The possible implication of the S250C variant of the autoimmune regulator protein in a patient with...

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The possible implication of the S250C variant of the autoimmuneregulator protein in a patient with autoimmunity andimmunodeficiency: in silico analysis suggests a molecular pathogenicmechanism for the variant

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OEmanuele Bellacchio a,1, Alessia Palma b,1, Stefania Corrente c, Francesco Di Girolamo d, E. Helen Kemp e,Gigliola Di Matteo f, Laura Comelli g, Rita Carsetti b, Simona Cascioli b,Caterina Cancrini c, Alessandra Fierabracci b,⁎a Research Laboratories, Bambino Gesù Children's Hospital IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italyb Immunology and Pharmacotherapy Area, Bambino Gesù Children's Hospital IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italyc University Department of Paediatrics, Bambino Gesù Children's Hospital IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italyd Department of Laboratory Medicine, Bambino Gesù Children's Hospital IRCCS, Piazza S. Onofrio 4, 00165 Rome, Italye Department of Human Metabolism, The Medical School, University of Sheffield, Sheffield S10 2RX, United Kingdomf Department of Systems Medicine, Tor Vergata University of Rome, Viale Oxford 81, 00133 Rome, Italyg Proteomics Laboratory Istituto di Fisiologia Clinica, CNR Via Moruzzi, 1, 56124 Pisa, Italy

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Abbreviations: Abs, autoantibodies; ACA, adrenal cortdrome; April, a proliferation inducing ligand; APECED, autofactor (TNF) family; BAFF-R, B-cell activating factor of theCD40L, gene for ligandof cluster of differentiation 40; CD95hypomethylated DNA; CVID, common variable immunodeprotein 3; GADA, glutamic acid decarboxylase (isoform 65ible costimulator; IFN-ω, interferon omega; IL-2R alpha, idrome; IRF-8, gene for interferon regulatory factor 8; mmajor histocompatibility complex; MECs, thymic medullaphosphate-oxidase complex; nr, normal range; ns, nanosBank; PE, phycoerythrin; PHD1 and PHD2, plant homeodgene for protein tyrosine phosphatase non-receptor typetransducer and activator of transcription 5b; STAT3, signaand calciummodulator and cyclophilin ligand interactor;gene.⁎ Corresponding author at: Autoimmunity Laboratory,

E-mail address: [email protected] (A. Fi1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.gene.2014.07.0640378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Bellacchio, E., et al.,autoimmunity and..., Gene (2014), http://dx

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Article history:Received 16 September 2013Received in revised form 14 July 2014Accepted 24 July 2014Available online xxxx

Keywords:AireS250C Aire variantMolecular modelingImmunological disorderPathogenesis

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RRECTAutoimmunity can develop from an often undetermined interplay of genetic and environmental factors.

Rare forms of autoimmune conditions may also result from single gene mutations as for autoimmunepolyendocrinopathy–candidiasis–ectodermal dystrophy, an autosomal recessive disease associated with mutat-ed forms of the autoimmune regulator gene. Itwas proposed that genetic variability in the autoimmune regulatorlocus, in particular heterozygous loss-of-function mutations, might favor the development of organ-specific au-toimmunity by affecting the presentation of self-antigens in the thymus. Indeed, heterozygous mutations of theautoimmune regulator gene were reported in patients with organ-specific autoimmunity. Also, in primary im-munodeficiencies, a breakdown in central/peripheral tolerance frequently produces associationwith autoimmu-nity. The causative linkmay involve a common genetic background and several gene defects have been identifiedas putative culprits. We report a unique patient, a 14 year old male from Lazio region, affected by common var-iable immunodeficiency associated with autoimmune manifestations (alopecia, onychodystrophy) and hetero-zygote for the S250C variant located in the SAND domain of the autoimmune regulator gene protein. To ourknowledge this is the first report of the S250C variant in a patient bearing this unusual combination of autoim-munity and immunodeficiency.

ex Abs; AIRE, autoimmune regulator gene; Aire, autoimmune regulator protein; ALPS, autoimmune lymphoproliferative syn-immune polyendocrinopathy–candidiasis–ectodermal dystrophy; BAFF, B cell activating factor belonging to the tumor necrosis

TNF receptors; BCR, B cell receptor; °C, Celsius degree; CD, cluster of differentiation; CD19, gene for cluster of differentiation 19;L, gene for cluster of differentiation 95; CGD, chronic granulomatous disease; cm, centimeter; CpG, cytosine–phosphate–guanineficiency disorder; Cys, cysteine; dL, deciliter; DNA, deoxyribonucleic acid; FBS, fetal bovine serum; Foxp3, gene for forkhead box) Abs; GATA-2, gene for GAT2 transcription factor; IA2, insulinoma-associated antigen 2; ICA, islet cell Abs; ICOS, gene for induc-nterleukin 2 receptor alpha; Ig, immunoglobulin; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked syn-Abs, monoclonal antibodies; mg, milligram; K, Kelvin degrees; Kg, kilogram; KU, kilo-unit; MD, molecular dynamics; MHC,ry epithelial cells; min, minutes; mL, milliliter; MSH5, gene for MutS homolog 5; NADPH, nicotinamide adenine dinucleotideecond; PBMC, peripheral blood mononuclear cells; PCA, parietal cells Abs; PCR, polymerase chain reaction; PDB, Protein Dataomains 1 and 2; pKa, negative logarithm of the acid dissociation constant; PID, primary immunodeficiency diseases; PTPN22,22; RAG, recombinant activating genes; RFLP-PCR, restriction fragment length polymorphism-PCR; Ser, serine; STAT5b, signall transducer and activator of transcription 3; STIM1, stromal interacting molecule 1; TACI, gene for transmembrane activatorTg, thyroglobulin; TNF, tumor necrosis factor; TPO, thyroperoxidase; Treg, regulatory T cells;WASP, Wiskott–Aldrich syndrome

Immunology and Pharmacotherapy Area, Italy.erabracci).

The possible implication of the S250C variant of the autoimmune regulator protein in a patient with.doi.org/10.1016/j.gene.2014.07.064

http://dx.doi.org/10.1016/j.gene.2014.07.064

mailto:[email protected]


http://www.sciencedirect.com/science/journal/03781119


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Please cite this article as: Bellacchio, E., et al.,autoimmunity and..., Gene (2014), http://dx

To obtain insights into the possiblemolecular effects of the S250C variant, we have carried out an in silico analysisof the SAND domain structure of the autoimmune regulator protein. In particular, homology modeling hasallowed us to observe that the cysteine introduced by the S250C variant is surrounded by cationic residues,and by means of molecular dynamics simulations together with pKa calculations, we have shown that these res-idues remain stably proximal to cysteine-250 lowering its pKa and thus conferring high chemical reactivity to themutated residue. We propose that the enhanced reactivity of cysteine-250, which is likely to impair the proteinfunction but probably insufficient to produce alone a phenotype as a heterozygous S250C variant due to compen-sationmechanisms,might becomemanifestwhen combinedwith other genetic/environmental factors. These re-sults can provide the rationale for the patient's unusual phenotype, shedding new light into the pathogenesis ofthe clinical association of autoimmunity and immunodeficiency.

© 2014 Published by Elsevier B.V.

