The Ever-Increasing Array of Novel Inborn Errors of Immunity: an Interim Update by the IUIS Committee

The most recent updated classification of inborn errors of immunity/primary immunodeficiencies, compiled by the International Union of Immunological Societies Expert Committee, was published in January 2020. Within days of completing this report, it was already out of date, evidenced by the frequent publication of genetic variants proposed to cause novel inborn errors of immunity. As the next formal report from the IUIS Expert Committee will not be published until 2022, we felt it important to provide the community with a brief update of recent contributions to the field of inborn errors of immunity. Herein, we highlight studies that have identified 26 additional monogenic gene defects that reach the threshold to represent novel causes of immune defects.


Introduction
Inborn errors of immunity (IEI) are generally considered to result from monogenic germline defects that manifest as increased susceptibility to severe and/or recurrent infectious diseases, autoimmune or autoinflammatory conditions, atopic manifestations, and hematopoietic or solid tissue malignancies [1]. Over the past decade, the discovery of new IEIs has been occurring at an impressive rate. Indeed, the 2011 biennial update published by the IUIS Committee update listed 191 IEIs; this number increased to 430 in the 2019 update [2,3]. This near-exponential increase in gene discovery is being driven by the accessibility and affordability of nextgeneration sequencing, and the efficient application of these technologies to elucidate the molecular etiology of unsolved cases of IEIs that are likely to result from single-gene defects [4].
Over the last 12 months, we have witnessed the ongoing rapid identification, and occasionally detailed molecular, biochemical, and cellular characterization, of genetic variants that cause, or are at least associated with, human diseases impacting host defense or immune regulation. Here, we will summarize reports on variants detected in 26 genes which we consider represent novel IEI (Table 1). Many additional genetic variants have been reported recently. However, those listed here have been adjudicated by the IUIS Committee to meet the strict criteria to be considered disease-causing [57]. These criteria include: 1. The patient's candidate genotype is monogenic and must not occur in individuals without the clinical phenotype; 2. Experimental studies must indicate the genetic variant impairs, destroys, or alters expression or function of the gene product; 3. The causal relationship between the candidate genotype and the clinical phenotype must be confirmed via a relevant cellular phenotype, including-where possible-rescue of a functional defect by reconstitution with the wildtype gene, or via a relevant animal phenotype [57].
Notably, several of these genes are already included in previous IUIS updates, namely IL6ST, STAT2, CEBPE, and RIPK1 [2,3]. However, they are listed here because the variant identified is pathogenic via a distinct mechanism and/or different mode of inheritance; i.e., autosomal recessive (AR) vs autosomal dominant for IL6ST [9] or RIPK1 [42,43], partial deficiency vs complete deficiency for IL6ST [10,11], or AR loss of function vs AR gain of function for CEBPE [24] or STAT2 [40,41]. Furthermore, the GOF variants reported for CEBPE appear to represent the first described germline neomorphic mutation in inborn errors of immunity where the variant allele has completely novel functions not seen for the wild type gene [24]. Thus, these findings underscore the importance of appropriately interpreting genetic variants identified by next-generation sequencing, not discarding variants of unknown significance simply because they do not match the expected zygosity or clinical phenotype of previously reported studies, and to rigorously validate the impact of novel variants on the function of the encoded protein.

