in the absence of common features associated with IEIs. The term primary atopic disorders (PADs) was coined in 2018 to describe this group of heritable genetic disorders presenting with dysregulated pathogenic allergic effector responses irrespective of initial sensitization24,25. IEIs and PADs are not mutually exclusive. All PADs have allergy symptoms as a substantial feature but may have other features of PIDs such as susceptibility to infections and inflammation. PADs are clinically and genetically heterogeneous with more than 46 known monogenic causes identified to date (Table 2.1) 4,22,61. The pathogenic underpinnings vary depending on the affected gene, spanning disruption of skin epithelial barrier function, through disruptions in cellular metabolism or actin cytoskeleton function, to defects in transcription factors (TFs) or signalling molecules important for immune cell development or function 4. In this chapter, we focus on different aspects of the clinical and molecular underpinnings of PADs because (i) the affected pathways provide important insights into the pathogenesis of allergy; and (ii) these conditions are likely underdiagnosed since allergies are traditionally believed to be complex polygenic disorders. 22 Table 2.1. Monogenic allergic disorders (i.e. PADs) and their targeted treatments. Gene Protein function Inheritance Allergic & other key manifestations Treatment strategies Epithelial barrier disruption DSG1 Desmosomal protein69 AR, LOF Severe dermatitis, FA, ­IgE, metabolic wasting Secukinumab70,71 CDSN Component of corneodesmosomes72 AR, LOF Peeling skin, diffuse ichthyosis, erythroderma, pruritus, FA, ­IgE Protein replacement therapy (Pre-clinical)73 DSP Desmosomal protein74 AD, LOF Severe dermatitis, erythroderma, multiple FA, eosinophilia, ­IgE, metabolic wasting75 IVIG + systemic acitretin for dermatitis75 Ustekinumab for erythroderma76 SPINK5 Protease inhibitor crucial to epithelial tissue homeostasis77 AR, LOF Erythroderma, AR, asthma, FA, angioedema, EGID, eosinophilia, ­IgE, trichorrhexis invaginatum, ichthyosis78 IVIG Dupliumab Omalizumab Ustekinumab79 80 Cytokines and cytokine signaling defects STAT6 (GOF) A signal transducer for many cytokines and growth factors through the cytoplasm81 AD, GOF Eczema, EGID, FA, eosinophilia, ­IgE, infections, connective tissue abnormalities, recurrent anaphylaxis, lymphoma82,83 Dupilumab83 Ruxolitinib82 STAT3 (LOF) A signal transducer for many cytokines and growth factors through the cytoplasm81 AD, DN LOF Eczema, EGID, FA, eosinophilia, ­IgE, infections, connective tissue abnormalities, lymphomas84 HSCT85 Omalizumab86 ERBB2IP Negative regulator of Transforming growth factor beta (TGF-b) through interaction with SMADs87 AD, LOF Eczema, FA, EGID, eosinophilia, ­IgE, connective tissue abnormalities Supportive treatment IL6ST Signal transducer of the IL-6 receptor complex, important in the Janus kinase/Signal transducer and activator of transcription (JAK/STAT) pathway88 AR, LOF Eczema, FA, eosinophilia, ­IgE, infections, connective tissue abnormalities89 Supportive treatment88 ZNF341 Transcription factor that promotes STAT3 transcriptionr90 AR, LOF Eczema, eosinophilia, ­IgE, infections, connective tissue abnormalities90 Supportive treatment90 TGFBR1 Receptor for TGF-b91 AD FA, eczema, asthma, AR, EGID, eosinophilia, ­IgE, vascular and connective tissue abnormalities92 Supportive treatment TGFBR2 Receptor for TGF-b 91 AD FA, eczema, asthma, AR, EGID, eosinophilia, ­IgE, vascular and connective tissue abnormalities92 Supportive treatment IL6R IL-6 receptor93 AR, LOF Eczema, eosinophilia, ­IgE, infections94 Supportive treatment94 STAT5B (LOF) A signal transducer for many cytokines and growth factors through the cytoplasm95 AD or AR, LOF Eczema, ­IgE, growth failure, autoimmunity Cyclosporine therapy for eczema96 23 Gene Protein function Inheritance Allergic & other key manifestations Treatment strategies IL2RA Component of the IL-2 receptor, also known as CD2597 AR, LOF Eczema, severe autoimmune enteritis, viral infections, lymphoproliferation Rapamycin98 FOXP3 Main transcription factor for the differentiation of Regulatory T cells (Tregs)99 XL, LOF AD, asthma, FA, EGID, eosinophilia, ­IgE, infections, autoimmunity, enteropathy Rapamycin HSCT 100,101 STAT3 (GOF) A signal transducer for many cytokines and growth factors through the cytoplasm81 AD, GOF Eczema, lymphadenopathy, multiorgan autoimmunity, infections, short stature Tocilizumab ± JAK inhibitors102-104 STAT1 A signal transducer for many cytokines and growth factors through the cytoplasm105 AD, GOF Eczema, infections, autoimmunity, enteropathy, short stature, vascular abnormalities HSCT 106 JAK inhibitors (Ruxolitinib)107,108 JAK1 Tyrosine kinase, associates with cytokine and growth factor receptors to transmit signals via phosphorylation of STAT proteins109 AD, GOF AD, asthma, FA, eosinophilia, poor growth JAK inhibitors (Ruxolitinib110 and Tofacitinib111) STAT5b (GOF) A signal transducer for many cytokines and growth factors through the cytoplasm95 Somatic, GOF AD, FA, urticaria, eosinophilia, diarrhea Ruxolitinib112 TBX21 Important transcription factor for the differentiation of T help 1 (Th1) cells and the production of Interferon gamma (IFN-g)113 AR, LOF Upper airway hyperresponsiveness, eosinophilia, mycobacterial disease Supportive treatment114 Actin cytoskeleton defects WAS Initiates actin polymerization by binding to Arp2/3 complex subunit 1B XL, LOF Eczema, eosinophilia, ­IgE, infections, thrombocytopenia, autoimmunity, malignancy HSCT 115 Gene therapy116,117 WIPF1 Key regulator of actin cytoskeleton remodelling118 AR, LOF Eczema, ­IgE, diarrhea, infections, thrombocytopenia HSCT 119 ARPC1B Component of the Arp2/3 complex that plays roles in actin polymerization120 AR, LOF Eczema, FA, asthma, eosinophilia, ­IgE, vasculitis, infections, bleeding HSCT 121 DOCK8 Guanine nucleotide exchange factor (GEF) which plays roles in immune cell migration, phagocytosis and adhesion, actin cytoskeleton regulation122, Toll-like receptor signaling and STAT3 activation123 AR, LOF AD, FA, AR, asthma, EGID, eosinophilia, ­IgE, infections, autoimmunity, malignancy124 Supportive HSCT 125-129 Dupliumab for AD130 NCKAP1L Subunit of the WAVE regulatory complex and a regulator of actin polymerization131 AR, LOF Atopic disease, ­IgE, infections, antibody abnormalities, hepatosplenomegaly, lymphadenopathy, autoimmunity132 NA Antigen presentation defects RFXANK A subunit of the regulatory factor X (RFX) complex, which assembles on promoters of MHCII genes AR, LOF Eczema, allergic cutaneous reaction to antibiotics, transient hypereosinophilia, infections, HSCT 135 24 Gene Protein function Inheritance Allergic & other key manifestations Treatment strategies and regulates their transcription133 chronic diarrhea, failure to thrive, autoimmunity134,135 Attenuated antigen receptor signaling CARD11 T and B cell-specific scaffold protein that interacts with BCL10 and MALT1 upon engagement of BCR/TCR with antigens136 AD, DN AD, asthma, AR, FA, eosinophilic esophagitis, eosinophilia, ­IgE, infections, autoimmunity, lymphoma169 Supportive treatment Dupilumab, omalizumab for AD137 CARD14 Scaffold protein that promotes the activation of NF-κB pathway by recruiting BCL10 and MALT1138 AD, LOF Asthma, allergy, atopic dermatitis, ­IgE Supportive treatment139 MALT1 Protease regulating NF-κB signaling140 AR, LOF Eczema, ­IgE, poor growth, infections141 HSCT 142-144 CARMIL2 Regulator of actin polymerization in T cells145 AR, LOF AD, asthma, FA, cold urticaria, ­IgE, infections HSCT 146 LIG4 DNA ligase involved in double stranded DNA (dsDNA) break repair during V(D)J recombination147 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 147 DCLRE1C Endonuclease involved in the non-homologous end-joining of DNA segments during V(D)J recombination148 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 149 RAG1 Catalytic component of the Recombination activating gene (RAG) complex involved in introducing dsDNA breaks during V(D)J recombination150 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 151 RAG2 Core component of the RAG complex involved in introducing dsDNA breaks during V(D)J recombination150,152 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 151 ADA Deaminating enzyme involved in purine metabolism that plays a key role in T and B cell development153 AR, hypomorphic LOF AD, AR, asthma, FA, urticaria, eosinophilia, ­IgE154, diarrhea, infections, multiorgan abnormalities HSCT 155 Enzyme replacement therapy156 Gene therapy157 IL7RA Alpha chain of IL-7 receptor158 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 159,160 IL2RG Gamma chain of the receptor of many cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-2161,162 XL Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 163 Gene therapy164 ZAP70 Tyrosine kinase, associates with T cell receptor complex upon engagement with antigen presenting cells165 AR, LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 166 CHD7 Chromatin remodeler that regulates gene expression AR, likely LOF* Erythroderma, eosinophilia, ­IgE, enteropathy, infections * HSCT 168 PBMC transplantation168 25 Upward arrow indicates elevation. AD (fourth column), atopic dermatitis; AD (third column), autosomal dominant; AR (fourth column), allergic rhinitis; AR (third column), autosomal recessive; DN, dominant-negative; EGID, eosinophilic GI disease; FA, food allergy; GOF, gain of function; HSCT, hematopoietic stem cell transplantation; LOF, loss of function; XL, X-linked. *Omenn syndrome and/or Omenn syndrome–like presentations, resulting from hypomorphic LOF variants that retain residual function. Gene Protein function Inheritance Allergic & other key manifestations Treatment strategies during thymic development167 Donor lymphocyte infusions169 Thymus transplantation170 TBX1 Crucial transcription factor for thymus formation during fetal development171 Haploinsufficient Erythroderma, eosinophilia, ­IgE level enteropathy, infections * Thymus transplantation170 Altered cellular metabolism PGM3 Carbohydrate-modifying enzyme involved in glycosylation of proteins172 AR, LOF Eczema, FA, asthma, eosinophilia, ­IgE level, bronchiectasis, infections, developmental delay, autoimmunity, malignancy, connective tissue abnormalities84 HSCT 173 CARD11 facilitates glutamine uptake through Alanine, serine, cysteine transporter 2 (ASCT2) AD, DN AD, asthma, AR, FA, EGID, eosinophilia, ­IgE level, infections, autoimmunity, lymphoma169 Glutamine supplementation (pre-clinical)170 Granulocyte dysregulation KIT Important for proper mast cell functioning including survival, differentiation, and chemotaxis174 AD, GOF Systemic mastocytosis Imatinib (Tyrosine kinase inhibitor)175 Avapritinib176 (phase II) PLCG2 Important enzyme for activation function of B cells, NK cells, mast cells, and macrophages177 AD, GOF Cold urticaria, bacterial infection, autoimmunity, skin granulomas Supportive treatment178 ADGRE2 A member of the epidermal growth factor seven transmembrane subfamily of G protein-coupled receptors (GPCRs) that acts as a cell surface receptor, promotes cell adhesion in neutrophils, macrophages, and mast cells179 AD, GOF Vibratory urticaria179 NA NLRP3 Intracellular receptor involved in the recognition of pathogen- and danger-associated molecular patterns180 AD Urticarial rash, eosinophilia, central nervous system involvement, arthropathy181 Canakinumab Anakinra Rilonacept182 26 2.1.1 PADs informing our understanding of fundamental human biology PADs have played a crucial role in informing our understanding of fundamental human biology and immune system functioning. One well-characterised PAD is Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome, which includes severe allergic inflammation as a key feature183. In 2001, pathogenic variants in Forkhead box P3 (FOXP3) were found to cause IPEX183, leading to the identification of FOXP3 as the essential transcription factor for regulatory T cell (Treg) development and the maintenance of tolerance184. Modulating Treg activity has since emerged as an exciting treatment for both autoimmune and neoplastic disorders185,186. Another PAD that has informed our understanding of allergic inflammation is autosomal dominant Hyper IgE syndrome (HIES) or Job Syndrome, caused by heterozygous dominant-negative variants in STAT3 (signal transducer and activator of transcription 3)187,188. Common features of STAT3 loss-of-function (LOF) include recurrent bacterial and fungal respiratory and skin infections, eczema, eosinophilia and elevated IgE189. Studying STAT3-HIES has uncovered STAT3’s key role in Th17 differentiation and function and has demonstrated the importance of Th17 cells in protecting against mucocutaneous fungal and bacterial infections in humans189,190. Collectively, PADs have been instrumental in driving our fundamental understanding of human biology and the pathogenesis of allergic disease. 2.1.2 Studying PADs reveals the genetic architecture of common allergies As we widen our lens from individual patients to the population level, understanding monogenic causes of severe allergic inflammation can inform our understanding of common complex diseases such as food allergy and asthma, which carry an immense health and economic 27 burden191. Genetic association studies support the hypothesis that variation in several PADs-causing genes contribute to common allergic diseases (Table 2.2)192,193. As an example, Netherton syndrome is a PAD caused by pathogenic biallelic variants in Serine protease inhibitor Kazal-type 5 (SPINK5) which encodes a protease inhibitor in desmosomes194. Autosomal recessive loss of SPINK5 destabilizes desmosomes, resulting in defective barrier function, and clinically manifests as excessive desquamation and severe ichthyosis, along with atopy and elevated IgE194. Interestingly, SPINK5 single nucleotide variants are associated with polygenic atopic dermatitis195,196, asthma197-199 and food allergy200. Although the link between defective skin barrier and allergic inflammation is not clearly elucidated, it can be related to the inappropriate introduction of allergens to the antigen presenting cells of the skin in the context of damage and inflammation201. As such, a series of studies are investigating the effectiveness of the administration of daily emollients in preventing atopic dermatitis and food allergy in high-risk children202, with promising results203. This is an example of how characterizing the mechanisms behind allergic disease in monogenic PADs may help to uncover new targets for preventing and treating common allergic disease. 28 Table 2.2. Number of published genetic association studies linking variation in known PAD genes to atopic disorders in the broader population. Atopic Dermatitis Asthma Allergic Sensitization* Food Allergy Allergic Rhinitis Elevated Total IgE Eosinophil Count Eosinophilic Esophagitis CARD11 3 44-46 CARMIL2 1 47 CDSN 1 48 CHD7 5 47, 49-52 DSG1 1 53 FOXP3 3 54-56 2 54, 55 1 56 IL2RA 4 47, 57-59 8 58-65 1 54 2 58, 59 3 47, 49, 51 IL6R 4 47, 57-59 5 58, 59, 61, 66, 67 2 58, 59 IL7R 3 57-59 1 58 2 58, 59 JAK1 1 47 4 47, 49-51 MALT1 1 47 SPINK5 3 68-70 3 68, 71, 72 2 68, 73 1 73 1 68 2 74, 75 STAT3 2 57, 76 1 62 STAT5b 2 58, 59 3 58, 59, 61 2 58, 59 TBX21 5 60-63, 77 1 47 TGFBR1 3 59, 61, 64 TGFBR2 1 54 WAS 1 49 *Allergic sensitization is defined as positive skin prick test, elevated specific IgE towards a certain allergen/ or allergic sensitization to specific allergen. The superscripts correspond to references in the bibliography. 2.2. Clinical red flags for the diagnosis of PADs The nuanced differentiation of PADs from prevalent polygenic allergic diseases is not only challenging for clinicians but carries significant implications for affected patients. The advent of 29 next-generation sequencing (NGS) has revolutionized the identification of monogenic allergic diseases and opening the doors to precision-based therapies tailored to the patients’ specific molecular defects. Such advancements underscore the necessity for clinicians to proficiently recognize PADs, ensuring that patients receive the most informed therapeutic options. Our review in the Journal of Allergy and Clinical Immunology aims to underscore this imperative, urging clinicians to consider PADs in patients presenting with severe, early-onset, or coexisting allergic conditions, even in the absence of conventional IEI-associated indicators22. This section delves into identifying key clinical features and warning signs (Figure 2.1) that necessitate PADs assessment in both adult and pediatric patients, advocating for the timely diagnosis and management to significantly enhance patient outcomes. 2.2.1 Atopy Patients who present solely with allergic immune dysregulation without a history of infection present a particular challenge for clinicians to differentiate between a monogenic or polygenic cause without genetic investigations67. An example of this is CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS)68,204. While some CADINS patients have a history of significant infections, autoimmunity, and/or malignancy, others may present with isolated allergic disease such as atopic dermatitis68,205. Based on these findings, Ma et al. suggest that patients with severe atopic dermatitis and a family history suggesting autosomal dominant inheritance should undergo genetic testing to evaluate for CADINS. It is also important to recognize that some IEI patients may initially present with atopic disease and only later develop other features of immune dysregulation and/or infection, as seen in some IPEX patients206. For 30 example, in a systematic review of patients with IPEX and IPEX-like syndrome, 17 patients were found to initially present with atopic disease, specifically dermatitis and food allergy, before any other manifestations206. Wiskott-Aldrich syndrome (WAS) is another IEI that is known to encompass atopic disease, with its classic clinical triad of eczema, thrombocytopenia, and severe/recurrent infection207. Absence or presence of eczema is one of seven entities of the clinical scoring tool used to define different clinical phenotypes of WAS208. The clinical findings of WAS are varied, with the absence of infection and immunologic abnormalities in some patients208. Another complicating factor is when a patient has no infectious history to suggest an IEI but presents with a combination of different immune dysregulation phenotypes such as patients with Janus kinase 1 (JAK1) gain-of-function (GOF), and Signal transducer and activator of transcription 5 B (STAT5b) GOF which can present primarily with severe atopy and eosinophilia accompanied by growth failure and/or autoimmunity209,210. It is important to keep in mind that although we have presented examples of how these IEIs present with atopic disease, each clinical presentation is heterogenous, and atopic disease may or may not be the most prominent feature. For example, the presentation in Zeta chain of T cell receptor associated protein kinase 70 (ZAP70) deficiency is usually patients under 2 years of age with typical findings of SCID211. The heterogeneity of this PAD can lead to extremes in severity, ranging from a classical SCID presentation to an Omenn-syndrome-like presentation with eczematous skin lesions and elevated laboratory indicators of allergic inflammation211. This highlights the importance of considering PADs in the absence of infection and maintaining a low threshold to pursue genetic testing in patients suffering from severe allergic inflammation. 31 2.2.2 Autoimmunity Autoimmunity often coexists with allergic manifestations in patients with PADs and is particularly prominent in T cell receptor signalling defects and actinopathies201. One such example is Dedicator of cytokinesis 8 (DOCK8) deficiency, which causes combined immunodeficiency and is characterized by recurrent infections including cutaneous viral and pulmonary infections, severe allergic disease, and autoimmunity212. DOCK8 functions as a guanine nucleotide exchange factor, with roles in actin cytoskeleton regulation and nuclear translocation and transcription of STAT3-dependent genes124. Several autoimmune complications have been described in DOCK8-deficient patients, such as hemolytic anemia, vasculitis, uveitis, hypothyroidism, nephritis, and systemic lupus erythematous213. Autoimmunity in DOCK8 deficiency is likely due to partial defects in T cell function214. Similarly, IPEX syndrome is an X-linked recessive PAD commonly associated with autoimmune manifestations such as type 1 diabetes mellitus, thyroiditis, chronic dermatitis, cytopenias, and severe enteropathy, with manifestations beginning as early as the perinatal/neonatal period101. Notably, autoimmune endocrinopathy is not seen in all cases of IPEX syndrome, and some patients may present solely with severe atopy215. Actinopathy that leads to severe allergic inflammation in conjunction with autoinflammation is caused by variants in ARPC1B, which encodes for a protein that plays an important role in actin polymerization and cellular motility. Several studies have reported individuals with ARPC1B deficiency presenting with combined immunodeficiency combined with vasculitis, gastroenteritis, extensive atopic dermatitis, food-induced anaphylaxis, elevated IgE, eosinophilia, failure to thrive, and platelet abnormalities216, 217,218. Although it is not completely understood, the exaggerated Th2 responses 32 and atopic features seen in these individuals may be explained by abnormal Treg function218. Thus, autoimmunity in addition to severe allergic inflammation should raise suspicion for a PAD, and further investigations are warranted. 2.2.3 Malignancy Malignancy is seen in a number of PADs, notably DOCK8 deficiency212, CADINS219 and WAS220. In keeping with other forms of IEIs, cancer most commonly affects PAD patients in the setting of chronic viral infections and/or inflammation220. In a study of 136 patients with DOCK8 deficiency, close to 17% were reported to have an epithelial cell or hematological cancer in childhood213. For example, Buchbinder et al. describe a case of a 22 year old Caucasian male with a history of B lymphoblastic leukemia who presented with a new diagnosis of Epstein-Barr virus (EBV)-associated diffuse large B cell lymphoma (DLBCL)220. This diagnosis of malignancy was the first overt sign of a monogenic IEI for this patient, as he lacked elevated IgE220 and eosinophilia124, which are typically hallmark laboratory findings in DOCK8-deficient patients. DOCK8 deficiency has also been reported in patients with malignancies such as squamous cell carcinoma associated with human papilloma virus (HPV) and EBV-associated smooth muscle tumors, as well as others that are not thought to be associated with viral infections such as microcystic adnexal carcinoma124. DOCK8 deficiency serves as a reminder that a history of malignancy (in particular those with childhood onset, viral-association, or hematologic) in a patient with underlying atopy should prompt consideration of an underlying PAD. 33 2.2.4 Short stature Short stature and poor growth in the context of allergic inflammation are notable red flags for PADs, and can be seen in IPEX syndrome, Mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALT1) deficiency221, DOCK8 deficiency222, JAK1 GOF210, and STAT5b GOF223, among others. This is in part attributable to chronic allergic inflammation, which has a negative association with linear growth.224 In certain PADs, growth can also be impaired secondary to the molecular defect’s role in signaling pathways shared between growth factors and cytokines involved in allergic inflammation. STAT5b is responsible for the majority of growth hormone (GH)-induced Insulin-like growth factor (IGF)-1 production223. When growth hormone binds to its receptor, this activates the JAK/STAT signaling pathway including STAT5b. Dysregulated STAT5b activation as seen in patients with STAT5b variants causes growth failure due to IGF-1 deficiency and growth hormone insensitivity223. Patients often present with a normal birth weight, but severe postnatal growth failure including resistance to growth hormone treatment223,225. Clinicians must therefore consider PADs when assessing patients with impaired growth and laboratory/clinical evidence of allergic inflammation. 2.2.5 Infections Obtaining a detailed infectious history is another critical component to PAD evaluation. The spectrum and severity of infections provides useful clues regarding the potential molecular etiology of the patient’s immune disorder. While certain PADs are associated with broad susceptibility to infections, others predispose to a narrow range of infections. Omenn syndrome falls into the former category, with patients suffering from life-threatening infections from a 34 variety of pathogens. Omenn syndrome is a rare form of severe combined immunodeficiency (SCID) caused by hypomorphic variants in genes important for T cell signaling and function, leading to oligoclonal expansion of T cells skewed towards type 2 inflammation226,227. Aleman et al. reviewed 49 patients with Omenn syndrome and over 70% suffered from recurrent infections, with more than 50% of these infections caused by either Candida albicans or Pneumocystis carinii228. In addition to these serious infections, Omenn syndrome patients also present with generalized erythroderma, alopecia, chronic diarrhea, pneumonitis, and failure to thrive227. Patients’ erythroderma may be misdiagnosed as atopic dermatitis early in the disease course as skin manifestations can worsen over time. Maintaining a low threshold to consider Omenn syndrome is therefore critical to avoid unnecessary diagnostic delays227. Cutaneous infections are a classic feature of many PADs, reflecting impaired skin barriers secondary to anatomical defects or chronic inflammation, as well as factors inherent to innate and/or adaptive immune dysfunction. Biallelic pathogenic variants in MALT1 cause combined immunodeficiency due to defective CARD11–BCL10–MALT1 (CBM) signalosome assembly, downstream NF-κB activation and MALT1 paracaspase proteolytic activity142,229. Affected patients suffer from neonatal-onset severe and persistent dermatitis, elevated IgE and eosinophilia. Skin infections may be caused by a variety of pathogens, including Staphylococcus aureus, varicella-zoster virus, herpes simplex virus and Candida albicans230. These patients also suffer from pulmonary and gastrointestinal infections230. 