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1. Introduction

Autoimmune diseases derive from a complex interaction of geneticand environmental factors (Bogdanos et al., 2012; Fierabracci et al.,2012a). Rare autoimmune conditions may also result from singlegene mutations as in the case of autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) (OMIM#240300)(Proust-Lemoine et al., 2012). The clinical diagnosis of this syndromeis confirmed by the presence of 2 of the clinical symptoms: chronic mu-cocutaneous candidiasis, hypoparathyroidism and Addison's disease(Fierabracci, 2011a). This is an autosomal recessive disease caused bymutations in the autoimmune regulator (AIRE) gene (Fierabracci,2011a). The encoded Aire protein functions as a non-classical transcrip-tion factor and is strongly implicated in the regulation of organ-specificantigen expression in thymic medullary epithelial cells (MECs) andthe imposition of T cell tolerance (Anderson and Takahama, 2012;Fierabracci, 2011b). It can putatively bind two different DNA sequencemotifs through different domains (Purohit et al., 2005): the planthomeodomains (PHD) binding to the sequence motif ATTGGTTA viaamino acid residues 299–355 (PHD1) and 434–475 (PHD2), and theatypical SAND domain binding to the sequence motif TTATTA via thespecific amino acid residues 189–196.

It has been suggested that genetic variability in the AIRE locus and inparticular heterozygous loss-of-function mutations might favor the de-velopment of certain organ-specific autoimmune disorders by affectingthe presentation of self-antigens in the thymus and borderline tolerance(Fierabracci, 2011b). Indeed, abnormal immunological mechanismshave been identified in the parents of APECED patients, suggestingthat a subclinical immune deficit is also present in heterozygotes withAIRE mutations (Sedivá et al., 2002). In addition, heterozygous AIREmutations have been reported in patients affected by organ-specificautoimmune disorders including: chronic hypoparathyroidismand thy-roiditis (Cervato et al., 2010); adrenalitis and thyroiditis (Toth et al.,2010); autoimmune hepatitis (Gossard and Lindor, 2012; Lankischet al., 2005, 2009); alopecia areata and universalis (Tazi-Ahnini et al.,2002); and systemic sclerosis associated with autoimmune thyroiditis(Ferrera et al., 2007). Interestingly, a unique autoimmune phenotypedistinct from APECED, manifesting predominantly as thyroiditis, wasreported in a kindredwith a G228WAire variant acting in an autosomaldominant-negative manner (Su et al., 2008).

Patients with primary immunodeficiency diseases (PID), which affectdistinct components of the innate and adaptive immune systems, fre-quently display autoimmune manifestations (Arason et al., 2010; Guptaand Louis, 2013). Several gene defects have been identified as possiblecauses of autoimmunity in PID such as Foxp3 (forkhead box protein 3)(Katoh et al., 2013) in immunodysregulation polyendocrinopathy enter-opathy X-linked syndrome (IPEX) (d'Hennezel et al., 2012). However,themechanisms for autoimmunity in other PID such as common variableimmunodeficiency disorders (CVIDs) are not well defined (Arason et al.,2010; Gupta and Louis, 2013; Teachey, 2012).

CVID represents a heterogeneous group of conditions in which afailure to produce immunoglobulin and protective antibodies causesrecurrent respiratory and gastrointestinal infections eventually associated

The possible implication of th.doi.org/10.1016/j.gene.2014.

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with autoimmunity and cancer predisposition (Arason et al., 2010).Marked reductions in serum IgG and IgA levels, and even in IgM levels,are detected in approximately 50% of patients. Themost common featureis the failure of antibody production following exposure to pathogens andimmunization. Several hypotheses have been advanced as to how defectsin immunoglobulin production could give rise to autoimmunity. The un-derlying mechanisms could be the lack of removal of autoreactive B lym-phocytes due to ineffective B cell receptor (BCR) signaling, altered ligandinteraction, accelerated expansion of autoreactive B cells, or increasedlevels of B cell activating factor belonging to the tumor necrosis factor(TNF) family/April (a proliferation inducing ligand) (Arason et al.,2010). Genes harboring CVID-associated mutations include: induciblecostimulator (ICOS) (Yong et al., 2009); transmembrane activator and cal-ciummodulator and cyclophilin ligand interactor (TACI) (Martinez-Galloet al., 2013; Zhang et al., 2007); B-cell activating factor of the TNF recep-tors (BAFF-R) (Losi et al., 2005); CD19 (Schäffer et al., 2007); and MSH5,a gene encoded in theMHCclass III region (Sekine et al., 2007). All togeth-er, these defects account for 10–15% of CVID cases (Arason et al., 2010).Therefore, for the majority of patients the underlying cause is still un-known. Interestingly, the products of these genes act at different levelsof B cell biology. In particular, we must underline that the role of defectsof TACI receptors has been disputed since TACImutations are also foundsometimes in healthy subjects who are not hypogammaglobulinemic.Nevertheless, hypogammaglobulinemic relatives of CVID patients havebeen shown to have detectable and selective in vitro B cell defects(Martinez-Gallo et al., 2013).

In the current study, we report the heterozygous S250C Aire variantin a patient affected by CVID and autoimmune manifestations, and wediscuss how this might underlie a possible novel genetic mechanismfor autoimmunity in CVID. We also present a molecular modelingstudy that provides a possible explanation of the defect in the Aire pro-tein function associated with the S250C variant.

2. Materials and methods

2.1. Immunological parameters

Serum immunoglobulin levels, memory/naïve T and B cell subsetanalysis, T and B cell in vitro responses to different stimuli and in vivospecific responses to vaccines were assessed according to standardprotocols.

For B cell phenotype analysis peripheral blood mononuclear cells(PBMC) were separated by Ficoll-Hypaque (Histopaque, Sigma-Aldrich Chemical C, St. Louis, MO, USA) from 5 to 10 mL, sodium-heparinized venous blood samples, washed twice in PBS and resus-pended in PBS/2% FBS (fetal bovine serum, Hyclone, South Logan, UT,USA). Single cell suspensions were incubated in the dark for 20 minat 4 °Cwith directly conjugatedmonoclonal antibodies (mAbs) directedagainst the following human surfacemolecules: CD19 (1:20, Cychrome-conjugated), CD27 (1:20, PE-conjugated), and IgM (1:300, Alexa Fluor647-conjugated). All antibodieswerepurchased fromBecton&Dickinson(BD, Pharmingen, San Diego, CA, USA). After labeling cells were washedtwice in PBS/2% FBS, and data were acquired with a FACScanto II (BD).

e S250C variant of the autoimmune regulator protein in a patient with07.064


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Flow cytometry profiles were analyzed using FACSDiva software (BD). Aminimum of 50,000 events were collected per data set.

2.2. Autoantibody assays

Anti-interferon ω (IFN-ω) autoantibodies (Abs) were detected byimmunoprecipitation of the patient's serum using 35S-labeled IFN-ω aspreviously described (Cervato et al., 2010). Serum was also tested forAbs to thyroglobulin (Tg) and thyroperoxidase (TPO) by chemilumines-cence, gastric parietal cells (PCA), adrenal cortex (ACA) and islet cell Absby indirect immunofluorescence, and glutamic acid decarboxylase (iso-form 65) (GADA), protein tyrosine phosphatase IA2, insulin, tyrosinase,tyrosine-related protein 1 (TRP1), tyrosine-related protein 2 (TRP2),tyrosine hydroxylase, melan A (MART1) andmelanocyte-specific proteinPMEL Abs by radioligand binding assays (Kemp et al., 2011).

2.3. DNA isolation and DNA sequencing of AIRE, PTPN22 and TACI

Genomic DNA was extracted from a whole blood sample of the pa-tient and his parents as well as 79 normal controls by QIAmp DNAblood mini kit (Qiagen, Hilden Germany). We screened the entire cod-ing sequence of the AIRE gene (GenBank ID: AJ009610) in DNA sampleswith primers designed on the flanking intronic regions of each of the 14coding exons using OLIGO primer analysis software (Molecular BiologyInsights, Inc.) to even identify any intronic mutation with splicing effect(Fierabracci et al., 2012b). All 5 exons and introns of TACI were se-quenced according to published protocols (Barroeta Seijas et al.,2012). Analysis of the protein tyrosine phosphatase non-receptor type22 (PTPN22) R620W (C1858T) single nucleotide polymorphismwas ac-complished using a RFLP-PCR method (Bianco et al., 2010).

