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Insulin alleles and autoimmune regulator (AIRE) gene expression both influence insulin expression in the thymus
Journal of Autoimmunity 25 (2005) 312e318 www.elsevier.com/locate/issn/08968411
Insulin alleles and autoimmune regulator (AIRE) gene expression both influence insulin expression in the thymus Lidia Sabater a, Xavier Ferrer-Francesch a, Mireia Sospedra a, Pepi Caro a, Manel Juan a,b, Ricardo Pujol-Borrell a,b,* a
Laboratory of Immunology for Research Applied to Diagnosis (LIRAD), Blood and Tissue Bank, Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Crtra. del Canyet s/n, 08916 Badalona, Spain b Division of Immunology, Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain Received 26 March 2005; revised 30 July 2005; accepted 1 August 2005
Abstract It is well established that the polymorphisms at the 5# of the insulin gene (IDDM2) confers susceptibility to type 1 diabetes, probably by modifying the level of insulin expression in the thymus that in turn influences immunological tolerance to insulin as self-antigen. AIRE is a transcription regulator which controls the expression of many peripheral antigens within the thymus, among them insulin. Results presented here confirm that insulin gene copies from both parental chromosomes are expressed in human thymus and that IDDM2 class III protective alleles are indeed associated with a higher level of insulin message expression. However, differences in insulin mRNA expression among different thymi were far wider than those determined by the class I and class III insulin gene alleles and maintained a clear correlation with AIRE expression. These results confirm the effect of IDDM2 alleles on insulin expression in the thymus, but suggest that the levels of AIRE may exert a stronger influence than IDDM2 alleles themselves. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Autoimmunity; Insulin; Type 1 diabetes; Tolerance; Thymus
1. Introduction Several autoantigens have been identified as targets of the autoimmune response causative of type 1 diabetes (T1D) but among them, insulin is prominent because it is islet beta cell specific, anti-insulin antibodies constitute the first evidence of islet autoimmunity in children, and T-cell response to insulin plays a central role in pathogenesis [1e3]. The possible contribution of the insulin gene to diabetes predisposition was first established in 1984 by Bell at al. [4], later confirmed in a genome-wide search for T1D susceptibility genes [5] and * Corresponding author. Laboratory of Immunology for Research Applied to Diagnosis (LIRAD), Blood and Tissue Bank, Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Crtra. del Canyet s/n, 08916 Badalona, Spain. Tel.: C34 93 497 8892; fax: C34 93 497 8843. E-mail address: [email protected] (R. Pujol-Borrell). 0896-8411/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2005.08.006
assigned the IDDM2 susceptibility locus. The risk mapped in a highly polymorphic VNTR minisatellite located at ÿ596 of insulin gene coding sequence (Fig. 1a). Alleles at this locus have been classified depending on the number of repeats of the ACAGGGGTSYGGGG consensus motif as class I (26e 63 repeats), class II (63e140 repeats) and class III (141e 209 repeats) [6,7] but only class I and class III alleles are found in European populations. Class I homozygous carry an increased relative risk (RR) that ranges between 1.9 and 5.0 [8]. The mechanism by which this polymorphism confers disease susceptibility remained elusive until it was shown that it influences the level of insulin expression in the thymus and that in this way it may affect immunological central tolerance to insulin [9,10]. The effect of IDDM2 alleles acting in cis on the expression of insulin in the thymus may be affected by many other genes acting in trans and is compounded by parental imprinting, all adding complexity to the analysis [7].
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Fig. 1. Genotyping strategy based on the known insulin haplotypes conferring risk and protection for type 1 diabetes. (a) Insulin gene and IDDM2 map. Arrows indicate the 138 bp region in the 3# UTR of the insulin gene amplified by INS primers that include the PstI polymorphism. In class III alleles the PstI restriction results in two bands of 84 bp and 54 bp. (b) Competitor fragment for QC-PCR. The insulin primers were incorporated into the M13(-72) ends of the pBluescript polylinker with hybrid primers and the construct was cloned in pZerO2 plasmid, then restricted by the NsiI enzyme. The amplification of the internal competitor gives a band of 354 bp with the co-amplification of cDNA of thymus and two bands of equal molecular weight 177 bp after PstI restriction.
The occurrence of a wide representation of peripheral selfantigens, among them insulin, in human thymus has been called ‘‘promiscuous gene expression’’ and it is now a well-established phenomenon [11]. The identification of the gene AIRE as a cause of the autoimmune polyendoendocrine syndrome 1 (APS1) or APECED [12,13], and the evidence that it acts as a broad-spectrum transcription factor regulator that determines the expression of peripheral antigens in the thymus and in some lymphoid organs, had an important impact in the understanding on how tolerance to endocrine autoantigens is established and maintained [14,15]. We report here results from quantitative-competitive and real-time PCR analysis of insulin alleles and AIRE gene expression in human thymus, aimed at elucidating their relative contribution to determine the insulin level of expression in the thymus. These results are relevant for the understanding genetic predisposition to T1D and the role of central tolerance to peripheral antigens in organ-specific autoimmunity.
2.2. Oligonucleotides Primers for insulin and GAPDH were designed using Oligo 4.0 software (MedProbe, Oslo, Norway). Primer for insulin were: INS-F 5#-AAGCGTGGCATTGTGGAAC-3#, INS-R 5#-CAAGGGCTTTATTCCATCTCTC-3#. Location of the primers with respect to insulin locus is shown in Fig. 1. GAPDH primers were sense: 5#-TCTTCTTTTGCGTCGCCAG-3# and antisense: 5#-AGCCCCCAGCCTTCTCCA-3#.
2.3. PCR-RFLP C1,127 PstI locus The genotyping of thymic samples for the C1,127 PstI insulin locus by PCR-RFLP was previously reported [7]. Briefly, genomic DNA was isolated from thymus by standard procedures. Class III and class I alleles were amplified with the same primers INS-F, INS-R and the amplicon underwent PstI (New England Biolabs, Beverly, MA) enzymatic restriction for allelic classification.
2. Methods 2.1. Samples and subjects
2.4. RNA isolation and retrotranscription
Thymic tissue from 45 patients was obtained in the course of routine thoracic surgery on children with congenital heart abnormalities or therapeutic thymectomy for myasthenia gravis patients. This study has been reviewed and approved by the ethical committees of Hospital Universitari Germans Trias i Pujol and Hospital Universitari Vall d’Hebro´. The age of the donors ranged from 12 days to 34 years. Multiple blocks of tissue (0.5 cm) from each glans were snap frozen and kept at ÿ70 C until use.