35 2.2.6 Connective tissue abnormalities Connective tissue abnormalities are observed in many PADs secondary to increased Transforming growth factor beta (TGF-b) signaling. Patients affected by STAT3-HIES may experience abnormal dentition, joint hyperextensibility, osteopenia, bone fractures, scoliosis, facial dysmorphism, and vascular abnormalities231,232. Facial features such as prominent forehead, facial asymmetry, and large interalar distance may not be present in early life, as they can accumulate with disease progression and time232. This highlights the importance of re-evaluating a patient’s phenotype throughout the life course, as certain syndromic features may not be apparent early in life. The connection between increased TGF-b activation and defective STAT3 was recently clarified by Lyons et al233. They identified a PAD caused by ERBB2-interacting protein (ERBIN) deficiency with clinical similarities to STAT3-HIES. ERBIN is a protein that acts as an anchor for SMAD proteins, which are the main signal transducers for TGF receptors. This led to the discovery that STAT3 negatively regulates TGF-b signaling through ERBIN. Deficiencies in STAT3 and ERBIN were both found to impair STAT3–ERBIN–SMAD2/3 complex formation, resulting in deregulated TGF-b activity, increased expression of IL-4 receptor a (IL-4Ra) on lymphocytes, Th2 skewing, and the clinical manifestations of connective tissue abnormalities and atopy233. 36 Figure 2.1. Clinical red flags and warning signs that warrant assessment for PADs, and the benefits of timely diagnosis and treatment of these conditions. This figure highlights seven critical clinical indicators that may suggest the presence of PADs, a subset of IEIs characterized by immune dysregulation and allergic inflammation. Traditional indicators of IEIs have focused predominantly on infectious phenotypes, often overlooking other significant symptoms such as malignancy, autoimmunity, and allergy. Recognizing these red flags is essential for lowering the threshold for the assessment of other diverse subsets of IEIs, including PADs. Timely and accurate diagnosis, followed by appropriate treatment, can significantly improve patient management and lead to enhanced clinical outcomes. 2.3. Molecular mechanisms of allergic inflammation Atopy is defined as the genetic tendency to develop allergic diseases234,235, a group of conditions characterized by pathogenic type 2 inflammation235,236. Often described as reaching epidemic proportions191,235,236, the incidence of allergic diseases is now estimated to affect approximately 20-30% of the global population237,238. These are chronic conditions that disproportionally impact youth191, resulting in reduced quality of life, increased morbidity and mortality, and considerable socioeconomic burden on the affected individuals and their families238. While allergic conditions 37 are typically multifactorial and influenced by genetic susceptibility, allergic immune dysregulation can also be a manifestation of an underlying IEI caused by a damaging variant in a single gene. Therefore, providers in both allergy and clinical immunology face allergic inflammation in their practice, and seek to better understand, prevent, and treat it. In this section, we venture into the intricate pathophysiology and molecular bases of allergic diseases, spanning from common allergic diseases to the rarer PADs, with a particular focus on precision medicine and targeted treatment of allergic inflammation, based on our review paper published in the Journal of Allergy and Clinical Immunology: In Practice4. This review emphasizes the critical role of advanced genetic sequencing tools in identifying both polygenic susceptibility loci and monogenic variants, thereby unraveling targetable cellular and molecular pathways in allergic diseases. In this review, we provide a comprehensive overview of genes implicated in both polygenic and monogenic allergic diseases, their pathogenic mechanisms, and corresponding targeted therapeutic strategies. Furthermore, we provide clinicians with a strategic guide (Figure 2.2) for integrating clinical observations with diagnostic tools, enabling accurate diagnoses and personalized treatment for patients with common and rare disease. 38 Figure 2.2. A roadmap for evaluating patients who present to the allergy and immunology clinic with symptoms of allergic disease. Following standard assessments, screen for red flags of monogenic allergic disease. If the presentation is not concerning for a monogenic allergic disease, patients should receive standard management for their allergic condition, including the use of biomarkers or genetic testing as indicated for personalized management and/or targeted therapies (refer to Table 2.3 and +). Patients should be monitored periodically, and if red flags 39 develop overtime or they become refractory to treatment, reconsider the possibility of a monogenic condition. If the presentation suggests a possible monogenic allergic disease, patients should undergo an immune evaluation, and if labs are abnormal or there is persistent clinical concern, genetic testing*. Genetic testing may yield known or novel monogenic causes of allergic disease, informing management and the potential use of targeted therapies (refer to Table 2.1). (Figure created with BioRender.com). * Refer to citation239,240 + Refer to citation241,242 2.3.1 Epithelial barrier disruption Atopic dermatitis (AD) is an itchy inflammatory skin disorder that waxes and wanes, is often diagnosed in early childhood, and is associated with atopic comorbidities such as asthma and allergic rhinitis. The sequential progression to asthma, allergic rhinitis, and other allergic illnesses such as food allergies and eosinophilic esophagitis from AD describes the ‘atopic march’. 243,244 AD pathophysiology is strongly linked to epithelial barrier defects, genetic predisposition, and other environmental and immunological factors244,245, all of which contribute to an individual’s susceptibility to systemic allergen sensitization and atopic march trajectory.245-247 This positions AD as a paradigm of barrier dysfunction and atopy. Several studies have demonstrated a genetic risk associated with AD, with heritability estimated to be about 84%.248-250 The most commonly reported genetic variants associated with skin barrier dysfunction are LOF variants in filaggrin (FLG) (Table 2.1).249 FLG encodes a critical skin barrier protein and FLG LOF has been associated with increased prevalence and persistence of AD.251-257 FLG is translated as profilaggrin, a 400-kilodalton polypeptide that is post-translationally cleaved into a FLG monomer. The FLG protein is expressed by keratinocytes during 40 the late stages of terminal differentiation, where it has a multifunctional role in skin barrier function: FLG monomers aggregate and align keratin bundles, strengthening the stratum corneum, and FLG breakdown products contribute to epidermal hydration and barrier function.258 Approximately 500 FLG LOF variants have been identified by the gnomAD (genome aggregation database), an international aggregate database of exome and genome sequencing data supported by the Broad Institute.259 Several studies have shown that the prevalence of FLG LOF variants vary by ethnicity as do the frequency of variation of the FLG gene.251,252,254,260-262 In addition to FLG, variants in other molecules comprising the skin epithelial barrier have also been associated with AD.260,263-265 Finally, type 2 cytokines such as IL-4, IL-13, TSLP, and IL-33 can have a direct effect on diminishing FLG production such that genetic variation in these cytokines could have a profound effect on skin barrier function too.247,266-269 It is also important to realize that all FLG LOF variants do not cause a complete loss of profilaggrin protein, therefore a complete absence of FLG does not occur in the skin barrier.270 It is therefore likely that diminished FLG, caused either by genetic variation or by more common processes like elevated type 2 cytokines, may be a not-too-innocent bystander in barrier dysfunction and allergic disease. Further emphasizing the critical role of an intact skin barrier is the fact that barrier defects can also occur in monogenic IEIs, as seen in Netherton Syndrome (NS) caused by biallelic LOF variants in SPINK5.79,271 Clinical manifestations include ichthyosiform erythroderma, trichorrhexis invaginata (or bamboo hair), and allergic immune dysregulation (Table 2.1).79 Similarly, LOF variants in genes encoding desmosomal proteins cause monogenic disorders of ichthyosis and 41 atopy.201 Desmosomes mediate cell-cell adhesion between keratinocytes and are crucial for epithelial barrier integrity.272 Loss of desmoglein-1273 and desmoplakin75 causes severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome). Given the critical physiological role of a healthy skin barrier, restoring barrier function through the use of emollients and moisturizers is important for managing both monogenic skin barrier defects and AD in the general population79. Treatments targeting type 2 cytokines such as dupilumab have also been found to reverse AD-associated epidermal abnormalities274. Recent studies have implicated the Th17 axis in ichthyosis, with therapies blocking IL-17a and IL-12/IL-23 showing benefit in case reports of patients with NS and desmosomal deficiencies.71,76,275 2.3.2 Cytokines and cytokine signaling defects Cytokines and their corresponding signaling pathways are central to the initiation and perpetuation of allergic inflammation276. A plethora of genome-wide association studies (GWAS) studies over the years have identified single nucleotide polymorphisms (SNPs) in the genes encoding multiple cytokines (IL-4, IL-5, IL-13, IL-6, TSLP, IL-10, TGF-β), and cytokine receptors (IL-4Rα, IL-13Rα1, and IL-31Rα) to be associated with a host of allergic diseases such as allergic rhinitis277-282, allergic asthma280,283-286, and AD287-289, as well as elevated IgE levels283,290. Leveraging this biological understanding, several therapeutics in the form of monoclonal antibodies have been developed to target these cytokines and their receptors (Table 2.3), one example being IL-4Rα blocking antibody, dupilumab, which is used to treat several common allergic diseases such as allergic asthma291 and AD292. 42 Mirroring these population-based insights into allergic disease, monogenic immune defects involving cytokine signaling pathways can also result in severe allergic inflammation. This is elegantly demonstrated by single gene defects affecting JAK and STAT proteins24,201,293. Binding of cytokines such as IL-4 and IL-13 to their receptors activates JAK proteins to phosphorylate and activate the STAT family of transcription factors. Both LOF and GOF variants in STAT5 and STAT3, and GOF variants in JAK1 and STAT1 cause IEIs with allergic features. One of the best described monogenic atopic disorders is autosomal dominant hyper-IgE syndrome (AD-HIES) or Job syndrome, caused by heterozygous dominant-negative (DN) LOF of STAT3. LOF variants in STAT3 and the upstream and downstream players in the STAT3 pathway (e.g. ZNF341, DOCK8, IL6ST, IL6R, and ERBIN) result in severe allergic diseases, eosinophilia, and markedly elevated IgE levels, together with other immunodeficiency features293. More recently, Ma et al. described two cases of somatic heterozygous GOF variants in STAT5b within multiple hematopoietic lineages, who presented with neonatal hypereosinophilia, AD, urticaria, and diarrhea294. Since JAK1 is upstream of STAT5b, the oral JAK1/2 inhibitor Ruxolitinib has been used to successfully treat two patients with somatic GOF STAT5b variants as well as patients with JAK1 GOF295 who were unresponsive to conventional treatment with oral corticosteroids, resulting in marked improvement of clinical symptoms and hypereosinophilia112. Interestingly, several GWAS studies have reported SNPs in STAT5b that are associated with the risk of developing polygenic forms of allergic disease296-302. As the JAK/STAT pathway’s role in allergic inflammation becomes more evident, clinical trials have started testing JAK1 inhibitors to treat common allergic diseases, in hopes of achieving higher efficacy and the ability 43 for long-term use with fewer side effects in comparison to corticosteroids. In 2019, a phase II study reported that topical ruxolitinib cream may serve as a preferred treatment for AD over topical corticosteroids (Table 2.3)303. This serves as an example of how studying monogenic atopic disorders can inform treatment of common allergic diseases in the general population. Table 2.3. Genes implicated in common allergic conditions with available targeted therapy. Offender gene Associated allergic conditions304,305 Targeted therapy Mechanism Indications IL4RA Eosinophil counts, IgE levels, asthma, AR, AD304 Dupilumab Anti-IL-4Rα (C): Severe eosinophilic asthma, AD, CRSwNP (CT): EoE306 IL5 Eosinophil counts, asthma304 Mepolizumab Anti-IL5 (C): Severe eosinophilic asthma, CRSwNP, EGPA, HES (CT): EoE306 Resilizumab Anti-IL5 (C): Severe eosinophilic asthma (CT): EoE306 IL5RA Eosinophil and basophil counts, HES304 Benralizumab Anti-IL-5Rα (C): Severe eosinophilic asthma IL13 IgE levels, asthma, AD, AR304 Dupilumab Anti-IL-4Rα (C): Severe eosinophilic asthma, AD, CRSwNP (CT): EoE306 Lebrikizumab Anti-IL13 (CT): Moderate-severe AD307 Tralokinumab Anti-IL13 (CT): Moderate-severe AD3 TSLP Eosinophil and basophil counts, asthma, AD, EoE, CRSwNP, AR, EGPA304 Tezepelumab Anti-TSLP (CT): Asthma, AD305 IL31RA AD308 Nemolizumab Anti-IL-31Rα (CT): Moderate-severe AD305 FCER1A IgE levels304 Omalizumab Anti-IgE (C): Moderate-severe asthma, CSU, CRSwNP (CT): EoE306 JAK1/JAK2 JAK1: Eosinophil counts, AD187 JAK2: Eosinophil counts, AD, AR, asthma, EoE187 Ruxolitinib (topical) JAK 1,2 inhibitor (C): Severe AD Baricitinib (oral) JAK 1,2 inhibitor (CT): Severe AD305 44 Offender gene Associated allergic conditions304,305 Targeted therapy Mechanism Indications Upadacitinib (oral) JAK 1 inhibitor Abrocitinib (oral) JAK 1 inhibitor PDE4D Asthma309 Crisaborole (topical) PDE4 inhibitor (C) AD CHF 6001 (inhaled) PDE4 inhibitor (CT): Asthma310 CYSLTR1 Asthma311, AD312 Montelukast Leukotriene Receptor Antagonist (C): Asthma, AR (Montelukast) (CT): AD, CSU313,314 Zafirlukast Pranlukast C = approved clinical indication; CT=clinical trial AD = atopic dermatitis, AR = allergic rhinitis, CRSwNP = chronic rhinosinusitis with nasal polyposis, CSU = chronic spontaneous urticaria, EGPA = eosinophilic granulomatosis with polyangiitis, EoE = eosinophilic esophagitis, HES = hypereosinophilic syndrome, IFN = interferon 2.3.3 Actin cytoskeleton defects The actin cytoskeleton plays a crucial role in immune cell function, particularly in lymphocytes315. T cell activation316, propagation of T cell receptor (TCR) signaling317, clonal expansion318, and ultimately migration and adhesion319 all heavily rely on repetitive cytoskeletal rearrangement of actin filaments320. In fact, a host of predominantly haematopoietically expressed proteins including Wiskott-Aldrich syndrome protein (WASp)116, WASp-interacting protein (WIP)321, DOCK8122,124, and Hematopoietic protein 1 (HEM1), in addition to protein complexes such as Actin Related Protein 2/3 (Arp2/3) complex322, together regulate the actin cytoskeleton in T cells. In the steady-state, WASp is autoinhibited and stabilized by WIP, but following TCR stimulation, DOCK8 activates cell division cycle 42 (CDC42), which in turn activates WASp by releasing it from WIP. WASp is then able to associate with Arp2/3 complex, which allows downstream actin 45 polymerization to commence, ultimately facilitating the propagation of various signaling cascades and cellular functions in the T cell123,317. Arp2/3-mediated filamentous actin nucleation is also controlled by the WAVE regulatory complex, which contains HEM1. Deficiencies of WASp, WIP, DOCK8, HEM1 and Arp2/3 complex subunit 1B are collectively referred to as ‘immuno-actinopathies’323, and cause a broad range of manifestations that include atopy. X-linked WASp deficiency causes Wiskott-Aldrich syndrome (WAS), which presents with the characteristic triad of immunodeficiency, thrombocytopenia, and AD324. Biallelic loss of WIP and Arp2/3 complex subunit 1B also present as phenocopies of WAS, with the same triad of symptoms. Biallelic loss of DOCK8 causes severe allergic inflammation, significant eosinophilia as well as elevated IgE levels, chronic infections, and an increased risk of cancer and autoimmunity.124 325,326 Interestingly, SNPs in DOCK8 have also been linked to elevated IgE levels and the development of AD in the general population327. Severe allergic inflammation in actinopathies may be partially explained by defects in T cell polarization. For instance, WASp deficient CD4 T cells exhibit diminished Th1 cytokine secretion (IL-2, IFN-γ and TNF-α), but near-normal Th2 cytokine secretion (IL-4, IL-5, and IL-10)328. Additionally, WASp has been shown to epigenetically regulate the transcription of Th1 master gene TBX21 (encoding T-bet), also explaining the Th1-Th2 imbalance seen in WASp deficient patients319,329. Similarly, CD4 T cells from DOCK8 deficient patients are preferentially polarized to Th2 effector phenotype at the expense of Th1 and Th17 polarization, which is explained by concomitant defective STAT3 signaling in these patients330. As such, DOCK8 deficiency-related dermatitis and elevated IgE levels are successfully treated with dupilumab, which perhaps 46 counteracts the elevated IL-4 production caused by a Th2 skewed immune system130. In terms of curative treatments for WASp, WIP, Arp2/3 and DOCK8 actinopathies, since the expression of these proteins are predominantly limited to the hematopoietic compartment, hematopoietic stem cell transplantation (HSCT) has been successfully used to treat them (Table 2.1)115,119,121,125,331. HEM1-deficiency may also be amenable to HSCT, though no cases reported thus far have undergone transplant, possibly because it is a newly recognized disease. In addition, stem cell gene therapy for WASp deficiency has shown promising results for treating atopic disease in the affected patients116,117. 2.3.4 Antigen presentation defects T cell recognition of antigen displayed on major histocompatibility complex (MHC) class I and II molecules are an essential component of human immunity and maintaining self-tolerance. The genes encoding MHC molecules are found on chromosome 6, and in humans are called human leukocyte antigens (HLA).332 CD8 and CD4 T cell receptors recognize peptides bound to class I and II MHC molecules, respectively; however, MHC class I ligands are also important regulators of natural killer (NK) cell activity.333 NK cells interact with MHC class I using killer Ig-like receptors (KIR).333,334 HLA genes are highly polymorphic: multiple amino acid substitutions result in structural changes that alter peptide binding and antigen presentation for MHC molecules produced from different alleles.335,336 This dictates how antigens are presented to T cells and shapes the immune response that ensues, with certain HLA and KIR genetic variants skewing the resulting T cell response towards an allergic phenotype337. 47 Previous studies, including GWAS and immunophenotyping techniques, have found associations between HLA genes and the risk of AD, as well as other atopic diseases such as asthma, allergic rhinitis, and food allergies.338-343 While GWAS has localized genetic variation to the HLA region, because GWAS assays are based on the analysis of SNPs, imputation of the actual HLA allele has been difficult and not always accurate compared to next generation sequencing (NGS) high resolution HLA typing.344,345 Due to similar technologic issues, most studies have only reported the presence or absence of KIR genes. As a result, studies that report both HLA and KIR variation using older technologies need to be carefully scrutinized. More recently, studies using NGS technologies have shown associations with AD susceptibility as well as AD persistence, with specific HLA class I alleles and binding pocket epitopes as well as KIR genes being linked to either an increase or decrease in risk.346,347 348,349 As an example of a monogenic immune disorder affecting antigen presentation, MHC class II deficiency is caused by pathogenic variants in genes regulating transcription of MHC II genes (Table 2.1). 350 The clinical phenotype is primarily characterized by severe infections, however, features of type 2 inflammation such as hypereosinophilia, AD, or cutaneous drug reactions were reported in 10 of 35 patients with MHC II deficiency caused by a homozygous RFXANK deletion.351 Understanding the role of HLA genetics in allergic disease risk has brought targeted approaches to immune-mediated delayed drug hypersensitivity reactions (DHRs). Due to the strong association between the HLA-B∗57:01 genotype and DHRs to the antiviral abacavir, screening patients prior to initiating treatment is now recommended352-354. HLA associations with 48 DHRs to allopurinol, carbamazepine, vancomycin, anti-cytokine biologics, among others have also been described354-357. Evaluating how additional host or environmental factors (such as variations in T-cell-receptor and KIR allelic repertoires, concurrent infections, and medications) contribute to a patient’s risk of DHRs may further refine our ability to prevent and appropriately manage these life-threatening drug reactions354. 2.3.5 Attenuated antigen receptor signaling Multiple lines of evidence suggest that CD4+ helper T cell differentiation is influenced by the relative avidity of TCR peptide-MHC interactions. More specifically, several studies have corroborated the concept that a weaker TCR signal can bias both murine and human CD4+ T cells to differentiate into Th2 effector cells 358-361. Th2-based atopic phenotypes are observed in various murine models harboring germline variants in specific molecules involved in TCR signal transduction, including Zap70, Lat, Card11, and Malt1 362-365. In humans, homozygous null variants in homologous TCR signaling molecules naturally result in severe combined immunodeficiency (SCID), often linked to compromised thymocyte development and a complete loss of peripheral T cells 366-374. However, heterozygous hypomorphic variants in the same genes (e.g. ZAP70) that preserve protein expression but attenuate TCR signaling (without blocking it entirely) expose a predisposition to Th2 differentiation in human patients with moderate to severe atopic disease 366,368,369,375,376. Over the past ten years, the CARD11-BCL10-MALT1 (CBM) complex has emerged as a particularly important site for hypomorphic variants that give rise to PADs in humans, featuring dysregulated allergic effector responses (i.e. Th2 skewing, IgE production, eosinophilia) 49 irrespective of allergen-specific sensitization 24,377. Indeed, GWAS identified CARD11 as a significant risk locus for AD in the Japanese population 378, and mapped a peak association signal for peanut allergy in the MALT1 locus in subjects that avoided peanuts in early life 379. CARD11 encodes an essential lymphocyte-specific scaffold protein that connects antigen receptor engagement with the activation of key downstream transcription factors, including NF-kB, c-Jun N-terminal kinase (JNK) and mammalian target of rapamycin complex 1 (mTORC1) 380-384. Upon TCR-induced phosphorylation, CARD11 adopts an open conformation that recruits BCL10 and MALT1 in assembling a large, filamentous CBM signalosome best known for recruiting IkB kinase (IKK) to foster IkB degradation and nuclear translocation of NF-kB heterodimers that transcribe multiple genes involved in lymphocyte proliferation, survival, and effector function 385,386. The CBM complex is also required for TCR-dependent activation of JNK and mTORC1380,382, although molecular mechanisms governing these outputs remain only partly elucidated. We and others recently described an expansive cohort of patients with CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS) disease68,387,388. Although a sizable subset of CADINS patients present with immune dysregulation features including cutaneous viral infections, sinopulmonary infections, and autoimmune sequelae, severe atopic disease (e.g. AD, asthma, food allergies, eosinophilic esophagitis) remains the cardinal clinical manifestation, consistent with Th2 skewing and elevated IgE. As the name implies, CADINS is caused by heterozygous DN CARD11 variants that universally impair the ability of wild-type CARD11 to stimulate NF-kB following TCR/CD28 ligation, often accompanied by defective mTORC1 activation. Consistent with this observation, mice harboring hypomorphic or 50 DN Card11 or Malt1 variants develop increased serum IgE with age, often accompanied by AD, despite marked lymphocyte activation defects 362,364,389,390. Mechanisms of CADINS disease pathogenesis must be further clarified to resolve questions about variable penetrance and expressivity, potential genotype-phenotype correlations, and the importance of specific downstream signaling defects in both T and B cells. Nevertheless, both dupilumab and omalizumab have recently been shown to ameliorate AD symptoms in CADINS patients137,391, suggesting blockade of atopic effector molecules may suffice as a treatment paradigm in the absence of appropriate therapeutic targets for restoring optimal antigen receptor signaling. 2.3.6 Altered cellular metabolism Cellular metabolism is inextricably linked to immune function. For example, lipid metabolism produces bioactive mediators such as leukotrienes important in allergic disease development through their roles in bronchoconstriction and inflammation; with therapies targeting leukotrienes in clinical practice (Table 2.3)392. We now appreciate that dynamic changes in metabolic programming shape the differentiation and function of effector and regulatory T cells; newly activated T cells rapidly switch their metabolic balance from oxidative phosphorylation to aerobic glycolysis and glutaminolysis, which supplies macromolecules for rapid clonal expansion and regulates the synthesis of Th effector cytokines like IFN-g393-395. We can thus hypothesize that metabolic dysregulation contributes to the pathogenesis of common atopic disorders and certain IEIs, including those with allergic manifestations. One illustrative example is triggered by recessive hypomorphic variants in phosphoglucomutase 3 (PGM3), which give rise to a syndrome featuring both neurological deficits and immune dysregulation in the form of infections, 51 autoimmunity, and a remarkable array of allergic disease presentations 396-398. PGM3 is an essential enzyme in the hexosamine biosynthesis pathway, responsible for generating UDP-GlcNAc, a molecule required for complex N- and O-linked protein glycosylation. A huge increase in complex branched N-glycans on the plasma membrane accompanies the switch to aerobic glycolysis in healthy activated T cells 399. In fact, reduced N-glycan complexity on CD4 T cells can be utilized as a diagnostic hallmark of PGM3 deficiency using lectin-based flow cytometry 400. Whether this glycosylation defect underlies the skewed Th2/Th17 response observed in PGM3-deficient patients remains unclear. Notably, both glucose and glutamine feed the hexosamine pathway for UDP-GlcNAc synthesis; indeed, all three molecules are integral to the activation of mTORC1, a central governor of metabolic reprogramming and Th differentiation 401,402. Dramatic increases in glucose and glutamine influx foster the switch to aerobic glycolysis and Th1 differentiation, respectively facilitated by the upregulation of plasma membrane transporters GLUT1 and ASCT2 394. Here we see a striking parallel with CADINS disease, in which patient T cells harboring DN CARD11 variants show diminished ASCT2 expression, glutamine uptake and S6 phosphorylation (a marker of mTORC1 signaling) upon activation68. Moreover, culturing CADINS patient T cells in excess glutamine (plus NF-kB-activating cytokines) partially rescues proliferation and IFN-g secretion 68, ostensibly by overcoming limited ASCT2 expression. We do not yet know if exogenous UDP-GlcNAc would produce similar effects; nevertheless, these findings imply a shared atopic disease etiology in patients with defective CBM complex or PGM3 function, stemming from a related metabolic insufficiency that attenuates mTORC1 activity in T cells. This hypothesis introduces a 52 compelling therapeutic possibility whereby allergic disease might be ameliorated in both disorders by metabolite supplementation (e.g. glutamine, GlcNAc, etc). Intriguingly, dietary glutamine supplementation during infancy was shown to dramatically reduce the incidence of atopic disease in early childhood, and topical glutamine can suppress contact dermatitis 403,404. 