Joining the Dots with Discoveries of Novel Inborn Errors of Immunity
Many known inborn errors of immunity impact a defined signaling pathway such that mutations in components of these same pathways can represent clinical phenocopies of diseases causes by distinct genetic variants (genetic heterogeneity). In other words, physiological homogeneity can be identified for many genotypes underlying a given phenotype. Classic examples of this are Mendelian susceptibility to mycobacterial disease (MSMD), which results from impaired IFNγ-mediated immunity following exposure to mycobacterial species [58], and herpes simplex virus encephalitis (HSE) resulting from impaired TLR3-mediated anti-HSV1 immunity [59,60]. Thus, variants in genes affecting the production of IFNγ (e.g., IL12RB1, IL12RB2, IL23R, TYK2, IKBKG, SPPL2A, IRF8) or cellular responses to IFNγ (e.g., IFNGR1, IFNGR2, STAT1, JAK1) result in MSMD in otherwise healthy individuals [58]. Similarly, inactivating mutations in signaling components of the TLR3 signaling pathway (TLR3, UNC93B, TRIF, TRAF3, TBK1, IRF3) underlie HSE due to impaired type 1 IFN-mediated central nervous system (CNS) intrinsic immunity against HSV1 [59,60].
Recent discoveries have further linked common clinical phenotypes with unique genotypes that converge in a shared pathway. Thus, the non-redundant role of IFNγ-mediated immunity in host defense against mycobacterial infection [58] has been definitively established by the identification of individuals with inactivating biallelic mutations in not only IFNG itself [26] but also TBX21 [25], the transcription factor that regulates expression and production of IFNγ.
Interestingly, variants in the small nucleolar RNA SNORA31 predispose affected individuals to HSE. Mechanistically, patient's iPSC-derived cortical neurons were found to be highly susceptible to HSV-1 infection in vitro, and this could be restored by exogenous IFNβ [29,60]. However, responses of these cells to TLR3 and IFNβ, but not HSV1, are intact, revealing that SNORA31 functions to regulate cellintrinsic immunity to HSV-1 by a mechanism independent of TLR3 signaling [29,60]. The discovery of individuals with SNORA31 variants will facilitate further understanding of CNS-intrinsic host defense.
The discoveries of individuals with complete gp130deficiency due to null/nonsense bi-allelic mutations of IL6ST [11], or pathogenic dominant-negative heterozygous variants of IL6ST [9], and a phenotype of eczema, hyper-IgE, and eosinophilia, likely explain these features of autosomal dominant hyper-IgE syndrome due to STAT3 negative dominance [61] and further highlight the role of IL-6 signaling in restraining atopic and allergic responses. Furthermore, the lack of mucocutaneous candidiasis in patients with impaired signaling via receptors for IL-6 (IL6R, IL6ST mutations [9,11,50,62,63]; anti-IL-6 autoantibodies [64]), IL-23 (biallelic IL23R variants) [65] or IL-21 (biallelic IL21 or IL21R variants) [66] argues that individually these cytokines are not required for the STAT3-mediated generation of human Th17 cells and host defense against fungal infections. Rather, the combinatorial defect of impaired STAT3 signaling downstream of these receptors explains chronic mucocutaneous candidiasis in an individual with dominant-negative STAT3 mutations. These findings again reveal the capacity for inborn errors of immunity to provide convincing evidence for basic immunological concepts. Indeed, this is further exemplified b y t h e d i s c o v e r y t h a t v a r i a n t s o f A T G 4 A o r MAP1LC3B2 cause recurrent HSV2 infection of the CNS, thereby establishing hitherto non-redundant functions of the autophagy pathway in non-hematopoietic cell-mediated intrinsic anti-viral immune responses [30].

SARS-CoV2 and Inborn Errors of Immunity
The COVID19 pandemic of 2020 has clearly changed the world in many ways. It has also yielded opportunities to understand host requirements for immunity against SARS-CoV2 infection. A recent study of~650 individuals who developed severe COVID-19 found that~3.5% of patients harbored germline loss-of-function variants in genes previously found to be important for host defense against influenza or other viral infections (e.g., bi-allelic loss of function mutations of IRF7 or IFNAR1, heterozygous mutations in TLR3, TICAM1, TBK1, or IRF3) [67] due to the key role of these genes in the type 1 IFN signaling pathway [59,68]. An accompanying study found that, strikingly,~10% of patients with severe COVID-19 have high levels of neutralizing autoantibodies (autoAbs) against type 1 IFNs in their serum [48]. The impact of these autoAbs was evidenced by the inability to detect IFN in serum from these patients, and their capacity to prevent anti-viral immune responses in vitro [48] (Table 1). These studies defined a crucial and non-redundant role for type 1 IFNs in immune control of SARS-CoV2 infection, and thus prevention of severe COVID-19. Furthermore, they also established that autoAbs against type 1 IFN phenocopy an inborn error of immunity, as previously determined for autoAbs against IFNγ and susceptibility to mycobacterial disease, anti-Th17 cytokine (IL-17A, IL-17F, IL-22) autoAbs in individuals with chronic mucocutaneous candidiasis, or pyogenic infections due to anti-IL-6 autoAbs [64,69].

