Dominant-negative heterozygous mutations in AIRE confer diverse autoimmune phenotypes

Summary Autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disease characterized by severe and childhood onset organ-specific autoimmunity caused by mutations in the autoimmune regulator (AIRE) gene. More recently, dominant-negative mutations within the PHD1, PHD2, and SAND domains have been associated with an incompletely penetrant milder phenotype with later onset familial clustering, often masquerading as organ-specific autoimmunity. Patients with immunodeficiencies or autoimmunity where genetic analyses revealed heterozygous AIRE mutations were included in the study and the dominant-negative effects of the AIRE mutations were functionally assessed in vitro. We here report additional families with phenotypes ranging from immunodeficiency, enteropathy, and vitiligo to asymptomatic carrier status. APS-1-specific autoantibodies can hint to the presence of these pathogenic AIRE variants although their absence does not rule out their presence. Our findings suggest functional studies of heterozygous AIRE variants and close follow-up of identified individuals and their families.


INTRODUCTION
The autoimmune regulator (AIRE) is essential for the establishment of central immunological tolerance. AIRE supports the expression and presentation of tissue-restricted antigens (TRAs) for developing T cells in the thymus, 1 promoting removal of autoreactive T cells from the repertoire by negative selection. Furthermore, AIRE promotes the expression of natural T regulatory cells capable of suppressing autoreactive T cells in the periphery. 2 Patients with autoimmune polyendocrine syndrome type 1 (APS-1) (OMIM: 240300) illustrate the consequences of loss of AIRE. They develop autoimmunity in multiple organs, with primary adrenal insufficiency (Addison's disease), hypoparathyroidism, and chronic mucocutaneous candidiasis (CMC) as main components. 3 Enamel hypoplasia, alopecia, vitiligo, pneumonitis, hepatitis, autoimmune gastritis, and enteropathy are also common manifestations.
With an estimated prevalence of 1:100 000, APS-1 is a rare disease. [3][4][5] Diagnosis is based on clinical presentation (two of three main components) or disease-causing AIRE mutations. Type I interferon autoantibodies are present in 98% of patients and have proven effective as screening tools. 3,6 However, more subtle reduction in AIRE function can also predispose to autoimmunity. Patients harboring dominant-negative heterozygous mutations in AIRE, especially within the plant homeodomain 1 (PHD-1) domain, present with a milder phenotype and later onset with a propensity for pernicious anemia and vitiligo. 7 In mouse models replicating some of these mutations, the underlying mechanism was shown to be abrogated multimerization of AIRE, hindering its binding to DNA. 8 Interestingly, these variants are relatively common in the general population with an estimated prevalence of up to 1:1000. 7 Genome-wide association studies of autoimmune Addison's disease and pernicious anemia recently found a strong association to another AIRE variant located in the PHD2 domain (p.R471C), indicating that subtle changes in AIRE's function predispose to autoimmunity against the adrenal cortex, gastric mucosa, and pancreas. [9][10][11] Here, we expand the spectrum of variants in AIRE's PHD domains associated with autoimmune diseases, describing patients harboring heterozygous mutations within the PHD1 and PHD2 domains of AIRE. The genetic and phenotypic characterization of patients and their families emphasize AIRE's critical role in central immunological tolerance induction and indicates a correlation between autoimmune disease and AIRE function.

Patient identification and genetic variants in AIRE
From 2016, eight heterozygous point mutations were identified within the PHD1 and PHD2 domains of AIRE in 11 patients with symptoms of autoimmunity or immunodeficiencies. These were identified while undergoing whole-exome or genome sequencing at their treating hospitals, or by targeted AIRE sequencing of patients in the Norwegian Registry of Organ-Specific Autoimmune Diseases (Table 1). Investigations of the index patients' families identified nine additional heterozygous carriers.