2.3.7 Granulocyte dysregulation Granulocytes – eosinophils, basophils, neutrophils, and most notably mast cells – are critical effectors in the pathogenesis of allergic diseases. Mast cells and basophils release mediators such as lipids, proteases, and histamine upon cross-linking of their high affinity IgE receptor, which act on secondary effectors and tissues to elicit features of an allergic reaction405. Genes encoding proteins involved in this cascade, from the Fc fragment of the IgE receptor, to leukotrienes/leukotriene receptors and phosphodiesterases, have been linked to atopy, and serve as therapeutic targets for allergic disease (Table 2.3) 304,309,311,312. Monogenic disorders affecting mast cell signaling lead to clinical manifestations of mast cell mediator release (Table 2.1). Mastocytosis is caused by somatic or germline GOF variants in KIT, encoding for a tyrosine kinase receptor critical for mast cell survival174. Recently, early phase clinical trials of a selective kinase inhibitor targeting the most common activating KIT variant found in systemic mastocytosis showed promising response rates176. Autosomal dominant GOF variants in ADGRE2 cause vibratory urticaria due to degranulation of dermal mast cells activated through ADGRE2 G-protein-coupled receptor (GPCR) by physical stimuli in an IgE-independent manner179. Similarly, a link between Mas-related G protein-coupled receptor X2 (MRGPRX2) on cutaneous mast cells and AD was recently proposed406-408. MRGPRX2 is activated and causes mast cell degranulation 53 by a vast array of substances including host-defense peptides, neuropeptides, and cationic drugs407. Although no studies to-date link rare variants or SNPs in MRGPRX2 to monogenic or common allergic diseases in humans, studies have observed upregulation in MRGPRX2 expression and its agonists in the skin transcriptome of patients with AD in comparison to healthy controls408. This has led to MRGPRX2 being considered as a potential therapeutic target for AD and other allergic-like diseases mediated by this receptor406,409. 2.4. Transcription factor defects in IEIs with atopy My third review, published in Frontiers in Allergy, hones in on PADs attributable to genetic variations in transcription factors, which I have covered in the following section. Transcription factors recognize specific DNA sequences through their DNA binding domain (DBD) to control transcription 410. They act as ‘master regulators’ controlling immune cell lineage specification and governing specific signalling pathways 411. Transcription factors are often structurally constrained, evolutionarily conserved and depleted of common variation within their DBDs 412. The variant alleles can exert effects through LOF (e.g. non-functional protein product), GOF, (e.g. altering sequence recognition/strength or through constitutive activation), or dominant-negative (DN, e.g. interfering with the wild-type allele through disrupted dimerization or off-target binding) mechanisms—or potentially through a combination of a number of these pathogenic mechanisms413. Transcription factor defects can disrupt the expression of genes involved in immune system development, activation, and differentiation, leading to different IEI manifestations, including atopy 4,24,61. This area of study is particularly crucial as approximately a quarter of currently identified PADs arise from deleterious variants in genes encoding 54 transcription factors. In fact, all novel IEIs described in my thesis occur as a result of monogenic variants in crucial transcription factors that govern vast biological processes in the body, both in the immune system and beyond. 2.4.1 Transcription factors orchestrating allergic immune responses Transcription factors function as central regulators of cellular functions, governing gene transcription through several known mechanisms (Figure 2.3) 414. These are proteins that bind to specific DNA sequences known as transcription factor-binding sites that are situated in the promoter or enhancer regions of genes. Transcriptional regulation is mediated through recruitment of either co-activator or co-repressor accessory proteins which either enhance or inhibit transcriptional activity, respectively 415. Additionally, transcription factors can interact with chromatin remodelers to modify the structure of nucleosomes, which can affect the accessibility of DNA to transcriptional regulators. These proteins can also interact with RNA polymerase and either stimulate or inhibit its activity, leading to changes in transcriptional output. Lastly, transcription factors can integrate signals from various signaling pathways, such as growth factors, cytokines, or hormones, and modify gene expression accordingly. The specific mechanisms by which transcription factors regulate gene expression is context-dependent and as such varies between genes and cell types414,416,417. Given their central role in regulating gene expression, minor variations in their expression and/or function can precipitate substantial disturbances in the gene regulation network. Furthermore, transcription factors require precision in their expression levels due to dosage sensitivity 418. This highlights the exactness required in 55 transcription factor expression and function, and the significant potential for harm from pathogenic variants in transcription factor-encoding genes419. Transcription factors play central roles in immune system development, maintenance, and function. These factors not only regulate cell-fate determination, but also co-ordinate immune cell responses following exposure to both endogenous and exogenous stimuli such as pathogens and allergens420. In atopic disorders, a complex network of genes and their products are regulated by a host of transcription factors to induce allergic inflammation, which involves the recruitment and activation of various immune cells, such as mast cells, eosinophils, and T-helper 2 (Th2) lymphocytes. This ultimately leads to the production of pro-inflammatory cytokines, chemokines, and allergen specific IgE antibodies, which collectively contribute to the hallmark clinical features of allergic disease. Key Transcription factors involved in this process are GATA-3, STAT6, NF-κB, T-bet, AP-1, and NFAT 421. 56 Figure 2.3. Diverse mechanisms of gene expression regulation by transcription factors. (1) Transcription factors integrate external stimuli, coordinating cellular responses through gene expression changes. (2) Upon binding to a specific DNA sequence, a transcription factor can act as a bridge or docking site for chromatin remodeling complexes, aiding their recruitment to nearby nucleosomes. (3) By facilitating a process called "looping", transcription factors bring the promoter region, where transcription initiates, into proximity with enhancer or silencer regions, which house transcription factor binding sites. (4) Lastly, transcription factors can alter gene expression by interacting with co-activators and co-repressors via their protein-binding domains. In this multi-pronged manner, transcription factors form complex regulatory networks, shaping chromatin structure and guiding the transcriptional machinery. (Created with Biorender.com) 2.4.2 Transcription factors implicated in the pathophysiology of PADs Given the significant role that transcription factors play in modulating immune responses, it is not surprising that defects in transcription factors or their regulators have been linked to numerous human diseases, including approximately 25% of PADs 4,422. In some cases, the affected 57 transcription factors are important for regulating the balance between Th1 and Th2 responses, commonly referred to as the ‘Th1/Th2 paradigm’. An imbalance or skewing towards either Th1 or Th2 disrupts immune homeostasis and can give rise to immune-related disorders. As such, the basis of allergy can be partly understood through the lens of this delicate equilibrium, as a skewing towards Th2 immunity and allergic inflammation 423. However, it is essential to recognize that the pathogenesis of PADs is not solely restricted to dysregulation of Th1/Th2 balance. As already discussed in previous sections, genetic variants in several components of the immune system beyond those directly related to Th1 and Th2 cells can cause PADs. 2.4.2.1 STAT family of transcription factors Monogenic defects impacting the JAK-STAT signaling pathway comprise a key group of IEIs associated with atopy 22,24,25. The JAK-STAT family consists of 7 Transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5A/B, and STAT6) and 4 receptor associated kinases (JAK1, JAK2, JAK3, TYK2) that regulate various cellular functions such as immunity, growth, differentiation, and survival 424,425. Despite each of these transcription factors possessing distinct functions, they exhibit sequence similarities and analogous activation mechanisms. The JAK-STAT signaling cascade is initiated upon ligand binding to an extracellular cytokine or growth factor receptor, leading to activation of the JAK kinase, phosphorylation of the receptor, and recruitment and activation of STAT proteins. The STAT proteins then dimerize and migrate to the cell nucleus, where they bind to specific DNA sequences (response elements) in the promoter regions of target genes and regulate their transcription. The combinatorial activation of STAT transcription factors in response to a cytokine stimulus enables the JAK-STAT pathway to transmit over 50 unique 58 cytokine signals and transcriptional outputs 426-430. When one STAT’s activity is significantly altered, the transcriptome induced by the cytokines upstream of its action will change. This leads to abnormal immune responses that frequently skew towards Th2, resulting in the atopic phenotype seen in IEIs caused by genetic variants in STATs including STAT1, STAT3, STAT5b and STAT6431-434. The phenotypic variability seen between and within STAT defects stems not just from the variant’s functional impact—whether LOF, GOF, or DN—but also from which transcription factors’ domain was genetically altered. Thus, understanding how these variants directly affect transcription factor functionality, and consequently the JAK-STAT pathway, is crucial to comprehending the broad spectrum of clinical manifestations these diseases present. One of the most well-studied IEIs associated with the JAK-STAT pathway is autosomal dominant hyper-IgE syndrome (AD-HIES)/STAT3 deficiency, characterized by eczema, eosinophilia, elevated IgE levels, mucocutaneous candidiasis, and connective tissue abnormalities (27, 28, 29). AD-HIES is caused by DN variants in STAT3, typically missense or in-frame insertion/deletion variants impacting the highly conserved DNA-binding or Src Homology 2 (SH2) domain of the protein amongst other conserved regions (28, 29). Such changes do not alter the expression levels of STAT3 protein, but do hinder STAT3's ability to regulate gene transcription downstream of several key cytokines, notably IL-6, IL-10, IL-11 and IL-21 (Figure 2.4 A) 435. Defective IL-6 signaling leads to blunted inflammatory responses and impaired Th17 cell differentiation, with clinical features of recurrent staphylococcal and fungal infections. CD4+ T cells show Th2 skewing and production of IL-4, IL-5, IL-13, contributing to the atopic phenotype seen in these patients of eczema and eosinophilia (30). IL-21 is important for B cell maturation and class switching, while both IL-21 59 and IL-10 play a role in suppressing IgE production; impaired IL-10 and IL-21 responses thus leads to humoral defects and elevated IgE435,436. Lastly, impaired IL-11 signaling likely contributes to the delayed primary tooth exfoliation and distinctive facial features associated with AD-HIES435,437. Pathogenic germline variants in other molecules that regulate the expression and function of STAT3 have now been shown to be associated with severe atopic disease, signifying the central role for STAT3 in these disorders. These include Erbin (encoded by ERBB2IP), ZNF341 (encoded by ZNF341), and transforming growth factor beta (TGF-β) receptors (TGFBR1 and TGFBR2)233,438. While LOF in certain STATs leads to allergic immune dysregulation as observed in AD-HIES, enhanced STAT TF activity can also cause severe atopy, with a notable example being the recently described IEI autosomal dominant STAT6 GOF (Figure 2.4 B). The clinical phenotype associated with STAT6 GOF includes treatment-resistant atopic dermatitis, hypereosinophilia, eosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis434,439-441. The condition is caused by heterozygous missense variants located in several protein domains including the DNA-binding, linker-, and SH2 domain of STAT6. Despite their diverse locations, most of these variants are positioned near the TF-DNA interface. By increasing the electro-positivity at this interface, they are predicted to enhance STAT6 binding to DNA, thus conferring the GOF mechanism of pathogenicity. STAT6 is fundamentally involved in allergic inflammation processes. Its pivotal role includes mediating the effects of IL-4 and IL-13, cytokines essential for Th2 cell differentiation, B cell proliferation, survival, and class switching to IgE. In T cells, STAT6 activation upregulates GATA3 expression, a critical regulator of Th2 60 differentiation, which subsequently amplifies the expression of cytokines IL-4, IL-5, and IL-13. These cytokines stimulate allergic responses by activating mast cells and eosinophils. Thus, Th2 skewing and elevated levels of IL-4/IL-5/IL-13 produced by these cells could be the driving factor behind the allergic phenotype observed in STAT6 GOF disease. 61 Figure 2.4. Mechanisms by which variants in transcription factor-encoding genes cause inborn errors of immunity associated with atopy. (A) Signal transducer and activator of transcription 3 (STAT3) is integral to the transduction of multiple cytokine signals, including IL-6, IL-10, IL-11, IL- 62 21, IL-22, and IL-23. Autosomal dominant loss-of-function (LOF) in STAT3 causes disruption in these signalling pathways, leading to abnormalities in several cytokine signalling pathways. Notably, the impairment of IL-6 and IL-23 signaling hinders Th17 cell differentiation, increasing host susceptibility to mucosal bacterial and fungal infections. Additionally, IL-21 function is compromised, affecting B cell maturation and isotype switching, thereby leading to elevated IgE levels. Impaired IL-11 signaling is associated with connective tissue abnormalities. Moreover, T helper 2 (Th2) skewing is observed in CD4+ T cells, contributing to atopy. Lastly, impaired IL-10 signaling is associated with impaired development of regulatory T cells (Tregs). (B) STAT6 plays a fundamental role in allergic inflammation, mediating the effects of cytokines essential for Th2 cell differentiation, B cell proliferation, and class switching to IgE. In T cells, STAT6 activation upregulates GATA Binding Protein 3 (GATA3) expression, amplifying cytokines IL-4, IL-5, and IL-13, which stimulate allergic responses, explaining the allergic phenotype observed in STAT6 gain-of-function (GOF). (C) T-bet, the master regulator of Th1 differentiation, suppresses Th2 cell lineage commitment by inhibiting GATA-3 function. T-bet deficiency disrupts this equilibrium, leading to increased chromatin accessibility of Th2 genes by GATA-3, resulting in the excessive production of Th2 cytokines like IL-5 and IL-13. (D) Forkhead box P3 (FOXP3) is an important TF for the development, stability, and suppressive function of Tregs. FOXP3 LOF leads to a failure in repressing the Th2 transcriptional program, generating Th2-like Tregs that are incapable of suppressing extrinsic Th2 cells. These Th2-like Treg exhibit increased intra-chromosomal interactions in the Th2 locus, leading to type 2 cytokine production. (Created with Biorender.com) 2.4.2.2 T-box transcription factor TBX21 Transcription factors such as T-box transcription factor TBX21 (T-bet) and GATA3 play pivotal roles in directing Th1 and Th2 differentiation, respectively. Aberrations in their signaling pathways or variants in genes encoding these transcription factors may contribute to the development of PADs by disrupting the delicate equilibrium between Th1 and Th2 responses. The Th1/Th2 counter-regulatory theory is supported by a recent study describing an individual with an autosomal recessive LOF variant in TBX21 114, encoding T-bet, the master regulator of Th1 differentiation 442. T-bet deficiency results in the loss of IFN-γ production, and in turn is linked to the excessive production of IL-5 and IL-13 by Th2 cells, leading to the development of upper airway allergic inflammation and eosinophilia (Figure 2.4 C) 114. The Th2 skewing and the resulting 63 atopic phenotype observed in this patient can be attributed to the known mechanism of action of T-bet. T-bet acts as a repressor of Th2 cell lineage commitment by preventing GATA3 from binding to its target sequence and suppressing GATA3 expression 443. Through these mechanisms, T-bet effectively suppresses the production of Th2 cytokines (IL-4, IL-5, IL-9, and IL-13), which are crucial for Th2 cell function and the development of allergic responses. Therefore, it is expected that the loss of T-bet leads to an overactive Th2 response and the subsequent development of atopic conditions such as upper airway allergic inflammation and eosinophilia 444. 2.4.2.3 Forkhead Box P3 FOXP3 (Forkhead Box P3) is a transcription factor that plays a critical role in maintaining immune homeostasis. It primarily controls the differentiation and function of Tregs. Pathogenic variants in FOXP3 cause IPEX syndrome445, an IEI characterized by autoimmunity, lymphoproliferation and severe atopy. Affected individuals often present with severe atopic dermatitis, high IgE, and eosinophilia 24. Currently over 70 variants in FOXP3 associated with IPEX have been reported 446, with the majority impacting the forkhead (FKH) DNA-binding domain of FOXP3 at the C-terminal end of the protein. Some variants are located in other FOXP3 regions, including the N-terminal proline-rich (PRR) or leucine-zipper (LZ) domains, whereas others are located upstream of the gene or in the polyadenylation site, thus influencing mRNA expression and/or stability. While lack of FOXP3 expression has been linked to severe phenotypes, disease severity does not always correlate with protein expression. Many of the affected individuals have missense variants that lead to normal or reduced expression of the variant, disrupting the regulatory activity of FOXP3 by changing its DNA binding sites, interactions with other molecules, or its ability to dimerize 101. 64 FOXP3-deficient Tregs still develop in the thymus, however they lose their regulatory function and acquire effector T cell (Teff) attributes447,448. The balance of T cell fate between Treg and Teff phenotype is in part related to metabolic programming which FOXP3 mediates. Teff cells are highly metabolically active, undergoing glycolysis and oxidative phosphorylation, whereas Treg metabolism favours fatty acid oxidative and keeps glycolysis under strict control. FOXP3 deficiency impairs its ability to regulate the metabolic kinase mammalian target of rapamycin (mTOR), leading to augmented glycolysis and degeneration of Tregs into Teff cells without suppressive function448. Several studies provide further insight into how alterations or loss of FOXP3 drives atopy (Figure 2.4 D). One study recapitulated in a mouse model a human IPEX syndrome-causing variant (M370I) impacting the FKH domain of FOXP3449. Compared to wild-type Tregs, M370I Tregs were much less efficient at inhibiting GATA3 expression during T cell activation, thus skewing extrinsic CD4+ cells towards Th2 differentiation. Moreover, M370I Tregs were unable to repress Th2 transcriptional programs intrinsically, leading to generation of ‘Th2-like’ Tregs with an effector Th2 phenotype and type 2 cytokine production. This is in keeping with previous work demonstrating FOXP3’s role in preventing degeneration of Tregs into effector T cells. A separate study similarly demonstrated that downregulation of FOXP3 in human Tregs is associated with strong and selective upregulation of Th2 signature genes, such as GATA3, IL-4, IL-5 and IL-13, supporting Th2 as the default differentiation pathway of FOXP3-negative Tregs 450. 65 2.5. Conclusion This chapter has provided a multi-dimensional perspective on PADs. The first section of this chapter laid a crucial foundation by delving into the clinical manifestations of PADs. It highlighted key clinical red flags, emphasizing the necessity of a genetic workup in cases where a monogenic allergic disease is suspected. This perspective is vital for clinicians, as it aids in the early identification and accurate diagnosis of these complex conditions. The second part of this chapter shifted our focus to the molecular level, unpacking the intricate pathophysiology underpinning PADs and the use of targeted precision therapies for the treatment of these diseases. The third section of this chapter narrowed its lens to examine PADs resulting from transcription factor defects. This specific focus sheds light on a critical aspect of these disorders, allowing for a deeper understanding of their genetic origins and how these contribute to the broader spectrum of allergic inflammation and immune dysregulation. 66 CHAPTER 3: Dominant-negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterized by immunodysregulation, craniofacial anomalies, hearing impairment, athelia, and developmental delay. 3.1. INTRODUCTION AND RATIONALE The Ikaros Zinc Finger (IKZF) family comprises five transcription factors – Ikaros (encoded by IKZF1), Helios (IKZF2), Aiolos (IKZF3), Eos (IKZF4), and Pegasus (IKZF5) – which play critical roles in both neurodevelopment451-456 and the development, maintenance, and regulation of hematopoietic cells457-459. Their role has been best characterized in lymphocyte development and function. Ikaros, Helios, Aiolos and Eos regulate the differentiation and function of CD4 T helper cell subsets by modulating cytokine pathways in a cell-specific manner458. In particular, Helios helps maintain the phenotype, stability, and function of regulatory T cells (Tregs), a T cell population critical for immune tolerance and autoimmunity prevention51,460. Helios stabilizes Treg function by suppressing IL2 transcription460. Loss of Helios results in the de-repression of the IL2 locus, allowing the production of IL2 and other effector cytokines by Tregs51,460. Expression of Helios extends beyond the immune system. During embryogenesis, Helios is expressed at varying levels in many tissues, including brain, eye, muscle, respiratory and gastrointestinal systems, kidney, skin, and all hematopoietic centers. In contrast, in adults, there is biased Helios expression in T cell lineages, the spleen, and the thymus461,462. During neurodevelopment, Helios is expressed in several brain structures including the lateral ganglionic eminence, the cingulate, insular and retrosplenial cortex, the hippocampus, and the accessory olfactory bulb in mice456,463. Helios-deficient mice develop a host of neurodevelopmental features, notably deficits in motor skill acquisition resulting from impaired striatal 67 neurogenesis463, and neuropsychiatric abnormalities resulting from dysfunction in the striatum and hippocampus454-456,463. In addition, Ikzf2 is postnatally expressed in outer hair cells in the cochlea and regulates outer hair cell maturation. Mice carrying a homozygous missense variant in Ikzf2 (Ikzf2cello/cello) have sensorineural hearing loss452. The dynamic, broad expression of Helios during development, and its role in Tregs, makes it a strong candidate for causing disease. Indeed, three recent studies described monoallelic and biallelic variants in IKZF2 causing immunodeficiency and immune dysregulation in nine individuals57,58,464. These variants largely fall outside known functional domains of the protein. Helios contains an N-terminal DNA-binding domain that directly interacts with gene regulatory elements, and a C-terminal protein-binding domain, which mediates homo- and heterodimerization with other Ikaros members and interaction with several transcriptional regulators465,466. The previously reported IKZF2 variants mainly affect the ability of Helios to interact with other transcriptional regulators and proteins, resulting in immune dysregulation and enhanced susceptibility to infections in the absence of syndromic features. Here, we describe a novel syndrome in two unrelated children caused by heterozygous de novo IKZF2 variants that exclusively affect the highly conserved zinc fingers in the DNA binding domain of Helios. Building on the known role of Helios in immune regulation and its widespread expression during development, embryogenesis and hearing, we hypothesized that changes in its zinc finger domain, crucial for DNA binding, might lead to a distinct syndrome that includes immunodeficiency and a wide range of other clinical features, such as facial differences, developmental abnormalities, and sensorineural hearing loss. Additionally, given Helios's critical 68 role in maintaining the stability and function of Tregs, it seemed plausible that heterozygous de novo IKZF2 variants could disrupt T cell balance, leading to T cell lymphopenia and frequent infections. As anticipated, the core phenotype in these two individuals comprised T cell lymphopenia, recurrent respiratory infections, craniofacial differences, cleft palate, sensorineural hearing impairment, athelia, and developmental delay. Given the broad clinical manifestations of the patients described here, we hypothesized that a dominant-negative effect could amplify and diversify the symptoms seen in these patients, going beyond the restricted immune dysfunction typically seen in patients with the previously described loss-of-function (LOF) variants. We hypothesized that the variant protein, by binding to its usual partners including members of the closely related Ikaros family of transcription factors, might impair their function as well through the formation of dysfunctional complexes, resulting in a phenotype that goes beyond Helios LOF. Indeed, our functional data supports this idea, indicating that the pathophysiology of this syndrome is underpinned by a dominant-negative mechanism—where a variant protein disrupts the normal function of the wild-type protein—leading to a more severe phenotype than that observed with previously described LOF IKZF2 variants. 