Total RNA from thymus was isolated from several thymic tissue blocks by the ChomczynkieSacchi method with minor modifications, and it was treated with DNA-freeÔ DNase Treatment (Ambion Inc., Austin, TX) to remove remaining genomic DNA. Residual contamination by genomic DNA was ruled out by the failure of the reaction when the retrotranscription step was omitted (data not shown). Nucleic acid concentration was determined by optical density and the integrity by agarose gel electrophoresis. RNA samples (2 mg) were
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retrotranscribed with oligo(dT)25 (1 mM final concentration, Genset, Paris, France) for 1 h at 37 C in a volume of 20 ml using First Strand Buffer 5!, dNTPs (1 mM, Pharmacia Biotech, Uppsala, Sweden) RNAsin Recombinant Ribonuclease Inhibitor (40 U, Promega, Madison, WI, USA) and SuperScript II Reverse Transcriptase (200 U, Invitrogen, Carldsbad, CA, USA) following the manufacturer’s protocol. Retrotranscription was arrested by heating at 90 C for 2 min. cDNA samples were diluted 1:5 and 1:50, and 2-ml aliquots of each dilution were amplified for the expression of the housekeeping gene GAPDH to normalize sample cDNA concentrations.
2.5. Quantitative-competitive PCR (QC-PCR) A fragment of the pBluescript II KS(C) polylinker which was modified to contain the insulin primers was cloned in pZerO, expanded and used as internal standard for QC-PCR. For each thymus, samples containing 33,000, 3300, 33 and no copies of the internal competitor, and an equal amount of each cDNA from thymus were subjected to QC-PCR amplification. A sample of genomic DNA was always run in parallel (200 ng) as control. Competitive PCR mixtures for insulin consisted of 1 ml of DZ buffer (50 mM KCl, 10 mM
TriseHCl pH 8.8, 1.5 mM MgCl2 and 0.1% Triton X-100), 0.5 mM of INS-F and INS-R primer, 200 mM of each dNTP (Pharmacia, Biotech), 0.4 U/ml of Dynazyme (Finnzymes, Oy, Espoo, Finland), 1 ml of the sample, and 1 ml of the internal standard at known concentration, in a total volume of 10 ml. Cycling parameters were 38 cycles of 95 C for 20 s and 65 C for 30 s with a final extension step at 72 C for 5 min. PCR products were labeled by adding [32P]dATP (5 mCi per tube) in the last cycle of amplification and were electrophoresed on an 8% polyacrylamide gel. The radioactivity counts were measured by exposing the dry gels to imaging plates (Eastman Kodak, Rochester, NY, USA) using a radiomaging system (PhosphorimagerÒ, Bio-Rad, Hercules, CA, USA) and readings were analyzed using Quantity-OneÒ software (Bio-Rad). The value of PstI undigested amplicon visible as a 354 bp band in the standard lane (Fig. 2b) was used to calculate a digestion efficiency correction factor. To calculate the number of insulin allele-specific copies (class I and class III), the radioactivity readings corresponding to the PstI digested plus the undigested band were transferred to a graphic of radioactivity vs. number of molecules generated from the competition fragment standard readings for which the starting number of molecules was known. The QC-PCR assays were run in duplicate and each thymus was analyzed at least twice with consistent results.
Fig. 2. Insulin gene allele transcription in human thymus. (a) Radioactive PCR products from genomic DNA (DNA) and reverse-transcribed RNA (RNA) after PstI restriction for typing and calculating the ratio of class III to class I alleles insulin transcription. A gel corresponding to six individuals class III/class I heterozygotes and one class I/class I homozygote is shown. 138 bp. band corresponds to the class I allele PstI undigested and the 84 bp and 54 bp bands correspond to class III PstI digested band (d, m, y correspond to day, month, year). (b) QC-PCR (quantitative-competitive PCR) for insulin mRNA quantification is shown for three individuals. The 34-year-old one was affected by myasthenia gravis. Three lanes with 10-fold dilutions of the competitor were included for every case to provide an internal standard curve for quantification. A genomic DNA control was always included. For more details, see text.
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2.6. Real-time PCR assay
315
amplification; fluorescence was acquired during annealing in single mode.
cDNAs from thymic tissue, including four samples not previously analyzed by QC-PCR, were prepared and assayed for insulin, AIRE and GAPDH mRNA expression by real-time quantitative PCR (LightCycler PCR, Roche Diagnostics GmbH, Mannheim, Germany). Standards for each measurement were prepared by amplification of the corresponding cDNAs from thymus (GAPDH and AIRE) and pancreas (insulin) with specific primers and purified with the GFX kit (Pharmacia Biotech) from a 1.5% NuSieve agarose gel. The standards were quantified by densitometry compared with a titration 100 bp Ladder standard (Biotools, Madrid, Spain) in a 2% LE-Seakem agarose gel stained with EtBr.
3. Results 3.1. IDDM2 Class III allele determines a higher level of expression In order to assess the insulin expression as a function of IDDM2 alleles in the thymus, DNA samples from 45 glands were typed for insulin PstI PCR-RFLP (restriction fragment length polymorphism) polymorphism. Owing to the tight linkage disequilibrium of the PstI PCR-RFLP with the IDDM2 locus (VNTR) and to the low frequency of Class II alleles in European populations, class I from class III IDDM alleles were assigned on the basis of this RFLP analysis [6,7]. Fifteen out of 45 glands (33%) were found to be heterozygotes for IDDM2, 28 (62%) class I homozygotes and only 2 (4%), class III homozygotes. Allelic frequencies in our population were 79% and 21% for class I and class III alleles, respectively. Insulin expression in the thymus was detected by radioactive RTePCR in the thymi from the 15 IDDM2 heterozygotic individuals. Due to sample availability limitations, further experiments were carried out in only 10 samples. After PstI restriction of insulin radioactive amplimers, the ratios of intensity of the digested (class III alleles) to the undigested band (class I alleles) were calculated for both the thymic cDNA and the genomic DNA samples (Fig. 2a). Ratios were maintained in genomic DNA throughout all the samples, validating them as good controls. The cDNA class III/class I corrected ratios were greater than 1 in all glands, confirming the expected higher level of transcription of the class III allele insulin gene. The ratio varied widelydfrom 1.5 to 4.4dsuggesting that factors other than the VNTR polymorphism may influence transcription of insulin in the thymus (Table 1). As an exception, a 6-year-old patient with Down’s syndrome had an inverted class III/class I ratio. In the thymus from a 36-years-old patient with myasthenia gravis, a ratio
2.6.1. GAPDH and insulin protocol (for AIRE expression see below) A total reaction volume of 10 ml contained 3 ml of the cDNA (DNAse I treated), 0.5 mM of each primer, 3 mM of MgCl2 and 1 ml of FastStar Master mix SybrGreen I 10! (Roche). The PCR program was set at 10 min denaturation step, followed by a variable number of amplification cycles depending on the amplimers (50 cycles for insulin and 35 cycles for GAPDH) at 95 C for 15 s, 5 s annealing at 63 C and 20 s extension at 72 C. Fluorescence was acquired in single mode during the extension step. The melting curve was set at 95 C for 0 s, 63 C for 15 s and 95 C for 0 s with a slope of 0.1 C/s in continuous mode of fluorescence acquisition. 2.6.2. AIRE protocol Reaction volume of 10 ml containing 1 ml of FastStar DNA Master Hybridization Probes (Roche Diagnostics GmbH, Basel, Switzerland), 2 mM of MgCl2, and 0.5 ml of the mix (primers and Taqman probe) from Assays-on-DemandÔ Gene expression Hs00230833 (Applied Biosystems, Foster City, CA, USA). 2.6.3. PCR program Ten minutes denaturation and 50 cycles of 95 C for 2 s (slope 20 C/s), 60 C for 20 s (slope 10 C/s) for
Table 1 Thymic expression of insulin alleles assessed by RTePCR and quantitative-competitive PCR Reference
Age at time of surgery
Diagnosis
Corrected class III/ class I cDNA ratio
Ins copies class III alleles QC-PCR
Ins copies class I alleles QC-PCR
Class III/class I ratio QC-PCR
Total no. of copies QC-PCR
1 2 3 4 5 6 7 8 9 10
12 days 26 days 8 months 11 months 11 months 3 years 4 years 6 years 11 years 34 years
Normal Normal Normal Normal Normal Normal Normal Down’s syndrome Normal MG
2.6 1.9 2.4 1.8 2.3 4.4 3.6 0.7 2.8 1.3
75 141 154 79 57 108 51 26 89 76
50 75 51 37 55 89 35 186 49 36
1.5 1.8 3.0 2.1 1.0 1.2 1.4 0.1 1.8 2.1
125 216 205 116 112 197 86 212 138 112
The cDNA ratios were calculated from radioactive RTePCR experiments using the counts from the digested insulin class III allele and the undigested class I allele bands; digestion efficiency was corrected using the values from the digestion of genomic DNA which were very reproducible both intra- and inter-individually. The number of insulin class III and class III allele copies were estimated by quantitative-competitive PCR using calibrated standards of the internal competitor (see Fig. 2b and text). MG, myasthenia gravis.
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of 1.3 was found, but the total insulin message was markedly reduced (Table 1). Allele-specific measurement of insulin mRNA in thymi from heterozygotes was also assessed by QC-PCR using an internal competitor that served as reference to calculate the actual number of cDNA copies and as control of the efficiency of PstI restriction. cDNA samples previously normalized for GAPDH gene expression were amplified in parallel by radioactive PCR (Figs. 1b and 2b). This technique confirmed the higher expression of class III alleles in the normal thymic samples. Expression ranged from 51 insulin copies to 154 copies for class III alleles, and from 35 to 75 insulin copies for class I alleles. The Down’s syndrome case was again the exception; 26 insulin copies from the class III allele and 186 insulin copies from the class I allele were detected, confirming an inverted ratio (Table 1). Class III to class I copies were always R1 but there was no strict correlation with the ratios obtained by direct radioactive RTePCR.
3.2. Total insulin message, IDDM2 alleles and AIRE expression in the thymus Total insulin message was measured by real-time PCR in glands from 9 class I/III heterozygotes and also from one class I and one class III homozygote (Fig. 3A). Because AIRE is being postulated as the transcription factor responsible for promiscuous expression in thymus of peripheral antigens, and insulin is one of these antigens, we investigated the possible correlation between the expression of these two genes. Due to sample availability, seven of the glands in this experiment were the same as in previous studies, but four had to be substituted with samples from another four glands; these are indicated in Fig. 3A. Results show that total insulin message measured by realtime PCR was very variable among glands from individuals
A
with the same IDDM2 genotype. Interestingly, AIRE gene was expressed at a higher level (2.5 times G 1.6) but correlated statistically with insulin (r Z 0.4969; p Z 0.0154) in a linear regression test (GraphPad Prism Software, San Diego, CA) (Fig. 3B). In the seven samples in which insulin was measured by both QC-RTePCR and real-time PCR there was a good correlation between the results obtained by the two techniques (r Z 0.7; p Z 0,01). It should be noticed that the highest insulin expression corresponded to the individual affected by Down’s syndrome and also that his expression of AIRE was one of the highest among all the samples tested.
4. Discussion The renewed interest on the role of the thymus in establishing and maintaining tolerance to peripheral self-antigens prompted us to analyze the expression of insulin, a key autoantigen in T1D, in human thymus. Pugliese et al. [9] and Vafiadis et al. [10] demonstrated a correlation of IDDM2 alleles with the level of insulin transcription in the thymus but since both authors have used a similar technique, we considered it of interest to confirm these results by alternative methods and also to try to relate the level of insulin expression to AIRE. In our population sample of 45 subjects the IDDM2 allelic frequencies were similar to those described in the literature [7,8]. Allele-specific insulin transcription in the thymus was analyzed in heterozygous subjects both by RTePCR using genomic DNA as control and by quantitative-competitive RTe PCR, and both indicated that class III alleles were transcribed at a higher level than class I, even if the two techniques did not give the exactly the same ratios. The clear exception was the one case of a Down’s syndrome patient in whom the ratio was inverted; this is not totally unexpected since in these patients the thymic function is markedly deficient [16]. The
B
Ref
1
2 4
6
7
8
9
Fig. 3. Insulin and AIRE gene expression in human thymus by real-time PCR. (A) Expression of insulin and AIRE in several glands, IDDM genotype and age at the time of the surgery is given in the X-axis. Note that the 6-year-old patient, labeled with an asterisk, is affected by Down’s syndrome). White bars represent AIRE expression and black bars, insulin. Numbers below the X-axis legend indicate the thymic gland reference in Table 1. (B) Correlation between total insulin message and AIRE gene expression in human thymus p Z 0.0154. Values are given as gene copies normalized by GAPDH content ! 100.
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precise mechanism by which the polymorphism at the ÿ596 G-rich IDDM2 minisatellite influences insulin transcription is not known. It has been proposed that repeats form variable quaternary structures that are recognized with variable efficiency by the controlling transcription factors [17]. The large interindividual levels of insulin expression observed in our small series led us to study its relationship with the expression of AIRE. This gene codes for a transcription factor regulator that controls peripheral self-antigen expression in the thymus. Inactivating mutations of AIRE are the cause of the polyendocrine autoimmune syndrome type 1 (APS1), which in 18% of the cases includes type 1 diabetes [18]. The finding of a direct correlation between AIRE and insulin expression in the thymus suggests that AIRE regulates insulin expression and adds support to the proposed role of this transcription regulator as the crucial factor in promoting ‘‘promiscuous’’ gene expression in the thymus. The correlation could be due to a more trivial reason such as variable degrees of RNA degradation. This is improbable since RNA extractions were carefully performed from a pool of several blocks from every gland, so as to ensure that the RNA was representative of the whole gland and the corresponding cDNAs were rigorously normalized for GAPDH. Cellular colocalization is probable but, since correlation is maintained throughout a collection of sample that includes glands from different ages which differ markedly in the proportion of cortex to medulla and in the activation state of the medullary cells, this implies that there is a correlation of the expression of the two genes within each cell, which in itself constitutes evidence of AIRE influencing insulin, even if indirect. More importantly, we have now performed a similar analysis for a number of antigens and glands, and the type of correlation detected for insulin and AIRE does not occur with other antigens such as Tg (Ferrer-Francesch, in preparation). On the other hand, a recent study suggested on the basis of colocalization and multiple correlations that some of the tissue-restricted antigens that are promiscuously expressed by thymus, among them insulin, are regulated by AIRE while others are not [19]. The role of AIRE in autoimmunity could well go beyond APS1. Recent experiments in the RIP-mHEL ! 3A9 TCR mouse model of diabetes demonstrated a geneedose effect of AIRE in the expression of insulin in the thymus [20]. The existence of such a dose effect has not been demonstrated in humans as APS1 follows a classical autosomal recessive inheritance mode. It is striking, however, that APS1 is particularly frequent in Finland and Sardinia, two ‘‘hot spots’’ for T1D. Our results do not provide direct evidence for the existence of an effect of AIRE on insulin expression in the thymus, but indirectly suggests that it could exist and be even greater than the influence of IDDM2 alleles. It remains to be elucidated how AIRE affects the expression of so many peripheral self-antigens in the thymus and, in the case of insulin, how the effect of VNTR promoter polymorphisms may be modulated by AIRE. Our results are in line with growing evidence for the importance of the fine-tuning of self-antigen expression in the thymus in the pathogenesis of autoimmune diseases.
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Acknowledgments L.S. was supported by ‘‘Fundacio´ per a la Recerca Biome`dica’’ of Germans Trias i Pujol Hospital, and X.F.-F. by the Network of Transplantation of the Instituto de Salud Carlos III, Spanish Ministry of Health (RC0301). The work was supported by grant SAF-2000-050 of the Ministry for Science and Technology of Spain, National Plan for Health and Pharmacy and by Eurothymaide, 6th FP of the EU. We thank Dr. Murtra and the heart surgery team of Hospital Universitario Vall d’Hebro´n for providing the surgical thymic samples. We especially thank George S. Eisenbarth for reviewing the manuscript. References [1] Moriyama H, Abiru N, Paronen J, Sikora K, Liu E, Miao D, et al. Evidence for a primary islet autoantigen (preproinsulin 1) in the non-diabetic obese mouse. Proc Natl Acad Sci USA 2003;100:10371e6. [2] Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 2005;435:220e3. [3] Kent SC, Chen Y, Bregoli L, Clemmings SM, Kenyon NS, Ricordi C, et al. Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope. Nature 2005;435:224e8. [4] Bell GI, Horita S, Karam JH. A polymorphic locus near the human insulin gene is associated with insulin-dependent diabetes mellitus. Diabetes 1984;33:176e83. [5] Davies JL, Kawaguchi Y, Bennett ST, Copeman JB, Cordell HJ, Pritchard LE, et al. A genome-wide search for human type 1 diabetes susceptibility genes. Nature 1994;371:130e6. [6] Lucassen AM, Julier C, Beressi JP, Boitard C, Froguel P, Lathrop M, et al. Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VNTR. Nat Genet 1993;4:305e10. [7] Bennett ST, Wilson AJ, Cucca F, Nerup J, Pociot F, McKinney PA, et al. IDDM2-VNTR-encoded susceptibility to type 1 diabetes: dominant protection and parental transmission of alleles of the insulin gene-linked minisatellite locus. J Autoimmun 1996;9:415e21. [8] Undlien DE, Bennett ST, Todd JA, Akselsen HE, Ikaheimo I, Reijonen H, et al. Insulin gene region-encoded susceptibility to IDDM maps upstream of the insulin gene. Diabetes 1995;44:620e5. [9] Pugliese A, Zeller M, Fernandez Jr A, Zalcberg LJ, Bartlett RJ, Ricordi C, et al. The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes. Nat Genet 1997;15:293e7. [10] Vafiadis P, Bennett ST, Todd JA, Nadeau J, Grabs R, Goodyer CG, et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nat Genet 1997;15:289e92. [11] Kyewski B, Derbinski J, Gotter J, Klein L. Promiscuous gene expression and central T-cell tolerance: more than meets the eye. Trends Immunol 2002;23:364e71. [12] Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, et al. Positional cloning of the APECED gene. Nat Genet 1997;17:393e8. [13] Aaltonen J, Bjo¨rses P, Perheentupa J, HorelliÿKuitunen N, Palotie A, Peltonen L, et al. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 1997;17:399e403. [14] Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, et al. Projection of an immunological self shadow within the thymus by the aire protein. Science 2002;298:1395e401. [15] Liston A, Lesage S, Wilson J, Peltonen L, Goodnow CC. Aire regulates negative selection of organ-specific T cells. Nat Immunol 2003;4:350e4. [16] Prada N, Nasi M, Troiano L, Roat E, Pinti M, Nemes E, et al. Direct analysis of thymic function in children with Down’s syndrome. Immunol Ageing 2005;2:4.
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[17] Lew A, Rutter WJ, Kennedy GC. Unusual DNA structure of the diabetes susceptibility locus IDDM2 and its effect on transcription by the insulin promoter factor Pur-1/MAZ. Proc Natl Acad Sci USA 2000;97(23):12508e12. [18] Eisenbarth GS, Gottlieb PA. Autoimmune polyendocrine syndromes. N Engl J Med 2004;50:2068e79.
[19] Derbinski J, Ga¨bler J, Brors B, Tierling S, Jonnakuty S, Hergenhahn M, et al. Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels. J Exp Med 2005;202:33e45. [20] Liston A, Gray DHD, Lesage S, Fletcher AL, Wilson J, Webster KE, et al. Gene dosage-limiting role of AIRE in thymic expression, clonal deletion and organ-specific autoimmunity. J Exp Med 2004;200(8):1015e26.
Insulin alleles and autoimmune regulator (AIRE) gene expression both influence insulin expression in the thymus Lidia Sabater a, Xavier Ferrer-Francesch a, Mireia Sospedra a, Pepi Caro a, Manel Juan a,b, Ricardo Pujol-Borrell a,b,* a
Laboratory of Immunology for Research Applied to Diagnosis (LIRAD), Blood and Tissue Bank, Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Crtra. del Canyet s/n, 08916 Badalona, Spain b Division of Immunology, Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain Received 26 March 2005; revised 30 July 2005; accepted 1 August 2005
Abstract It is well established that the polymorphisms at the 5# of the insulin gene (IDDM2) confers susceptibility to type 1 diabetes, probably by modifying the level of insulin expression in the thymus that in turn influences immunological tolerance to insulin as self-antigen. AIRE is a transcription regulator which controls the expression of many peripheral antigens within the thymus, among them insulin. Results presented here confirm that insulin gene copies from both parental chromosomes are expressed in human thymus and that IDDM2 class III protective alleles are indeed associated with a higher level of insulin message expression. However, differences in insulin mRNA expression among different thymi were far wider than those determined by the class I and class III insulin gene alleles and maintained a clear correlation with AIRE expression. These results confirm the effect of IDDM2 alleles on insulin expression in the thymus, but suggest that the levels of AIRE may exert a stronger influence than IDDM2 alleles themselves. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Autoimmunity; Insulin; Type 1 diabetes; Tolerance; Thymus
1. Introduction Several autoantigens have been identified as targets of the autoimmune response causative of type 1 diabetes (T1D) but among them, insulin is prominent because it is islet beta cell specific, anti-insulin antibodies constitute the first evidence of islet autoimmunity in children, and T-cell response to insulin plays a central role in pathogenesis [1e3]. The possible contribution of the insulin gene to diabetes predisposition was first established in 1984 by Bell at al. [4], later confirmed in a genome-wide search for T1D susceptibility genes [5] and * Corresponding author. Laboratory of Immunology for Research Applied to Diagnosis (LIRAD), Blood and Tissue Bank, Institut d’Investigacio´ en Cie`ncies de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Crtra. del Canyet s/n, 08916 Badalona, Spain. Tel.: C34 93 497 8892; fax: C34 93 497 8843. E-mail address: [email protected] (R. Pujol-Borrell). 0896-8411/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2005.08.006
assigned the IDDM2 susceptibility locus. The risk mapped in a highly polymorphic VNTR minisatellite located at ÿ596 of insulin gene coding sequence (Fig. 1a). Alleles at this locus have been classified depending on the number of repeats of the ACAGGGGTSYGGGG consensus motif as class I (26e 63 repeats), class II (63e140 repeats) and class III (141e 209 repeats) [6,7] but only class I and class III alleles are found in European populations. Class I homozygous carry an increased relative risk (RR) that ranges between 1.9 and 5.0 [8]. The mechanism by which this polymorphism confers disease susceptibility remained elusive until it was shown that it influences the level of insulin expression in the thymus and that in this way it may affect immunological central tolerance to insulin [9,10]. The effect of IDDM2 alleles acting in cis on the expression of insulin in the thymus may be affected by many other genes acting in trans and is compounded by parental imprinting, all adding complexity to the analysis [7].
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Fig. 1. Genotyping strategy based on the known insulin haplotypes conferring risk and protection for type 1 diabetes. (a) Insulin gene and IDDM2 map. Arrows indicate the 138 bp region in the 3# UTR of the insulin gene amplified by INS primers that include the PstI polymorphism. In class III alleles the PstI restriction results in two bands of 84 bp and 54 bp. (b) Competitor fragment for QC-PCR. The insulin primers were incorporated into the M13(-72) ends of the pBluescript polylinker with hybrid primers and the construct was cloned in pZerO2 plasmid, then restricted by the NsiI enzyme. The amplification of the internal competitor gives a band of 354 bp with the co-amplification of cDNA of thymus and two bands of equal molecular weight 177 bp after PstI restriction.
The occurrence of a wide representation of peripheral selfantigens, among them insulin, in human thymus has been called ‘‘promiscuous gene expression’’ and it is now a well-established phenomenon [11]. The identification of the gene AIRE as a cause of the autoimmune polyendoendocrine syndrome 1 (APS1) or APECED [12,13], and the evidence that it acts as a broad-spectrum transcription factor regulator that determines the expression of peripheral antigens in the thymus and in some lymphoid organs, had an important impact in the understanding on how tolerance to endocrine autoantigens is established and maintained [14,15]. We report here results from quantitative-competitive and real-time PCR analysis of insulin alleles and AIRE gene expression in human thymus, aimed at elucidating their relative contribution to determine the insulin level of expression in the thymus. These results are relevant for the understanding genetic predisposition to T1D and the role of central tolerance to peripheral antigens in organ-specific autoimmunity.
2.2. Oligonucleotides Primers for insulin and GAPDH were designed using Oligo 4.0 software (MedProbe, Oslo, Norway). Primer for insulin were: INS-F 5#-AAGCGTGGCATTGTGGAAC-3#, INS-R 5#-CAAGGGCTTTATTCCATCTCTC-3#. Location of the primers with respect to insulin locus is shown in Fig. 1. GAPDH primers were sense: 5#-TCTTCTTTTGCGTCGCCAG-3# and antisense: 5#-AGCCCCCAGCCTTCTCCA-3#.
2.3. PCR-RFLP C1,127 PstI locus The genotyping of thymic samples for the C1,127 PstI insulin locus by PCR-RFLP was previously reported [7]. Briefly, genomic DNA was isolated from thymus by standard procedures. Class III and class I alleles were amplified with the same primers INS-F, INS-R and the amplicon underwent PstI (New England Biolabs, Beverly, MA) enzymatic restriction for allelic classification.
2. Methods 2.1. Samples and subjects
2.4. RNA isolation and retrotranscription
Thymic tissue from 45 patients was obtained in the course of routine thoracic surgery on children with congenital heart abnormalities or therapeutic thymectomy for myasthenia gravis patients. This study has been reviewed and approved by the ethical committees of Hospital Universitari Germans Trias i Pujol and Hospital Universitari Vall d’Hebro´. The age of the donors ranged from 12 days to 34 years. Multiple blocks of tissue (0.5 cm) from each glans were snap frozen and kept at ÿ70 C until use.
Total RNA from thymus was isolated from several thymic tissue blocks by the ChomczynkieSacchi method with minor modifications, and it was treated with DNA-freeÔ DNase Treatment (Ambion Inc., Austin, TX) to remove remaining genomic DNA. Residual contamination by genomic DNA was ruled out by the failure of the reaction when the retrotranscription step was omitted (data not shown). Nucleic acid concentration was determined by optical density and the integrity by agarose gel electrophoresis. RNA samples (2 mg) were
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retrotranscribed with oligo(dT)25 (1 mM final concentration, Genset, Paris, France) for 1 h at 37 C in a volume of 20 ml using First Strand Buffer 5!, dNTPs (1 mM, Pharmacia Biotech, Uppsala, Sweden) RNAsin Recombinant Ribonuclease Inhibitor (40 U, Promega, Madison, WI, USA) and SuperScript II Reverse Transcriptase (200 U, Invitrogen, Carldsbad, CA, USA) following the manufacturer’s protocol. Retrotranscription was arrested by heating at 90 C for 2 min. cDNA samples were diluted 1:5 and 1:50, and 2-ml aliquots of each dilution were amplified for the expression of the housekeeping gene GAPDH to normalize sample cDNA concentrations.
2.5. Quantitative-competitive PCR (QC-PCR) A fragment of the pBluescript II KS(C) polylinker which was modified to contain the insulin primers was cloned in pZerO, expanded and used as internal standard for QC-PCR. For each thymus, samples containing 33,000, 3300, 33 and no copies of the internal competitor, and an equal amount of each cDNA from thymus were subjected to QC-PCR amplification. A sample of genomic DNA was always run in parallel (200 ng) as control. Competitive PCR mixtures for insulin consisted of 1 ml of DZ buffer (50 mM KCl, 10 mM
TriseHCl pH 8.8, 1.5 mM MgCl2 and 0.1% Triton X-100), 0.5 mM of INS-F and INS-R primer, 200 mM of each dNTP (Pharmacia, Biotech), 0.4 U/ml of Dynazyme (Finnzymes, Oy, Espoo, Finland), 1 ml of the sample, and 1 ml of the internal standard at known concentration, in a total volume of 10 ml. Cycling parameters were 38 cycles of 95 C for 20 s and 65 C for 30 s with a final extension step at 72 C for 5 min. PCR products were labeled by adding [32P]dATP (5 mCi per tube) in the last cycle of amplification and were electrophoresed on an 8% polyacrylamide gel. The radioactivity counts were measured by exposing the dry gels to imaging plates (Eastman Kodak, Rochester, NY, USA) using a radiomaging system (PhosphorimagerÒ, Bio-Rad, Hercules, CA, USA) and readings were analyzed using Quantity-OneÒ software (Bio-Rad). The value of PstI undigested amplicon visible as a 354 bp band in the standard lane (Fig. 2b) was used to calculate a digestion efficiency correction factor. To calculate the number of insulin allele-specific copies (class I and class III), the radioactivity readings corresponding to the PstI digested plus the undigested band were transferred to a graphic of radioactivity vs. number of molecules generated from the competition fragment standard readings for which the starting number of molecules was known. The QC-PCR assays were run in duplicate and each thymus was analyzed at least twice with consistent results.
Fig. 2. Insulin gene allele transcription in human thymus. (a) Radioactive PCR products from genomic DNA (DNA) and reverse-transcribed RNA (RNA) after PstI restriction for typing and calculating the ratio of class III to class I alleles insulin transcription. A gel corresponding to six individuals class III/class I heterozygotes and one class I/class I homozygote is shown. 138 bp. band corresponds to the class I allele PstI undigested and the 84 bp and 54 bp bands correspond to class III PstI digested band (d, m, y correspond to day, month, year). (b) QC-PCR (quantitative-competitive PCR) for insulin mRNA quantification is shown for three individuals. The 34-year-old one was affected by myasthenia gravis. Three lanes with 10-fold dilutions of the competitor were included for every case to provide an internal standard curve for quantification. A genomic DNA control was always included. For more details, see text.
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2.6. Real-time PCR assay
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amplification; fluorescence was acquired during annealing in single mode.
cDNAs from thymic tissue, including four samples not previously analyzed by QC-PCR, were prepared and assayed for insulin, AIRE and GAPDH mRNA expression by real-time quantitative PCR (LightCycler PCR, Roche Diagnostics GmbH, Mannheim, Germany). Standards for each measurement were prepared by amplification of the corresponding cDNAs from thymus (GAPDH and AIRE) and pancreas (insulin) with specific primers and purified with the GFX kit (Pharmacia Biotech) from a 1.5% NuSieve agarose gel. The standards were quantified by densitometry compared with a titration 100 bp Ladder standard (Biotools, Madrid, Spain) in a 2% LE-Seakem agarose gel stained with EtBr.
3. Results 3.1. IDDM2 Class III allele determines a higher level of expression In order to assess the insulin expression as a function of IDDM2 alleles in the thymus, DNA samples from 45 glands were typed for insulin PstI PCR-RFLP (restriction fragment length polymorphism) polymorphism. Owing to the tight linkage disequilibrium of the PstI PCR-RFLP with the IDDM2 locus (VNTR) and to the low frequency of Class II alleles in European populations, class I from class III IDDM alleles were assigned on the basis of this RFLP analysis [6,7]. Fifteen out of 45 glands (33%) were found to be heterozygotes for IDDM2, 28 (62%) class I homozygotes and only 2 (4%), class III homozygotes. Allelic frequencies in our population were 79% and 21% for class I and class III alleles, respectively. Insulin expression in the thymus was detected by radioactive RTePCR in the thymi from the 15 IDDM2 heterozygotic individuals. Due to sample availability limitations, further experiments were carried out in only 10 samples. After PstI restriction of insulin radioactive amplimers, the ratios of intensity of the digested (class III alleles) to the undigested band (class I alleles) were calculated for both the thymic cDNA and the genomic DNA samples (Fig. 2a). Ratios were maintained in genomic DNA throughout all the samples, validating them as good controls. The cDNA class III/class I corrected ratios were greater than 1 in all glands, confirming the expected higher level of transcription of the class III allele insulin gene. The ratio varied widelydfrom 1.5 to 4.4dsuggesting that factors other than the VNTR polymorphism may influence transcription of insulin in the thymus (Table 1). As an exception, a 6-year-old patient with Down’s syndrome had an inverted class III/class I ratio. In the thymus from a 36-years-old patient with myasthenia gravis, a ratio
2.6.1. GAPDH and insulin protocol (for AIRE expression see below) A total reaction volume of 10 ml contained 3 ml of the cDNA (DNAse I treated), 0.5 mM of each primer, 3 mM of MgCl2 and 1 ml of FastStar Master mix SybrGreen I 10! (Roche). The PCR program was set at 10 min denaturation step, followed by a variable number of amplification cycles depending on the amplimers (50 cycles for insulin and 35 cycles for GAPDH) at 95 C for 15 s, 5 s annealing at 63 C and 20 s extension at 72 C. Fluorescence was acquired in single mode during the extension step. The melting curve was set at 95 C for 0 s, 63 C for 15 s and 95 C for 0 s with a slope of 0.1 C/s in continuous mode of fluorescence acquisition. 2.6.2. AIRE protocol Reaction volume of 10 ml containing 1 ml of FastStar DNA Master Hybridization Probes (Roche Diagnostics GmbH, Basel, Switzerland), 2 mM of MgCl2, and 0.5 ml of the mix (primers and Taqman probe) from Assays-on-DemandÔ Gene expression Hs00230833 (Applied Biosystems, Foster City, CA, USA). 2.6.3. PCR program Ten minutes denaturation and 50 cycles of 95 C for 2 s (slope 20 C/s), 60 C for 20 s (slope 10 C/s) for
Table 1 Thymic expression of insulin alleles assessed by RTePCR and quantitative-competitive PCR Reference
Age at time of surgery
Diagnosis
Corrected class III/ class I cDNA ratio
Ins copies class III alleles QC-PCR
Ins copies class I alleles QC-PCR
Class III/class I ratio QC-PCR
Total no. of copies QC-PCR
1 2 3 4 5 6 7 8 9 10
12 days 26 days 8 months 11 months 11 months 3 years 4 years 6 years 11 years 34 years
Normal Normal Normal Normal Normal Normal Normal Down’s syndrome Normal MG
2.6 1.9 2.4 1.8 2.3 4.4 3.6 0.7 2.8 1.3
75 141 154 79 57 108 51 26 89 76
50 75 51 37 55 89 35 186 49 36
1.5 1.8 3.0 2.1 1.0 1.2 1.4 0.1 1.8 2.1
125 216 205 116 112 197 86 212 138 112
The cDNA ratios were calculated from radioactive RTePCR experiments using the counts from the digested insulin class III allele and the undigested class I allele bands; digestion efficiency was corrected using the values from the digestion of genomic DNA which were very reproducible both intra- and inter-individually. The number of insulin class III and class III allele copies were estimated by quantitative-competitive PCR using calibrated standards of the internal competitor (see Fig. 2b and text). MG, myasthenia gravis.
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of 1.3 was found, but the total insulin message was markedly reduced (Table 1). Allele-specific measurement of insulin mRNA in thymi from heterozygotes was also assessed by QC-PCR using an internal competitor that served as reference to calculate the actual number of cDNA copies and as control of the efficiency of PstI restriction. cDNA samples previously normalized for GAPDH gene expression were amplified in parallel by radioactive PCR (Figs. 1b and 2b). This technique confirmed the higher expression of class III alleles in the normal thymic samples. Expression ranged from 51 insulin copies to 154 copies for class III alleles, and from 35 to 75 insulin copies for class I alleles. The Down’s syndrome case was again the exception; 26 insulin copies from the class III allele and 186 insulin copies from the class I allele were detected, confirming an inverted ratio (Table 1). Class III to class I copies were always R1 but there was no strict correlation with the ratios obtained by direct radioactive RTePCR.
3.2. Total insulin message, IDDM2 alleles and AIRE expression in the thymus Total insulin message was measured by real-time PCR in glands from 9 class I/III heterozygotes and also from one class I and one class III homozygote (Fig. 3A). Because AIRE is being postulated as the transcription factor responsible for promiscuous expression in thymus of peripheral antigens, and insulin is one of these antigens, we investigated the possible correlation between the expression of these two genes. Due to sample availability, seven of the glands in this experiment were the same as in previous studies, but four had to be substituted with samples from another four glands; these are indicated in Fig. 3A. Results show that total insulin message measured by realtime PCR was very variable among glands from individuals
A
with the same IDDM2 genotype. Interestingly, AIRE gene was expressed at a higher level (2.5 times G 1.6) but correlated statistically with insulin (r Z 0.4969; p Z 0.0154) in a linear regression test (GraphPad Prism Software, San Diego, CA) (Fig. 3B). In the seven samples in which insulin was measured by both QC-RTePCR and real-time PCR there was a good correlation between the results obtained by the two techniques (r Z 0.7; p Z 0,01). It should be noticed that the highest insulin expression corresponded to the individual affected by Down’s syndrome and also that his expression of AIRE was one of the highest among all the samples tested.
4. Discussion The renewed interest on the role of the thymus in establishing and maintaining tolerance to peripheral self-antigens prompted us to analyze the expression of insulin, a key autoantigen in T1D, in human thymus. Pugliese et al. [9] and Vafiadis et al. [10] demonstrated a correlation of IDDM2 alleles with the level of insulin transcription in the thymus but since both authors have used a similar technique, we considered it of interest to confirm these results by alternative methods and also to try to relate the level of insulin expression to AIRE. In our population sample of 45 subjects the IDDM2 allelic frequencies were similar to those described in the literature [7,8]. Allele-specific insulin transcription in the thymus was analyzed in heterozygous subjects both by RTePCR using genomic DNA as control and by quantitative-competitive RTe PCR, and both indicated that class III alleles were transcribed at a higher level than class I, even if the two techniques did not give the exactly the same ratios. The clear exception was the one case of a Down’s syndrome patient in whom the ratio was inverted; this is not totally unexpected since in these patients the thymic function is markedly deficient [16]. The
B
Ref
1
2 4
6
7
8
9
Fig. 3. Insulin and AIRE gene expression in human thymus by real-time PCR. (A) Expression of insulin and AIRE in several glands, IDDM genotype and age at the time of the surgery is given in the X-axis. Note that the 6-year-old patient, labeled with an asterisk, is affected by Down’s syndrome). White bars represent AIRE expression and black bars, insulin. Numbers below the X-axis legend indicate the thymic gland reference in Table 1. (B) Correlation between total insulin message and AIRE gene expression in human thymus p Z 0.0154. Values are given as gene copies normalized by GAPDH content ! 100.
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precise mechanism by which the polymorphism at the ÿ596 G-rich IDDM2 minisatellite influences insulin transcription is not known. It has been proposed that repeats form variable quaternary structures that are recognized with variable efficiency by the controlling transcription factors [17]. The large interindividual levels of insulin expression observed in our small series led us to study its relationship with the expression of AIRE. This gene codes for a transcription factor regulator that controls peripheral self-antigen expression in the thymus. Inactivating mutations of AIRE are the cause of the polyendocrine autoimmune syndrome type 1 (APS1), which in 18% of the cases includes type 1 diabetes [18]. The finding of a direct correlation between AIRE and insulin expression in the thymus suggests that AIRE regulates insulin expression and adds support to the proposed role of this transcription regulator as the crucial factor in promoting ‘‘promiscuous’’ gene expression in the thymus. The correlation could be due to a more trivial reason such as variable degrees of RNA degradation. This is improbable since RNA extractions were carefully performed from a pool of several blocks from every gland, so as to ensure that the RNA was representative of the whole gland and the corresponding cDNAs were rigorously normalized for GAPDH. Cellular colocalization is probable but, since correlation is maintained throughout a collection of sample that includes glands from different ages which differ markedly in the proportion of cortex to medulla and in the activation state of the medullary cells, this implies that there is a correlation of the expression of the two genes within each cell, which in itself constitutes evidence of AIRE influencing insulin, even if indirect. More importantly, we have now performed a similar analysis for a number of antigens and glands, and the type of correlation detected for insulin and AIRE does not occur with other antigens such as Tg (Ferrer-Francesch, in preparation). On the other hand, a recent study suggested on the basis of colocalization and multiple correlations that some of the tissue-restricted antigens that are promiscuously expressed by thymus, among them insulin, are regulated by AIRE while others are not [19]. The role of AIRE in autoimmunity could well go beyond APS1. Recent experiments in the RIP-mHEL ! 3A9 TCR mouse model of diabetes demonstrated a geneedose effect of AIRE in the expression of insulin in the thymus [20]. The existence of such a dose effect has not been demonstrated in humans as APS1 follows a classical autosomal recessive inheritance mode. It is striking, however, that APS1 is particularly frequent in Finland and Sardinia, two ‘‘hot spots’’ for T1D. Our results do not provide direct evidence for the existence of an effect of AIRE on insulin expression in the thymus, but indirectly suggests that it could exist and be even greater than the influence of IDDM2 alleles. It remains to be elucidated how AIRE affects the expression of so many peripheral self-antigens in the thymus and, in the case of insulin, how the effect of VNTR promoter polymorphisms may be modulated by AIRE. Our results are in line with growing evidence for the importance of the fine-tuning of self-antigen expression in the thymus in the pathogenesis of autoimmune diseases.
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Acknowledgments L.S. was supported by ‘‘Fundacio´ per a la Recerca Biome`dica’’ of Germans Trias i Pujol Hospital, and X.F.-F. by the Network of Transplantation of the Instituto de Salud Carlos III, Spanish Ministry of Health (RC0301). The work was supported by grant SAF-2000-050 of the Ministry for Science and Technology of Spain, National Plan for Health and Pharmacy and by Eurothymaide, 6th FP of the EU. We thank Dr. Murtra and the heart surgery team of Hospital Universitario Vall d’Hebro´n for providing the surgical thymic samples. We especially thank George S. Eisenbarth for reviewing the manuscript. References [1] Moriyama H, Abiru N, Paronen J, Sikora K, Liu E, Miao D, et al. Evidence for a primary islet autoantigen (preproinsulin 1) in the non-diabetic obese mouse. Proc Natl Acad Sci USA 2003;100:10371e6. [2] Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, et al. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Nature 2005;435:220e3. [3] Kent SC, Chen Y, Bregoli L, Clemmings SM, Kenyon NS, Ricordi C, et al. Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope. Nature 2005;435:224e8. [4] Bell GI, Horita S, Karam JH. A polymorphic locus near the human insulin gene is associated with insulin-dependent diabetes mellitus. Diabetes 1984;33:176e83. [5] Davies JL, Kawaguchi Y, Bennett ST, Copeman JB, Cordell HJ, Pritchard LE, et al. A genome-wide search for human type 1 diabetes susceptibility genes. Nature 1994;371:130e6. [6] Lucassen AM, Julier C, Beressi JP, Boitard C, Froguel P, Lathrop M, et al. Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VNTR. Nat Genet 1993;4:305e10. [7] Bennett ST, Wilson AJ, Cucca F, Nerup J, Pociot F, McKinney PA, et al. IDDM2-VNTR-encoded susceptibility to type 1 diabetes: dominant protection and parental transmission of alleles of the insulin gene-linked minisatellite locus. J Autoimmun 1996;9:415e21. [8] Undlien DE, Bennett ST, Todd JA, Akselsen HE, Ikaheimo I, Reijonen H, et al. Insulin gene region-encoded susceptibility to IDDM maps upstream of the insulin gene. Diabetes 1995;44:620e5. [9] Pugliese A, Zeller M, Fernandez Jr A, Zalcberg LJ, Bartlett RJ, Ricordi C, et al. The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes. Nat Genet 1997;15:293e7. [10] Vafiadis P, Bennett ST, Todd JA, Nadeau J, Grabs R, Goodyer CG, et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nat Genet 1997;15:289e92. [11] Kyewski B, Derbinski J, Gotter J, Klein L. Promiscuous gene expression and central T-cell tolerance: more than meets the eye. Trends Immunol 2002;23:364e71. [12] Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Heino M, et al. Positional cloning of the APECED gene. Nat Genet 1997;17:393e8. [13] Aaltonen J, Bjo¨rses P, Perheentupa J, HorelliÿKuitunen N, Palotie A, Peltonen L, et al. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 1997;17:399e403. [14] Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, et al. Projection of an immunological self shadow within the thymus by the aire protein. Science 2002;298:1395e401. [15] Liston A, Lesage S, Wilson J, Peltonen L, Goodnow CC. Aire regulates negative selection of organ-specific T cells. Nat Immunol 2003;4:350e4. [16] Prada N, Nasi M, Troiano L, Roat E, Pinti M, Nemes E, et al. Direct analysis of thymic function in children with Down’s syndrome. Immunol Ageing 2005;2:4.
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