c.901G>A (p.V301M), c.916G>A (p.G306R), c.926T>C (p.I309T), c.977C>T (p.P326L), and c.982C>T (p.328W) were located within PHD1 (Figure 1), while c.1102C>G (p.368A) and c.1235 C>T (S412L) variants were located between PHD1 and PHD2. A heterozygous c.1399G>C (p.G467R) change was found in PHD2. An overview of the AIRE mutations described is given in Table 2. Of these variants, c.926T>C p.I309T had not been reported previously with this particular amino acid change in the publicly available databases. The others have been reported with varying allele frequencies, c.901G>A (p.V301M) being the most common with an allele frequency of 1.06e-3 (Tables 3 and S1). The phenotypes ranged from no autoimmune manifestation to severe autoimmune disease accompanied by autoantibodies (Tables 4 and 5 and Figure S1). p.V301M, previously described as a dominant-negative mutation in humans in vitro, was found in three different individuals/families with a variable clinical presentation. In family I, the index patient (II-I) presented with anaphylaxis, angioedema, abdominal pain, migraine type headache, gastritis, intestinal dysfunction, CMC, food intolerance, and pernicious anemia. Several of these disease components are also common in the classical form of APS-1, in particular gastritis, pernicious anemia, and CMC. Pernicious anemia is commonly seen in other patients with PHD1 AIRE mutations. However, the patient was negative for type I interferon-and the Th17 cytokine-targeted autoantibodies tested. Her father (I-I) also harbored the heterozygous p.V301M mutation, presenting with symptoms of hives and heat intolerance, while her son (III-I) was diagnosed with urticaria pigmentosa since birth, severe diarrhea, and a pituitary adenoma. Of note, her daughter without this AIRE variant also had food intolerances with abdominal pain and diarrhea.
The young boy in family II with p.V301M was diagnosed with enteropathy. Interestingly, he was positive for autoantibodies against both interferon (IFN)-u and interleukin (IL)-22, autoantibodies which are found in almost all patients with classical APS-1. In family III, the index patient with p.V301M presented at the age of 3 years and 5 months with a history of recurrent upper respiratory tract infections and skin infections since birth, with a severe course of cellulitis. She also had angular cheilitis and chronic gingivitis. Upon physical examination, she was at À2 SD for weight and height. Blood analysis revealed a severe chronic intermittent neutropenia of <500 mcl. No anti-neutrophil antibodies were found but bone marrow and peripheral blood analysis was in line with a potential autoimmune neutropenia. Other immunological investigations, including immunoglobulin levels, immunophenotyping, and STAT1 phosphorylation, were all normal. Skeletal survey and the amylase pattern were normal as well. Bone mineral content was normal for a six-year-old individual. She was started on filgrastim 5 mg/kg three times per week, which improved her condition significantly. At 11 years of age, gastrointestinal endoscopy revealed microscopic colitis, iScience Article which was successfully treated with mesalazine. More recently, she has been suffering from intermittent arthritis.
The heterozygous AIRE mutation p.G306R was found in two non-related patients, a girl from Greece and a boy from the United States. The index patient in family IV (II-I) was diagnosed with hypoparathyroidism and was also positive for autoantibodies against IFN-u. Her mother (I-II) and uncle (II-I) also harbored the same mutation but had neither autoimmune clinical manifestations nor autoantibodies. Patient V is a 14-year-old male patient referred to the Mayo Clinic for evaluation of refractory status epilepticus and type 1 diabetes. During pregnancy, his mother experienced hypertension, but the patient was born at term without neonatal complications. Early psychomotor development was normal. Multifocal seizures started at age 5 years of age and were often severe requiring hospitalization. When he was 14 years old, he was diagnosed with type 1 diabetes that is currently well controlled. Several family members have autoimmune disorders including thyroid disorders. The patient has pernicious anemia and high GAD65 antibodies consistent with a type 1 diabetes, but also with autoimmune epilepsy. The patient has deep set eyes and short philtrum but is otherwise without dysmorphic features. Whole-exome sequencing identified c.916G>A, a de novo AIRE variant (p.G306R) in exon 8 as the mother and father did not carry this variant.