69 3.2. SUBJECTS AND METHODS 3.2.1. Sequencing, genomic data analysis, and variant validation Proband 1 Proband was identified and recruited into this study through referrals to Dr. Anna Lehman and the Care4Rare research program, after the standard-of-care clinical tools were insufficient for a definitive diagnosis. Inclusion criteria focused on individuals with complex, undiagnosed conditions that suggest a genetic basis, especially those who have exhausted standard diagnostic avenues. Whole-genome libraries were prepared from peripheral blood DNA from Proband 1 and parents at The Centre for Applied Genomics (Toronto), using Illumina TruSeq PCR-Free library prep kit. The libraries were sequenced to a targeted depth of 40X using the Illumina HiSeq. Reads were mapped to human reference genome GRCh37 using BWA-MEM-v.0.7.17467. Single nucleotide variants (SNVs) and insertions/deletions (Indels) discovery was performed using Genome Analysis Toolkit (GATK HaplotypeCaller-v.3.8)468. SNVs and Indels were filtered out if QD < 2.0. Variants were annotated using VarSeq™ v2.2.1 (Bozeman, MT: Golden Helix). We filtered for rare variants (gnomAD v2.1.1 minor allele frequencies ≤ 1%) (https://gnomad.broadinstitute.org/). We further filtered for coding non-synonymous variants, and non-coding variants that had a SpliceAI score or had pathogenic or variant of uncertain significance records in ClinVar 469. Among these, de novo, compound heterozygous, homozygous, hemizygous, or parentally inherited in genes with evidence of involvement in the immune system were prioritized according to allele counts in gnomAD, damaging in silico predictions, inheritance, relevant phenotypes or functions, and gene constraint metrics (pLI ≥ 0.9, Missense Z-score > 3 70 for missense variants). Bioinformatic tools such as SIFT, PolyPhen2470, CADD471, dbscSNV472, and SpliceAI were used to assess the possible impacts on the protein. Structural variants (SVs) were called using CNVnator-v.0.3473, LUMPY-v.0.2.13474, ERDS-v.1.1475, and Manta-v.1.1.1476, and were annotated using Annovar477. Variants were filtered out if were present in inhouse control databases (allele count >5) or were called by only one tool. SVs were prioritized based on inheritance, zygosity, presence in the reference population databases and other variant databases (gnomAD-SV-v2.1478, ClinVar479, dbVar480, Decipher481, DGV482, LOVD-3.0483), gene constraint metric (pLI ≥ 0.9), and relevant gene function or phenotypes. Proband 2 Following the initial discovery related to proband 1, the details of the genetic findings and clinical presentation were shared on GeneMatcher, a platform designed to connect researchers and clinicians worldwide who are working on similar genetic disorders. Shortly after sharing this information, our team was contacted by another research group who had identified a patient with a phenotype remarkably similar to that of proband 1, including a variant affecting the DNA-binding domain of IKZF2. Exome sequencing was performed by a clinical commercial lab on genomic DNA extracted from whole blood for Proband 2 and his parents. Enrichment was carried out using NimbleGen SeqCap EZ Exome Library SR protocol (Roche NimbleGen) and captured libraries were sequenced on the Illumina HiSeq. Sequencing reads were mapped to the human reference assembly (UCSC hg19) using Edico DRAGEN BioIT. Variants were called using Edico DRAGEN haplotype-based caller. CNV analysis of exome sequencing data was performed to identify exonic copy number changes. Variants were annotated using Emedgene. Synonymous 71 variants, intronic variants not affecting splicing sites, and common benign variants were excluded from interpretation unless previously reported in the literature as possibly pathogenic variants. The candidate variants were re-annotated using VarSeq™ v2.2.1 (Bozeman, MT: Golden Helix) to keep the annotations consistent with those generated for Proband 1. 3.2.2. RT-PCR and sanger sequencing The mRNA was isolated from lymphoblastoid cell lines (LCLs) derived from Proband 1 and a healthy control, and cDNA was synthesized using Superscript III First-strand synthesis SuperMix (Thermo Fisher Scientific). Reverse transcription PCR (RT-PCR) was carried out with 2x FroggaMix Plus (FroggaBio) using a forward primer (catgacaagcacaaattcag) that spans exon 3-4 junction of the two main transcripts of IKZF2, and a reverse primer (ttgtggcagcgttccttgtg) that maps to the exon 6 region. The resulting PCR products were loaded on an agarose gel, and subsequently Sanger sequenced. 3.2.3. Protein 3D structure Protein 3D structure for the wild-type (WT) protein was predicted by Alphafold2484. Protein structure for the HeliosGly136_Ser191dup was modeled with AlphaFold2484, and executed on Google Colab using the default parameters485. We utilized PyMOL (http://www.pymol.org/) to generate HeliosGly153Arg protein structure image by mutagenizing Gly153 on the WT protein structure. 3.2.4. Protein expression analysis Helios expression was assessed by immunoblotting. Briefly, 8x105 HEK293 cells were seeded in 6-well culture plates in 2 mL of Dulbecco modified Eagle medium (DMEM) with 10% FBS (Gibco, 72 Life Technologies), 1% Penicillin-Streptomycin (Gibco, Life Technologies), 2mM L-glutamine (HyClone, Thermo Fisher Scientific), and 1mM sodium pyruvate (Gibco, Life Technologies) and incubated for 24 hours at 37°C prior to transfection. Each well was transfected with 3 ug of each plasmid (EV, WT, or variant) using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Whole cell lysates were prepared 24 hrs post transfection by lysing cells in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) supplemented with the Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentrations were measured using Pierce™ Coomassie Plus (Bradford) Assay Reagent. Lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). Membranes were blocked using 5% BSA in Tris-buffered saline supplemented with Tween-20, incubated with anti-Helios (Cell Signalling Technology, cat# 89270, 1:1000), anti-FLAG (Sigma Aldrich, cat# F1804, 1:1000), and anti-β-Actin (Cell Signalling Technology, cat# 3700, 1:20,000) primary antibodies in blocking buffer overnight at 4˚C, and lastly incubated with anti-rabbit (IgG DyLight 800, Rockland Immunochemicals) and anti-mouse (IgG IRDye 680RD, LI-COR) secondary antibodies at a concentration of 1:20,000 for 1hr at room temperature. The membranes were imaged using a LI-COR Odyssey infrared scanner (LI-COR Biosciences). The bands were quantified using Image Studio™ Lite (LI-COR) and normalized to their corresponding β-Actin expression levels. For total protein staining, Revert™ 700 Total Protein Stain Kit was used (LI-COR Biosciences). 73 3.2.5. Luciferase reporter assay The -580 to +57 region of the human IL2 promoter was cloned from genomic DNA obtained from HEK293 cells and the fragment inserted into PGL4.14 (Promega) firefly luciferase reporter plasmid using XhoI and HindIII restriction sites. Briefly, 1.5x105 HEK293 cells were seeded in 24-well culture plates in 0.5 mL of DMEM with 10% FBS, 1% Penicillin-Streptomycin, 2mM L-glutamine, and 1mM sodium pyruvate and incubated at 37°C for 24 hours prior to transfection. Cells were transfected with 250 ng of IL2 promoter plasmid, 250 ng of a plasmid encoding either empty vector (EV), WT and/or variants, and 10ng of PGL4.74 Renilla luciferase (Promega) control plasmid using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Specifically, cells received either EV, WT, or variant plasmids (250 ng) along with the promoter and renilla plasmid or were co-transfected with increasing ratios of variant to WT plasmid (respectively 0.5:1, 1:1, 2:1, 4:1 and 8:1) adding up to a total amount of 250 ng of expression plasmid. Cell lysates were prepared using 1x Glo Lysis Buffer (Promega) and transferred to white flat-bottom 96-well plates in technical triplicates. Dual-Glo Luciferase Assay Kit (Promega) was used according to manufacturer’s recommendations and luciferase activity was measured using the Infinite M200 plate reader (Tecan) by integrating luminescence over 10 seconds per well. 3.2.6. Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Statistical significance was evaluated by paired one-way ANOVA with the Dunnett's test to account for multiple 74 comparisons, paired two-tailed T-test, and Pearson correlation analysis. The following annotations were used to represent significance: p-val < 0.05 (*), < 0.01 (**), < 0.001 (***). 3.3. RESULTS 3.3.1. Identification of two unrelated individuals with syndromic developmental and immune abnormalities Proband 1 Proband 1 is a female child born at 38 weeks of gestation after an unremarkable pregnancy. Details of her medical history are provided in Table 3.1 and summarized here. Her birth weight was within the lower normal range (10th centile). Shortly after birth, she experienced respiratory distress due to excessive mucus, which resolved without intervention. She continues to experience increased lower respiratory secretions and chronic rhinorrhea. Investigations have shown normal ciliary ultrastructure and mild bronchiectasis. Table 3.1. Clinical summary and variant description of Probands 1 and 2. Proband 1 Proband 2 Variant Information Genomic change (GRCh37) NC_000002.11:g.213900960_213921017dup NC_000002.11:g.213914554C>G cDNA change NC_000002.11(NM_016260.3):c.406+540_574+13477dup NM_016260.3:c.457G>C Protein change NP_057344.2:p.Gly136_Ser191dup NP_057344.2:p.Gly153Arg Exon(s) Affected Exon 5 Exon 5 Variant Description Exon 5 is duplicated in tandem, resulting in the inframe duplication of ZFs 2 and 3 and two linker sequences in the DNA binding domain Missense variant is located in ZF2 in the DNA binding domain Variant detection method Trio genome sequencing– not detected by exome sequencing Trio exome sequencing Inheritance De novo De novo General Information Ancestry European European (Italian, Dutch) Consanguinity No No Sex Female Male 75 Proband 1 Proband 2 Pregnancy and Birth Antenatal complications • Unremarkable pregnancy • Normal growth and anatomy on ultrasounds at 18-20 weeks of gestation • Concern for gestational diabetes, treated with diet Gestational age • Spontaneous vaginal delivery at 38 weeks • Caesarean birth at 35 weeks for breech presentation with premature rupture of membranes Neonatal findings • Abnormal results on Auditory Brainstem Response (ABR) test • Respiratory congestion due to excessive mucus – ongoing issue since birth • Excessive weight loss due to feeding difficulties related to cleft palate • Abnormal results on Auditory Brainstem Response (ABR) test • Respiratory distress after birth and was intubated/on supplemental oxygen for 8-weeks in the NICU Birth weight (kg) 2.86 (10th percentile) 2.24 (30th percentile) Most Recent Examination Age 4-6 years 6-8 years Weight (kg) 15.5 (10th percentile) 21.2 (10th percentile) Height (cm) 106.5 (25th percentile) 119.6 (10th percentile) Neurobiological Features Developmental delay/ intellectual disability • Global developmental delay affecting language, fine motor skills, and processing speed and emotional regulation • Autism (CARS score 43 at 2.5y) – repetitive behaviors, head banging, poor eye contact, sensory-seeking/sensory-adverse behaviors, emotional outbursts Motor development • Delayed fine motor skills • Borderline-mild delay in gross motor skills Language and speech delay • Receptive language is at the low end of normal for age • Expressive language is one year delayed • Phonological speech disorder • Significant language/communication delays • Nonverbal MRI scan • Normal inner ear structures • Cystic lesion within sella • Normal inner ear structures Other Findings Urological No • Hypospadias Hearing/ENT • Bilateral severe-to-profound sensorineural hearing loss • Chronic clear rhinorrhea and nasal congestion • Bilateral moderate sensorineural hearing loss • Mild asymmetric right bony choanal stenosis Vision • Blepharophimosis • Severe photophobia • Yellow crusting on eyelashes due to increased mucous production • Variable esotropia • Fusion of the right eyelid • Lagophthalmos • The cornea of both eyes had stellate epithelial and anterior stromal central opacity Feeding • Cleft palate causing feeding difficulties early in life – corrected by surgery • Feeding through NG tube starting 4 months of age until receiving a G-tube at 13 months • Mainly on oral diet starting 3 years of age • Cleft palate • Persistent feeding difficulties, received a G-tube at 3 months of age Craniofacial differences • Bitemporal narrowing • Ankyloglossia • Upturned nose • High forehead • Macrotia • Depressed nasal bridge • Brachycephaly • Midface hypoplasia • Narrow and long palpebral fissures • Short philtrum • Simplified ears • Prominent chin 76 Proband 1 Proband 2 Teeth • Poor enamel • Thickened gums • Malaligned teeth • Dental caries Skeletal • No anomalies on examination and x-ray • Juvenile osteochondrosis of the head of the left femur Additional findings • Athelia (absent nipples) • Mild bronchiectasis with increased lower airway secretions • Athelia (absent nipples) • Chronic lung disease - discoid atelectasis • Sleep problems Immune Phenotype Newborn screening • Abnormal TREC assay – T cell lymphopenia • Abnormal TREC assay – T cell lymphopenia Susceptibility to infections • Recurrent respiratory infections • Recurrent pneumonia and ear infections Atopic disease • Atopic dermatitis diagnosed at 10 months • None noted Autoimmune disease • Autoimmune hemolytic anemia diagnosed at 2 months of age • None noted Additional findings • Lymphopenia: low total CD4 and CD8 T cells • Normal IgA and IgM levels • Unable to accurately assess endogenous IgG production – patient on IVIG • Normal tetanus and diphtheria vaccine titers documented off IVIG • Lymphopenia: low total CD4, CD8 and B cells • Mildly low IgG with normal IgM and IgA • Poor Hib, tetanus, and S. pneumoniae vaccine titers Perinatally, she experienced failure to thrive and feeding difficulties. She was diagnosed with cleft palate at 2 months old. She failed her newborn hearing screen and was diagnosed with sensorineural hearing loss at 6 weeks of age by auditory brainstem response (ABR) testing. Her visual reinforcement audiometry test at 17 months old indicated moderately severe to profound hearing loss in the right ear, and moderately severe to severe hearing loss in the left ear (Figure 3.1 A), which has since progressed to severe-to-profound hearing loss in both ears. Brain MRI and CT scan at 9 months old was unremarkable and indicated no inner ear anomalies. She received cochlear implants between ages 2.5-3.5 years old that have aided her hearing and language development. Proband 1 continues to experience developmental delays across multiple domains, including language, fine motor skills, processing speed, and emotional regulation. Notable craniofacial features include microcephaly, deep-set eyes, blepharophimosis, blepharitis, 77 photophobia, bitemporal narrowing, ankyloglossia, poor enamel, thickened gums, upturned nose, depressed nasal bridge, high forehead, and macrotia. She also has athelia (Figure 3.1 C-G). Figure 3.1. Clinical phenotypes of Proband 1 and 2. (A) Proband 1’s visual reinforcement audiometry (VRA) test completed at 17 months of age using insert headphones and warble tones. Air conduction thresholds from this test revealed moderately severe to profound hearing loss in the right ear, and moderately severe to severe hearing loss in the left ear. (B) Air conduction thresholds from Proband 2’s auditory brainstem response (ABR) test at 7 years of age indicated moderate to moderately severe hearing loss for the right ear and moderately severe to severe hearing loss for the left ear. (C-D) Face of Proband 1 at 10 months-1.5 years. In C and D note high forehead, macrotia, deep-set eyes, blepharophimosis, blepharitis (yellow crust on lashes), and 78 bitemporal narrowing. In D and E note inflammation and excoriation marks from atopic dermatitis involving the whole face. In E note upturned nose and depressed nasal bridge. (F) Mouth of Proband 1. Note misaligned teeth and thickened gums. (G) Chest of Proband 1. Note athelia (absent nipples). (H-J) Face of Proband 2 at 6-7.5 years. Note brachycephaly, midface hypoplasia, narrow and long palpebral fissures, lagophthalmos (incomplete closure of eyelids), depressed nasal bridge, short philtrum, simplified ears, prominent chin, abnormal dentition, and misaligned teeth. (K) Hand of Proband 2. Note callused digits. The photos are shared with the families' consent. At two months of age, she was diagnosed with an unusual presentation of autoimmune hemolytic anemia. Her direct antiglobulin test (DAT) was strongly positive with mild red cell agglutination and a pan-reactive antibody screen. Bone marrow failure was ruled out by bone marrow biopsy. Proband 1 was also diagnosed with T cell lymphopenia from birth, detected by both an abnormal T cell receptor excision circle (TREC) assay and abnormal flow cytometry documenting low T cell numbers (total CD3, CD8, CD4) (Table 3.2). Serum immunoglobulins showed normal levels of IgG, IgA, and IgM, but elevated IgE levels (Table 3.2). At 10 months of age, she developed severe atopic dermatitis, affecting her face, limbs, and trunk, which responded to topical corticosteroids and emollients. Proband 1 experiences frequent respiratory infections. To support her immune function, she is treated with monthly intravenous immunoglobulin therapy and daily oral antibiotic prophylaxis with good clinical benefit as documented by fewer respiratory infections requiring health service utilization. Table 3.2. Representative laboratory tests of Proband 1 and 2. Proband 1 (0.5-5 yo) Reference (0.5-5 yo) Proband 2 (7 yo) Reference (7 yo) Immune Subsets Lymphocytes (x109/L) 3.1 2.50 – 16.50 1.14 NA CD3+ T cells (x109/L) 1.10* 2.40 – 6.90 0.70* 1.20 – 2.60 CD4+ T cells (x109/L) 0.59* 1.40 – 5.10 0.47* 0.65 – 1.50 CD8+ T cells (x109/L) 0.18* 0.60 – 2.20 0.16* 0.37 – 1.10 CD19+ B cells (x109/L) 0.67* 0.70 – 2.50 0.23* 0.27 – 0.86 TREC (Copy number/3mL) 3.08* >75 Very Low* Normal Neutrophils (x109/L) 2.05 1 – 9 0.51 0.16 – 0.79 Monocytes (x109/L) 0.83 0.50 – 1.80 0.11* 0.19 – 0.85 79 *Indicates abnormal values that are not within the reference range. ^Patient receiving IgG replacement with IVIG. Proband 2 Proband 2 is a male child linked to this research project through GeneMatcher.org 486. He was born at 35 weeks of gestation. He experienced respiratory distress shortly after birth, and he was mechanically ventilated for the first 36 hours of life. He required supplemental oxygen for most of his 8-week stay in the neonatal intensive care unit. CT scan identified mucosal thickening and inflammation within the nasal cavity, in addition to mild asymmetric right bony choanal stenosis. His birth weight was within the normal range (30th percentile). Similar to Proband 1, he was diagnosed with cleft palate and had feeding difficulties. He required gastrostomy tube placement at 3 months of age. He failed newborn hearing screening and later his ABR test indicated moderate to severe hearing loss affecting both ears (Figure 3.1 B). His brain MRI demonstrated normal inner ear structures. Proband 2 has a history of recurrent pneumonia with chronic right upper lobe atelectasis, chronic nasal congestion, and frequent wheezing. He has a history of recurrent sinus infections and otitis media. He was diagnosed with T cell lymphopenia, based on abnormal newborn TREC assay and abnormal flow cytometry revealing low T (total CD3 and CD4) and B cell numbers. Assessment of humoral immune function revealed mildly low serum IgG with normal IgM and IgA levels, and additionally poor responses to routine vaccines (Table 3.1). Proband 1 (0.5-5 yo) Reference (0.5-5 yo) Proband 2 (7 yo) Reference (7 yo) Antibody Responses IgG (mg/dL) 1030^ 270 – 1140 474* 635 – 1284 IgA (mg/dL) 10 <80 54.7 32 – 191 IgM (mg/dL) 99 6 – 145 101 44 –190 IgE (IU/mL) 100.8* <7.5 9.6 <90 Tetanus antitoxin (IU/mL) 0.70 0.10 – 1.00 0* >0.1 Diphtheria antitoxin (IU/mL) 0.83 0.5 – 1.10 0.1 >0.1 80 Physical features of Proband 2 include brachycephaly, midface hypoplasia, narrow and long palpebral fissures, fusion of the right eyelid, lagophthalmos, corneal opacity, short philtrum, simplified ears, prominent chin, abnormal dentition (dental caries), athelia, and hypospadias (Figure 3.1 H-K). Proband 2 has also been diagnosed with autism (Childhood Autism Rating Scale score = 43) with repetitive behaviors, head banging, reduced eye contact, sensory-seeking/sensory-adverse behaviors, and emotional outbursts. He is currently nonverbal with a mild delay in gross motor skills (Table 3.1). 3.3.2. Identification of two monoallelic de novo IKZF2 variants Genome sequencing for Proband 1 and her unaffected parents revealed a 20 kb de novo heterozygous intragenic tandem duplication (2:213900960-213921017) in IKZF2 spanning exon 5 and parts of the flanking introns 4 and 5 of the Refseq transcript NM_016260.3 (NC_000002.11(NM_016260.3): c.406+540_574+13477dup) (Table 3.1; Figure 3.2A and 3.2C). Two bases were inserted at the join-point which was confirmed by Sanger sequencing (Figure 2C). This duplication is not present in reference population databases (DGV, gnomAD) or other variant databases (ClinVar, LOVD, Decipher, dbVar). It was detected by four of four in silico tools (Manta, Lumpy, CNVnator, ERDS), and was supported by evidence from split reads crossing the breakpoint junction, discordantly mapped read pairs, and increased read depth (Figure 3.2C). 81 Figure 3.2. Family pedigrees, RT-PCR, Sanger results, and schematic of the IKZF2 variants on the gene and protein. (A and B) The pedigrees of Proband 1 and 2. For individuals tested by genome and/or Sanger sequencing, the genotypes are noted. WT indicates a wild-type allele. Black boxes 82 show individuals with ICHAD. Proband 1 harbors a de novo intragenic tandem duplication and Proband 2 harbors a de novo missense variant (C) The Integrative Genomics Viewer visualization of the duplicated region, with discordant read pairs and increased read depth providing evidence of the genomic duplication in Proband 1 (on top). Sanger sequencing chromatogram of the tandem duplication breakpoint junction on DNA and a GT insertion (at the bottom) (D) The gel image on the left shows the results of the RT-PCR. The two bottom bands (b and c) in the first and second lanes represent the amplicons from the two wild-type transcripts (T1, NM_016260.3; T2, NM_001079526.2; wt, wild-type), while the two top bands that are only visible in the second lane (a and b) correspond to the products from the two transcripts with exon 5 duplication (dup, exon 5 duplication). On the right side of the figure, a schematic of the RT-PCR products is shown alongside the Sanger trace from the region marked on the four RT-PCR products. The second sequence maps to the exon 5-exon 5 junction on altered T2. (E) Schematic of the wild-type and altered gene (top) and protein (bottom). (F) Helios protein and its functional domains (ZFs 1-6). The de novo tandem duplication (p.Gly136_Ser191dup) in Proband 1 and the de novo missense variant (p.Gly153Arg) in Proband 2 are shown on top of the protein, and the previously reported variants in the literature are shown at the bottom of the protein. Both variants presented in this study are located in the DNA binding domain of Helios. ZF, zinc finger; Refseq NM_016260.3; Underlined variants are homozygous; Orange arrow indicates missense variant; Black arrow indicates stop gain variant. Comparison of common SNP allelic frequencies located in this region in Proband 1, and her parents indicated that the duplication arose on the maternal allele. Loading the RT-PCR products on the gel revealed four bands, two of which corresponded to the expected wild-type products for the two main transcripts of IKZF2, while the other two bands were approximately 168 bp larger than the corresponding wild-type products, indicating the presence of the duplication of exon 5 that encodes 56 amino acids in both transcripts (Figure 3.2 D). Subsequent Sanger sequencing of the RT-PCR products revealed a complete duplication of exon 5 on the coding sequence, and confirmed that the duplication occurs without any alteration in the reading frame or splicing (Figure 3.2 D and Figure 3.3). These findings indicate that this variant results in an in-frame duplication of residues Gly136 to Ser191 (NP_057344.2:p.Gly136_Ser191dup) (Table 3.1; Figure 3.2 E and 3.2 F). 83 Figure 3.3. Sanger sequencing results from the four bands corresponding to the RT-PCR products. At the top, a schematic of the RT-PCR products is shown. In the middle, a portion of the Sanger sequencing trace is shown, displaying four peaks corresponding to the regions indicated on the four RT-PCR products. The four sequences at the bottom are obtained from the Sanger trace from the marked region. The second sequence, T2(dup) maps to the exon5-exon5 junction in NM_001079526.2 transcript supporting presence of the exon5 duplication. We also detected the exon5-exon5 junction in NM_016260.3 transcript (Data not shown). T1, NM_016260.3; T2, NM_001079526.2. The terms 'wt' and 'dup' denote the wild-type and exon5-duplicated transcripts, respectively. Exon 5 of IKZF2 encodes N-terminal Cys2His2 (C2H2) zinc fingers (ZF) 2 and 3 and two linker sequences. ZFs 2 and 3 are highly conserved and are critical for DNA binding activity of Helios (Figure 3.2 E and 3.2 F)487,488. Figure 3.4 B illustrates a predicted structure of the wild-type and the altered protein. Notably, somatic tandem duplications in exon 5 of IKZF2 have been reported previously in lymphoma patients (Table 3.3)489. Table 3.3. Three somatic IKZF2 tandem duplications were reported previously in three cases of Peripheral T-cell lymphoma Not Otherwise Specified (PTCL-NOS). Case ID Chromosome Start End Reference PTCL012 chr2 213,895,993 213,915,694 19,701 PMID: 31092896 PTCL013 chr2 213,914,002 213,917,426 21,433 PMID: 31092896 PTCL062 chr2 213,913,371 213,992,982 79,611 PMID: 31092896 84 Exome sequencing for Proband 2 and his unaffected parents (Family 2) revealed a de novo heterozygous p.Gly153Arg missense variant in IKZF2 (NM_016260.3:c.457G>C) (Figure 3.2 B and 3.2 F). This variant is absent in reference population databases including gnomAD, TOPMed, Decipher, ClinVar, and LOVD. All five in silico tools (SIFT=”Damaging”, Polyphen-2=”Probably damaging”, REVEL=”0.5”, FATHMM-XF=”1.0”, and CADD=”32”) predict that this missense variant has a damaging impact on the protein. Gly153 is a highly conserved residue across vertebrates and the paralogs of IKZF2 (Figure 3.4 A). The missense variant is in ZF2 on the +2 residue which is one of four residues (+6, +3, −1 and +2 in reference to the beginning of α helix) critical for DNA recognition and binding (Figure 3.4 A)490. Figure 3.4 C shows the predicted structure of ZF2 and the Gly153 and Gly153Arg. Pathogenic variants affecting the four residues of the α helix have been reported in Ikaros and Aiolos491. Specifically, residues in the +2 position in the ZF domains have an important function in DNA binding by recognizing and making contact with the complementary strand of DNA490. 