2.4. Homology modeling of the S250C Aire variant

The structure of the SAND domain of the Aire protein in the aminoacid interval 189–280 was built by homology modeling with theprogram Swiss-PdbViewer (v4.1) (http://www.expasy.org/spdbv)(Guex and Peitsch, 1997).

2.5. Molecular dynamics simulations

Themodeled structure of the SANDdomain of wild type Aire proteinand its S250C mutant was prepared for molecular dynamics (MD)simulations under periodic boundary conditions in water, with Na+

and Cl− ions added to obtain electroneutrality and ionic strength of0.1 mol/L, using the following procedure: a first cycle of minimizationfollowed by equilibration was made allowing only the movement ofwater molecules, ions, and the amino acids resulting as insertionswith respect to the structure of the template used in the homologymodeling (Aire protein residues G197, G242, 248-SSS-250, and 263-GAAPGGG-269; the first equilibration was carried out at high tempera-ture, 500 K, to enhance sampling of conformational space by the aminoacids forming the insertions); in a second minimization/equilibrationcycle, the atom groups that were allowed to move, in addition towater molecules and ions, were extended to all protein side chains,and the equilibration was performed at T= 310 K. Then, a further min-imization was carried out allowing all the atoms composing the molec-ular system to move freely. Each minimization and equilibrationprocedure was respectively carried out for 50 and 500 ps. The qualityof the model was assessed with ANOLEA (Melo and Feytmans, 1998).The MD simulations were then performed without restraints for10.5 ns (ns) at T = 310 K. Temperatures were maintained constant byLangevin Dynamics during all equilibrations and the final MD simula-tions. The atomic coordinates were recorded every 5 ps, and snapshotsof the MD trajectories in the time range 0.5–10.5 ns were used for thecalculations of the pKa of Cys-250 (in the mutant protein), and Cys-223 (in the wt protein) the latter representing the unique cysteine

Please cite this article as: Bellacchio, E., et al., The possible implication of thautoimmunity and..., Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.

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residue naturally occurring in the SAND domain of the Aire protein(the first 0.5 ns of simulation was used to equilibrate the molecularsystem after the last minimization). MD calculations were performedwith NAMD (v. 2.7b1) (Kalé et al., 1999; Phillips et al., 2005) with theCHARMM22 protein force field (MacKerell et al., 1998). Simulationswere all carried out with an integration step of 1 fs.

3. Results

3.1. Clinical phenotype

The patient, now 14 years old [height 151 cm (50° centile), weight50 kg (75° centile) at the last examination when 13 years old], wasthe second son of non-consanguineous parents (actual age of the father51 years and of themother 44 years) without any family history for im-munodeficiencies and autoimmune diseases. His parents and brotherwere apparently healthy. The patient and his family were from theLazio region. At the age of 10 years, the patient was referred to theImmunoinfectivology Unit of the Bambino Gesù Children's Hospital,Rome, Italy, for upper respiratory recurrent infections which had mani-fested since the first years of life. From the age of 12 years, the patientdeveloped alopecia areata and onychodystrophy with general as-pects of nail pitting and hyperkeratosis. Associated symptomswere allergic rhinitis and lactose intolerance with recurrent abdom-inal pain (Gijsbers et al., 2012).

In repeated testing, the patient presented hypogammaglubinemiawith IgA and IgG deficiency (Table 1). Normal T cell distribution and Tcell proliferation to different stimuli (antigens and mitogens) were evi-dent. B cell phenotype analysis both basal and after CpG (cytosine–phosphate–guanine hypomethylated DNA) stimulation (Capolunghiet al., 2008) revealed a B cell defect characterized by low levels ofswitched memory cells (Fig. 1). In vivo defective responses to anti-pneumococcal and tetanus vaccines were observed (Table 1). Thediagnosis of CVID was made. The patient received, when necessary,antibiotics and local treatments for alopecia and no intravenous im-munoglobulin treatment.

3.2. Detection of autoantibodies

Abs against IFN-ω (3.39% BNDG, 1.67 index), Tg [18.1 U/mL; normalrange (nr) 9–60], TPO (36.3 U/mL; nr 0–60), PCA, ACA, islet cells Abs(ICA), GADA (0.1 U/L; normal value b 1.1), Abs to IA2 (0.1 U/mL; nor-mal value b 1.1), insulin (4%; normal value b 7%), tyrosine hydroxylase(Ab index 1.01; nr 0.86–1.16), TRP1 (Ab index 1.05; nr 0.90–1.15), TRP2(Ab index 0.96; nr 0.93–1.15), andMART1 (Ab index 1.12; nr 0.90–1.13)tested negative in the patient's serum. Abs against tyrosinase (Ab index2.45; nr 0.89–1.21) and melanocyte-specific protein PMEL (Ab index2.10; nr 0.88–1.10) were detected in the patient's serum. Previously,both thesemelanocyte-specific proteins have been identified as autoan-tibody targets in patients with alopecia areata (Kemp et al., 2011).

3.3. Detection of AIRE, PTPN22 and TACI mutations

For the presence of an APECED-like condition characterized by im-munodeficiency with B cell defects and autoimmune conditions alope-cia and onychodystrophy, analysis of mutations in TACI (TNFRSF13B,MIM 604907), AIRE and PTPN22 was conducted. No mutations in TACIwere detected, although heterozygous single nucleotide polymor-phisms of unknown non-pathogenic significance were present includ-ing: c.81GNA (p.T27T) (exon 2); g.23976ANC (c.445+25ANC) (intron3); g.32823TNC (c.632−60TNC) (intron 4); and c.831TNC (p.S277S)(exon 5). Screening of PTPN22 did not detect the heterozygous singlenucleotide polymorphism R620W (C1858T), which is frequentlyreported in alopecia and other autoimmune conditions (Biancoet al., 2010).


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t1:1 Table 1t1:2 Serological immunological parameters of the patient in some repeated testing. The table shows hypogammaglobulinemia in the patientwith IgA and IgG deficiency and defective responset1:3 to anti-pneumococcal vaccines.

t1:4 Age IgA IgG IgG1 IgG2 IgG3 IgG4 IgM IgE Isoemoagglutinin Response toanti-pneumococcalvaccine

t1:5 10 yearst1:6 (at diagnosis)

b5 mg/dLnr 70–250

569 mg/dLnr 700–1400

73 mg/dLnr 40–165

Anti-A 1:16(borderline)

After first doseAb 7 mg/L

t1:7 10.2 years b5 mg/dLnr 70–320

578 mg/dLnr 700–1500

524 mg/dLnr 400–1150

41.7 mg/dLnr 98–480

0.15 mg/dLnr 15–149

0.64 mg/dLnr 3–210

80 mg/dLnr 50–155

t1:8 10.9 years b5 mg/dLnr 70–320

512 mg/dLnr 700–1500

54 mg/dLnr 50–155

After first doseAb 30 mg/L

t1:9 11.6 years After second doseAb 17 mg/L

t1:10 11.9 years b5 mg/dLnr 70–320

476 mg/dLnr 700–1500

464 mg/dLnr 400–1150

71 mg/dLnr 98–480

13.1 mg/dLnr 15–149

1.37 mg/dLnr 3–210

92 mg/dLnr 50–155

t1:11 12.5 years b5 mg/dLnr 70–320

599 mg/dLnr 700–1500

93 mg/dLnr 50–155

b2 KU/dLnr b 100 KU/dL

t1:12 12.10 years b5 mg/dLnr 70–320

527 mg/dLnr 700–1500

473 mg/dLnr 370–1280

59 mg/dLnr 106–610

18.9 mg/dLnr 18–163

0.56 mg/dLnr 4–230

77 mg/dLnr 50–155

After second doseAb 10 mg/L

t1:13 nr = normal range; altered parameters are in bold.