Three amino acids upstream, a heterozygous p.I309T variant was found in an 18-year-old male patient (II-I) (family VI) with multiple autoimmune presentations and recurring fever. At 8 years of age, endoscopy and intestinal biopsy revealed increased intra-epithelial lymphocytes, crypt hyperplasia, and blunted villi, although anti-transglutaminase antibodies were negative. He was started on gluten-free diet. At 13 years of age, liver biopsy revealed autoimmune hepatitis successfully treated with glucocorticoids and azathioprine. 12 He had growth retardation (weight and height <3p), intermittent neutropenia (970-1500/mm 3 ), and lymphopenia (560-2460/mm 3 ), but bone marrow aspiration was normal. At 16 years of age, he was started on anti-tuberculous therapy because of pulmonary infiltrations and high purified protein derivative as 20 mm. Autoantibodies were positive for anti-parietal antigen, anti-IFN-u, anti-21OH, and anti-TPH. He Family members with mutation 20 With symptoms 18 Family members without mutations 5 With symptoms 3    Th17 (IL-17+) T cells. In addition to a more aggressive intranasal anti-inflammatory and rising regimen, the patient was started on a trial of immune globulin replacement therapy for specific antibody deficiency which has helped reduce the frequency of sinus infections.
In family VIII, a heterozygous p.R328W change in AIRE was found in a mother and two children. They all had vitiligo, as did the sibling without the p.P328W variant. However, while the carriers of p.R328W had severe childhood onset vitiligo, the non-carriers had mild adult-onset vitiligo. The same variant was also seen in family IX where the index patient (II-I) had a history of loose, frequent stools since birth. This was associated with weight loss at around 3 months of age leading to additional evaluation. Stool alpha-1 antitrypsin and pancreatic elastase-1 were both normal. Stool reducing substances were positive (3+) on several occasions. Genetic testing identified heterozygosity for the p.R328W variant in AIRE. Her weight improved after addition of a hypoallergenic baby formula to her diet. Parathyroid, hepatic, and thyroid functions were normal. Her mother (I-I) has a history of transient B12 deficiency during her early 20s that resolved. She also had a history of abnormal glucose tolerance during pregnancy, eczema, asthma, and food allergy. Targeted variant testing revealed heterozygosity for the p.R328W variant. The index patient's maternal grandmother tested negative for the variant while the index patient's father is not available for testing. Her parathyroid hormone, calcium, vitamin B12, and blood counts were all normal at the latest examination.
In family X, a p.P368A mutation was found in the mother and a daughter (Patient II-I) with a broad clinical picture, ranging from migraine type headache, chronic constipation, poor appetite, recurrent fever, proteinuria, postural orthostatic tachycardia syndrome, atypical compound melanocytic nevi (BRAFV600E mutation with retained BAP-1 expression), and to elevated hemoglobin A1C at 5.8% and fasting glucose  iScience Article of 104. She was also positive for autoantibodies against IL-17A, often found in patients with APS-1. The patient's mother reports vaginal candidiasis and onychomycosis beginning in childhood with significantly reduced frequencies as an adult.
Patient XI, a Danish male, presented with disseminated Mycobacterium kansasii infection with multiple (including liver) granulomas, abscess formation, and purulent arthritis at age 68 years. Autoantibodies directed against IFN-g, IL-6, IL-1a, IL-17F, TNF-a, and IFN-u were absent. A concomitant bone marrow biopsy showed granuloma formation, but no signs of malignancy. Four years earlier, the patient was diagnosed with immune thrombocytopenic purpurea and leukopenia (Table 4), subsequently also with hypogammaglobulinemia, the latter fulfilling the criteria for common variable immune deficiency (total IgG <4.5 g/L) leading to immunoglobulin substitution. Immunological work-up revealed total absence of B cells (0% of lymphocytes) and severely reduced CD4 + T cell numbers: 0.05 x 10 9 /L (normal range: 0.3-1.7 x 10 9 /L), the latter consisting mostly of CD45R0+ memory cells (98% of CD4 + T cells). A high degree of T cell activation was also recorded (HLA-DR+: 79% of CD3 + T cells). The CD8 + T cell concentrations were normal, but peripheral gd CD3 + T cells were not detectable, and a test for human immunodeficiency virus was negative. The patient did not display classical APS-1 symptoms, nor vitiligo. Anti-nuclear antibodies were negative but IgM rheumatoid factor (titer 44), 21OH, and SSC autoantibodies were positive ( Table 4). The patient had normal serum potassium and sodium and a normal morning plasma cortisol (292 nmol/L). Within the last two years, the patient was diagnosed and treated for a B cell lymphoma, which is now in full remission. Next-generation sequencing revealed a missense mutation in AIRE c.1399G>C (p.G467R) in the PHD2 domain. The variant is situated in a well-defined tertiary structure of PHD2, spanning the residues R433-S476. 13 The background frequency of this mutation is 0.04% and Universal Mutation Database prediction classified it as disruptive.