85 Figure 3.4. Multiple alignments of ZFs 2 and 3 and the linker sequences in orthologs and paralogs of IKZF2. (A) The tandem duplication (p.Gly136_Ser191dup) affects ZFs 2 and 3 and linker sequences which are highly conserved across vertebrates and the paralogs, IKZF1, IKZF3, and IKZF4. The underlined sequence shows ZFs 2 and 3, and the remaining sequence represents the linkers. The missense variant (p.Gly153Arg) affects an amino acid which is situated at position 2 in reference to the start of α helix of ZF2 and has a critical role in DNA recognition and binding. (B) The Alphafold predicted structure of ZFs 1-4. The left structure shows four ZFs (ZFs 1-4), and the right structure shows six ZFs including the duplication (ZFs 1, 2, 3, 2, 3, 4). ZF, zinc finger. (C) The Alphafold predicted structure of human ZF2. ZF2 is shown in red and the amino acid Gly153 shown in dark blue. The left structure shows the wild-type residue, Gly153, and the right structure shows the altered residue, Gly153Arg. A similar amino acid substitution, p.Gly159Arg, affecting the same homologous amino acid in IKZF3 (Aiolos) (Figure 3.5) has been functionally shown to be pathogenic, as it impairs binding of Aiolos homodimers and Aiolos-Ikaros heterodimers to their corresponding canonical DNA sequences in a dominant-negative manner492. Figure 3.5. Amino acid sequence alignment of human Helios (IKZF2) and human Aiolos (IKZF3) using BlastP. The residues highlighted in red are HeliosGly153 and AiolosGly159. HeliosGly153 is homologous to AiolosGly159. AiolosGly159Arg is a previously known pathogenic variant. 86 3.3.3. HeliosGly136_Ser191dup protein expression is reduced while HeliosGly153Arg protein expression is comparable to WT Helios To understand the impact of p.Gly136_Ser191dup on protein expression, we utilized Human Embryonic Kidney (HEK293) cells which lack basal expression of Helios and subsequently transfected them with pCMV6-XL4 plasmids containing either FLAG-tagged wild-type IKZF2 (WT), p.Gly136_Ser191dup (Dup) IKZF2, or an empty vector (EV). As expected, EV-transfected cells lacked Helios expression as assessed by both FLAG and Helios antibody probes, confirming absence of detectable endogenous expression of Helios protein in HEK293 cells. In contrast, in WT IKZF2 transfected cells, a prominent band at ~70 kDa was detected by FLAG and Helios antibodies, coinciding with the expected size of the major isoform of the Helios protein (Figure 3.6 A and 3.6 B). HeliosGly136_Ser191dup was expressed at ~75 kDa, reflecting the 56 amino acid duplication of ZFs 2 and 3. However the relative expression of the variant was significantly reduced to ~50% of the WT Helios protein, as quantified by densitometry (Figure 3.6 A-C). To model the heterozygous state of Proband 1, we co-transfected cells with both WT and Dup IKZF2 plasmids. The expression of HeliosGly136_Ser191dup remained significantly lower than the WT protein (Figure 3.6 D). We also show that the expression of the WT protein is independent of Heliosp.Gly136_Ser191dup, as the amount of the WT protein co-expressed with the variant remains unaffected compared to when it is co-expressed with the EV (Figure 3.6 E). However, we show that the co-expression of Heliosp.Gly136_Ser191dup with the WT protein leads to significantly higher expression levels of Heliosp.Gly136_Ser191dup compared to its co-expression with EV. This suggests that the presence of the WT protein may affect the stability of the variant protein (Figure 3.6 F). 87 Figure 3.6. Assessment of variant protein expression in HEK293 cells. (A and B) Immunoblot of lysates obtained from HEK293 cells transfected with with pCMV6-XL4 plasmids expressing FLAG-tagged wild-type IKZF2 (WT), p.Gly136_Ser191dup variant (Dup), and/or empty vector control (EV). Protein was detected using anti-FLAG antibody in A and anti-Helios antibody in B. An anti-β actin antibody was used as loading control. A, B are representative blots from five independent experiments. (C, D, E and F) Band intensities of FLAG and β-actin fluorescence signals were quantified using Image Studio™ Lite Quantification Software (Licor). Data presented are mean ± SEM from five independent experiments. Paired two-tailed t-test was used for statistical analysis: 88 **p £ 0.01; ***p £ 0.001. (G and H) Immunoblot of lysates obtained from HEK293 cells transfected with pCMV6-XL4 plasmids expressing FLAG-tagged WT IKZF2, p.Gly153Arg (G153R), and/or EV. Protein was detected using anti-FLAG antibody in G and anti-Helios antibody in H. An anti-β-actin antibody was used as loading control. G and H are representative blots from three independent experiments. (I) Quantification of band intensities of FLAG and β-actin fluorescence signals using Image Studio™ Lite. Data presented are mean ± SEM from three independent experiments. Paired two-tailed t-test was used for statistical analysis; ns, not significant. The expression of HeliosGly153Arg was indistinguishable from WT protein, as quantified by densitometry (Figure 3.6 G-I). To recapitulate the heterozygous state of Proband 2, we co-transfected cells with both WT and p.G153R (G153R) plasmids. Total Helios protein expression remained unchanged and statistically comparable to that of the WT Helios and HeliosGly153Arg alone (Figure 3.6 I). This suggests the expression of WT and HeliosGly153Arg are independent of each other. In addition, to confidently interpret changes to protein expression levels for both variants, total protein staining was used to ensure even loading of the gel (Figure 3.7). 89 Figure 3.7. Total protein staining using Revert 700 total protein stain. Total protein was extracted from HEK293 cells transfected with pCMV6-XL4 plasmids expressing FLAG-tagged wild-type IKZF2 (WT), p.Gly136_Ser191dup (Dup), p.Gly153Arg (G153R), and/or empty vector control (EV). Samples was separated by gel electrophoresis and stained using Revert 700 total protein stain (bottom image), and anti-FLAG and beta-actin antibodies (top image). The molecular weight markers are indicated on the left, and the protein bands are indicated on the right. 3.3.4. HeliosGly136_Ser191dup and HeliosGly153Arg cannot effectively repress IL2 promoter activity and interfere with WT protein function in a dominant-negative manner Exon 5 encodes ZFs 2 and 3, which are critical domains for DNA recognition and binding capability of Helios 458. Helios binds to and epigenetically modifies the Interleukin 2 (IL2) promoter, supressing its expression 460. We employed a luciferase reporter assay to assess the ability of both variant proteins to suppress IL2 expression. We co-transfected HEK293 cells with a firefly luciferase reporter plasmid containing the -580 to +57 region of the human IL2 promoter, EV, or WT/variant plasmids, and Renilla luciferase plasmid (Figure 3.8 A). We measured firefly luciferase luminescence as an indicator of IL2 promoter activity and normalized this value to Renilla luciferase luminescence to account for differences in transfection efficiency. HeliosWT (represented by 0% variant to WT plasmid ratio) was able to repress IL2 promoter activity significantly more effectively than HeliosGly136_Ser191dup and HeliosGly153Arg expressed alone (100% variant to WT plasmid ratio). When the WT plasmid was co-transfected with increasing ratios of p.Gly136_Ser191dup or p.Gly153Arg plasmids (respectively 0.5:1, 1:1, 2:1, 4:1 and 8:1 ratios of variant to WT plasmids), we observed a significant increase in the promoter activity of IL2, which indicated a decrease in the ability of the variant proteins to repress the IL2 promoter in a dominant-negative manner (Figure 3.8 B and 3.8 C). 90 Figure 3.8. Luciferase reporter assay to assess variants’ ability to repress IL2 promoter activity. (A) Schematic representing the experiment. HEK293 cells were transfected with an empty pCMV6-XL4 (EV) plasmid or a plasmid encoding the wild-type (WT) and/or the indicated ratios of IKZF2 variants, a pGL4.14 firefly luciferase reporter plasmid carrying a region of the human IL2 promoter, and pGL4.74 renilla luciferase plasmid as transfection control. (B and C) After 24 hours, cells were lysed and measured for firefly luciferase and renilla luciferase activity. Data presented are mean ± SEM from three independent experiments, and each independent experiment is an average of three technical replicates. Dotted lines indicate 95% confidence intervals for Pearson's Correlation test. 91 3.4. DISCUSSION In this study we describe a novel multisystem disorder in two unrelated patients caused by heterozygous pathogenic de novo variants affecting the DNA binding domain of Helios. Both variants result in syndromic features including craniofacial differences, athelia, early-onset bilateral sensorineural hearing loss, cleft palate, developmental delays, combined with immune system abnormalities including T cell lymphopenia and recurrent respiratory infections. We propose the acronym ICHAD, short for Immunodysregulation, Craniofacial anomalies, Hearing impairment, Athelia, and Developmental delay. Although some functional roles of Helios remain poorly understood, its broad expression during embryogenesis454,462,463 likely underlies the developmental phenotypes observed in both probands. There are phenotypic similarities between the patients and various murine models of Helios deficiency. For example, both probands have developmental abnormalities of their eyelids. Proband 1 has blepharophimosis and blepharitis (Figure 3.1 C and 3.1 D), and Proband 2 has fusion of his right eyelid, lagophthalmos, and corneal opacity (Figure 3.1 H and 3.1 J). Replicating this human phenotype, Ikzf2−/− mice have smaller eye openings493, and incomplete palpebral closure due to inflammation of the sebaceous glands in the eyelids456, while Ikzf2cello/cello mice exhibit microphthalmia and a ‘cloudy eye’ phenotype452. In addition, replicating the early-onset bilateral sensorineural hearing loss seen in both patients (Figure 3.1 A and 3.1 B), Ikzf2cello/cello mice show elevated ABR thresholds and postnatal hearing impairment, as explained by the role of Helios in the maturation of outer hair cells of the cochlea452. 92 Consistent with the role of Helios in the development of T lymphocytes and regulation of the immune system, both probands have T cell lymphopenia and enhanced susceptibility to infections, while Proband 1 is also affected by immune dysregulation in the form of atopy and autoimmunity. Similar immune features are seen in global and selective murine models of Helios deficiency. Mice with Helios-deficient Tregs develop progressive systemic immune activation over time494, and Ikzf2−/− mice develop autoimmune disease by 6–8 months of age51. Helios deficiency in mice has also been linked to neurodevelopmental abnormalities. Martín-Ibáñez et al. have shown that Helios has a regulatory role in development of the striatum, cortex, and hippocampus in mice, and Ikzf2−/− mice show a decrease in striatopallidal neurons of the dorsomedial striatum that control motor skill learning during embryonic development 454,463. Ikzf2−/− mice also exhibit schizophrenic-like behaviors related to dysfunction in the striatum and hippocampus455. When considered in aggregate, the murine models support a role of Helios impairment in causing developmental delays. Since both HeliosGly136_Ser191dup and HeliosGly153Arg proteins are expressed (Figure 3.6), haploinsufficiency is unlikely to be the mechanism of disease. Both patients are heterozygous for variants that affect critical and highly conserved zinc fingers (ZF) in the DNA binding domain of Helios that recognize unique DNA motifs and thus determine the overall sequence specificity and affinity of Helios495. As such, we hypothesized that both variants would alter the function of WT Helios in a dominant-negative manner. The pathogenicity and dominant-negative impact of p.Gly136_Ser191dup and p.Gly153Arg are supported by our luciferase reporter assay using the IL2 promoter which showed that co-transfecting WT plasmid with increasing amounts of variant 93 plasmids resulted in significant increase in the transcriptional activity of the IL2 locus, thereby demonstrating that the variant proteins fail to suppress IL2 transcription in a dominant-negative manner (Figure 3.8). We anticipate this assay will be useful in assessing the functional impact of additional novel IKZF2 variants likely to emerge. Other evidence supporting a dominant-negative mechanism comes from similar tandem duplications involving ZFs 2 and 3 of Helios that have been reported in cases of peripheral T-cell lymphoma and a dominant-negative function has been hypothesized for these variants (Table 3.3)489. Somatic variants in Helios with dominant-negative function have been detected in hematopoietic malignancies496. Furthermore, the discordance of the number of ZFs between HeliosGly136_Ser191dup and the WT protein may affect the size and structure of the altered Helios homodimers and heterodimers, consequently impacting their ability to bind to canonical DNA sequences. The dominant-negative effect of HeliosGly136_Ser191dup may result from variant protein forming dysfunctional dimers with WT protein. Finally, based on the role of the gene and zinc finger duplications as an evolutionary process in the functional diversification of the poly-zinc-finger gene family497, we speculate that the duplication of zinc fingers in our study may have a neomorphic effect by recognizing novel DNA binding sites. However, we did not experimentally investigate this, and further studies would be necessary to confirm/refute this hypothesis. Evidence for pathogenicity of HeliosGly153Arg can be drawn from study of human diseases related to missense variants in other Ikaros family members. Gly153 is a highly conserved residue across paralogs (Figure 3.4 A) and is homologous to Gly159 in Aiolos (Figure 3.5). Recently, Yamashita et al. reported a pathogenic missense variant (p.Gly159Arg) in Aiolos, resulting in T 94 and B cell defects in the affected individuals due to dominant-negative and neomorphic effects492. AiolosGly159Arg was expressed comparably to WT and formed homodimers with WT Aiolos but failed to recognize the canonical Aiolos DNA sequences. Furthermore, AiolosGly159Arg interfered with Ikaros function by forming defective heterodimers that were unable to bind to canonical Aiolos-Ikaros DNA binding sequences. AiolosGly159Arg also showed a neomorphic effect by binding to novel sequences. Based on the homology between Gly153 in Helios and the pathogenic Gly159 in Aiolos, we propose that the Gly153Arg substitution not only exerts a dominant-negative effect but may also result in a neomorphic effect as does AiolosGly159Arg. Nevertheless, the focus of our study was on the dominant-negative effect, and further investigations are needed to confirm this proposition. In conclusion, our data demonstrate that dominant-negative-acting variants in Helios, a key transcription factor involved in hematopoietic development and embryogenesis, are associated with a syndromic disorder involving developmental and immune abnormalities. 95 CHAPTER 4: A multimorphic variant in ThPOK causes a novel inborn error of immunity characterized by T cell immune developmental abnormalities, immunodysregulation, atopy, and organ fibrosis. 4.1 INTRODUCTION AND RATIONALE T-helper-inducing POZ/Kruppel-like factor (ThPOK), encoded by ZBTB7B, is a transcription factor that belongs to the zinc finger and BTB/POZ domain-containing (ZBTB) family of proteins62. ThPOK acts both as an activator and a suppressor of transcription through direct binding to promoter regions of target genes and by interaction with various co-regulators63. In recent years, ThPOK has garnered significant attention in immunological research, primarily through insights gained from murine models. The most studied role of ThPOK is its function in directing the differentiation of T cell lineages, particularly in promoting the development of CD4+ T cells and inhibiting the development of CD8+ T cells in the thymus64. This discovery was initially made based on a spontaneous mutant mouse strain with a biallelic missense variant in the second zinc finger domain of ThPOK. These so-called ‘helper-deficient’ (HD) mice show a selective absence of mature CD4+ T cells and increased frequency of CD8+ T cells. While the primary focus on ThPOK has been its immune functions, its role extends to other biological processes, although these are less well-understood. For example, ThPOK is highly expressed in the skin and downregulates type I collagen gene expression in murine fibroblasts, binding specifically to the regulatory regions of type I collagen genes (Col1a1 and Col1a2)66. Despite advances in our understanding of the role of ThPOK in immunity, the direct implications of ZBTB7B variants in human health have remained largely unexplored. To date 96 disabling genetic changes in ThPOK have not been implicated as a cause of human monogenic disease. Here, we describe a patient with a de novo heterozygous variant (NM_001256455.2:c.1080A>C, p.K360N) in ZBTB7B. Given the pivotal role of ThPOK in regulating T cell lineage commitment and its known interactions with collagen gene regulation in murine models, we hypothesized that pathogenic variants in ZBTB7B might disrupt T cell maturation, leading to an imbalance between CD4+ and CD8+ T cell populations, but could also influence fibroblast function and collagen gene expression, potentially contributing to tissue fibrosis. Indeed, the patient exhibited a complex syndromic phenotype including CD4 T lymphopenia, CD8 T lymphocytosis, early-onset allergic disease, fibroinflammatory interstitial lung disease, and growth failure. Our functional investigations demonstrated multimorphic activity of ThPOKK360N. ThPOKK360N demonstrated both loss-of-function, by impairing T cell development and failing to inhibit fibroblast profibrotic pathways (antimorph and hypomorph), and neomorphic effects through altered gene regulation and DNA-binding specificity. Functional assays in patient derived primary fibroblasts directly linked the variant to patient’s clinical manifestations of fibrosis, with the variant inducing a profibrotic gene expression in fibroblasts, while overexpression of the WT protein reversed this phenotype. This description of a novel IEI caused by a multimorphic variant in ThPOK confirms ThPOK's role in CD4 development in an intact human context, while also revealing an unanticipated role for ThPOK in tissue fibrosis. 97 4.2 SUBJECTS AND METHODS 4.2.1 Study design The patient with a previously uncharacterized disease was identified after presenting to the immunology clinic. Trio whole genome sequencing identified a de novo heterozygous variant in ZBTB7B that results in a missense change in the DNA-binding domain of ThPOK. The objective of this study was to elucidate the pathophysiology of this novel syndromic disease characterized by immune developmental abnormalities, immunodysregulation, allergy, and organ fibrosis. We performed extensive phenotyping of the patient’s peripheral blood cells by single-cell RNA sequencing (scRNA-seq) and conventional flow cytometry to reveal the immune cell abnormalities associated with the disease. To further analyze the functional defects of T cells, we employed a retroviral approach to test whether the variant protein could rescue ThPOK deficiency or alternatively interfere with normal ThPOK functions in CD4 T cells during infection responses. To assess the fibrotic phenotype, we employed an in vitro system, utilizing lentiviral transduction to introduce both the wild-type (WT) and variant genes into primary pulmonary fibroblasts derived from healthy controls and the patient. This approach successfully replicated the patient’s profibrotic phenotype in a relevant cellular model, mirroring findings seen through clinical imaging, histopathology, and functional pulmonary tests. This method also established direct causality, as the overexpression of the WT gene reversed the fibrotic gene signature in these cells. We also did treatment testing using two antifibrotic agents one of which successfully reversed the fibrotic gene signature in patient primary pulmonary fibroblasts in an in-vitro model 98 of fibrosis. Lastly, we uncovered the altered DNA binding specificity and transcriptional activity of the variant by multiple approaches, including EMSA, HT-SELEX, luciferase assay, and ChIP-seq. 4.2.2 Study approval All study participants and/or their parents/guardians provided written informed consent. Research study protocols were approved by the University of British Columbia Clinical Research Ethics Board (H15-00641). 4.2.3 Genomic analysis Genomic DNA from the patient, mother and father were sequenced with paired-end reads on the Ilumina platform by GeneDx. Average mean sequencing coverage was reported to be at least 40x across the genome, with a minimum threshold of 30x for each sample. Bi-directional sequencing reads were assembled and aligned to the reference sequence based on NCBI Refseq transcripts and human genome build GRCh37/UCSC hg19. Using a custom-developed analysis tool (XomeAnalyzer), data were filtered and analyzed to identify sequence variants, repeat expansions, and most deletion and duplications greater than 1kb. A missense variant in ZBTB7B was reported as the only candidate with a potential relationship to the disease phenotype, which was only observed in the patient. 4.2.4 Histopathology A wedge biopsy was obtained from the peripheral edge of right upper lobe of the lung in a surgical open biopsy procedure. The immunohistochemistry staining of the lung tissue was performed in BC Children’s Hospital Pathology laboratory with clinically validated antibodies and protocols. 99 4.2.5 Expression plasmid cloning The wild-type (WT) Myc-DDK tagged ZBTB7B plasmid (cat #RC234388), corresponding to the human tagged ORF clone NM_001256455.2, was purchased from OriGene. The gene insert containing the point mutation corresponding to the patient’s variant (c.A1080C, NM_001256455.2) was ordered from GenScript Biotech and inserted into the pCMV6-Entry plasmid (cat #PS100001, OriGene) using AsiSI and SacII. The sequence integrity of the full-length plasmid was confirmed using Sanger Sequencing and long-read sequencing (Oxford Nanopore Technologies) through the Plasmidsaurus platform (plasmidsaurus.com, Eugene, OR). 4.2.6 Immunoblotting ThPOK expression was assessed by immunoblotting. Briefly, 1x106 HEK293 cells were seeded in 6-well culture plates in 2 mL of Dulbecco modified Eagle medium (DMEM) with 10% FBS (Gibco, Life Technologies), 2mM L-glutamine (HyClone, Thermo Fisher Scientific), and 1mM sodium pyruvate (Gibco, Life Technologies) and incubated overnight at 37°C prior to transfection. Each well was transfected with 3 ug of relevant plasmids using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Whole cell lysates were prepared 24 hrs post transfection by lysing cells in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) supplemented with the Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentrations were measured using Pierce™ Coomassie Plus Assay Reagent (Thermo Fisher Scientific). Laemmli Sample Buffer (Bio-Rad Laboratories) supplemented with β-mercaptoethanol was added to the cell lysates and boiled at 37°C for 5 minutes. Lysates were separated by 10% SDS-PAGE and transferred onto 100 polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were blocked using 5% BSA in Tris-buffered saline supplemented with Tween-20, incubated with anti-ThPOK (D9V5T) Rabbit mAb (Cell Signalling Technology, cat# 13205, 1:1000), anti-Myc-Tag (9B11) Mouse mAb (Sigma Aldrich, cat# F1804, 1:1000), and anti-β-Actin (8H10D10) Mouse mAb (Cell Signalling Technology, cat# 3700, 1:20,000) primary antibodies in blocking buffer overnight at 4˚C, and lastly incubated with anti-rabbit (IgG DyLight 800, Rockland Immunochemicals) and anti-mouse (IgG IRDye 680RD, LI-COR) secondary antibodies at a concentration of 1:20,000 for 1hr at room temperature in blocking buffer. The membranes were imaged using the Odyssey DLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences). The bands were quantified using EmpiriaStudio (LI-COR Biosciences) and normalized to their corresponding β-Actin expression. 4.2.7 Luciferase reporter assay A 1406 bp region of the promoter sequence of human Suppressor of cytokine signaling 1 (SOCS) including a part of exon 1 (chr16:11,255,899-11,257,304 on GRCh38/hg38) was ordered from GenScript Biotech and cloned into pGL4.20 [luc2/Puro] (Promega) firefly luciferase reporter plasmid using KpnI and HindIII restriction sites. The sequence integrity of the full-length plasmid was confirmed using Sanger Sequencing and long-read sequencing (Oxford Nanopore Technologies) through the Plasmidsaurus platform. Briefly, 1.5x105 HEK293 cells were seeded in 24-well culture plates in 0.5 mL of DMEM with 10% FBS, 2mM L-glutamine, and 1mM sodium pyruvate and incubated at 37°C for 24 hours prior to transfection. We analyzed the effects of the empty vector (EV), wild-type (WT), and p.K360N variant plasmids, both individually and in combination, on the activity of the SOCS1 promoter. To test the effect of each plasmid in 101 isolation, cells were transfected with 250 ng of a single type of plasmid, which either contained an EV, the WT ZBTB7B gene, or the p.K360N variant. To model the heterozygous state of where the WT protein is present with p.K360N, co-transfections were conducted where cells received a combination of WT and p.K360N plasmids in increasing ratios of WT to p.K360N (0.5:1, 1:1, 2:1, 4:1, and 8:1) totaling to 250 ng. Additionally, all cells were also transfected with 250 ng of SOCS1-luciferase reporter plasmid and 10 ng of PGL4.74 renilla luciferase control plasmid (Promega). The transfection was carried out using Lipofectamine™ 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 24 hours, cell lysates were prepared using 1x Glo Lysis Buffer (Promega) and transferred to white flat-bottom 96-well plates in technical triplicates. Dual-Glo Luciferase Assay Kit (Promega) was used according to manufacturer’s recommendations and luciferase activity was measured using the Infinite M200 plate reader (Tecan) by integrating luminescence over 10 seconds per well. In the analysis step, the firefly luciferase activity was normalized against renilla luciferase which controlled for variation in transfection efficiency. The normalized firefly luciferase activity was further divided by the normalized value from the EV condition, providing a relative measurement against the EV baseline. 