In Search for the Missing Link in APECED-like Conditions: Analysis of the AIRE Gene in a Series of 48 Patients

Autoimmune diseases are a heterogeneous group of disorders of the immune system. They can cluster in the same individual, revealing various preferential associations for polyendocrine autoimmune syndromes. Clinical observation, together with advances in genetics and the understanding of pathophysiological processes, has further highlighted that autoimmunity can be associated with immunodeficiency; autoimmunity may even be the first primary immunodeficiency manifestation. Analysis of susceptibility genes for the development of these complex phenotypes is a fundamental issue. In this manuscript, we revised the clinical and immunologic features and the presence of AIRE gene variations in a cohort of 48 patients affected by high polyautoimmunity complexity, i.e., APECED-like conditions, also including patients affected by primary immunodeficiency. Our results evidenced a significant association of the S278R polymorphism of the AIRE gene with APECED-like conditions, including both patients affected by autoimmunity and immunodeficiency and patients with polyautoimmunity compared to healthy controls. A trend of association was also observed with the IVS9+6 G>A polymorphism. The results of this genetic analysis emphasize the need to look for additional genetic determinants playing in concert with AIRE polymorphisms. This will help to improve the diagnostic workup and ensure a precision medicine approach to targeted therapies in APECED-like patients.


Introduction
Autoimmune diseases are a heterogeneous group of disorders of the immune system. Environmental factors, family history and/or genetic susceptibility underlie their etiopathogenesis [1]. These disorders are due to a loss of tolerance to self-proteins or autoantigens that can be organ specific or systemic [2]. Organ-specific autoimmune diseases are due to target cell destruction determined by autoreactive T lymphocytes and can cluster in the same individual revealing various preferential associations; this is the case of polyendocrine autoimmune syndrome Type I (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome (APECED)), Type II and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome [3]. Indeed, although phenotypically different and confirmed by different diagnostic procedures, autoimmune disorders can share similar immune and genetic defects, a phenomenon called 'autoimmune tautology' [2], i.e., the cooccurrence of polyautoimmunity or multiple autoimmune syndrome (MAS) and familiarity for autoimmunity [4].
Clinical observation has further highlighted that autoimmunity can even share some common characteristics and mechanisms with other conditions that initially were considered independent polar opposites. Indeed, this was suggested by the high prevalence of autoimmune manifestations in primary immunodeficiencies (PID) and the observation that autoimmunity may even be the first manifestation [5].
Identifying susceptibility genes for these complex phenotypes and unraveling their putative effects in their etiopathogenesis is a relevant issue. Further increased awareness and use of genetic screening of confirmatory functional studies, together with immunological markers, can lead to a precision medicine workup for early specific diagnosis in highly vulnerable patient categories [6].
Both purely autoimmune conditions and PIDs can exhibit defects in central and peripheral tolerance influenced by mutations in genes that regulate immunological tolerance [5]. In addition to human leukocyte antigen (HLA) haplotypes [7], several single-nucleotide polymorphisms (SNPs) were discovered to underlie the pathogenesis of autoimmune phenotypes [8]. Examples of common susceptibility genes involved in immune regulation include cytotoxic T lymphocyte-associated antigen 4 (CTLA4), which suppresses T-cell activation [9][10][11], forkhead box P3 (FOXp3), involved in the differentiation of T regulatory cells (Tregs) [12,13], and the interleukin-2 receptor (IL-2R)α/CD25 gene, which affects the development and function of Tregs [14]. Further, polymorphisms of the tumor necrosis factor (TNF)-α gene, located on chromosome 6p21.3, increase the risk of association of insulindependent diabetes mellitus (Type 1 diabetes, T1D) and autoimmune thyroid disease [15] and the association of alopecia areata and vitiligo [16]. Among the others, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene is associated with several autoimmune diseases; this encodes for a more active phosphatase, namely the Lyp variant R620W, which is a potent inhibitor of T-cell activation [17,18].
Regarding complex autoimmunity phenotypes, the APECED syndrome (OMIM#240300) [19] is a rare autosomal recessive disease caused by mutations in the autoimmune regulator (AIRE) gene [20,21]. The encoded AIRE protein is a transcription factor with an important role in regulating the escape of autoreactive T cells from the thymus in perinatal age and the development of Tregs [19,22]. Classic diagnostic criteria for APECED is the presence of two of the following manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CHP) and Addison's disease (AD) [23]. Indeed, CMC is often the first clinical manifestation in APECED patients in which multiple organ-and non-organ-specific autoimmune conditions may subsequently develop during their lifetime [23]. Anti-interferon omega (IFNω) antibodies circulating at high titers are serological hallmarks of the syndrome [24].
The presentation of self-antigens in the thymus that might favor the development of certain organ-specific autoimmune disorders is also conceived to be influenced by genetic variability in the AIRE locus and the presence of heterozygous loss-of-function mutations of the AIRE gene [25,26]. In this regard, AIRE variants have indeed already been reported in the DNA of patients affected by organ-specific autoimmune disorders [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Of note in parents of APECED patients harboring heterozygous AIRE mutations, immunological dysregulation was detected in the peripheral blood by elevated levels of IgA and activated T lymphocytes [28]. Furthermore, AIRE gene monoallelic mutations located in the first plant homeodomain (PHD1) zinc finger with autosomal dominant inheritance were found associated with autoimmune diseases characterized by a later onset, milder phenotype and reduced penetrance; however, manifestations in these conditions did not satisfy the clinical diagnostic criteria for APECED [47]. A milder phenotype was reported in a 'non-classical late onset' APECED due to a dominant-negative monoallelic mutation (G228W) located in the SAND domain of the AIRE gene in an Italian family with high incidence of Hashimoto's thyroiditis (HT) [48]. Instead, heterozygous recessive AIRE gene mutations may, although minimally, contribute to the occurrence of sporadic non-mendelian autoimmunity in the general population [49]. Of note, genome-wide association studies (GWAS) conducted in European cohorts of patients affected by pernicious anemia revealed rs74203920 missense variant leading to R471C substitution (p.Arg471Cys) in the second PHD (PHD2) of the AIRE gene among the identified risk loci [50]. Two independent signals rs74203920 and the intronic rs2075876 of the AIRE gene were also detected as significantly associated with Addison's disease in the Swedish population. The last SNP was in linkage disequilibrium with SNP rs1800520 coding for the S278R variant [51].
In a preliminary investigation, we demonstrated the trend of increased association of AIRE gene variants, particularly the S278R polymorphism, in patients affected by autoimmune polyendocrinopathies than in healthy controls [46]. The association of the S278R AIRE polymorphism was also reported with other autoimmune conditions, including hepatitis, alopecia areata, systemic sclerosis associated with HT and sporadic AD [34][35][36][37][40][41][42].
In light of the foregoing, the present study aimed to analyze the AIRE gene in a different group of patients affected by even higher polyautoimmunity complexity compared to the previously published cohort [46]. The present screened APECED-like population included variable associations of endocrine and non-endocrine and even immune-dysregulatory conditions manifested as immunodeficiency symptoms/confirmed PIDs and allergies. We also estimated the frequency of the detected AIRE gene variants and discussed their putative involvement in the pathophysiological process leading to their clinical and immunological features.