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Analysis of the patient's AIRE gene detected the S250C variant inexon 6 which caused substitution of a serine residue at position 250(Ser-250) by a cysteine residue (Cys-250) (Fig. 2a). The heterozygousmutation was also detected in the patient's father (Fig. 2b). The DNAof 79 healthy controls tested negative when screened for the S250Cvariant.

3.4. Molecular modeling of the S250C mutant of the Aire protein

To obtain insight into the possible effects of the S250C variant in theAire protein,we built a homologymodel of the SANDdomain of the pro-tein. The multiple sequence alignment of Aire homologues from differ-ent organisms in this region is shown in Fig. 3a. In order to identifythe most suitable structurally solved template among homologues, wefirst produced the structural alignment of the SAND domains availablefrom the Protein Data Bank (PDB, accession codes 1UFN, 1H5P and1OQJ) and retrieved their corresponding multiple sequence alignment.Subsequently, the Aire SAND domain sequence was added to the latterin a profile–profile alignment fashion. The overall alignment was im-proved bymanual adjustments taking care inmaintaining the samemu-tual alignment among the PDB sequences as in their original structuralalignment and avoiding to introduce any gap/insertion inside theknown α-helix or β-sheet elements. The final alignment is shown inFig. 3b. The PDB structure 1UFN, corresponding to the SAND domainof mouse Sp110 nuclear body protein, exhibited the best alignmentwith Aire SAND domain (the percentage of amino acid identity in eachpairwise alignment was 26%, 24% and 16% respectively for 1UFN, 1H5Pand 1OQJ), therefore we chose this structure (its first NMR conformer)as the template for homologymodeling of Aire SAND domain; the relatedpairwise alignment is shown in Fig. 3c.We found this approachof aligningthe Aire SAND sequence to a structurally based and unmodifiable pre-alignment of PDB sequences (used as a restraint) to be more reliablethan carrying out direct pairwise alignments of Aire with the individualPDB sequences. In fact, direct pairwise alignments could apparently pro-duce slightly higher sequence identity percentages, but they harbor aclear incongruousness since a few less conserved regions of Aire becomeassigned to non-corresponding structural regions in the different PDBstructures of SAND domains. Despite the overall 26% amino acid identityindicates remote relationship (less than 30% amino acid identity) be-tween the two sequences and might underlie errors in the alignment, itmust be added that this score is penalized by the way we calculated it(as the number of identical residues in the two sequences divided bythe length of Aire SAND domain sequence and multiplied by 100) sincethe C-terminal part of the SAND domain of Aire contains a number ofamino acid insertions with respect to the sequence of the template


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(1UFN). However, these insertions in Aire can be identified by visualinspection as the low-complexity segments formed by residues 248-SSS-250 and 263-GAAPGGG-269 (please see Fig. 3c). Once theselow-complexity segments are not taken into account, the length of thetwo sequences becomes comparable, and the alignment of the less ho-mologous region of Aire SAND domain (residues 242–280)with the tem-plate (1UFN) becomesmore straightforward. On the other hand, it can beobserved that the alignment between the remaining parts of the SANDdomain of Aire (residues 189–241) with the template appears plain.The modeled SAND domain of Aire is shown in Fig. 4.

Taking into account the relatively low conservation of the short seg-ment encompassing Ser-250 (Fig. 3a) and that the S250C amino acidchange, per se, does not imply a dramatic change in the physical chem-ical properties of the site, we investigated whether the novel cysteineintroducedby themutation could present enhanced chemical reactivity,a property arising from certain interactions with the local protein envi-ronment. We made this analysis because reactive cysteines are knownto impact the function of proteins and we thought that if this propertycan be identified in the S250C Airemutant it could provide an indicationof one of the factors concurring to the phenotype observed in the pa-tient. In particular, this type of residue can become highly reactive ifthe local protein environment promotes the deprotonation of the thiolside chainwith formation of the highly nucleophilic thiolate anion. Con-ditions reported to cause decreased pKa values and the associated in-creased reactivity of cysteines are those enabling interactions of thethiol group of the cysteine side chain with cationic groups or aromaticside chains or metal centers (Shlomai, 2010). Since the SAND domainfeatures an abundance of basic residues, which are likely related to theputative DNA binding ability of the protein, we examined whetherone or more of such cationic residues can interact with Cys-250 andlower significantly the pKa of the thiol group in the mutant proteinthus providing an indication of enhanced chemical reactivity as a possi-blemolecular rationale for the observed phenotype. Here, we show thatin the modeled structure of the S250C mutant of the SAND domain thelocal environment around Cys-250 is populated by a number of basicresidues (Fig. 4). We also undertook MD simulations on both the wildtype and the S250C variant of Aire SAND domain, performing calcula-tions of pKa values using H++ (v3.1) (Anandakrishnan et al., 2012)(URL: http://biophysics.cs.vt.edu/H++) on protein conformers obtain-ed at various times. Interestingly, the pKa values for the side chain ofCys-250 was significantly lower (Fig. 5) than the 8.5–8.8 pKa valuerange expected for ordinary cysteine side chains within proteins(Cantor and Schimmel, 1980), hence revealing enhanced reactivity forthe cysteine introduced in the S250C variant. On the other hand, pKa cal-culations made as a mean of comparison on Cys-223, which is the


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Fig. 1. Patient's B cell phenotype. Representative dot plots of PBMC from a healthy control (upper panels) and the patient (bottompanels) stainedwith anti-CD19 Cychrome, anti-CD27 PEand anti-IgM Alexa Fluor 647-labeled monoclonal antibodies. Memory B cells are identified as CD19+CD27+ cells (left panels). The staining with anti-IgM and anti-CD27 antibodies dif-ferentiated switchedmemory B cells (CD27+IgM) from IgMmemory B cells (CD27+IgM+) (right panels). Total memory, switched and IgMmemory B cells in the B cell gate are indicatedas percentages for both the patient and the control. The patient shows a low relative percentage of switchedmemory B cells. For reference the normal values related to the age of the pa-tient are for memory B cells 14–40%, for switched memory B cells 9–32% and for IgM memory B cells 8–30%.


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pKa values that arewell above the pKa range of typical protein cysteines,thus indicating that this cysteine is even much less reactive thanordinary cysteines. pKa calculations were repeated using PROPKA 3.0(Olsson et al., 2011), which yielded a trend of pKa values for Cys-223and Cys-250 similar to that found with H++ (data not shown). DuringMD simulations we did not notice important differences in the confor-mations of the wild type and S250C mutant. The stabilities of MD simu-lations were assessed by examining the RMSD of the protein backboneatoms (Fig. 6).

4. Discussion

PID are often present in associationwith several autoimmune condi-tions. Inmonogenic immunodeficiencies such as APECED, an autosomalrecessive disorder due to mutations in the AIRE gene (Fierabracci,2011b) and IPEX, to mutation in the Foxp3 gene, and severe combinedimmunodeficiency, caused by mutations in the RAG genes, a definedgene defect is recognized. In other disorders, such as CVID, the patho-genesis of autoimmunity remains unknown and several gene defectswere envisaged as possible contributing factors.