4.2.8 Electrophoretic mobility shift assay (EMSA) Preparation of whole cell lysates was performed as previously described in the immunoblotting section above. A section of the SOCS1 promoter containing the WT ThPOK consensus DNA-binding sequence (5’ CAAATTACAGCCCCGACCACCGACCGCCCACCATC 3’) was used to generate the oligonucleotide probe used in this assay. Based on this sequence, custom single-stranded IRDye 700–labeled oligonucleotides were ordered from Integrated DNA Technologies, and 102 double-stranded oligonucleotides were generated by annealing using 10x annealing buffer made in house by combining 100 uL of 100 1M Tris-HCl, pH 7.5, 200 uL of 5M NaCl, 20 uL of 0.5M EDTA and 680 uL of molecular biology grade water. The annealing conditions were 98°C for 3 min followed by cooling overnight at room temperature. Supershift assays were performed with 5 ug of nuclear protein lysate incubated on ice for 30 min with either anti-ThPOK (D9V5T) Rabbit mAb (Cell Signalling Technology, 1:1000) or Rabbit IgG Isotype Control (Invitrogen, Thermo Fisher Scientific) and then incubated at room temperature for 20 min with the labelled DNA probes and reagents provided in the Odyssey® EMSA Kit (LI-COR Biosciences) according to the manufacturer’s recommendations. Reagents used from the commercial EMSA Kit specifically included: 10x Binding Buffer, 25 mM DTT with 2.5% Tween® 20, Poly (dI•dC), sheared salmon sperm DNA, 50% Glycerol, 1% NP-40, and 1 M KCl, 10X Orange Loading Dye. Protein-oligonucleotide-antibody mixtures were then subjected to electrophoresis in 5% acrylamide/Bis-acrylamide 29:1 gel in 1x Tris-borate-EDTA (TBE) migration buffer (Thermo Scientific) for 120 min at 70 V at room temperature. Imaging was done using the Odyssey DLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences). Table 4.1. List and sequence of IRDye labelled WT and variant probes used in EMSA. SOCS1 promoter probe 5’caaattacagcccCGACCACCgaccgcccaccatc 3’ sense – IRDye700 5’ gatggtgggcggtcggtggtcggggctgtaatttg 3’ anti-sense Variant Probe 1 5’ caaattacagcccCATCCCCCgaccgcccaccatc 3’ sense – IRDye700 5’ gatggtgggcggtcgggggatggggctgtaatttg 3’ anti-sense Variant Probe 2 5’ caaattacagcccCCTCCACCgaccgcccaccatc 3’ sense – IRDye700 5’ gatggtgggcggtcggtggagggggctgtaatttg 3’ anti-sense 103 The bolded letters within the sequences represent the core DNA-binding consensus sequence. The dinucleotides highlighted in green and red denote regions corresponding to wild-type (WT) and variant dinucleotide changes, respectively, as identified by high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). 4.2.9 High-Throughput Systematic Evolution of Ligands by EXponential enrichment (SELEX) WT and variant ThPOK (ZBTB7B_WT and ZBTB7B_K360N) were cloned into an eGFP expression vector pF3A-ResEnz-egfp. The transcription factor samples were expressed by using a TNT SP6 High-Yield Wheat Germ Protein Expression System Kit (Promega). High-Throughput Systematic Evolution of Ligands by EXponential enrichment (HT-SELEX) was modified from our previous approach498 to use Abcam Anti-GFP antibody ab290 immobilized to Protein G Mag Sepharose® Xtra Cytiva 28-9670-70) in the step where the protein–DNA complexes are separated from unbound DNA. The assay similarly used IVT-produced proteins, and the selection reactions were carried out in a buffer of 140 mM KCl, 5 mM NaCl, 1 mMK2HPO4, 2 mM MgSO4, 100 uM EGTA, 1 mM ZnSO4, and20 mM HEPES-HCl (pH 7). After selections, the ligands were amplified by PCR and then subjected to Illumina sequencing. Mung Bean Nuclease treatment also was used to reduce the ssDNA background during the ligand selection. Data analysis was performed as described499, where automatic detection of a sequence pattern defining local maxima was followed by semi manual generation of seeds that were then used to construct multinomial-1 or multinomial-2 position frequency matrices for the transcription factor target specificity. 104 4.2.10 Chromatin Immunoprecipitation Sequencing To establish GFP-tagged ZBTB7B expressing HEK293 Flp-In-TRex cell lines, parental HEK293 Flp-In-TRex cells were transfected with separate expression vectors each carrying the WT of the variant ZBTB7B open reading frames (FuGENE® HD Transfection Reagent, Promega) in 6 well plates and after 48 hours transferred to Hygromycin selection media (0.2ug/ul). Colonies of each line were pooled and used for further experiments. 24 hours prior to cross linking for chromatin immunoprecipitation, doxycycline (100ng/ml) was added to cells and GFP expression was confirmed with fluorescent microscopy. Chromatin immunoprecipitation was performed as previously described500. In brief, cells from 15cm plates at 100% confluency were cross-linked for 10 min in 1% formaldehyde followed by 10 min of quenching with 2M glycine. After washing the cells with cold PBS, cells were collected and pelleted. Using a three-step lysis process, chromatin was released and then sonicated to produce DNA fragment length range of 200–300 bp using a Bioruptor (Diagenode). GFP-tagged proteins were immunoprecipitated with a polyclonal anti-GFP antibody (ab290, Abcam) and Dynabeads Protein G (Invitrogen). Crosslinks were reversed at 65°C overnight and bound DNA fragments were purified (QIAquick PCR Purification Kit, Qiagen). ChIP libraries were prepared using NEBNext Ultra II DNA kit. Sequencing was performed with 150n paired end at 2x107 reads per sample. For each variant of ZBTB7B, two biological replicates and two input control samples were sequenced using the NovaSeq 6000 Illumina sequencer. In the data processing step, adapter sequences were trimmed from ChIP-seq reads with Cutadapt (v2.1) (https://doi.org/10.14806/ej.17.1.200) and mapped to the human genome (hg38) with Bowtie2 (v2.4.1)501 using the –very-sensitive option. Reads with a map quality of less 105 than 30 were discarded along with PCR duplicates and reads for which one or both of the paired ends could not be mapped. A single set of control reads was generated using the four-input control ChIP-seq replicates by merging them with SAMTools (v1.9)502 and subsampling to one quarter of the total reads. MACS2 (v2.2.9.1)503 was used to call peaks for each of WT and variant ThPOK ChIP-seq replicates using the merged input read set as the control. For each set of peaks, the 2000 with the highest enrichment scores were used for downstream analyses. For each wild-type ThPOK ChIP replicate, we scanned the peak sequences to identify peaks that contained a match to the HT-SELEX wild-type ThPOK motif using FIMO (v5.5.0)504 with default settings. Using BEDTools intersect (v2.30.0)505 we overlapped the motif-containing ChIP peak summits for each replicate with a BED file containing the locations of human gene promoter regions (1000 bp upstream of the transcription start site to 500 bp downstream). To establish a set of high-confidence candidates for direct regulation by WT ThPOK we took the intersection of genes identified from each replicate. We then performed the same analysis using the variant ThPOK ChIP replicates and the variant ThPOK HT-SELEX motif. We searched for overrepresented pathways in the set of high-confidence ThPOK regulated genes and variant ThPOK regulated genes using the PANTHER statistical overrepresentation test with PANTHER pathways506. We checked each set in its entirety, each set excluding genes that overlapped with the other set, and the intersection of the two sets. We report all overrepresented PANTHER pathways with a false discovery rate P < 0.05. 106 4.2.11 Isolation and culture of primary pulmonary fibroblasts Fresh lung tissue was collected from the patient undergoing open lung biopsy for histopathological evaluation of interstitial lung disease. The tissue was immediately transported to the laboratory in ice-cold Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin (Thermo Fisher Scientific). We followed an optimized published method for generating a fibroblast-enriched single-cell suspension combining mechanical and enzymatic dissociation507. Concentrations for all the reagents used are reported in the original study507. In brief, the tissue was minced into small pieces using sterile scissors and forceps in Dulbecco's phosphate-buffered saline supplemented with Amphotericin B (Gibco). The tissue was then digested with a mixture of Collagenase P (Roche) and Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich) for 75 minutes at 37°C with gentle agitation. The digested tissue was then mechanically dissociated by pipetting up and down using a 10 ml serological pipette, followed by filtration through a 40 um cell strainer (Corning) to obtain a single-cell suspension. The suspension was then incubated with TrypLE Express (Thermo Fisher Scientific) and red blood cell lysis buffer (Sigma-Aldrich) to disrupt intercellular adhesions and remove red blood cells, respectively. The processed cells were resuspended in Fibroblast Growth Medium 2 (PromoCell) and plated onto tissue culture-treated dishes (Corning) for culture. The cells were maintained in a humidified incubator at 37°C with 5% CO2 and the medium was changed every 2-3 days. Once the cells reached 80-90% confluency, they were passaged using 0.25% Trypsin-EDTA (Gibco) and replated at a density of 5,000-10,000 cells/cm2. The cells were used for downstream applications. 107 4.2.12 Lentiviral transduction of primary pulmonary fibroblasts To generate lentivirus vectors, WT, and p.K360N cDNA from the previously described expression plasmids were cloned into a GFP-tagged Lenti vector (cat #PS100071, OriGene) using EcoRI-HF and NotI-HF (New England BioLabs). The sequence-verified lentiviral plasmids were packaged using 3rd generation packaging plasmids and transfected into HEK293T cells using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific). Viral supernatant was harvested and filtered through a 0.45 um PES filter (Thermo Scientific Nalgene) and concentrated using an Amicon Ultra-15 100 kDa centrifugal filter (Millipore). The concentrated lentivirus solution was then aliquoted and stored at -80°C until use. To establish patient-derived and healthy control-derived pulmonary fibroblasts that stably express WT and p.K360N, patient and healthy control cells were infected with EV, WT, or p.K360N lentiviral particles and 5ug/ml polybrene (Sigma-Aldrich) through spinfection at 1000 x g for 2 hours at 32°C, cultured, and expanded in Fibroblast Growth Medium 2 (PromoCell) for 3 days before undergoing sorting based on GFP expression using BD FACS Aria (BD Biosciences) cell sorter. 4.2.13 RNA sequencing To investigate the global transcriptome of the transduced patient and healthy control primary pulmonary fibroblasts stably expressing EV, WT, or p.K360N ZBTB7B, 150,000 GFP+ cells were sorted based on GFP expression using BD FACS Aria (BD Biosciences) cell sorter directly into 500 ul of cold Buffer RLT Plus (Qiagen) supplemented with 2-Mercaptoethanol according to the Qiagen RNeasy Plus Mini Kit instructions. Following sorting, the volume of Buffer RLT Plus was adjusted so that there was exactly 350 ul of Buffer RLT Plus to 100 ul of sorted sample volume. 108 RNA isolation was done according to the RNeasy Plus Mini Kit instructions (Qiagen). Sequencing was done on rRNA depleted RNA libraries using PE150 Illumina NovaSeq Sequencing, targeting 100M individual reads (50M read pairs/clusters) per sample. Three biological replicates were included for each condition, which were independently transduced, sorted, and sequenced. Sequenced reads were aligned to a reference sequence using Spliced Transcripts Alignment to a Reference aligner. E1 Assembly and expression were estimated using Cufflinks E2 through bioinformatics apps on Illumina BaseSpace. Expression was normalized to reads between samples using the edgeR package in R (R Foundation). Normalized counts were filtered to remove low counts using the filterByExpr function in edge. Principal component analysis (PCA) was done on log2 (normalized counts+0.25) in R using the PCA function. Differential expression analysis was accomplished using Limma. Differentially expressed genes were defined as those with adjusted p-value less than 0.05. Pathway analysis was done by first performing Gene set enrichment analysis (GSEA) with 1000 permutations using the Molecular Signatures Database Hallmark module. Signal-to-noise ratio was used for gene ranking and the obtained P-values were further adjusted using the Benjamini-Hochberg method. Pathways with an adjusted P-value <0.05 were considered significant. Leading edge genes from significant pathways between WT ZBTB7B and p.K360N ZBTB7B transduced cells were identified. Expression levels of these genes were then determined in each group. Sample level enrichment analyses (SLEA) scores were computed for each significant pathway. Briefly, z-scores were computed for gene sets of interest for each sample. The mean 109 expression levels of significant genes were compared to the expression of 1000 random gene sets of the same size. The difference between observed and expected mean expression was then calculated and represented on heatmaps generated using Morpheus (https://software.broadinstitute.org/morpheus). 4.2.14 Single cell-RNA sequencing and TCR repertoire analysis Using single cell RNA-Sequencing (scRNA-seq), whole transcriptome Analysis (WTA) and T cell receptor (TCR) repertoire analysis was conducted on PBMCs from the patient pre and post dupilumab treatment and three age-matched healthy controls. The BD Rhapsody Single Cell platform was used, including the Rhapsody Enhanced Cartridge Reagent Kit (BD), the BD Rhapsody Cartridge Kit (BD), the Rhapsody cDNA Kit (BD), the Rhapsody WTA Amplification Kit (BD), the Human Single-Cell Multiplexing Kit (BD), the TCR/BCR Multiomic Assay for WTA kit (BD), and the BD Rhapsody TCR/BCR Amplification Kit (BD) according to manufacturer’s recommendations and protocols. Briefly, thawed PBMCs were rested overnight and then stimulated with ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL Technologies) or left untreated for 16 hours. Libraries for TCR full-length mRNA sequencing, whole transcriptome analysis, and AbSeq analysis were prepared following the TCR/BCR Full Length, mRNA WTA, BD® AbSeq, and Sample Tag Library Preparation Protocol. Briefly, cells from each donor were labelled with sample tags and a panel of BD® AbSeq Ab-oligos (BD) sequentially, washed with stain buffer, and pooled together in cold sample buffer to obtain ~60,000 cells in 620ul for each of the unstimulated and stimulated samples. Two nanowell cartridges were primed and subsequently loaded with the pooled samples for 15 min at room temperature on the Rhapsody Express 110 instrument (BD). Cell capture beads were prepared and loaded onto the cartridge. The cartridge was washed, cells were lysed, and beads were retrieved. Reverse transcription was performed to convert mRNA into cDNA, followed by treatment with exonuclease I to remove unincorporated primers and dNTPs. Following this, several PCR amplification and cleanup procedures were performed to amplify specific regions of interest and incorporate necessary sequencing adapters and indices for downstream analysis. Finally, index PCR was then carried out to add sample-specific barcodes and sequencing adapters to all the PCR products for WTA, TCR, AbSeq, and Sample Tag libraries. The resulting index PCR products underwent purification, including single-sided cleanup, to remove unincorporated primers and nucleotides. Quality control was performed using Agilent DNA High Sensitivity Kit (Agilent Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies) on the intermediate and final sequencing libraries, including estimating the concentration of each sample, measuring the average fragment size of the libraries, and following sequencing recommendations. Libraries were diluted to 350-650 picomolar range per sample and sequenced using Illumina NovaSeq 150bp PE sequencing targeting 8,000M individual reads (4,000M read-pairs) with PhiX spike-in of 20%. FASTQ files were processed using the BD Rhapsody Targeted Analysis Pipeline and Seven Bridges (www.sevenbridges.com) according to manufacturer’s recommendations. The R package Seurat was utilized for all downstream analysis. Scaling and clustering was performed on each pool of samples independently. Dimensionality reduction using PCA was done on the most variable genes, and UMAP was based on the first 20 PCs. Cell identities were annotated manually, or via cell surface antibody sequencing (AB-seq), to first identify major cell types (T cells, B cells, 111 NK cells and Monocytes) and then defining subtypes (CD4 T cells, CD8 T cells, Double Positive T cells, and their subsequent Naïve and Memory subgroups). For differential gene expression analyses, we utilized the Seurat implementation of negative binomial test, assuming an underlying negative binomial distribution in RNA-Seq data while leveraging the UMI counts to remove technical noise. Pseudotime analysis was done using the monocle3 package. 4.2.15 Flow cytometry To carry out immunophenotyping and intracellular cytokine detection, PBMCs from the patient and age-matched healthy controls were stimulated with Phorbol myristate acetate (PMA) and Ionomycin for 4 hours at 37˚C in the presence of GolgiStop (BD Biosciences). After stimulation, the cells were stained with a cocktail of antibodies against surface markers for 20 minutes on ice and then fixed with Foxp3 Fixation/Permeabilization working solution (Invitrogen, Thermo Fisher Scientific) from the eBioscience Foxp3 Transcription Factor Staining Buffer Set (Invitrogen, Thermo Fisher Scientific) for 20 minutes. The fixed cells were subsequently stained for 45 minutes with antibodies targeting intracellular cytokines or transcription factors in 1x Permeabilization Buffer (Invitrogen, Thermo Fisher Scientific). The samples were then washed and analyzed using the BD FACSymphony flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences). The antibody panels used for staining are listed in the table below. 112 Table 4.2. List of antibodies used in the T cell phenotyping research flow cytometry panel. Marker Fluorophore Dilution Catalog # Company Viability eF780 1:100 65-0865-14 Invitrogen CD3 BUV805 1:100 612896 BD Horizon CD4 BUV563 1:100 612923 BD Horizon CD8 BV570 1:100 301037 Biolegend CD45RA PerCP-Cy5.5 1:100 563429 BD Pharmingen CCR7 APC-R700 1:100 565868 BD Horizon IFN-gamma BV605 1:50 562974 BD Horizon TNF-alpha BUV395 1:50 563996 BD Horizon IL-4 PE 1:50 554485 BD Pharmingen IL-5 BV421 1:50 504311 Biolegend IL-13 PE-Cy7 1:50 501914 Biolegend IL-17A AlexF488 1:10 560488 BD Pharmingen IL-21 AlexF647 1:10 560493 BD Pharmingen 113 4.2.16 Treatment testing and qPCR of primary fibroblasts Primary patient pulmonary fibroblasts were used to evaluate the effects of various treatments. Initially, 5x105 cells were plated in each well of a 6-well plate (Corning) containing Fibroblast Growth Medium 2 (PromoCell). The cells were incubated overnight under standard conditions. Subsequently, they were either left untreated or treated with recombinant human TGF-β1 (R & D Systems) at a concentration of 5 ng/ul for 24 hours to induce a fibrotic state, a well-established in vitro model of fibrosis as documented in existing literature. Following the TGF-β1 treatment period, cells were subjected to different drug treatments for an additional 48 hours. These treatments were all purchased from MedChemExpress (www.medchemexpress.com) and included Nintedanib (0.1uM final concentration in media), and Pirfenidone (1mM final conscentration in media). For the groups designated to continue with TGF-β1 exposure, fresh medium supplemented with TGF-β1 was added concurrently with the drug treatments. This experimental setup allowed for the assessment of drug efficacy in both the presence and absence of an induced fibrotic environment. Post treatment, total RNA was extracted from treated and untreated cells using a RNeasy Plus Mini Kit (Qiagen) and converted to cDNA using an iScript cDNA synthesis kit (BioRad Laboratories). Transcript abundance was measured using a Universal SYBR Green Super Mix (Bio-Rad) and a 7300 Real-Time PCR System (Applied Biosystems). Relative transcript abundance was quantified relative to Actin-b (ACTB) using the 2-ΔΔCT method. 4.2.17 Dose response assessment for pirfenidone and nintedanib Primary patient pulmonary fibroblasts were used to evaluate the effects of various treatments. Initially, 5x105 cells were plated in each well of a 6-well plate (Corning) containing Fibroblast 114 Growth Medium 2 (PromoCell). The cells were incubated overnight under standard conditions. Subsequently, they were either left untreated or treated with recombinant human TGF-β1 (R & D Systems) at a concentration of 5 ng/ul for 24 hours to induce a fibrotic state, a well-established in vitro model of fibrosis as documented in existing literature. Following the TGF-β1 treatment period, cells were subjected to different drug treatments for an additional 48 hours. These treatments which were purchased from MedChemExpress (www.medchemexpress.com) which included Nintedanib (testing range of 0.01uM, 0.1uM, 1uM, and 10uM) and Pirfenidone (testing range of 0.25mM, 0.5mM, 1mM, and 2mM). For the groups designated to continue with TGF-β1 exposure, fresh medium supplemented with TGF-β1 was added concurrently with the drug treatments. Post treatment, total RNA was extracted from treated and untreated cells using a RNeasy Plus Mini Kit (Qiagen) and converted to cDNA using an iScript cDNA synthesis kit (BioRad Laboratories). Transcript abundance was measured using a Universal SYBR Green Super Mix (Bio-Rad) and a 7300 Real-Time PCR System (Applied Biosystems). Relative transcript abundance was quantified relative to Actin-b (ACTB) using the 2-ΔΔCT method. 4.2.18 Lactate dehydrogenase release assay for assessing cytotoxicity Cytotoxicity was assessed using the LDH-Glo™ Cytotoxicity Assay (Promega Corporation) according to the manufacturer’s recommendations. This assay quantitatively measures lactate dehydrogenase (LDH) release from damaged cells into the culture medium. Supernatants were obtained from cells that undergone treatment testing (outline above). At the end of the treatment period, 5 uL of supernatant from each well was diluted with 495 uL of LDH storage buffer, achieving a 100x dilution. Subsequently, 50 µL of this diluted sample was transferred to a 115 new 96-well plate. To each well, 50 uL of reconstituted LDH detection reagent was added. The plates were incubated at room temperature for 30 minutes, protected from light. Luminescence was measured using the Infinite M200 plate reader (Tecan) by integrating luminescence over 1000 milliseconds (ms) per well. Percent cytotoxicity was determined by comparing the LDH release from experimental wells to a maximum LDH release control. The control consisted of cells lysed with 40 uL of 10% Triton X-100 in a total medium volume of 2000 uL. The LDH release from each experimental well was divided by the maximum LDH release value to calculate percent cytotoxicity. A standard dilution curve of LDH, ranging from 32 mU/mL to 0 mU/mL, was employed to confirm that the measured values fell within the assay’s reliable detection range. 4.2.19 Statistical Analysis Statistical significance was evaluated by various statistical methods specified in figure legends for each panel of every figure. The following annotations were used to represent significance: p-val < 0.05 (*), p-val < 0.01 (**), p-val < 0.001 (***). 4.3 RESULTS 4.3.1 A novel de novo heterozygous ZBTB7B variant associated with symptoms of immunodeficiency, allergic disease, and interstitial lung disease The patient is a 5-year-old male born to healthy non-consanguineous healthy parents at 33 weeks gestational age due to preterm rupture of membranes and maternal preeclampsia. His complex clinical course was notable for global developmental delay, failure to thrive (height and weight 116 below 3rd percentile) with feeding dependent on a gastrostomy tube, bilateral sensorineural hearing loss, non-healing corneal ulceration causing visual impairment, hepatic steatosis, increased echogenicity of multiple tissues (i.e. pancreas, liver, and kidneys), growth hormone deficiency, allergic disease, combined immunodeficiency (CD4 T cell deficiency), and severe interstitial lung disease requiring supplemental oxygen (Figure 4.1 A). His allergic disease manifestations were expansive: atopic dermatitis, cow’s milk protein colitis requiring elemental formula, multiple food allergies, anaphylaxis following general anesthesia, elevated IgE and eosinophilia. While no respiratory support was required postnatally, he was admitted at 4 months of age for respiratory distress secondary to rhinovirus/enterovirus bronchiolitis requiring prolonged hospitalization. He had persistent chest imaging abnormalities (Fig 4.1 B-C) and tachypnea and was started on continuous oxygen supplementation at 16 months of age following an abnormal overnight oximetry study. Lung biopsy at 3.75 years demonstrated chronic inflammation and fibrosis of the lung, characterized by lipoproteinosis and notable subpleural cystic remodeling and honeycomb changes (Fig 4.1 D-I). The gross examination of the biopsy tissue revealed patchy hemorrhages and enlarged air spaces. Microscopic examination revealed a complex fibroinflammatory process affecting the airways and lung parenchyma, with preserved regions of normal lung architecture interspersed (Figure 4.1 D). There were regions noted with lymphoid aggregates with germinal centers and a predominance of T-cell and B-cell infiltrates, as evidenced by CD3 and CD20 immunostaining (Figure 4.1 E and 4.1 F). Fibrotic changes were especially pronounced in the subpleural areas, manifesting as dense fibrosis with cystic alterations resembling honeycombing 117 (Figure 4.1 G and 4.1 H). Within these remodeled areas, there was also an accumulation of lipoproteinaceous material and cellular debris within the airspaces (Figure 4.1 I). This lipoproteinosis featured cholesterol granulomas and the presence of neutrophils. Giant cells containing large lipid droplets and eosinophilic material were noted sporadically. Although T cell receptor excision circle (TREC) counts were normal at birth, CD4 lymphopenia identified at 4 months of age. Detailed immunophenotyping at 28 months of age revealed CD4 lymphopenia, reduced naïve CD4 T cell percentage, with the opposite finding in CD8 T cells of lymphocytosis and elevated naïve percentage, and B cell lymphocytosis. Trio whole genome sequencing of the proband and parents identified a novel heterozygous de novo variant in ZBTB7B (NM_001256455.2:c.1080A>C, p.K360N), which was subsequently confirmed by Sanger sequencing (Figure 4.1 J-L). The variant results in a substitution from a positively charged lysine residue to a polar uncharged asparagine residue at a highly conserved region of the gene, altering the first zinc finger in the DNA-binding domain of ThPOK (Figure 4.1 J). 