Conclusions
Discoveries over the past 12 months in the field of inborn errors of immunity have further identified nonredundant functions of key genes in human immune cell development, host defense, and immune regulation. In some cases, these functions go well beyond what may have been expected or anticipated based on animal models (e.g., TBX21 [25]). They have also already highlighted the heterogenous phenotypes that can result from variants in the same gene (e.g., CDC42 [33][34][35][36][37][38][39]52]), indicated that significant diseases can arise from mono-allelic or bi-allelic loss of function (IL6ST [9], RIPK1 [42,43]) or bi-allelic loss-or gain-of-function (CEBPE [24], STAT2 [40,41]) variants in the same gene, or from autoAb phenocopies of monogenic lesions (e.g., COVID19 and anti-IFN Abs) [48], and identified novel somatic mutations as pathogenic causes of immune disorders (UBA1) [47]. Importantly, they have also provided opportunities for therapeutic interventions, such as JAK inhibitors to treat STAT2 gain of function [40,41] or SOCS1 deficiency [22], IFNγ to treat mycobacterial disease [25,26], or early IFN-β or IFN-α2a treatment of SARS-CoV2 infection in COVID-19 patients with autoantibodies against IFN-α or IFN-ω [67] or impaired type 1 IFN responses [70]. This snapshot of genetic discoveries underpinning human immune disorders further highlights the critical contributions of inborn errors of immunity to our broader understanding of basic, translational, and clinical immunology.  • Partial rescue of some of the functional defects by expression of WT SLP76  • Phenocopies aspects of IL6R and IL11R deficiencies (due to unresponsiveness to these cytokines) • Effects of variants well-characterized, including a partial rescue of patient amniocytes • Bronchiectasis • Congenital heart defects (e.g., hypertrophic cardiomyopathy) • Variable neutropenia (severe or intermittent) Crohn disease (one patient) • Developmental delay • Increased AMPK activity Validation • Almost recapitulates the mouse model [12,13]   • mutant allele reduces the binding of CTNNBL1 to AID, with impaired nuclear translocation of AID; • defective SHM rescued in mutant Ramos cells by WT CTNNBL1 Table 3 Subtable 3 [18] TNFSF13 (APRIL; 1 patient)

Refs
Severe COVID-19 • High levels of neutralizing autoAbs against type 1 IFNs (primarily IFNα, IFNω) • Severe, life-threatening infection with SARS-CoV-2 • Not all Tables in the 2020 classifications [2,3] are listed in the " Table" column because not all Tables are represented by the new variants detailed above • Mutations in PAX1 previously reported to cause OTFCS2 (but CID/SCID not reported) [49] • Variants in IL6ST have been previously listed to cause an IEI due to recessive partial LOF alleles [50] • Dominant-negative mutations in IL6ST all target the intracellular domain of gp130, truncating the STAT3 binding sites as well as recycling motif, leading to sustained expression of a dead receptor (as opposed to recessive alleles, when detected in heterozygous carriers are benign) • Fnip1 ko mice also previously reported [12,13]; humans very similar • Variants in CEBPE have been previously listed to cause an IEI due to recessive LOF alleles [51] • Mutations in CDC42 previously identified in individuals with neurodevelopmental delay (Takenouchi-Kosaki syndrome) [52]; in these patients with autoinflammation and CDC42 mutations no such features were noted, except mild facial dysmorphism in some [33][34][35][36][37][38][39] .• Variants in STAT2 have been previously listed to cause an IEI due to recessive LOF alleles [53,54] • The same amino acid was found to be affected (R148Q/W) for both families affected by STAT2 GOF [53,54] • Variants in RIPK1 have been previously listed to cause an IEI due to recessive LOF alleles [55,56]; the heterozygous dominant mutations in RIPK1 reported here all affect the D324 amino acid residue that is important for cleavage Authors Contribution SGT wrote the drafts of the manuscript, prepared the table, and revised the original manuscripts for resubmission. All coauthors contributed to and edited drafts of the original and revised manuscripts and table, and approved the final submitted version.
Funding The members of the Inborn Errors of Immunity committee would like to thank the International Union of Immunological Societies for funding, as well as CSL Behring, Baxalta, and Shire/Takeda for providing educational grants to enable us to compile this interim update to novel causes of immune diseases. This work was also supported in part by the Intramural Research Program of the NIAID, NIH. SGT is supported by an Investigator Grant (Level 3) awarded by the National Health and Medical Research Council of Australia. IM is a senior clinical investigator of FWO Vlaanderen (EBD-D8974-FKM).
Data Availability Not applicable.

Declarations
Ethics Approval This work is a review of recently-reported genetic variants that represent novel inborn errors of immunity. No human research studies were performed in order to produce this review. Thus, no approvals by appropriate institutional review boards or human research ethics committees were required to undertake the preparation of this report.
Consent to Participate Not applicable as this is a review of recentlyreported genetic variants.

Consent for Publication
The authors consent to publish the content of this review. However, as noted above, as this is a review of recentlyreported genetic variants that represent novel inborn errors of immunity, we did not require consent to publish from participants.

Conflict of Interest
The authors declare that they have no conflict of interest.
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