The Aire protein is considered to play important roles in the estab-lishment of self-tolerance because it upregulates the transcription ofcertain organ-specific self-antigens in MECs (vide supra) and has arole in the negative selection of organ-specific thymocytes. Earlyhypotheses on how AIRE might be involved in the regulation of


autoimmunity included: determining the organization of the thymicstroma (Fierabracci, 2011a); controlling thymocyte tolerization; regu-lating B cell and T cell responses to antigenic stimuli; inducing apoptosisof parenchymal cells thereby enhancing cross-presentation of their an-tigens; and impinging on the differentiation of CD4+CD25+ regulatoryT (Treg) cells. Our patients are affected by CVID, onychodystrophy andalopecia areata, with the latter two clinical symptoms being frequentsecondary manifestation in APECED. There is a central role for T lym-phocytes inmediatingdamage to anagen hair follicles in alopecia areata,which is considered to be an autoimmune disease (Tazi-Ahnini et al.,2002). Although the major T cell autoantigen in alopecia areata hasnot been identified, several characterized autoantibody targets includetyrosine hydroxylase and the melanocyte-specific proteins tyrosinaseand PMEL (Kemp et al., 2011). Our patient showed slightly raised auto-antibody levels against tyrosinase and PMEL, which could be markersfor melanocyte destruction in the hair follicle by, for example, cytotoxicT cells. Since the patient harbors a novel heterozygous variant of theAire protein (S250C), we pointed out a possible pathogenetic mecha-nism and discuss the possible genotype–phenotype correlation.

Ser-250, the amino acid involved by the amino acid change, is com-prised in a short segment of the SAND domain of the Aire protein that ischaracterized by a significant amino acid variability as well as differentlengths across phylogenically close species (Fig. 1a). The relatively lowconservation of this short segment together with its compositionand hydrophilic character suggests that it might constitute a solvent-exposedflexible loop rather than a defined secondary structure element



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Fig. 2. Detection of the S250C AIRE gene variant. (a) Electropherograms of representativefragments of exon 6 of the AIRE gene relative to a normal control and the patient. The anal-ysiswas performed after informed consent. Thewild type allele is shown for exon 6 in thecontrol (upper panel). The patient (bottom panel) is heterozygous, having one copy of themutated c.748 ANT allele (exon 6). None of the already described AIREmutations was de-tected in all the 14 exons of the gene. 79 normal controls tested negative for the novelmu-tation. (b) Phylogenetic tree relative to the studied family showing the singleheterozygous state for the novel c.748 ANT variant in the father and the single heterozy-gosity c.748 ANT in the patient. The c.748 ANT variant alters the sequence of the Aire pro-tein causing a substitution of a serine residue with a cysteine at amino acid sequenceposition 250. N = wild type.


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of the SAND domain. However, the S250C variant is found in a patientexhibiting an important phenotype and, to reconcile this observationwith the low conservation of Ser-250 and flanking residues, it wouldbe necessary to invoke the engagement of this narrow region in func-tions that are strictly species-dependent, such as, for example, that theAire protein of phylogenetically close organisms may specifically inter-act with distinct DNA sequences. A more probable scenario would bethat the S250C variant can introduce a significant alteration in the struc-ture of the Aire protein, but the relatively modest differences between aserine and a cysteine residue together with the occurrence of this vari-ant in a non-strictly conserved position of the protein appear as aninsufficient argument to explain the observed phenotype. However,


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dramatic effects on the SAND domain structure and/or function mightarise if the novel cysteine residue is rendered highly reactive by thelocal environment of the protein. Therefore, to support this hypothesis,we built the SAND domain of the Aire protein by homology modelingand performedMD simulations on the structure carrying the S250C var-iant to understandwhether the reactivity of Cys-250 could be increasedby the surrounding protein residues.

Our results show that a number of basic residues are proximal toCys-250 (Fig. 4) hence they can generate the electrostatic forces thatfavor deprotonation of the thiol cysteine side chain with formation ofthe thiolate form. Indeed, pKa calculations on snapshots obtained at var-ious times along the MD simulation provided values for the thiol sidechain of Cys-250 (Fig. 5) that are significantly below the 8.5–8.8 pKa

value range typically expected for cysteines in proteins (Cantor andSchimmel, 1980), and that are indicative of enhanced cysteine reactivi-ty. On the other hand, the pKa calculations of Cys-223, which is theunique naturally occurring cysteine in the SAND domain of the protein,yield much higher values (Fig. 5) that denote very scarce reactivity forthis residue. These results, together with the ample exposure ofCys-250 on the protein surface, indicate that the cysteine introducedby the S250C variant is likely to react at physiological pH. Reactivecysteines undergo various oxidative reactions, which include S-nitrosylation and S-glutathionylation, and those forming sulfenicand sulfinic acids and disulfides (Spadaro et al., 2010). These chemicalmodifications can have very important effects on the protein structure/function considering the transcriptional activity assigned to the Aire pro-tein and mediated by its SAND domain, although Aire's SAND domainseems to bind to DNA in a rather non-specific fashion (Mathis andBenoist, 2009). Indeed, there are various examples of transcription factorswhose activity is modulated by the redox form of reactive cysteines di-rectly in the DNA binding region, such as in the case of Fos and Jun(Abate et al., 1990), p53 (Parks et al., 1997) and the bovine papillomavirustype 1 E2 protein (McBride et al., 1992) and also via a disulfide-mediateddimerization influencing the DNA binding ability of proteins like in thecase of Twist1 (Danciu andWhitman, 2010).

The very high pKa calculated for Cys-223 is understandable con-sidering that the SAND domain of Aire is proposed to be involved inthe homooligomerization of the protein (Halonen et al., 2004) and,to prevent the free Cys-223 from participating to undue disulfide-mediated cross-linking of Aire proteins brought into proximity duringhomooligomerization, the local protein environment surrounding Cys-223 must have been designated in such a way to completely suppressthe reactivity of this residue.

Reactions involving the cysteine residue introduced by the S250Cvariant can be detrimental considering that, in addition to the proposedinvolvement of the SANDdomain of Aire in homooligomerization and inDNA binding, this domain has also been proposed to play a role in thenuclear localization of the protein (Ramsey et al., 2002). Of note is theevidence that point mutations that affect the subcellular localization ofAire include residues that are very close to Ser-250, such as Lys-253(Ramsey et al., 2002), and Pro-252, Lys-243 and Arg-247, which werealso found to disturb the localization of the wild type Aire proteinwhen this is co-expressed with the mutants (Ilmarinen et al., 2005),thus mutations in this region might produce dominant negative effects.

Interestingly, we can remark that the discovered heterozygous vari-ant may act through a new pathogenetic mechanism in a dominantmanner of genetic transmission, as opposite to the effect that AIREgene mutations exert in homozygosis or compound heterozygosis inthe recessive APECED condition. A similar dominant negative effectwas reported for the G228W Aire variant, which affects the SAND do-main and causes a unique autoimmune phenotype distinct fromAPECED (vide supra) (Su et al., 2008). Of note Kumar et al. (2001) dem-onstrated that dimers and tetramers of the Aire protein, but not themonomers, can bind to G-doublets with the ATTGGTTA motif and theTTATTA-box. This implies that each single Aire protein both in dimersand tetramers must cooperate in DNA binding, since monomeric



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Fig. 3. Multiple sequence alignment of the SAND domain of the Aire protein with homologues. (a) Multiple sequence alignment of the Aire protein (UniProt entry O43918, amino acidrange 189–280, with homologues from different organisms). Bars on top of the alignment indicate residue conservation (calculated with Rate4Site (Mayrose et al., 2004) on the samealignment, and normalized to the range comprised between 0, minimal conservation, and 1, maximum conservation), with black and gray colors respectively marking values aboveand below the value 0.6 chosen as a conservation threshold. (b) Multiple sequence alignment of the SAND domain of human Aire protein (residues 189–280) and SAND domains fromavailable PDB structures (PDB entries 1UFN, 1H5P and 1OQJ). The residue conservation scores (blosum62) computed from the individual pairwise alignments between Aire and each se-quence extracted from the SAND domain PDB structures are plotted on top of themultiple sequence alignment and are also represented by the intensity of the blue color highlighting theamino acids in the corresponding PDB sequences (darker blue indicates higher conservation; amino acid residues identical to those in Aire are also highlighted in bold letters). (c) Pairwisesequence alignment employed for the homology modeling of the SAND domain of human Aire protein (residues 189–280) using Sp110 from mouse as the template (PDB entry 1UFN).Residue conservation is reported on top of the alignment. Identical residues within the two proteins are highlighted by bold and grayed letters. Secondary structures extracted with DSSPfrom the template protein (PDB 1UFN) are reported at the bottom of the alignment. (For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.)