118 119 Figure 4.1. Identification of a monoallelic missense variant in ZBTB7B in a pediatric patient with inborn error of immunity and fibroinflammatory lung disease. (A) A summary of patient’s clinical features. (B) Chest x-ray at 3 years and 8 months showing hyperinflated lungs, coarse reticular shadowing, and interstitial septal thickening. (C) Chest CT (coronal view) at 4 years showing ground glass opacity and subpleural honeycombing changes. (D) Low-power view lung wedge biopsy (H&E stain). (E) CD20 immunohistochemistry highlights (in brown) the B cell population of the lymphoid aggregates in the interlobular septa. (F) CD3 immunohistochemistry highlights (in brown) the T cell population of the lymphoid aggregates in the interlobular and alveolar septa. (G) Subpleural honeycomb change is evident on trichrome stain, which highlights the interstitial fibrosis in blue. (H) The lung architectural abnormalities are patchy (non-uniform), as demonstrated by relatively normal parenchyma in the top half of this image, and interstitial fibrosis and alveolar widening in the lower half (trichrome stain). The patchy changes can also be appreciated in the low-power photomicrographs (A, B, C). (I) Some alveoli contain aggregates of alveolar macrophages surrounding cholesterol clefts (arrowhead), as well as amorphous eosinophilic material (arrow) (H&E stain). All images have scale bars embedded in the respective panels. (J) Illustration of the ThPOK protein, with four C-terminal zinc fingers responsible for DNA-binding to the consensus sequence and the N-terminal protein-binding BTB/POZ domain. Location of the missense variant (NM_001256455.2:c.1080A>C, p.Lys325Asn) is shown (red dot). ZF, zinc finer. (K) Family pedigree of the patient. Filled symbols = affected individuals; unfilled symbols = unaffected individuals. WT, wild-type. (L) Sanger sequencing results of all family members indicating heterozygous substitution at position 1080. 4.3.2 ThPOKK360N displays altered DNA binding specificity We assessed the effect of the NM_001256455.2:c.1080A>C, p.K360N variant on protein expression using HEK293 cells lacking endogenous expression of ThPOK. Expression of the variant protein was assessed both in isolation and in combination with wild-type (WT) ThPOK, modelling the patient’s heterozygous state. Expression of ThPOKK360N remained comparable to the WT protein both in isolation and in the heterozygous state, suggesting that the presence of the variant does not impact ThPOK protein (Figure 4.2 A). Next, we evaluated the effect of the variant on function of ThPOK as a transcription factor and its regulatory influence on SOCS1 expression. Building on Luckey et al.'s identification of 120 ThPOK-binding sites within the mouse Socs1 promoter region and their demonstration of ThPOK's dose-dependent augmentation of Socs1 transcriptional activity508, we developed a luciferase reporter assay utilizing a segment of the human SOCS1 promoter containing the previously identified DNA binding motif of ThPOK 498. We established that ThPOKK360N is impaired in its ability to upregulate SOCS1 transcriptional activity compared to the WT ThPOK (Figure 4.2 B). When co-transfecting cells to mimic the heterozygous state with both WT and p.K360N, the transcriptional activity of SOCS1 exhibited an intermediate increase, indicative of a partial dominant-negative effect of the variant on the WT protein's transcriptional enhancement (Figure 4.2 B). We then investigated the potential dominant-negative effect of ThPOKK360N on WT ThPOK by performing a dose-increase assay. We systematically increased the ratio of the variant plasmid relative to the WT plasmid and assessed the impact on SOCS1 transcriptional activity using the luciferase reporter system. This showed a clear inverse relationship, as the proportion of the variant plasmid increased, there was a corresponding decrease in SOCS1 transcriptional activity (Figure 4.2 C). The quantitative assessment of this relationship using Pearson correlation and linear regression analysis yielded a high r2 value of 0.9262, with a highly significant p-value of 0.0005, confirming a significant suppressive effect of the variant on WT-mediated transcription (Figure 4.2 C). We hypothesized that changes to the transcriptional activity of ThPOKK360N may be a result of altered DNA-binding profile. To assess the DNA-binding ability of ThPOKK360N, we employed an Electrophoretic Mobility Shift Assay (EMSA) using a tagged 35 nucleotide probe from the SOCS1 promoter that included the established ThPOK DNA binding motif. To ensure the observed 121 binding was specific, a supershift assay with an anti-ThPOK antibody was performed. Results confirmed that WT protein could effectively bind to this probe, while the ThPOK variant exhibited no detectable binding activity, indicating a loss of DNA-binding capability attributable to the missense change in the DNA-binding zinc finger (Figure 4.2 D). Additionally, a competition assay with a 100-fold excess of unlabeled competitor probe substantially diminished the supershifted WT ThPOK-DNA complex band, further verifying the specificity of the binding (Figure 4.2 E). To define and compare the DNA binding profiles of WT ThPOK and ThPOKK360N, we utilized High-Throughput Systematic Evolution of Ligands by EXponential Enrichment (HT-SELEX). The HT-SELEX results further corroborated changes in DNA binding specificity of ThPOKK360N compared to the WT protein, as illustrated in Figure 4.2 F. Positional weight matrices (PWM) revealed a shift in nucleotide preference in positions 3 and 4, specifically changing ‘GA’, preferred by the WT, to ‘CT’ or ‘AT’, preferred by ThPOKK360N. Notably, both cytosine (C) and adenine (A) were similarly preferred at position 4 for ThPOKK360N (Figure 4.2 F). Additionally, we generated a scatterplot to visually represent the relative abundance of 8-mer sequences from the third selection cycle of the HT-SELEX experiments for both the WT and ThPOKK360N. This revealed that sequences with high affinity for either the WT or ThPOKK360N generally had low incidences in the counterpart's selection. This distinct separation underscores the divergent DNA binding specificities between the WT and ThPOKK360N, highlighting the significant impact of the amino acid change on the protein's DNA interaction profile (Figure 4.2 G). Given that the original 'GA' dinucleotide at positions 3 and 4 in the WT binding sequence is highly conserved and exhibits low flexibility, changes to this sequence are likely to significantly 122 impact binding specificity. To test this, we created two variant oligonucleotide probes that incorporated the dinucleotide modifications CT or AT, preferred by ThPOKK360N, to test using EMSA (Table 4.1). Our results demonstrated that ThPOKK360N selective binding to Variant Probe 1 with no detectable interaction with the WT protein. ThPOKK360N also exhibited a strong binding affinity for Variant Probe 2. Notably, there was detectable, albeit partial, recognition of Variant Probe 2 by the WT protein (Figure 4.2 H). Integrating HT-SELEX motifs with ChIP-seq data identified high-confidence gene targets for ThPOK and the K360N variant. A gene was designated as a high-confidence target based on the co-occurrence of ChIP-seq peaks in both replicates within the promoter regions (spanning from -1000 to +500 bp relative to the transcription start site) that also harbored corresponding HT-SELEX motif hits (Figure 4.2 I). Our analysis revealed distinct gene targets regulated by the WT and the variant protein, as illustrated by the Venn diagram (Figure 4.2 I), with limited overlap between the gene sets targeted by each protein, suggesting that the WT and variant proteins engage in differential regulatory networks. This pattern indicates potentially unique transcriptional roles for each protein, which may underlie differences in cellular responses or phenotypic outcomes. 123 Figure 4.2. Functional and molecular characterization of ThPOKK360N. (A) Comparative analysis of ThPOKK360N and WT ThPOK protein expression using immunoblotting in HEK293 cells. Protein was detected using anti-ThPOK and anti-Myc antibodies. Β-actin was assessed as loading control. Representative blot from three independent experiments. (B and C) Luciferase reporter assay using a region of SOCS1 promoter in HEK293 cells transfected with various combinations and ratios of vectors indicated below the plot. Firefly luciferase activity was normalized to Renilla 124 luciferase to account for transfection efficiency variations, with values further normalized to the empty vector (EV) condition, offering a relative comparison to the EV baseline. Data are presented as mean ± SEM, derived from three independent experiments, each comprising an average of three technical replicates. For panel B, statistical analysis was conducted using one-way ANOVA for repeated measures, with Sidak's method applied for multiple comparisons correction. For panel C, a regression analysis was performed to model the dose-response relationship, with dotted lines representing 95% confidence intervals for the Pearson correlation coefficient, indicating the strength and certainty of the linear relationship between dose and response. (D) EMSA showing in vitro DNA binding of WT ThPOK and ThPOKK360N. Nuclear lysates of HEK293 cells transfected with an expression vector encoding each protein were incubated with infrared fluorescent dye labeled SOCS1 probe. Anti-ThPOK antibody generated a supershift of the DNA–protein complexes (red arrow). Representative image from three independent experiments. (E) Competitor EMSA assay using an unlabeled (cold) probe identical to the labelled probe sequence. (F) Position weight matrices for WT ThPOK and ThPOKK360N, showing the relative frequency of nucleotide occurrence at each position within the binding site. (G) 8-mer plot illustrating the relative binding affinities of WT ThPOK and the ThPOKK360N to different 8-base DNA sequences. Each point on the plot represents a unique 8-base DNA sequence, and the plot shows how strongly each protein variant binds to these sequences, providing insights into their DNA-binding preferences and specificities. (H) EMSA showing in vitro DNA binding of WT ThPOK and ThPOKK360N to variant probes designed using binding sequences revealed by EMSA. (I) Combined analysis of HT-SELEX derived motifs and ChIP-seq data, showing the overlap of identified DNA sequences with gene regulatory regions. 4.3.3 ThPOKK360N causes impaired T cell development and activation ThPOK plays a significant role in the development of lymphoid cells, particularly in the lineage commitment of CD4 T cells 62-64,508,509. Its central role in this process is underscored by its position downstream of the lineage commitment E protein transcription factor, E2A (TCF3) 510,511, as depicted in Figure 4.3 A. To define how ThPOKK360N impacts T cell development, we first performed immunophenotyping of the patient’s lymphoid compartment. Clinical flow cytometry data, collected over multiple time points, consistently revealed T cell abnormalities; including persistent CD4 lymphopenia, CD8 lymphocytosis, and a lower CD4/CD8 ratio, all while largely maintaining a normal absolute CD3 count in comparison to age-matched controls (Figure 4.3 B). 125 To understand the molecular underpinnings of the observed developmental T cell defect, we conducted single-cell RNA sequencing combined with antibody sequencing (scRNA-seq/Ab-seq) on the patient and two healthy controls, both with and without T cell receptor (TCR) stimulation. The T cell subset proportions, as measured using surface marker sequencing, reflected those observed in clinical flow cytometry: the patient exhibited low CD4 T cells and high CD8 T cells (Figure 4.3 C). Despite these differences, dimensionality reduction using UMAP on the global transcriptomic signature did not place the patient's T cells in a separate cluster from those of the healthy controls (Figure 4.3 D). However, pseudotime analysis of the RNA signature revealed that the patient's T cells lagged in their differentiation state compared to those of the healthy controls. Most T cells from the patient shared an early maturity stage and had a similar transcriptomic profile with each other, whereas T cells from the healthy controls spanned a spectrum of transcriptomic states (Figure 4.3 E). Transcriptomic signature differences were evaluated separately in key T cell subpopulations. Initially, we examined double positive (DP) T cells, reflecting potential developmental defects in the final stages of T cell maturation in the thymus. We noted that the lineage commitment gene TCF3 was significantly upregulated in the patient's DP T cells compared to healthy controls (Figure 4.3 F). The accumulation of TCF3 suggests a defect in the downstream mediators of CD4 T cell commitment, namely ThPOK and GATA3511-513. Subsequently, we investigated gene signatures in naïve CD4 and CD8 T cells. We observed few differences between the naïve cells in the patient and healthy controls. However, we noted significantly fewer upregulated genes and significantly more downregulated genes in the patient's naïve CD4 and 126 CD8 T cells following TCR stimulation (Figure 4.3 G-J). This lack of activation was particularly evident with a loss of proliferative signaling pathways in CD4 T cells (e.g., the MYC pathway) and a loss of effector CD8 T cell pathways (e.g., interferon pathways) (Figure 4.3 H and 4.3 J). These results suggest a significant role for ThPOK in the activation of mature single-positive T cells. To establish this link experimentally, we stably transduced T cells, activated by TCR, with Empty Vector (EV), WT, and p.K360N ThPOK, and cultured them for 3 days. We observed that many pathways (e.g., MYC proliferation, and interferon effector) were significantly upregulated in WT-transduced T cells, but these pathways failed to upregulate in p.K360N-transduced cells (Figure 4.3 K). Hence, we determined that the defects in T cell activation were attributable to the dominant-interfering effect of ThPOKK360N. 127 128 Figure 4.3. ThPOK's impact on T cell development and activation. (A) Schematic representation of ThPOK's role downstream of TCF3 (E2A) in CD4 T cell lineage commitment. (B) Clinical flow cytometry of patient’s CD3+, CD4+, and CD8+ cells over time compared to pediatric healthy control range. (C) scRNA-seq/Ab-seq analysis reflecting the proportion of CD4 single positive, CD8 single positive, double positive (DP) and double negative (DN) T cells in the patient and two healthy controls. HC, healthy control. (D) UMAP analysis of T cells from the patient and two age-matched healthy controls. (E) Pseudotime analysis of T cell differentiation in the patient compared to two age-matched healthy controls. (F) Volcano plot comparing the differential expression of genes between DP cells of the patient in comparison to two age-matched healthy controls using scRNA-seq. (G-J) Transcriptome and Hallmark pathway analysis of post-TCR stimulated naïve CD4 (in G and H) and CD8 T cells (in I and J) in the patient compared to two age-matched healthy controls. (K) Pathway analysis of the differentially expressed genes assessed by RNA sequencing in healthy control T cells transduced with EV, WT, and p.K360N ThPOK. 4.3.4 ThPOKK360N contributes to Th2 skewing in patient CD4 T cells ThPOK has been found to have additional significant roles in the development and function of other T cell subsets beyond the CD4/CD8 lineage decision; it is involved in maintaining the integrity and effector potential of post-thymic CD4+ T cells, as well as in the development and/or acquisition of effector functions by various CD4 helper T cell subsets 63,514-516. The patient’s severe atopic manifestaYons, which encompassed early-onset and severe allergic condiYons, transient eosinophilia (Figure 4.4 A) and persistently elevated IgE levels (Figure 4.4 B), prompted an examinaYon of the T helper 2 (Th2) cell response under sYmulaYon. We conducted an immunophenotyping study using flow cytometry to analyze the T cell compartment using blood drawn from the patient at three different points over two years. For comparative analysis, we included three age-matched pediatric healthy controls and four adult healthy controls. Our results demonstrate significant alterations in the T cell compartment of the patient relative to both pediatric and adult controls. The frequency of total CD4 T cells was 129 significantly reduced in the patient compared to healthy controls. Conversely, the representation of CD8 T cells was significantly increased, suggesting a shift in the CD4/CD8 T cell ratio (Figure 4.4 C). Within the CD4 T cell population, there was a significant increase in the frequency of memory T cells alongside a substantial decrease in naive CD4 T cells in the patient (Figure 4.4 D). This shift indicates a more aged T cell profile, which could reflect chronic immune activation or differential homeostatic regulation. Upon stimulation with PMA and ionomycin, a marked increase in Th2 cell activity was observed in the patient. This was characterized by significantly elevated production of Th2 effector cytokines, including IL-4, IL-5, and IL-13, compared to both pediatric and adult HCs (Figure 4.4 E and 4.4 F). These findings suggest that the patient exhibits distinct immune dysregulation, particularly in the T cell compartment, with a pronounced shift towards memory phenotypes and Th2 effector function. 130 Figure 4.4. Elevated IgE levels and enhanced Th2 responses in patient CD4+ T cells. (A) Absolute eosinophil count measured by the clinical laboratory. (B) Serum IgE levels determined by the clinical laboratory. (C) Representative flow cytometry plots illustrating the proportions of CD4 and CD8 T cells in the patient compared to age-matched pediatric and adult healthy controls 131 (HCs). Scatter dot plots summarize results from three independent blood draws for the patient compared to three age-matched pediatric and four adult healthy controls (HCs). (D) Representative flow cytometry plots showing the proportions of memory and naive CD4 T cells in the patient, identified by CCR7 and CD45RA markers, compared to age-matched pediatric and adult HCs. Scatter dot plots summarize results from three independent blood draws for the patient compared to three age-matched pediatric and four adult HCs. (E-F) Representative flow cytometry plots and analysis of Th2 cytokine production (IL-4, IL-5, and IL-13) in memory CD4+ T cells post-PMA/ionomycin stimulation. Scatter dot plots display comparative results. Data are mean ± SEM; statistical significance assessed by one-way ANOVA, with Dunnett’s method applied for multiple comparisons correction. 4.3.5 ThPOKK360N disrupts profibrotic gene suppression in pulmonary fibroblasts The patient carrying ThPOKK360N had evidence of multi-organ fibrosis, and it has previously been shown that murine ThPOK binds to the regulatory regions of the type I collagen genes, Col1a1 and Col1a2 and inhibits their expression. Using HEK293 cells, which express COL2A1 but lack endogenous expression of ZBTB7B (ThPOK), we compared the effects of ThPOKK360N to WT ThPOK. Our results show that while WT ThPOK suppresses COL2A1 expression as expected, ThPOKK360N does not, leading to significantly higher COL2A1 levels similar to the control with an empty vector (EV) (Figure 4.5 A). This suggests that ThPOKK360N is a LOF variant. Additionally, cells expressing both WT and ThPOKK360N, mimicking a heterozygous state, also showed significantly increased COL2A1 expression compared to WT alone (Figure 4.5 A), indicating a dominant interfering effect of ThPOKK360N on the WT protein in repressing COL2A1 expression. This led us to further validate our findings through stable lentiviral transduction, highlighting ThPOK's critical role in regulating collagen gene expression. ThPOK is highly expressed in fibroblasts (https://www.proteinatlas.org/). In the context of the patient presenting with severe interstitial lung disease characterized by advanced fibroinflammatory changes and considering ThPOK's role 132 as a repressor of Col1a1 and Col1a2 (highly expressed in fibroblasts)66, we utilized primary human pulmonary fibroblasts as a model system. Healthy control (HC) pulmonary fibroblasts were lentivirally transduced to stably express either the WT ThPOK or ThPOKK360N, with the inclusion of an EV as a control (Figure 4.5 B). By overexpressing the WT and the variant gene in these cells, our goal was to identify specific pathways and molecular mechanisms potentially involved in the development and progression of severe interstitial lung disease seen in the patient, and to provide insights into the cellular processes influenced by ThPOK in relation to its regulatory role in fibroinflammatory pathology and fibrosis. Principal Component Analysis (PCA) of bulk RNA sequencing data showed clear and distinct clustering for each group. PC1 and PC2 accounted for 52.6% and 32.9% of the total variance, respectively, highlighting significant differences in gene expression profiles influenced by ThPOK status (Figure 4.5 C). Differential analysis between EV-transduced and WT ThPOK-transduced fibroblasts shows significant gene expression changes upon WT-ThPOK transduction, many of which are noticeably not disrupted to the same level in ThPOKK360N -transduced cells (Figure 4.5 D). Interestingly, overexpression of the ThPOKK360N led to a noticeable change in the gene expression profile where genes that were downregulated by the WT protein were upregulated in the presence of the variant, and vice versa (Figure 4.5 E). This shift indicates that the missense change in the first zinc finger of ThPOK significantly changes its regulatory impact, potentially altering its DNA-binding affinity, interaction with other proteins, or responsiveness to cellular signals. We then performed Sample Level Enrichment Analysis (SLEA) on the transcriptome data which revealed multiple significant pathways that were differentially regulated in healthy control 133 pulmonary fibroblasts expressing the WT ThPOK in comparison to ThPOKK360N (Figure 4.5 F). Notably, there was a downregulation of critical pathways including interferon alpha and gamma signaling, as well as the inflammatory response pathway. Conversely, upregulation was observed in several pathways, such as estrogen response late, Myc targets, and epithelial mesenchymal transition. The upregulation of the epithelial mesenchymal transition, a pathway integral to fibrosis and wound healing, was especially interesting as it underlined the variant's role in potentially exacerbating fibrotic conditions, highlighting a key area for further investigation. This is supported by the differential expression of several pro-fibrotic genes –ACTA2517-525, TGFB1523,526,527, LTBP2524,528,529, and BDNF530, and COL1A1525,531, and MDK532– that have been implicated in pulmonary fibrosis by multiple studies (Figure 4.5 G). These genes have significantly increased transcript abundance in fibroblasts stably expressing ThPOKK360N to compared to WT transduced controls, meaning that their expression is suppressed by WT ThPOK but upregulated or not adequately suppressed by ThPOKK360N. These findings suggest that ThPOKK360N represents a LOF in its ability to suppress fibrosis and profibrotic gene expression in fibroblasts, thereby highlighting the crucial role of ThPOK in maintaining cellular homeostasis against fibrotic processes. 134 Figure 4.5. ThPOKK360N disruption of ThPOK-mediated fibrosis suppression in fibroblasts. (A) Expression of COL2A1 measured by qPCR in HEK293 cells transfected with various constructs: EV, WT ThPOK, p.K360N variant, or a combination of WT and the variant. Data are mean ± SEM; n=4 experiments; statistical significance assessed by one-way ANOVA, with Dunnett’s method applied for multiple comparisons correction. (B) Experimental schema showing healthy control primary human pulmonary fibroblasts, transduced with WT ThPOK, ThPOKK360N, or EV. (C) PCA plot of healthy control primary human pulmonary fibroblasts transduced with WT ThPOK, ThPOKK360N, or EV. (D) Heatmap illustrating gene expression variations across all three groups: EV, WT ThPOK, and ThPOKK360N in healthy control primary human pulmonary fibroblasts. Three biological replicates were included for each group, each replicate consisting of independently transduced, sorted, processed, and sequenced samples. (F) Sample Level Enrichment Analysis of healthy control lung fibroblasts transduced with EV, WT ThPOK, and ThPOKK360N. (G) Expression of profibrotic genes in healthy control lung fibroblasts transduced with EV, WT ThPOK, and ThPOKK360N. (H) Experimental schema showing patient-derived primary pulmonary fibroblasts, 135 transduced with WT ThPOK, ThPOKK360N, or EV. (I) Expression of profibrotic genes measured by RNA-sequencing in patient-derived primary lung fibroblasts overexpressing WT ThPOK (J) Expression of ACTA2, a known profibrotic marker, measured by qPCR to evaluate the effect of Pirfenidone and Nintedanib on profibrotic gene expression in TGF-β-treated patient-derived pulmonary fibroblasts. Data are mean ± SEM; n=6 experiments; statistical analysis was conducted using one-way ANOVA for repeated measures, with post-hoc comparisons performed using Fisher’s LSD test. 4.3.6 Reversal of fibrotic gene signature in patient pulmonary fibroblasts is achieved by over-expression of WT ThPOK or Pirfenidone treatment We aimed to parallel our previous work where overexpression of the variant in HC primary pulmonary fibroblasts induced fibrotic gene signature changes. This time, we utilized patient-derived primary pulmonary fibroblasts to determine if stable overexpression of the WT ThPOK gene, achieved via lentiviral transduction, could reverse the observed fibrotic gene signature (Figure 4.5 H). Our results revealed that the profibrotic genes, which were differentially regulated in HC cells, exhibited similar differential regulation in the patient cells upon WT ThPOK expression. Notably, these genes displayed a significant decrease in transcript abundance in fibroblasts stably expressing the WT ThPOK gene, thereby indicating a successful rescue of the patient-derived cells from the fibrotic phenotype (Figure 4.5 I). Building on our previous findings, we aimed to test the efficacy of FDA-approved pulmonary fibrosis drugs – pirfenidone533-535 and nintedanib534-536 – on patient-derived pulmonary fibroblasts in their steady state, and also under fibrosis-induced conditions. This approach was designed to better mimic the fibrotic microenvironment of the patient's lungs, acknowledging that in vivo, cells are influenced by surrounding signals and are not isolated. For the induction of a fibrotic cell model, we employed transforming growth factor beta (TGF-b) 136 stimulation, a method well-established in the literature for simulating fibrosis in human fibroblasts525,535. In our experimental setup, we continued to focus on the expression of ACTA2, measured using quantitative PCR (qPCR), as our primary readout. This choice was guided by our previous observations of ACTA2's differential expression in lentiviral transduction models and its recognition in the literature as a critical marker of myofibroblasts and activated fibroblasts, key players in fibrotic disease progression across various organs, including the lungs517-525. We demonstrated that treatment with pirfenidone (1mM) over a 48-hour period significantly reduced ACTA2 expression in TGF-b-treated fibroblasts in comparison to untreated controls (Figure 4.5 J). This finding highlights the efficacy of pirfenidone in modulating the fibrotic gene signature. In contrast, a similar 48-hour treatment regimen with nintedanib (0.1uM) did not produce a comparable effect; it proved ineffective in reducing ACTA2 expression (Figure 4.5 J). This differential response may underscore the drugs’ distinct mechanisms of action of these drugs and their varied impact one fibrotic pathways in patient-derived pulmonary fibroblasts. To note, we determined the optimal treatment doses by conducting dose-response experiments and assessing cytotoxicity in our model system using the lactate dehydrogenate (LDH) release cytotoxicity assay (4.6 A to 4.6 D). The biologically relevant dose was selected based on established literature and the resulting dose-response curve. 137 Figure 4.6. Evaluating the efficacy and toxicity of pirfenidone and nintedanib in TGF-β-treated patient primary pulmonary fibroblasts. (A and C) Dose-response graphs illustrating the expression of ACTA2, a profibrotic marker, in response to four different doses of pirfenidone or nintedanib. Expression levels were measured using qPCR to evaluate the effect of pirfenidone or nintedanib on profibrotic gene expression in patient-derived pulmonary fibroblasts treated with recombinant human TGF-β1 (5 ng/µL for 24 hours) to induce fibrosis. Following TGF-β1 induction, cells were exposed to varying concentrations of pirfenidone or nintedanib for an additional 48 hours. Data represent mean ± SEM from two independent experiments. (B and D) Cytotoxicity associated with different doses of pirfenidone or nintedanib, assessed by lactate dehydrogenase (LDH) release assay using supernatants from A and C. 138 4.4 DISCUSSION We describe the first human with an inborn error of immunity caused by disruption of ZBTB7B which encodes the transcription factor, ThPOK. This index patient who was heterozygous for a de novo variant in ZBTB7B presented with a complex syndromic phenotype including combined immunodeficiency, severe atopy, fibroinflammatory interstitial lung disease, sensorineural hearing loss, global developmental delay, and growth failure. Our investigations align with the established guidelines for genetic studies in single patients, as proposed by Jean-Laurent Casanova20. Trio genome sequencing identified the NM_001256455.2:c.1080A>C, p.K360N ZBTB7B variant as de novo, not present in unaffected parents or siblings. Its absence in the gnomAD database underscores its rarity, and a pLI score of 0.98 suggests significant intolerance to heterozygous loss-of-function, aligning with the severe phenotype observed. ThPOKK360N exhibits multimorphic effects, altering both protein function of the transcription factor and its ability to regulate gene expression. Despite normal protein expression, ThPOKK360N displays a hypomorphic dominant LOF effect, impairing T cell development and function, and failing to inhibit fibroblast profibrotic pathways. Additionally, its neomorphic properties, evidenced by altered DNA-binding specificity identified through HT-SELEX and ChIP-seq, result in distinct binding patterns and regulation of novel gene targets, as also shown by fibroblast transcriptomic analysis. Functionally, we linked the variant to the clinical phenotype by demonstrating its effects in two relevant cellular models. Lentiviral transduction experiments in HC primary pulmonary fibroblasts with the variant induced profibrotic gene expression, while the overexpression of WT ThPOK in patient cells reversed this effect. In lentiviral transduction of HC T cells, the variant 139 recapitulated the abnormalities seen in patient's T cell activation and function, as confirmed by single-cell RNA sequencing. These results establish a direct causal relationship between the variant and key clinical manifestations. The discovery of a monogenic defect in ThPOK in the patient has offered an exciting opportunity to explore the role of ThPOK in human immunology, providing a direct in vivo context that was previously inaccessible and primarily inferred from the helper-deficient (HD) mice and in vitro human studies. ThPOK is best known for its role as a ‘master regulator’ of CD4 lineage commitment in the thymus, where it has been shown to be crucial for CD4+ versus CD8+ lineage decisions, particularly in MHC-II-restricted CD69+ CD4+ CD8low intermediate thymocytes62-64,509,516,537-540. This was first discovered through the HD mouse model characterized by the selective absence of mature CD4+ T cells in the periphery and increased numbers of CD8+ cells65. Our observations of persistent CD4 lymphopenia and CD8 lymphocytosis in the patient's T cell compartment parallel the phenotypic alterations seen in HD mice. The K360N variant's lysine-to-asparagine substitution in the first DNA-binding zinc finger, similar to the HD variant's arginine-to-glycine change in the second zinc finger, replaces key positively charged amino acids with uncharged amino acids that interact with the negatively charged DNA at the DNA-binding interface. This modification likely affects DNA binding, impacting the regulation of target genes (Cd4, Cd8a, Cd8b, Runx3, Socs1, Socs3, and Cish) essential for CD4 T cell development, suggesting a shared mechanism of altered gene regulation508,516,540. In addition, HD mice have markedly increased frequency of CD4+ CD8low intermediate cells, suggesting that class II-restricted thymocytes are arrested at this stage65. Our pseudotime analysis of patient’s T cells also reveal 140 that they predominantly occupy an early differentiation state, lagging in maturation compared to healthy controls. The significant upregulation of TCF3 in the patient's DP T cells also suggests a blockade in the CD4+ T cell commitment pathway. This aberrant expression pattern of TCF3, a key player in T cell lineage fidelity, further illustrates the mechanistic disruption caused by the p.K360N variant, reinforcing the variant's pathogenicity in T cell development. In the context of CD4+ T cell biology, our findings complement the established literature by demonstraYng that ThPOK not only orchestrates the development of CD4+ T cells in the thymus, but it also regulates the differenYaYon of several CD4 T helper cell subsets and the acYvaYon and memory potenYal of both CD4 and CD8 T cells in the periphery514-516. Notably, ThPOK plays a pivotal role in promoYng Th2 cell differenYaYon while concurrently prevenYng the aberrant trans-differenYaYon of Th1/Th2 cells into cytotoxic T cell phenotypes515,516. Our data present an unexpected finding regarding Th2 cells, where the paYent exhibited an increased Th2 response with a significant enhancement in effector cytokine producYon (IL-4, IL-5, and IL-13) upon PMA/Ionomycin sYmulaYon, suggesYng an unanYcipated role of ThPOK in modulaYng Th2 cell effector funcYons in humans. Our findings reveal an intriguing associaYon between the observed increase in Th2 cell responsiveness and the paYent's severe atopic manifestaYons, which encompass early-onset and severe allergic condiYons. The paYent’s allergic inflammaYon significantly improved upon treatment with dupilumab, an IL-4Rα antagonist. The disYnct Th2 cell hyperresponsiveness mediated by the ThPOK variant in the paYent, coupled with the severe allergic phenotype and response to treatment targeYng Th2 cytokine signaling, highlight ThPOK as a protein whose role in allergic diseases warrants further study. 141 ThPOK is also recognized to be important for CD4+ T cell acYvaYon, in parYcular preserving transcriptomic integrity and memory potenYal. In line with this, we observed a marked deficiency in the upregulaYon of genes essenYal for T cell receptor (TCR) sYmulated acYvaYon in naïve CD4+ T cells from the paYent compared to healthy controls, highlighYng a significant deficit in proliferaYve signaling pathways, such as the MYC pathway. The introducYon of WT ThPOK into T cells acYvated by TCR led to a notable upregulaYon of pathways criYcal for proliferaYon and effector funcYons, which were conspicuously absent in cells transduced with the p.K360N ThPOK variant. Similarly, in the realm of CD8+ T cell immunobiology, our research builds upon the known impact of ThPOK deficiency on clonal proliferaYon and effector molecule producYon. ThPOK deficiency is shown to markedly impair the clonal proliferaYon and the producYon of CD8 effector molecules, such as IL-2 and granzyme B, within long-lived CD8+ T memory (Tm) cells upon anYgenic rechallenge541. Similarly, we idenYfied a pronounced impairment in the acYvaYon of naïve CD8+ T cells in the paYent, evidenced by a significant reducYon in the upregulaYon of genes following TCR sYmulaYon. This was parYcularly notable in the downregulaYon of effector pathways, such as those mediated by interferons, which are crucial for the funcYonality of CD8+ T cells. These findings suggest a direct link between ThPOK funcYon and the acYvaYon of mature single-posiYve T cells. Our results thus provide a nuanced understanding of ThPOK's role, not only in the differenYaYon and development of T cell subsets but also in their acYvaYon and funcYonal response, thereby underscoring the complex regulatory mechanisms orchestrated by ThPOK in T cell immunobiology. 142 Beyond challenging the existing paradigm of ThPOK's role in immunity, our study significantly advances the understanding of ThPOK's function in human biology and other tissues and organ systems, both mechanistically and clinically. Through transcriptomic analysis of transduced primary pulmonary fibroblasts, we uncovered substantial differences in pathway regulation between healthy control pulmonary fibroblasts expressing WT ThPOK and those expressing the p.K360N variant. Notably, we observed an upregulation in the epithelial-mesenchymal transition pathway, which is particularly relevant due to its association with fibrotic conditions. In addition, overexpression of ThPOKK360N led to an exacerbated fibrotic gene signature, indicative of ThPOK's involvement in regulating fibrotic processes beyond the immune system. This was further evidenced by experiments with patient-derived pulmonary fibroblasts, where overexpression of WT ThPOK reversed the fibrotic gene signature, pointing to ThPOK's therapeutic potential in mitigating fibrotic diseases. Thus, our study suggests that genetic changes within ThPOK could have substantial consequences on the gene expression landscape in pulmonary fibroblasts, offering new insights into lung cell biology. Moreover, our exploration of FDA-approved pulmonary fibrosis drugs on patient-derived fibroblasts revealed that pirfenidone significantly reduced the expression of ACTA2, a marker of myofibroblasts and fibrosis, under both steady state and TGF-β-induced fibrotic conditions. The demonstration of pirfenidone's ability to modulate the fibrotic gene signature in patient-derived fibroblasts is particularly promising, suggesting a possible therapeutic approach for managing the fibrotic aspects of diseases associated with ThPOK dysfunction. This finding not only emphasizes the value of 143 targeted therapies in complex genetic diseases but also offers new insights into idiopathic pulmonary fibrosis and other fibrotic lung conditions. In conclusion, here we describe a novel human disease associated with ThPOK LOF and neomorphic function, establishing its causative link to the patient's clinical profile through complementary experimental approaches. This study not only broadens the scope of ThPOK's known functions in immune regulation but also reveals its tissue-dependent dual regulatory nature, impacting both immune cells and fibroblasts. While our findings offer significant insights, they are derived from a single patient, the only identified case to date, highlighting a limitation and the need for further discovery of similar cases to expand the disease phenotype. Our research opens avenues for further research which includes assessing the impact of ThPOK variants across different tissues and their potential role in non-immune pathologies and a longitudinal study to track the patient’s disease progression. Moreover, further investigation into ThPOK's interactions within the immune system, particularly its role in the skewed Th2 immune response, is essential. These future directions promise to deepen our understanding of ThPOK's multifaceted role in human health and disease, paving the way for improved clinical management and therapeutic strategies for patients with ThPOK variant-associated conditions. 144 CHAPTER 5: Conclusions and future directions By uncovering novel variants in IKZF2 and ZBTB7B, this research contributes significantly to our understanding of the intricate and evolving landscape of inborn errors of immunity (IEIs), particularly within the context of primary atopic disorders (PADs) and primary immune regulatory disorders (PIRDs). The discovery and characterization of these variants helps to expand the catalog of IEIs and highlights the expanding spectrum of clinical manifestations associated with these disorders, which extend beyond the traditionally emphasized enhanced susceptibility to infections. Supported by collaborative efforts, we have identified and validated these novel variants through rigorous genomic sequencing and bioinformatics analyses. The subsequent functional characterization of these variants at the molecular and cellular level has elucidated their impact on gene expression, protein function, and cellular pathways and processes in the human body. These insights pave the way for identifying new targets for precise treatments, helping to better manage and treat the affected individuals. The work presented in Chapter 2 provides three major pieces of medical reference literature aimed at advancing the diagnosis and treatment of monogenic allergic diseases, commonly termed Primary Atopic Disorders (PADs)2,4,22. To date, ~46 monogenic causes of PADs have been identified, with diverse pathogenic mechanisms affecting various biological processes and immune pathways4. This chapter emphasizes the importance of understanding the affected molecular and cellular pathways in the currently described PADs for two main reasons. Firstly, these insights are invaluable for unraveling the pathogenesis of allergy, offering a deeper understanding of how these monogenic disorders disrupt normal immune responses and lead to 145 unchecked allergic inflammation. Secondly, there is a pressing need to improve the diagnosis of PADs. Allergies are traditionally viewed as complex polygenic disorders, which may lead to the under-diagnosis of PADs. By shedding light on the monogenic causes and their clinical implications, this work aims to bridge the gap in the diagnosis and treatment of these conditions, potentially leading to more targeted and effective therapeutic interventions for patients suffering from PADs. To do so, we conducted a thorough review of the existing literature on the currently known PADs, compiling information on the disease mechanisms, clinical presentations, and laboratory findings of the affected individuals. Our work included a detailed examination of the diseases' pathophysiology, pinpointing the specific molecular and cellular dysfunctions involved. In an effort to enhance the detection and diagnosis of PADs, we highlighted distinctive 'clinical red flags' that should warrant further workup by clinicians. These red flags were signs, symptoms, or lab findings that were either unique to or commonly associated with these disorders. Additionally, we included information on current therapeutic approaches, specifically precision medicine interventions that had shown efficacy in managing these conditions. Lastly, we created a roadmap for evaluating patients who present to the allergy and immunology clinic with severe early-onset allergy, that may be candidates for genome sequencing. Importantly, we acknowledge that the 'clinical red flags' suggested in this thesis for the diagnosis of PADs will continue to evolve and expand as new PADs are discovered, which will influence the proposed roadmap for patient evaluation. The increasing availability and decreasing cost of Next-Generation Sequencing (NGS) in clinical settings will facilitate the identification of novel PADs. 146 This advancement will enable more studies to associate specific genes with immune function and dysfunction, further enriching our understanding and management of these complex conditions. In chapter 3, we described a novel multisystem disorder linked to heterozygous pathogenic de novo variants in the DNA binding domain of Helios in two unrelated individuals. In the context of existing knowledge, Helios, encoded by IKZF2, is recognized for its crucial role in the development, regulation, and function of regulatory T cells. Previous research has established a link between variants in IKZF2 and conditions involving immunodeficiency and immune dysregulation in humans. Our study builds upon this foundation by identifying two dominant-negative (DN) variants in the Helios DNA binding domain, which are associated with a broader and more complex clinical syndrome in humans. This syndrome, which we have termed ICHAD, encompasses immunodysregulation, craniofacial anomalies, hearing impairment, athelia, and developmental delay. This discovery underscores Helios' expansive role in human physiology, extending its influence beyond the immune system to encompass aspects of fetal development and neurodevelopment, in line with observations from murine models of Helios deficiency. This research has had several implications for the scientific community and clinical practice. The luciferase reporter assay, which we developed to assess the functional impact of the two variants in our study, has been further utilized to characterize additional IKZF2 variants for our collaborators in the Netherlands, who are studying familial hearing loss in a cohort of patients. In addition, our work has broadened the spectrum of Helios-related disorders, urging further exploration into the complex molecular mechanisms that underpin these conditions, including the role of Helios dysfunction in immune dysregulation and the development of 147 syndromic features. However, our study has faced limitations inherent to rare disease research, such as the small sample size of only two patients. It is important to note that the phenotypic characterization of ICHAD syndrome in our study might not encompass the full spectrum of clinical presentations that may occur as a result of DN variants in Helios. Furthermore, although our findings contribute to the knowledge of the clinical and genetic aspects of ICHAD syndrome, our study does not fully elucidate the mechanistic pathway through which DN variants in IKZF2 lead to the developmental defects observed, indicating a need for further detailed mechanistic research to assess the role of Helios in these newly described cellular and developmental processes. Further research is needed to understand the broader implications of Helios-related dysfunction across different tissues and organ systems, which will be crucial for developing more comprehensive diagnostic and therapeutic strategies for individuals with IKZF2 variants. In chapter 4, we described a novel multisystem disorder caused by a multimorphic variant affecting the DNA-binding domain of the transcription factor ThPOK. This discovery is particularly novel, as the role of ThPOK in humans has not been formally established, given that individuals with ThPOK deficiency or other pathogenic genetic changes in ThPOK have not previously been identified. In this study we report the first case of a human with a genetic change in ThPOK causing a complex syndrome compromising immunodeficiency (notably CD4 T cell deficiency), severe allergic disease, fibroinflammatory lung disease, sensorineural hearing loss, developmental delay, and growth failure. Trio whole genome sequencing revealed the de novo NM_001256455.2:c.1080A>C, p.K360N variant in ZBTB7B. ThPOKK360N demonstrated both loss-of-function (LOF), by impairing T cell development and failing to inhibit fibroblast profibrotic 148 pathways, and neomorphic effects through altered gene regulation and DNA-binding specificity. This discovery mirrors our group's recent characterization of a multimorphic variant in IRF4, which we described as a novel mechanism underlying human disease542. Specifically, this study identified the IRF4T95R variant as exhibiting a blend of hypermorphic, hypomorphic, and neomorphic activities. This variant's complex functionality—increased DNA binding affinity (hypermorphic), reduced transcriptional activity on canonical genes (hypomorphic), and aberrant gene regulation through noncanonical DNA site binding (neomorphic)—culminated in a unique clinical and immunological phenotype observed in patients with Combined Immunodeficiency (CID). This was the first study in the literature to formally describe mechanisms by which multimorphic variants significantly impact health and disease, especially in transcription factors that reshape genome-wide binding and transcriptional patterns. The identification of the c.1080A>C, p.K360N ZBTB7B variant as multimorphic (exhibiting antimorphic, hypomorphic, and neomorphic properties simultaneously) in nature extends this paradigm, suggesting that the diversity of variant-induced effects in immune regulation may be broader and more complex than previously appreciated, thereby opening new avenues for understanding the molecular mechanisms driving immune dysregulation and disease. In this study, we used functional assays in fibroblasts to link the variant to clinical manifestations of lung fibrosis in the patient. The expression of ThPOKK360N induced a profibrotic gene expression profile in lung fibroblasts from healthy controls, while restoring the wild-type (WT) gene expression in the patient's lung fibroblasts reversed this fibrotic gene signature. Notably, ThPOK is predominantly expressed in the skin and has been identified in mice as a 149 negative regulator of type I collagen gene expression, specifically targeting the regulatory regions of the Col1a1 and Col1a2 genes66. Despite this, there has been no direct evidence linking ThPOK to tissue fibrosis in either mice or humans, making our findings the first to show the role of this transcription factor in fibroblast function and regulation. The patient's clinical data, including the lung histopathology and chest imaging studies showing advanced lung fibrosis further corroborate our in vitro findings. Additionally, echogenicity and functional abnormalities have been noted in the patient’s liver and kidneys, although these have not been definitively characterized as fibrosis by the managing clinicians. Given this newly characterized involvement of ThPOK in fibroblast regulation, it prompts speculation about the potential presence of fibrosis in other organs related to this variant. However, the exploration of such a hypothesis is constrained by the ethical and clinical limitations on obtaining biopsies without a clear medical indication from the patient. This represents both a future direction for this research and a limitation in fully understanding the systemic impact of ThPOKK360N. Moreover, this study reveals a potential role for ThPOK in the development and function of T helper cells, with particular emphasis on its influence on Th2 cells and the pathogenesis of allergic inflammation. The patient presented with a broad range of allergic symptoms, including atopic dermatitis, cow's milk protein colitis, multiple food allergies, anaphylaxis post-general anesthesia, along with elevated IgE and eosinophilia. Cell phenotyping indicated an enhanced Th2 response upon PMA/Ionomycin stimulation, evidenced by increased production of IL-4, IL-5, and IL-13 compared to healthy controls. This is intriguing, given that ThPOK has been reported to promote Th2 differentiation in vivo and to inhibit the divergence of differentiated Th1/Th2 cells 150 into cytotoxic T cells, largely by suppressing the expression and activity of RUNX3. RUNX3 typically silences CD4 and IL-4 while simultaneously activating cytotoxic molecule expression 515,516. However, our observation of exaggerated Th2 responses, which we attribute to a DN LOF in ThPOK, presents a paradox compared to what is previously reported in the literature. This suggests that ThPOK's regulatory mechanisms in Th2 cell differentiation and effector function may be more complex than previously understood. Firstly, compensatory mechanisms may be at play, where other transcription factors or signaling pathways might augment Th2 differentiation or function in the absence of ThPOK. Additionally, the function of ThPOK in regulating Th2 cells may be highly context-dependent, influenced by factors such as the cellular microenvironment, the presence of other immune cells, or the specifics of the antigenic stimulus. This suggests a complex network of regulatory pathways where ThPOK's influence is one of many factors contributing to Th2 cell effector function and differentiation. Given this, future studies aimed at understanding Th2 skewing due to ThPOK LOF and its role in Th2 differentiation should focus on several key areas. Developing or utilizing ThPOK-deficient models, both animal and human cell lines, will be crucial for dissecting ThPOK's impact on Th2 cell behavior. Deeper cellular and genomic phenotyping of Th cells from these ThPOK deficient models will help identify specific gene expression changes and impacted signaling pathways. Functional assays to assess Th2 effector functions will also help to clarify how ThPOK LOF alters Th2 cell capabilities. Additionally, exploring potential compensatory mechanisms and interactions between ThPOK and other regulatory proteins will provide insights into the complex regulatory network governing Th2 cell 151 differentiation and function. These focused studies will advance our understanding of ThPOK's role in immune regulation and allergic disease pathogenesis. In summary, major contributions outlined in this thesis include: 1. Highlighting the importance of genome sequencing in the clinical diagnosis of primary atopic disorders, and the creation of a roadmap for evaluating patients presenting with severe early-onset allergy. 2. Identification of novel variants in IKZF2 and ZBTB7B, expanding the catalog of inborn errors of immunity (IEIs) and elucidation of the impact of these novel variants on gene expression, protein function, and cellular pathways, contributing to a deeper understanding of primary atopic disorders and primary immune regulatory disorders. 3. Establishment of a novel multisystem disorder termed ICHAD, linked to pathogenic de novo variants in IKZF2, and the development of a luciferase reporter assay to assess the functional impact of the IKZF2 variants, utilized by collaborators internationally. 4. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Turvey, Stuart E.
Characterizing the molecular and clinical impact of two novel human inborn errors of immunity caused by defects in IKZF2 and ZBTB7B
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