DISCUSSION
Here, we report 20 individuals from 11 kindreds with dominant heterozygous mutations in AIRE located within or close to the PHD1 and PHD2 domains, all with dominant-negative effect in vitro. Enteropathy was a common symptom among the patients, where several also presenting with autoantibodies against IFN-u.
Five variants were found in the PHD1 domain. The p.V301M variant was previously reported in a mother and her daughter where the mother presented with autoimmune Addison's disease, autoimmune thyroiditis, premature ovarian insufficiency, and autoantibodies targeting 21-hydroxylase, AADC, and IL-17F. Her daughter only had autoantibodies against IL-17F as a sign of autoimmunity. 7 We here report five additional patients from three families with heterozygous p.V301M and APS-1 manifestations such as gastritis, CMC, pernicious anemia, and enteropathy. Some had autoantibodies against IFN-u and IL-22, seen in almost all patients with APS-1. However, the corresponding mutation p.V303M in mice did not significantly alter the transcriptome of TRAs in mTECs or the development of regulatory T cells, hence questioning the dominancy of this mutation or highlighting the species background. 8 Interestingly, the previously published in vitro assay measuring dominancy showed a variable pattern of dominancy in the AIRE-regulated genes tested, suggesting a milder effect of this variant. 7 Furthermore, since V301M is relatively frequent, we would expect to see this mutation in patients with classical APS-1, but we do not. This indicates that p.V301M predisposes for other autoimmune manifestations than those typically associated with APS-1. Yet, p.V301M could work in concert with other genes and environmental triggers to elicit autoimmune manifestations.
Interestingly, several disease component and autoantibodies typically seen in classical APS-1 were observed, like autoantibodies against IL-22 and IFN-u in patients with the p.V301M change. The previously unreported p.G306R heterozygote variant was found in two families, where hypoparathyroidism, pernicious anemia, and vitiligo as well as autoantibodies against IFN-u were found. Vitiligo and autoantibodies against IFN-u and 21-OH was also found in a boy from Turkey harboring a heterozygous p.I309T change. The p.R328W was found to be dominant negative in two families with manifestations such as vitiligo and B12 deficiency. A different change at the same position, an arginine to a glutamine, was previously reported as dominant negative in a patient with vitiligo and autoantibodies against glutamic acid decarboxylase and with gastric parietal cell antibodies. 7 The young girl described herein had congenital diarrhea and will be interesting to follow-up with regards to the development of additional autoimmune manifestations.  8 An overview of all dominant-negative mutations described in AIRE is found in Figure 3.