Subjects
A total of 48 patients affected by APECED-like disease, including variable association of organ-and non-organ-specific autoimmune disorders and immunodeficiencyassociated conditions (16 males, 32 females with age ranges at presentation between 1 and 15.42 years), were recruited from the University Department of Pediatrics (DPUO), at Bambino Gesù Children's Hospital (OPBG) in Rome. The patients' sera were assayed for insulin-dependent diabetes mellitus (Type 1 diabetes (T1D))-related autoantibodies (Abs), i.e., glutamic acid decarboxylase (GADA) (isoform 65), tyrosine phosphatase-related islet antigen 2 (IA2) and insulin (IAA) Abs, for anti-adrenal Abs by radioimmunoassay (RIA), for thyroid-related Abs, i.e., TSH-receptor Abs (TRAb immunoassay, Immulite TSI, Siemens Healthcare, Tarrytown, NY, USA), thyroglobulin (Tg), and thyroperoxidase (TPO) and for celiac-disease-related transglutaminase (TRG) Abs by chemiluminescence (ADVIA Centaur analyzer, Siemens Healthcare, Erlangen, Germany), gliadin, extractable nuclear antigen (ENA), endomysial (EMA) Abs, anti-liver kidney microsomal (LKM) and parietal cells Abs (APCA) by indirect immunofluorescence (IFL). Non-organ-specific Abs anti-nuclear (ANA), anti-neutrophil cytoplasmic (ANCA), anti-double-stranded DNA (dsDNA), antireticulin (ARA), anti-mitochondrial (AMA) and anti-smooth muscle cell (ASMA) were also tested. IFNω Abs were assayed by RIA in collaboration with FIRS Laboratories RSR Ltd. (Cardiff, UK). Informed consent was obtained from all those who took part in the present study in accordance with the Declaration of Helsinki. The investigation was approved by the local Institutional Review Board (IRB) of Bambino Gesù Children's Hospital (OPBG), which regulates human sample usage for experimental studies (Study Protocol No.: 1385_OPBG_2017). A control group included 84 healthy blood donors (44 females and 40 males) [46]. Controls were recruited from the OPBG Blood Transfusion Centre; they had no history of autoimmunity and immunodeficiency and no autoantibodies were detected in their serum.

Molecular Studies
Genomic leukocyte DNA was extracted from whole blood samples of patients by the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's guidelines.

AIRE Gene Screening
All 14 exons and intronic regions of the AIRE gene were sequenced according to already described protocols (Genetic Analyzer 3500 Applied Biosystems HITACHI system, Thermo Fisher Scientific, Rodano, Italy) in the DNA of recruited patients [46].

Statistical Analysis
Differences in the number of subjects with S278R polymorphism or the IVS9+6 G>A intronic variation of the AIRE gene between patients and healthy controls were assessed by the χ2 (chi-square) test on variances and the GraphPad Prism Software (version 7, San Diego, CA, USA). A value of p < 0.05 was considered significant.

WT
The difference in prevalence of the S278R polymorphism between the patient group and the healthy controls was statistically significant (Figure 2A). A trend of increase in the prevalence of the IVS9+6 G>A polymorphism between the patient group and the healthy controls was observed ( Figure 2B). These data suggest the putative influence of AIRE gene polymorphisms in APECED-like conditions, which is particularly evident in Patient 3 (Table 1), where polymorphism S278R is present in compound heterozygosity with the known R471C AIRE mutation [30].
The difference in prevalence of the S278R polymorphism between the patient group and the healthy controls was statistically significant (Figure 2A). A trend of increase in the prevalence of the IVS9+6 G>A polymorphism between the patient group and the healthy controls was observed ( Figure 2B). These data suggest the putative influence of AIRE gene polymorphisms in APECED-like conditions, which is particularly evident in Patient 3 (Table 1), where polymorphism S278R is present in compound heterozygosity with the known R471C AIRE mutation [30].

Discussion
Clinical examination of the series of patients has helped to raise physicians' awareness of the possible development of different autoimmune manifestations at different ages in the same individual; autoimmunity may even be the first manifestation of PID [2]. Nowadays, PIDs include more than 430 entities [55,56], and they are associated with polyautoimmunity; this is especially applied to CVID and CID.
In light of the foregoing, preventing the development of polyautoimmunity is a fundamental task [6]. Furthermore, identifying a PID in a heterogeneous group of patients with several autoimmune disorders can also be a difficult task. In the presence of polyautoimmunity, immunologic evaluation should be included at the initial diagnostic workup in order to avoid significant delay of a specific diagnosis in vulnerable patients affected by genetic immune defects.

Discussion
Clinical examination of the series of patients has helped to raise physicians' awareness of the possible development of different autoimmune manifestations at different ages in the same individual; autoimmunity may even be the first manifestation of PID [2]. Nowadays, PIDs include more than 430 entities [55,56], and they are associated with polyautoimmunity; this is especially applied to CVID and CID.
In light of the foregoing, preventing the development of polyautoimmunity is a fundamental task [6]. Furthermore, identifying a PID in a heterogeneous group of patients with several autoimmune disorders can also be a difficult task. In the presence of polyautoimmunity, immunologic evaluation should be included at the initial diagnostic workup in order to avoid significant delay of a specific diagnosis in vulnerable patients affected by genetic immune defects.
In previous studies, a high frequency of certain polymorphisms of the AIRE gene, including S278R, were discovered in autoimmune patients including those with non-APECED autoimmunity [37,46] (vide supra).
In order to validate the influence of susceptibility genes in the pathogenesis of complex autoimmune phenotypes, in the present investigation, we searched for AIRE gene variants in a population of APECED-like patients. Seven patients were also diagnosed with PID, and in some of them, recurrent infections, CMC, failure to thrive and autoimmunity could be listed as the warning signs of PID. A high presence of allergies was also reported in patients with associated autoimmunity and immunodeficiency [57]. The results of the present study evidenced a significant association of the S278R polymorphism of the AIRE gene with APECED-like conditions, including both patients affected by purely polyautoimmune disorders and patients affected by immune-dysregulatory manifestations/confirmed PID. This could be indicative of common molecular mechanisms that underlie the association of different autoimmune symptoms and even their association with immunodeficiency conditions. A trend of association was also observed with the IVS9+6 G>A polymorphism compared to the healthy controls ( Figure 2B). Of note, the sera of two patients, Patients 11 and 34 in Table 1, tested positive for anti-IFNω Abs, known to be typical of the APECED syndrome.
In light of the foregoing, we highlight the importance of analyzing known susceptibility genes in cohorts of patients. We corroborate the evidence that common AIRE polymorphisms may partially contribute to high complex polyautoimmunity phenotype predisposition in APECED-like patients. AIRE polymorphism identification may indeed act as a marker to emphasize the need to look for additional or novel genetic determinants playing in concert in causing polyautoimmunity and autoimmunity-immunodeficiencyassociated conditions. Association studies based on the candidate gene approach and the recent advent of whole-exome sequencing will definitively help to elucidate the genetic risk factors responsible for these complex phenotypes. This will contribute to establishing an improved personalized diagnostic protocol and to ensure the development of targeted therapies in APECED-like conditions.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Bambino Gesù Children's Hospital, which regulates human sample usage for experimental studies (Study Protocol No.: 1385_OPBG_2017).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study. The Institutional Review Board approved the consent procedure.

Data Availability Statement:
The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.