Uproteins alone, even if functional, are devoid of DNA binding capability.Thus, if the variant S250C allele has impaired DNA binding ability, wecan speculate that even dimers composed of this variant with wildtype allele might not be able to bind DNA. This could be another expla-nation for the dominant negative effect of the discovered S250C variant.

In the light of the aforementioned, we can suggest that the heterozy-gous S250C variant produces an Aire protein exhibiting enhanced reactiv-ity of Cys-250, which may cause weakened DNA binding ability and/oraltered subcellular localization and/or impaired homooligomerization,and a specific disulfide-mediated oligomerization of the Aire protein.Any of such defective behaviors of the mutated Aire protein might havecontributed to enhanced escape of autoreactive T cells in early life, thuspossibly explaining the presence of autoimmunity in our CVID patient.


Of note the DNA of the father, who so far, to our knowledge, did notdevelop any clinical or preclinical autoimmune manifestation, harborsthe same heterozygous S250C amino acid change, implying a differentpenetrance of the dominant AIRE variant in causing autoimmunity. Wecan finally speculate that it has produced the pathological autoimmuneeffect by acting in concert with other gene variants and/or unknownenvironmental factors that the CVID patient might have encounteredin his life.

Initial attempts to demonstrate functional effects of the S250Cvariant were performed by immunoprecipitation on PBMC from the pa-tient and one control using in both cases two different anti-human Aireantibodies (H300, Santa Cruz Biotechnology, Santa Cruz, CA (Kumaret al., 2011) and kindly provided by Dr P. Peterson, Tampere University,


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Fig. 4.Homologymodel of the SANDdomain of the humanAire protein. The serine residue(site of the S250C variant) and Cys-223 (the unique cysteine residue in the SAND domainof the wild type protein) are shown as sticks and indicated by arrows. Basic residues (ar-ginines and lysines) are shown as blue sticks. Remaining residues are shown as gray lines.(For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)

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Finland). Nevertheless the success of the procedure was hampered bythe lowexpression of theAire protein, that should be stabilized in futurecell culture experiments i.e. by acetylation with CBP/p300 (Saare et al.,2012).

It must be finally pointed out that our hypothesis will require con-firmation by additional functional studies. However we believe thatcarrying out functional studies on a single genetic variant alone mightnot provide a straightforward explanation on the disease pathogenesis.As reported in the Introduction only in 10–15% of CVID patients the un-derlying genetic defect has been elucidated and these conditions canalso be associatedwith autoimmunity, resulting in complex phenotypesas in our patient. The genetic background of these disorders could onlybe in depth investigated through future exome sequencing approaches.

4.1. Conclusions

We report the heterozygous Aire variant S250C in a patient havingautoimmune alopecia, onychodystrophy and CVID. Our aim was to

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Fig. 5. pKa values of cysteine thiols calculated on snapshots from the molecular dynamicssimulations of the S250C variant and the wild type SAND domain of the Aire protein. Re-ported are the pKa values of Cys-250 (present in the S250C variant) and Cys-223 (theunique cysteine naturally occurring in the SAND domain of the wild type Aire proteinand used as reference). The horizontal dotted line marks the pKa threshold (pKa = 8.5)below which cysteines are characterized by increased reactivity. These results revealthat the cysteine introduced by the S250C variant exhibits a pKa value persisting belowthe threshold, thus implying that the local protein environment induces enhanced reactiv-ity in this cysteine residue along the entire MD simulation. On the other hand, the pKa ofthe naturally occurring Cys-223 stably remainsmuch above the threshold, thus indicatingthat Cys-223 behaves like ordinary protein cysteines that are devoid of high reactivity.


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ROpoint out a possible pathogenetic mechanism for this variant. By build-

ing a homology model of the SAND domain of the Aire protein and car-rying out MD simulations on the S250C variant, we discovered thatstable interactions with basic residues, conferring high reactivity toCys-250, can provide the rationale for the impairment of the proteinfunction; further experiments are required to confirm the results ofthe molecular modeling analysis. Our results draw the attention onAire S250C variant (Database of Single Nucleotide Polymorphisms(dbSNP)) as a protein product presenting potentially atypical behaviorcompared to the wild type protein. Taking these observations together,a hypothesis can be made for the association of this variant with auto-immunity and immunodeficiency in this patient. We propose that theS250C variant of the Aire protein normally results in effects devoid ofclinical significance, but under certain conditions the Aire variant mayemerge as a factor contributing to disease development. Neverthelessour work does not definitively prove that the Aire variant causes orinfluences the observed phenotype.We still might expect that other un-discovered gene defects can provide themechanisms for the occurrenceof autoimmunity in CVID patients as envisaged by other authors (Chenet al., 2013). We highlight a possible explanation for the association ofautoimmunity and immunodeficiency in this patient suggesting S250C(Database of Single Nucleotide Polymorphisms (dbSNP)) as a mutationin the Aire protein that might contribute with other undiscovered genedefects to disease development under certain conditions. Future exomesequencing approaches will elucidate whether other unknown geneticvariants acting also in concert with putative environmental factorsmight have contributed to the complex phenotype in this patient.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This studywas supported by the ItalianMinistry of Health.We thankFIRS Laboratories, RSR Ltd., Cardiff, UK for their expert assistance in test-ing IFN-ω antibodies.

References

Abate, C.,Patel, L.,Rauscher III, F.J.,Curran, T., 1990. Redox regulation of fos and jun DNA-binding activity in vitro. Science 249, 1157–1161.

Anandakrishnan, R.,Aguilar, B.,Onufriev, A.V., 2012. H++ 3.0: automating pK predictionand the preparation of biomolecular structures for atomistic molecular modeling andsimulations. Nucleic Acids Res. 40, W537–W541.

Anderson, G., Takahama, Y., 2012. Thymic epithelial cells: working class heroes for T celldevelopment and repertoire selection. Trends Immunol. 33, 256–263.



T

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656657658659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738

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UNCO

RREC

Arason, G.J., Jorgensen, G.H.,Ludviksson, B.R., 2010. Primary immunodeficiency and auto-immunity: lessons from human diseases. Scand. J. Immunol. 71, 317–328.

Barroeta Seijas, A.B.,Graziani, S., Cancrini, C., Finocchi, A., Ferrari, S.,Miniero, R., Conti, F.,Zuntini, R., Chini, L., Chiarello, P., Bengala, M., Rossi, P.,Moschese, V., Di Matteo, G.,2012. The impact of TACI mutations: from hypogammaglobulinemia in infancy to au-toimmunity in adulthood. Int. J. Immunopathol. Pharmacol. 25, 407–414.

Bianco, B.,Verreschi, I.T.,Oliveira, K.C.,Guedes, A.D.,Galera, B.B.,Galera, M.F.,Barbosa, C.P.,Lipay, M.V., 2010. PTPN22 polymorphism is related to autoimmune disease risk in pa-tients with Turner syndrome. Scand. J. Immunol. 72, 256–259.

Bogdanos, D.P., Smyk, D.S., Rigopoulou, E.I.,Mytilinaiou, M.G.,Heneghan, M.A., Selmi, C.,Gershwin, M.E., 2012. Twin studies in autoimmune disease: genetics, gender andenvironment. J. Autoimmun. 38, J156–J169.

Cantor, C.R., Schimmel, P.R., 1980. The conformation of biological macromolecules.Biophysical Chemistry — Part I. Freeman, W.H. and Company, New York, USA.

Capolunghi, F.,Cascioli, S.,Giorda, E.,Rosado, M.M.,Plebani, A.,Auriti, C.,Seganti, G.,Zuntini,R.,Ferrari, S.,Cagliuso, M.,Quinti, I.,Carsetti, R., 2008. CpG drives human transitional Bcells to terminal differentiation and production of natural antibodies. J. Immunol. 80,800–808.

Cervato, S., Morlin, L., Masiero, S., Greggio, N.,Meossi, C., Chen, S., del Pilar Larosa, M.,Furmaniak, J.,Rees Smith, B.,Alimohammadi, M.,Kämpe, O.,Valenzise, M.,Betterle, C.,2010. AIRE gene mutations and autoantibodies to interferon omega in patients withchronic hypoparathyroidism without APECED. Clin. Endocrinol. 73, 630–636.

Chen, K., Coonrod, E.M.,Kumánovics, A., Franks, Z.E., Durtschi, J.D.,Margraf, R.L.,Wu, W.,Heikal, N.M., Augustine, N.H., Ridge, P.G., Hill, H.R., Jorde, L.B., Weyrich, A.S.,Zimmerman, G.A.,Gundlapalli, A.V., Bohnsack, J.F.,Voelkerding, K.V., 2013. Germlinemutations in NFKB2 implicate the noncanonical NF-kB pathway in the pathogenesisof common variable immunodeficiency. Am. J. Hum. Genet. 93, 812–824.

Danciu, T.E.,Whitman, M., 2010. Oxidative stress drives disulfide bond formation betweenbasic helix–loop–helix transcription factors. J. Cell. Biochem. 109, 417–424.

Database of Single Nucleotide Polymorphisms (dbSNP). Bethesda (MD): National Center forBiotechnology Information,National Library ofMedicine. dbSNP accession: ss342534056(dbSNP Build ID: 134). Available from: http://www.ncbi.nlm.nih.gov/SNP/.

d'Hennezel, E.,Bin Dhuban, K.,Torgerson, T.,Piccirillo, C.A., 2012. The immunogenetics ofimmune dysregulation, polyendocrinopathy, enteropathy, X linked (IPEX) syndrome.J. Med. Genet. 49, 291–302.

Ferrera, F., Rizzi, M., Sprecacenere, B., Balestra, P., Sessarego, M., Di Carlo, A., Filaci, G.,Gabrielli, A., Ravazzolo, R., Indiveri, F., 2007. AIRE gene polymorphisms in systemicsclerosis associated with autoimmune thyroiditis. Clin. Immunol. 122, 13–17.

Fierabracci, A., 2011a. Recent insights into the role and molecular mechanisms of the au-toimmune regulator (AIRE) gene in autoimmunity. Autoimmun. Rev. 10, 137–143.

Fierabracci, A., 2011b. The role of heterozygous mutations of the autoimmune regulatorgene (AIRE) in non-APECED autoimmunity: a comment on recent findings. Clin.Endocrinol. 74, 532–533.

Fierabracci, A.,Milillo, A.,Locatelli, F.,Fruci, D., 2012a. The putative role of endoplasmic re-ticulum aminopeptidases in autoimmunity: insights from genomic-wide associationstudies. Autoimmun. Rev. 12, 281–288.

Fierabracci, A.,Bizzarri, C.,Palma, A.,Milillo, A.,Bellacchio, E.,Cappa, M., 2012b. A novel hetero-zygous mutation of the AIRE gene in a patient with autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy syndrome (APECED). Gene 511, 113–117.

Gijsbers, C.F.,Kneepkens, C.M.,Büller, H.A., 2012. Lactose and fructose malabsorption inchildren with recurrent abdominal pain: results of double-blinded testing. ActaPaediatr. 101, e411–e415.

Gossard, A.A., Lindor, K.D., 2012. Autoimmune hepatitis: a review. J. Gastroenterol. 47,498–503.

Guex, N.,Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environmentfor comparative protein modeling. Electrophoresis 18, 2714–2723.

Gupta, S., Louis, A.G., 2013. Tolerance and autoimmunity in primary immunodeficiencydisease: a comprehensive review. Clin. Rev. Allergy Immunol. 45, 162–169.

Halonen, M.,Kangas, H.,Rüppell, T.,Ilmarinen, T.,Ollila, J.,Kolmer, M.,Vihinen, M.,Palvimo,J., Saarela, J.,Ulmanen, I., Eskelin, P., 2004. APECED-causing mutations in AIRE revealthe functional domains of the protein. Hum. Mutat. 23, 245–257.

Ilmarinen, T., Eskelin, P., Halonen, M., Rüppell, T., Kilpikari, R., Torres, G.D., Kangas, H.,Ulmanen, I., 2005. Functional analysis of SANDmutations in AIRE supports dominantinheritance of the G228W mutation. Hum. Mutat. 26, 322–331.

Kalé, L.,Skeel, R.,Bhandarkar, M.,Brunner, R.,Gursoy, A.,Krawetz, N.,Phillips, J., Shinozaki, A.,Varadarajan, K.,Schulten, K., 1999. NAMD2: greater scalability for parallel molecular dy-namics. J. Comput. Phys. 151, 283–312.

Katoh, H.,Zheng, P.,Liu, Y., 2013. FOXP3: genetic and epigenetic implications for autoim-munity. J. Autoimmun. 41, 72–78.

Kemp, E.H.,Sandhu, H.K.,Weetman, A.P.,McDonagh, A.J., 2011. Demonstration of autoan-tibodies against tyrosine hydroxylase in patients with alopecia areata. Br. J. Dermatol.165, 1236–1243.

Kumar, P.G., Laloraya, M.,Wang, C.Y., Ruan, Q.G.,Davoodi-Semiromi, A.,Kao, K.J., She, J.X.,2001. The autoimmune regulator (AIRE) is a DNA-binding protein. J. Biol. Chem.276, 41357–41364.

Kumar, V.,Pedroza, L.A.,Mace, E.M.,Seeholzer, S.,Cotsarelis, G.,Condino-Neto, A.,Payne, A.S.,Orange, J.S., 2011. The autoimmune regulator (AIRE), which is defective in autoim-mune polyendocrinopathy–candidiasis–ectodermal dystrophy patients, is expressedin human epidermal and follicular keratinocytes and associateswith the intermediatefilament protein cytokeratin 17. Am. J. Pathol. 178, 983–988.

Lankisch, T.O., Strassburg, C.P., Debray, D.,Manns, M., Jacquemin, E., 2005. Detection ofautoimmune regulator genemutations in children with type 2 autoimmune hepatitisand extrahepatic immune-mediated disease. J. Pediatr. 146, 839–842.


ED P

RO

OF

Lankisch, T.O.,Mourier, O.,Sokal, E.M.,Habes, D.,Lacaille, F.,Bridoux-Henno, L.,Hermeziu, B.,Lenaerts, C., Strassburg, C.P., Jacquemin, E., 2009. AIRE gene analysis in children withautoimmune hepatitis type I or II. J. Pediatr. Gastroenterol. Nutr. 48, 498–500.

Losi, C.G.,Silini, A.,Fiorini, C.,Soresina, A.,Meini, A.,Ferrari, S.,Notarangelo, L.D.,Lougaris, V.,Plebani, A., 2005. Mutational analysis of human BAFF receptor TNFRSF13C (BAFF-R)in patients with common variable immunodeficiency. J. Clin. Immunol. 25, 496–502.

MacKerell, A.D.,Bashford, D.,Bellott, M.,Dunbrack, R.L.,Evanseck, J.D.,Field, M.J.,Fischer, S.,Gao, J.,Guo, H.,Ha, S.,Joseph-McCarthy, D.,Kuchnir, L.,Kuczera, K.,Lau, F.T.K.,Mattos, C.,Michnick, S.,Ngo, T.,Nguyen, D.T.,Prodhom, B.,Reiher, W.E.,Roux, B., Schlenkrich, M.,Smith, J.C., Stote, R., Straub, J.,Watanabe, M.,Wiórkiewicz-Kuczera, J., Yin, D.,Karplus,M., 1998. All-atom empirical potential for molecular modeling and dynamics studiesof proteins. J. Phys. Chem. B 102, 3586–3616.

Martinez-Gallo, M., Radigan, L., Almejún, M.B., Martínez-Pomar, N., Matamoros, N.,Cunningham-Rundles, C., 2013. TACI mutations and impaired B-cell function in sub-jects with CVID and healthy heterozygotes. J. Allergy Clin. Immunol. 131, 468–476.

Mathis, D.,Benoist, C., 2009. Aire. Annu. Rev. Immunol. 27, 287–312.Mayrose, I., Graur, D., Ben-Tal, N., Pupko, T., 2004. Comparison of site-specific rate-

inference methods for protein sequences: empirical Bayesian methods are superior.Mol. Biol. Evol. 21, 1781–1791.

McBride, A.A.,Klausner, R.D.,Howley, P.M., 1992. Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regula-tion of the DNA-binding activity in vitro. Proc. Natl. Acad. Sci. U. S. A. 89, 7531–7535.

Melo, F.,Feytmans, E., 1998. Assessing protein structures with a non-local atomic interac-tion energy. J. Mol. Biol. 277, 1141–1152.

Olsson, M.H.M.,Søndergaard, C.R.,Rostkowski, M., Jensen, J.H., 2011. PROPKA3: consistenttreatment of internal and surface residues in empirical pKa predictions. J. Chem. The-ory Comput. 7, 525–537.

Parks, D., Bolinger, R.,Mann, K., 1997. Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA. Nucleic Acids Res. 25,1289–1295.

Phillips, J.C.,Braun, R.,Wang, W.,Gumbart, J.,Tajkhorshid, E.,Villa, E.,Chipot, C.,Skeel, R.D.,Kalé, L., Schulten, K., 2005. Scalable molecular dynamics with NAMD. J. Comput.Chem. 26, 1781–1802.

Proust-Lemoine, E.,Saugier-Veber, P.,Wémeau, J.L., 2012. Polyglandular autoimmune syn-drome type I. Presse Med. 41, e651–e662.

Purohit, S.,Kumar, P.G.,Laloraya, M.,She, J.X., 2005. Mapping DNA-binding domains of theautoimmune regulator protein. Biochem. Biophys. Res. Commun. 327, 939–944.

Ramsey, C.,Bukrinsky, A.,Peltonen, L., 2002. Systematic mutagenesis of the functional do-mains of AIRE reveals their role in intracellular targeting. Hum. Mol. Genet. 11,3299–3308.

Saare, M.,Rebane, A., Rajashekar, B.,Vilo, J., Peterson, P., 2012. Autoimmune regulator isacetylated by transcription coactivator CBP/p300. Exp. Cell Res. 318, 1767–1778.

Schäffer, A.A., Salzer, U.,Hammarström, L.,Grimbacher, B., 2007. Deconstructing commonvariable immunodeficiency by genetic analysis. Curr. Opin. Genet. Dev. 17, 201–212.

Sedivá, A., Ciháková, D., Lebl, J., 2002. Immunological findings in patients with autoim-mune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) and theirfamily members: are heterozygotes subclinically affected? J. Pediatr. Endocrinol.Metab. 15, 1491–1496.

Sekine, H.,Ferreira, R.C.,Pan-Hammarström, Q.,Graham, R.R.,Ziemba, B.,de Vries, S.S.,Liu, J.,Hippen, K.,Koeuth, T.,Ortmann, W., Iwahori, A., Elliott, M.K.,Offer, S., Skon, C.,Du, L.,Novitzke, J., Lee, A.T., Zhao, N., Tompkins, J.D., Altshuler, D., Gregersen, P.K.,Cunningham-Rundles, C.,Harris, R.S.,Her, C.,Nelson, D.L.,Hammarström, L.,Gilkeson,G.S.,Behrens, T.W., 2007. Role for Msh5 in the regulation of Ig class switch recombi-nation. Proc. Natl. Acad. Sci. U. S. A. 104, 7193–7198.

Shlomai, J., 2010. Redox control of protein–DNA interactions: from molecular mecha-nisms to significance in signal transduction, gene expression, and DNA replication.Antioxid. Redox Signal. 13, 1429–1476.

Spadaro, D.,Yun, B.W.,Spoel, S.H.,Chu, C.,Wang, Y.Q.,Loake, G.J., 2010. The redox switch:dynamic regulation of protein function by cysteine modifications. Physiol. Plant. 138,360–371.

Su, M.A.,Giang, K., Zumer, K., Jiang, H.,Oven, I., Rinn, J.L.,Devoss, J.J., Johannes, K.P., Lu, W.,Gardner, J., Chang, A., Bubulya, P., Chang, H.Y., Peterlin, B.M., Anderson, M.S., 2008.Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutationin Aire. J. Clin. Invest. 118, 1712–1726.

Tazi-Ahnini, R., Cork, M.J., Gawkrodger, D.J., Birch, M.P., Wengraf, D., McDonagh, A.J.,Messenger, A.G., 2002. Role of autoimmune regulator (AIRE) gene in alopecia areata:strong association of a potentially function AIRE polymorphism with alopeciauniversalis. Tissue Antigens 60, 489–495.

Teachey, D.T., 2012. New advances in the diagnosis and treatment of autoimmunelymphoproliferative syndrome. Curr. Opin. Pediatr. 24, 1–8.Salzer, U.,Unger, S.,Warnatz, K., 2012. Common variable immunodeficiency (CVID):exploring the multiple dimensions of a heterogeneous disease. Ann. N. Y. Acad. Sci.1250, 41–49.

Toth, B.,Wolff, A.S.B.,Halasz, Z.,Tar, A.,Szüts, P., Ilyés, I.,Erdos, M.,Szegedi, G.,Husebye, E.S.,Zeher, M., Maródi, L., 2010. Novel sequence variation of AIRE and detection ofinterferon-ω antibodies in early infancy. Clin. Endocrinol. 72, 641–647.

Yong, P.F.,Salzer, U.,Grimbacher, B., 2009. The role of costimulation in antibody deficiencies:ICOS and common variable immunodeficiency. Immunol. Rev. 229, 101–113.

Zhang, L., Radigan, L., Salzer, U., Behrens, T.W., Grimbacher, B., Diaz, G., Bussel, J.,Cunningham-Rundles, C., 2007. Transmembrane activator and calcium-modulating cyclophilin ligand interactor mutations in common variable immu-nodeficiency: clinical and immunologic outcomes in heterozygotes. J. AllergyClin. Immunol. 120, 1178–1185.


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