Chronic diarrhea and enteropathy were frequently seen in these patients. In total, six of the patients reported here had such manifestations, and it was also reported in one of the patients with the p.C446G variant. 8 As AIRE is suggested to play a role in the development of Tregs, 14 these cells might contribute to enteropathy as this manifestation is common in patients with reduced T reg function. In the IPEX syndrome (Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked) caused by loss-of-function mutations in FOXP3 leading to loss of regulatory T cells is an apt example. These patients develop early-onset severe enteropathy, type 1 diabetes, and dermatitis. 15 The thymic architecture is not disturbed in mice models of this disease, suggesting that the major role of Foxp3 is promotion of Treg cell differentiation within the T cell lineage. 16 Also, patients with CTLA-4 insufficiency have compromised Treg function, leading to enteropathy and endocrinopathy. More than 50 heterozygous germline mutations have been reported, and incomplete penetrance and variable expressivity of genetic variants is common. 15 Enteropathy has been reported together with immunodeficiency, autoimmunity, and alopecia as a result of thymic stromal malfunction in patients with tetratricopeptide repeat domain 7A (TTC7A) defects. Like AIRE, TTC7A is highly expressed in mTECs. 17,18 Also, patients with thymoma can develop enteropathy 19,20 in addition to autoimmune conditions, most commonly myasthenia gravis. They have defective epithelial expression of AIRE and a failure to generate Tregs. 21 Interestingly, about 70% of patients with thymoma have autoantibodies neutralizing type I IFNs. 22,23 The incomplete penetrance of these dominant-negative mutations resembles what has been found in other diseases, like hypercholesterolaemia 24 and rhabdoid tumor predisposition syndrome. 25 Factors like the action of unlinked modifier genes, epigenetic changes, or environmental factors may underlie this variable penetrance. We have not investigated such factors, and the small sample number will make their role challenging to determine. Looking to the mouse models of the dominant-negative mutations, recessive mutations (causing classical APS-1 in humans) result in a lack of expression of the AIRE protein, while the dominant mutations increase the expression of AIRE. Furthermore, AIRE's ability to bind its own proximal enhancers is not altered, but they have reduced ability to induce changes in the accessibility to these loci. 8 The mechanistic aspects of the new dominant AIRE mutations described herein requires further attention; however, the previously described mechanisms support the notion of a dosage dependency of AIRE, where the degree of accessibility vary in a stochastic manner. Interestingly, two single-nucleotide variants in AIRE were recently found to increase the risk of isolated autoimmune Addison's disease, [9][10][11] supporting the concept that small changes in AIRE can predispose for autoimmune disease.
The classical APS-1 phenotype is rarely seen in patients with heterozygous dominant mutations although some have autoantibodies typical for APS-1. Instead, the clinical picture is diverse, with enteropathy, immunodeficiency, and vitiligo as common features. The fact that patients with the same mutation even in

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the same family have diverse or even no phenotype suggests that genetic, epigenetic, or environmental factors could alter the phenotype. As most of our patients are from Europe or America, a larger and more geographically diverse patient cohort is necessary to shed light on this. Looking at classical APS-1, AIRE mutations and phenotypes seem to be uncorrelated between countries and within families. Among four common AIRE mutations causing classical APS-1, two are found within certain populations; p.Y85C is common among Persian Jews while p.R139X is frequent among Sardinians. 26,27 p.Y85C has been associated with less candidiasis. A 13-base pair deletion in exon 8 is found within British, Dutch, German, Finnish, and Norwegian patients with APS-1 and is also the most frequent AIRE mutation in India and America. [28][29][30][31][32][33] p.R257X is commonly referred to as the major Finnish mutation but is also common among German, Swiss, British, Northern Italian, Russian, Polish, and Norwegian patients with APS-1. 33 Interestingly, an AIRE mutation often seen in Italy, C322fsx372, is also found in India as the most common mutation among Muslim probands. 32,34 There is no obvious correlation between mutations and phenotype, and large phenotypic variation among those with the same genotype, including siblings.
A close follow-up of patients with dominant-negative AIRE mutations and their families will reveal whether the risk of developing other and more severe autoimmune manifestations is increased. As whole-exome and genome sequencing are increasingly used in the diagnostic setting, we expect to see more heterozygous AIRE mutations in patients presenting with autoimmune disease and immunodeficiencies. Therefore, it is necessary to establish robust high-throughput in vitro assays and take advantage of data simulation programs to explore the impact of these mutations determining their pathogenic effects.

Limitations of the study
There are certain limitations to our study. The major drawback is the lack of mechanistic insight into how these mutations reduce AIRE's function and affects the presentation of tissue-restricted antigens in the thymus. Mouse models have been instrumental in answering these questions earlier 8 but are expensive and time-consuming. Cell lines that stably express AIRE are lacking, hence overexpression of AIRE in cell lines is currently the best model but comes with limitations when reflecting a biological system. The emergence of thymic organoids will likely be a good model system to study human variants. Our study also lacks an unbiased search for patients, as no systematic identification of patients across hospitals has been done.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: