Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells in genetically susceptible individuals. Growing evidence suggests that genetically determined variation in the expression of self-antigens in thymus may affect the shaping of the T-cell repertoire and susceptibility to autoimmunity. For example, both allelic variation and parent-of-origin effects influence the thymic expression of insulin (a known type 1 diabetes autoantigen), and insulin gene transcription levels in thymus inversely correlate with susceptibility in both humans and transgenic models. It is unclear why patients lose tolerance to IA-2 (insulinoma-associated tyrosine phosphatase-like protein, or islet cell antigen 512 [ICA512]), especially because IA-2 polymorphisms are not associated with type 1 diabetes. We report that alternative splicing determines differential IA-2 expression in islets compared with thymus and spleen. Islets express full-length mRNA and two alternatively spliced transcripts, whereas thymus and spleen exclusively express an alternatively spliced transcript lacking exon 13. This encodes for the transmembrane (TM) and juxta-membrane (JM) domains that comprise several type 1 diabetes target epitopes, supporting the concept that tolerance to IA-2 epitopes not expressed in lymphoid organs may not be achieved. We propose differential splicing as a regulatory mechanism of gene expression playing a permissive role in the development of autoimmune responses to IA-2. Our findings also show that candidate gene expression studies can help in dissecting the complex genetic determinants of a multifactorial disease such as type 1 diabetes.

IA-2 is a tyrosine phosphatase-like protein with a single transmembrane (TM) region (residues 577–600) and extracellular and intracellular domains (Fig. 1) (1). IA-2 is enriched in the secretory granules of pancreatic islet cells and neuroendocrine cells, including peptidergic neurons, pituitary cells, and adrenal chromaffin cells (2). It is unclear why the immune system reacts against IA-2 as part of the autoimmune responses associated with type 1 diabetes (36). The IA-2 gene is also expressed in the human thymus (7) in a manner similar to other genes coding for self-molecules with tissue-restricted expression, including insulin and glutamic acid decarboxylase (GAD) (711), two other autoantigens in type 1 diabetes. Self-antigen expression in thymus may play a key role in promoting immunological self-tolerance, and variation in such expression may have dramatic effects on tolerance. We have also obtained evidence that the self-antigens are detected not only in thymus but in peripheral lymphoid organs as well (spleen and lymph nodes) (12).

An alternatively spliced variant of the IA-2 transcript has been recently discovered through the sequencing of the ICA512.bdc clone (13) derived from a human pancreas library routinely translated in vitro as source of antigen in a specific assay for the detection of type 1 diabetes–associated IA-2 autoantibodies. This variant transcript lacks exon 13 (Δ exon 13), which codes for 73 amino acids (aa 557–629) encompassing the TM and JM domains of the IA-2 protein (Fig. 1). Several epitopes recognized by autoimmune responses in type 1 diabetes overlap with these domains; thus, we hypothesized that splicing of the IA-2 gene could be differentially regulated in lymphoid organs and pancreas, because such differences may influence immune responsiveness to IA-2. We investigated IA-2 expression in islets, thymus, and spleen from nondiabetic human tissue donors (no tissues were available from patients) of different ages (from 21 weeks of gestation to 52 years of age) and from diverse racial groups (Table 1). IA-2 mRNA expression and splicing were evaluated by reverse transcriptase–polymerase chain reaction (RT-PCR) using primers that amplify both full-length and Δ exon 13 transcripts, and the identity of the products was verified by sequencing. All nine pancreatic samples (four islet and five whole pancreas samples) expressed full-length IA-2 message, and five of the nine also expressed the Δ exon 13 transcript (Table 2). IA-2 mRNA was also detected in lymphoid organs, including 9 of 15 thymus and 11 of 15 spleen specimens (Table 2, Fig. 2A). Consistent with previous literature, not all of the lymphoid tissues tested expressed IA-2 message (7,10). This finding may have several explanations, including RNA degradation and genetic variability of trans- or cis-acting factors that may affect the expression of IA-2. Of the lymphoid tissues expressing IA-2, all thymus (9 of 9) and spleen (11 of 11) specimens exclusively expressed the Δ exon 13 transcript. The above also included five cases for which we could simultaneously evaluate IA-2 expression in pancreas, thymus, and spleen (cases 3222, 3236, 3444, 3446, and 3450). The analysis of tissues from the same subject also indicates that the observed differences between pancreas and lymphoid organs reflect tissue-specific splicing and not individual variation in the regulation of IA-2 splicing in lymphoid organs. We confirmed the above findings using a second primer pair and also detected another transcript intermediate in size between the full-length and the Δ exon 13 transcript (Fig. 2C). Sequence analysis confirmed that this is another alternatively spliced IA-2 transcript in which 129 bp of exon 14 are spliced out, resulting in the deletion of 43 amino acids (aa 653–695) in the intracellular domain (Figs. 1 and 2C and Table 3). This transcript was detected in 4 of 9 pancreatic samples compared with 0 of 11 thymic samples and 1 of 15 samples from spleens (very weak signal, case 3222) (Table 2). Thus, our findings show that IA-2 splicing results in the exclusive expression of the Δ exon 13 transcript in lymphoid organs, but they also show that individual variation in IA-2 splicing and expression exists in pancreas. Of note is the observation that individual variation was also found in five adrenal samples: three of five adrenal samples expressed full-length IA-2 mRNA, and four of five expressed the exon 13 variant; thus, two of five adrenal samples tested exclusively expressed the exon 13 variant (however, none of the adrenal samples expressed the exon 14 variant [data not shown]). Moreover, two brain samples expressed all three variants (data not shown). The presence of all IA-2 variants in pancreas, adrenal, and brain samples suggests that exclusive expression of the Δ exon 13 transcript may be typical of lymphoid tissues.

Because both alternatively spliced transcripts are read in frame downstream of the alternative splicing sites, they should be translated into protein. To verify the in vivo translation of the Δ exon 13 transcript, we stained frozen thymus and spleen sections with two rabbit antibodies recognizing either intracellular (aa 601–979) or extracellular portions (aa 389–576) of IA-2 (2). Distinct cells in thymus (Fig. 2D) and spleen (Fig. 2E) were specifically stained with both sera, demonstrating that IA-2 mRNA is translated into protein in lymphoid organs. Because the Δ exon 13 transcript is essentially the only IA-2 message detected in lymphoid organs, the staining observed can only derive from the translation of the Δ exon 13 transcript into protein. The IA-2–positive cells observed correspond to a subset of apparently tolerogenic self-antigen–presenting cells that we have recently characterized in lymphoid organs (12), and similar cells with tolerogenic function exist in the mouse thymus (9). This finding further supports the concept that differential expression may affect immune responsiveness to IA-2. We also performed Western blot analysis to verify that the alternative IA-2 transcripts are translated into proteins in the pancreas. Consistent with the mRNA expression studies, three bands of the predicted size for full-length IA-2 and the two splicing variants were detected by Western blotting pancreas protein extracts (Fig. 2F) with a rabbit antibody recognizing the extracellular domain (aa 389–575) (14).

We demonstrated that alternative splicing is responsible for differential IA-2 expression in pancreas compared with lymphoid organs. The exclusive expression of the Δ exon 13 transcript in lymphoid organs and the finding of individual variation in the pancreatic expression of the IA-2 variants suggest that differential IA-2 splicing in these tissues may be a permissive mechanism for the potential development of autoimmunity against IA-2. Such differences may affect immune responsiveness to specific epitopes and help explain why IA-2 and not many other islet proteins become targets of autoimmunity in type 1 diabetes. We speculate that immunological tolerance to linear or conformational epitopes typical of the full-length protein or of the Δ exon 14 variant may not be achieved if these are expressed in islets but not in thymus and spleen, where they appear to be expressed by tolerogenic self-antigen–presenting cells. The specific lack of expression of the TM/JM domains (exon 13) in lymphoid organs helps explain why epitopes from these domains are often targeted by autoimmune responses in type 1 diabetes. Indeed, autoantibodies against IA-2 epitopes encoded by exons 13 and 14 have been reported, and in some patients they can precede the appearance of autoantibodies against other intracellular epitopes (epitope spreading). In particular, the 605–682 and 605–620 epitopes (Fig. 1) are early autoantibody targets and are recognized by 56% of patients’ sera (1518). Two additional epitopes exist in the JM domain between residues 601–642 (Fig. 1), and a third epitope extends to the region preceding the 601 residue (poster presentation [17]). Moreover, type 1 diabetes in children is reportedly more strongly associated with autoantibodies against JM rather than intracellular epitopes (15). Only a few T-cell epitopes have been identified because of the limitations of current T-cell assays, which include poor reproducibility, limited ability to distinguish patients from control subjects, and the sometimes arbitrary choice of overlapping peptides to measure T-cell reactivity (1923). Moreover, T-cell reactivity against IA-2 epitopes encoded by exons 13 and 14 or against epitopes generated by alternative splicing of these exons has not yet been analyzed. However, there is evidence that the HLA-DR4–restricted naturally processed 654–674 epitope (exon 14) is recognized by autoreactive T-cells (24). Although the Δ exon 13 IA-2 transcript exclusively expressed in lymphoid organs contains this sequence, the lack of the TM domain may affect its processing and presentation in lymphoid organs. It is also possible that immunity against exon 14 epitopes may be driven by the islet-specific expression of the Δ exon 14 protein lacking amino acid residues 653–695. In theory, partial sequence sharing and cross-reactivity between Δ exon 14 and full-length proteins may result in a spreading of the immune response to exon 14 epitopes expressed by the full-length molecule.

Notably, all of the 25 subjects in this study were nondiabetic tissue donors who were randomly selected from the general population and who belonged to diverse races and age-groups. Because of its common occurrence in the population, differential IA-2 splicing and expression may help explain the reported difficulties in distinguishing patients from unaffected subjects when studying stimulated T-cell reactivity in vitro (19,20). In fact, measurable responses could be elicited even from normal subjects if the population is not fully tolerant to IA-2, as suggested by our findings. The observation that only minorities of individuals develop spontaneous autoimmune responses against IA-2 and diabetes may have several explanations. First, IA-2 autoantibodies are usually detected during the later stages of the prodromic period, suggesting that IA-2 autoimmunity may be secondary to inflammatory damage taking place during the earlier stages of the disease process. This may also be one of the reasons why type 1 diabetic patients do not apparently react against other tissues expressing IA-2 (i.e., adrenal), because TM and intracellular IA-2 epitopes may be unavailable to the immune system without inflammation and tissue damage. Second, other genetic factors (e.g., genes coding for HLA antigens, cytokines, etc.) and environmental exposures may be necessary to trigger autoimmunity. Consistent with our findings and this interpretation is the observation that differential splicing affects the expression of the proteolipid protein autoantigen in brain and thymus, which in turn affects susceptibility to experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis (25,26). Although differential splicing is similarly observed in both susceptible and control strains, only the susceptible mice bearing a predisposing major histocompatibility complex develop disease. Third, the observed individual variation in pancreatic IA-2 splicing and expression (only four of nine pancreatic samples expressed the Δ exon 14 variant that was not expressed in lymphoid organs) may also be a factor because individuals expressing the Δ exon 14 variant in the pancreas may be more prone to develop autoimmune responses.

In conclusion, we suggest that differential splicing of the IA-2 gene in pancreas compared with lymphoid organs could affect the immune system reactivity to IA-2 and help explain why IA-2 is an autoantigen in type 1 diabetes. Similar to the parent-of-origin effects affecting insulin gene expression in thymus (7,8) and peripheral lymphoid organs (27), differential IA-2 splicing appears to function as a mechanism regulating gene expression independent of inherited alleles at the insulin and IA-2 loci. Although recent investigations had excluded linkage with IA-2 polymorphisms (28), our findings show that expression studies for selected candidate genes in tissues relevant to the disease process can help in dissecting the complex genetics of a multifactorial disease such as type 1 diabetes.

Tissues.

We studied frozen specimens of pancreas, thymus, and spleen from fetal and neonatal autoptic cases provided by the University of Miami Brain and Tissue Bank for Developmental Disorders. We also studied tissues from adult cadaver donors (pancreas, spleen, and occasionally thymus) provided by the Cell Transplant Center, Diabetes Research Institute, University of Miami, Miami, Florida. A total of 25 cases were selected on the basis of tissue availability, and of these, 10 were fetal (estimated gestational age range 21–34 weeks) and 15 were postnatal donors (age range 1 day to 52 years old). The age, race, and sex for the individual cases studied are shown in Table 1. None of the tissue donors suffered from diabetes. Tissues were obtained according to the guidelines of the University of Miami Institutional Review Board, which gave its approval for the study.

Expression of IA-2 mRNA in pancreas and other tissues.

Total mRNA was isolated from pancreas or purified islet cells (positive control), thymus, and spleen with standard procedures (RNANOW-LM; Biogentex). cDNA was generated from total RNA by reverse transcription, and then subjected to PCR amplification (RT-PCR). All PCRs were run for 35 cycles in a Thermolyne thermocycler. PCR products were visualized on ethidium bromide–stained 2% agarose gels. We used two primer pair combinations with reproducible results. Primers IA-2-F 5′-AGA CAG GGC TCC AAA TCT TGC-3′ and IA-2-R 5′-GCC TGG GCT GCG TCG CTG AAC-3′ amplify both the full-length IA-2 mRNA (370 bp) and the Δ exon 13 transcript (151 bp). Primers IA-2-F and IA-2-R2 5′-GAT GTG ACC CAA CAA GCA GGG-3′ amplify the full-length (617 bp) transcript and both the Δ exon 13 and Δ exon 14 transcripts (398 and 488 bp, respectively). The IA-2-R2 primer spans an intron and thus is mRNA-specific. The location of the primers in the IA-2 cDNA sequence is shown in Table 3. Primer annealing temperature was 60°C. For each sample tested, we ran a parallel reaction omitting the reverse transcriptase enzyme to control for genomic DNA contamination. Negative control reactions in which no template was added to the reaction were also run with every experiment to control for PCR contamination. RNA quality was checked in all tissues studied by evaluating the expression of β-actin mRNA. Primer sequences and PCR conditions for β-actin were previously reported (7). All tissues studied expressed β-actin mRNA.

Sequencing of PCR products.

Selected PCR products were cloned into the TA Cloning Vector (Promega). DNA samples extracted from transformed colonies were then sequenced (both strands, using the SP6 and T7 primers) with an automated sequencer to confirm the identity of the amplified cDNAs.

Immunohistochemistry.

Frozen tissue blocks were sectioned at 5-μm thickness using a cryotome, and sections were mounted on glass slides and stored at –80°C until use. Sections were fixed just before use in 20% buffered formalin for 30 min at room temperature. Tissue sections were stained using the labeled streptavidin-biotin-peroxidase method and aminoethyl carbazole (AEC), an alcohol-soluble chromogen, as a substrate for the horseradish peroxidase enzyme. AEC generates a reddish deposit at the reaction site on tissue or cell preparations. Immunohistochemistry was performed with reagents from Zymed Laboratories (San Francisco, CA) according to the manufacturer’s instructions. Two rabbit sera directed against the intracellular and extracellular portions of the ICA512 (IA-2) molecule were used. Serum 8959 was raised against the intracellular portion (aa 601–979), serum 9218H was raised against the extracellular portion (aa 389–576), and both sera have been extensively used to characterize the ICA512 (IA-2) molecule (2,14). Negative controls included omitting the primary antibody, staining all tissue sections with a rabbit isotype control antibody, and staining lung sections that do not express IA-2 (data not shown).

Western blotting.

Proteins extracted from pancreatic tissue were Western blotted using a well-characterized rabbit antibody recognizing the extracellular domain (aa 389–575) of ICA512 (IA-2) (14). Western blot was carried out after electrophoretic transfer of proteins to Hybond ECL nitrocellulose membrane (Amersham, U.K.) at 100 V in 48 mmol/l Tris, 39 mmol/l glycine (pH 9.2), and 20% (vol/vol) methanol for 90 min. The membrane was blocked at 4°C overnight with a blocking solution consisting of 5% (wt/vol) fat-free dry milk in phosphate-buffered saline, 0.1% Tween-20 (PBS-T). The membrane was incubated with the above antibody diluted in blocking solution (1:1,000) for 1 h at room temperature, then washed three times with PBS-T, blocked for 30 min with blocking solution, and incubated for 1 h at room temperature with the anti-IgG horseradish-peroxidase conjugate. The membrane was washed again with PBS-T, and bands were visualized using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). Chemiluminescence was detected by autoradiography.

FIG. 1.

Structure of the IA-2 protein. Diagram illustrating the structure of the IA-2 protein and the relationships of exons 13 and 14 with protein domains and epitopes recognized by autoantibodies and T-cells in patients with type 1 diabetes. SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain.

FIG. 1.

Structure of the IA-2 protein. Diagram illustrating the structure of the IA-2 protein and the relationships of exons 13 and 14 with protein domains and epitopes recognized by autoantibodies and T-cells in patients with type 1 diabetes. SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; IC, intracellular domain.

FIG. 2.

Alternative splicing of the IA-2 gene and IA-2 protein expression. Panel A: IA-2 mRNA transcripts amplified with primers IA-2-F and IA-2-R (370 bp, full-length; 151 bp, Δ exon 13 transcript) from total RNA samples extracted from spleen (S), thymus (T), and pancreas (P). The molecular weight marker (M) bands shown correspond to 600, 500, 400, 300, and 200 bp, top to bottom. Note that all tissues were from the same subject (case 3444). Panel B: β-actin mRNA (439 bp) amplified from the same samples in panel A (28 PCR cycles). The molecular weight marker (M) bands shown correspond to 500 and 400 bp, top to bottom. Panel C: IA-2 mRNA transcripts amplified with primers IA-2-F and IA-2–R2 (617 bp, full-length; 488 bp, Δ exon 14 transcript; 398 bp, Δ exon 13 transcript) from total RNA extracted from purified pancreatic islet cells (IC) (case 999). The molecular weight marker (M) bands shown correspond to 700 (partially shown), 600, 500, 400, 300, and 200 bp, top to bottom. Panel D: Frozen section of human thymus stained with an antibody recognizing the intracellular portion of the ICA512 (IA-2) molecule (aa 601–979). Sparse cells in the thymus medulla are stained in red (with AEC), and thus they express IA-2 protein. Counterstained with hematoxylin, magnification ∼128×. Similar results were obtained using an antibody against the extracellular portion of the molecule (not shown). Panel E: Frozen section of human spleen stained with an antibody against the extracellular portion of the ICA512 (IA-2) molecule (aa 389–576). Sparse cells expressing IA-2 are stained in red (with AEC). Counterstained with hematoxylin, magnification ∼200×. Similar results were obtained using an antibody against the intracellular portion of the molecule (not shown). Panel F: Western blot of pancreas protein extracts showing three bands of the predicted size for the IA-2 protein variants encoded by the full-length and alternatively spliced transcripts.

FIG. 2.

Alternative splicing of the IA-2 gene and IA-2 protein expression. Panel A: IA-2 mRNA transcripts amplified with primers IA-2-F and IA-2-R (370 bp, full-length; 151 bp, Δ exon 13 transcript) from total RNA samples extracted from spleen (S), thymus (T), and pancreas (P). The molecular weight marker (M) bands shown correspond to 600, 500, 400, 300, and 200 bp, top to bottom. Note that all tissues were from the same subject (case 3444). Panel B: β-actin mRNA (439 bp) amplified from the same samples in panel A (28 PCR cycles). The molecular weight marker (M) bands shown correspond to 500 and 400 bp, top to bottom. Panel C: IA-2 mRNA transcripts amplified with primers IA-2-F and IA-2–R2 (617 bp, full-length; 488 bp, Δ exon 14 transcript; 398 bp, Δ exon 13 transcript) from total RNA extracted from purified pancreatic islet cells (IC) (case 999). The molecular weight marker (M) bands shown correspond to 700 (partially shown), 600, 500, 400, 300, and 200 bp, top to bottom. Panel D: Frozen section of human thymus stained with an antibody recognizing the intracellular portion of the ICA512 (IA-2) molecule (aa 601–979). Sparse cells in the thymus medulla are stained in red (with AEC), and thus they express IA-2 protein. Counterstained with hematoxylin, magnification ∼128×. Similar results were obtained using an antibody against the extracellular portion of the molecule (not shown). Panel E: Frozen section of human spleen stained with an antibody against the extracellular portion of the ICA512 (IA-2) molecule (aa 389–576). Sparse cells expressing IA-2 are stained in red (with AEC). Counterstained with hematoxylin, magnification ∼200×. Similar results were obtained using an antibody against the intracellular portion of the molecule (not shown). Panel F: Western blot of pancreas protein extracts showing three bands of the predicted size for the IA-2 protein variants encoded by the full-length and alternatively spliced transcripts.

TABLE 1

Characteristics of tissue donors

Age (weeks)SexRaceTissues
Fetal cases 
 3222 21 AA Thymus, spleen, pancreas 
 1278 22 Thymus 
 1279 22 Thymus 
 3446 23 AA Thymus, spleen, pancreas 
 2892 24 AA Thymus, spleen 
 3430 24 Thymus 
 3236 26 AA Thymus, spleen, pancreas 
 3223 27 Thymus, spleen 
 3444 27 Thymus, spleen, pancreas 
 3450 34 AA Thymus, spleen, pancreas 
Postnatal cases 
 3421 1 day Thymus 
 1829 2 days AA Thymus, spleen 
 1383 5 days AA Spleen 
 2073 5 days HC Thymus, spleen 
 3252 7 days AA Thymus 
 1263 1 month Thymus 
 853 20 years ND Islet cells 
 955 23 years Islet cells 
 1078 29 years Spleen 
 1111 32 years AA Thymus, islet cells 
 1135 36 years Spleen 
 999 40 years Islet cells 
 1134 41 years Spleen 
 1132 46 years Spleen 
 1083 52 years ND Spleen 
Age (weeks)SexRaceTissues
Fetal cases 
 3222 21 AA Thymus, spleen, pancreas 
 1278 22 Thymus 
 1279 22 Thymus 
 3446 23 AA Thymus, spleen, pancreas 
 2892 24 AA Thymus, spleen 
 3430 24 Thymus 
 3236 26 AA Thymus, spleen, pancreas 
 3223 27 Thymus, spleen 
 3444 27 Thymus, spleen, pancreas 
 3450 34 AA Thymus, spleen, pancreas 
Postnatal cases 
 3421 1 day Thymus 
 1829 2 days AA Thymus, spleen 
 1383 5 days AA Spleen 
 2073 5 days HC Thymus, spleen 
 3252 7 days AA Thymus 
 1263 1 month Thymus 
 853 20 years ND Islet cells 
 955 23 years Islet cells 
 1078 29 years Spleen 
 1111 32 years AA Thymus, islet cells 
 1135 36 years Spleen 
 999 40 years Islet cells 
 1134 41 years Spleen 
 1132 46 years Spleen 
 1083 52 years ND Spleen 

AA, African-American; C, Caucasian; H, Hispanic; ND, not determined.

TABLE 2

IA-2 mRNA splicing in pancreas, islet cells, thymus, and spleen

Δ exon 13Δ exon 14Full-length
Islet Cells 
 999 
 853 – 
 955 – 
 111 – 
Pancreas 
 3222 – – 
 3236 – – 
 3444 
 3446 – – 
 3450 
Total 5/9 4/9 9/9 
Thymus 
 1111 – – – 
 1263 – – 
 1278 – – – 
 1279 – – – 
 1829 – – 
 2073 – – – 
 2892 – – 
 3222 – – 
 3223 – – 
 3236 – – 
 3252 – – – 
 3421 – – – 
 3444 – – 
 3446 – – 
 3450 – – 
Total 9/15 0/15 0/15 
Spleen 
 1078 – – 
 1083 – – 
 1132 – – 
 1134 – – – 
 1135 – – – 
 1383 – – – 
 1829 – – – 
 2073 – – 
 2892 – – 
 3223 – – 
 3236 – – 
 3222 – 
 3444 – – 
 3446 – – 
 3450 – – 
Total 11/15 1/15 0/15 
Δ exon 13Δ exon 14Full-length
Islet Cells 
 999 
 853 – 
 955 – 
 111 – 
Pancreas 
 3222 – – 
 3236 – – 
 3444 
 3446 – – 
 3450 
Total 5/9 4/9 9/9 
Thymus 
 1111 – – – 
 1263 – – 
 1278 – – – 
 1279 – – – 
 1829 – – 
 2073 – – – 
 2892 – – 
 3222 – – 
 3223 – – 
 3236 – – 
 3252 – – – 
 3421 – – – 
 3444 – – 
 3446 – – 
 3450 – – 
Total 9/15 0/15 0/15 
Spleen 
 1078 – – 
 1083 – – 
 1132 – – 
 1134 – – – 
 1135 – – – 
 1383 – – – 
 1829 – – – 
 2073 – – 
 2892 – – 
 3223 – – 
 3236 – – 
 3222 – 
 3444 – – 
 3446 – – 
 3450 – – 
Total 11/15 1/15 0/15 
TABLE 3

IA-2 cDNA sequence, spliced sequences, primers used, and amino acid sequence

Partial cDNA sequence (nucleotides 1,704–2,321)* 
AG ACA GGG CTC CAA ATC TTG Cag aca gga gtg gga cag agg gag gag gca gct gca gtc ctt ccc caa act gcg cac agc acc tca ccc atg cgc tca gtg ctg ctc act ctg gtg gcc ctg gca ggt gtg gct ggg ctg ctg gtg gct ctg gct gtg gct ctg tgt gtg cgg cag cat gcg cgg cag caa gac aag gag cgc ctg gca gcc ctg ggg cct gag ggg gcc cat ggt gac act acc ttt gag tac cag gac ctg tgc cgc cag cac atg gcc acg aag tcc ttg ttc aac cgg gca gag ggt cca ccg gag cct tca cgg gtg agc agt gtg tcc tcc caG TTC AGC GAC GCA GCC CAG GCc agc ccc agc tcc cac agc agc acc ccg tcc tgg tgc gag gag ccg gcc caa gcc aac atg gac atc tcc acg gga cac atg att ctg gca tac atg gag gat cac ctg cgg aac cgg gac cgc ctt gcc aag gag tgg cag gcc ctc tgt gcc tac caa gca gag cca aac acc tgt gcc acc gcg cag ggg gag ggc aac atc aaa aag aac cgg cat cct gac TTC CTG CCC TAT GAC CAT GCC 
Partial amino acid sequence (aa 545–749) 
T G L Q I L Q T G V G Q R E E A A A V L P Q T A H S T S P M R S V L L T L V A L A G V A G L L V A L A V A L C V R Q H A R Q Q D K E R L A A L G P E G A H G D T T F E Y Q D L C R Q H M A T K S L F N R A E G P P E P SR V S S V S S Q F S D A A Q A S P S S H S S T P S W C E E P A Q A N M D I S T G H M I L A Y M E D H L R N R D R L A K E W Q A L C A Y Q A E P N T C A T A Q G E G N I K K N R H P D F L P Y D H A 
Partial cDNA sequence (nucleotides 1,704–2,321)* 
AG ACA GGG CTC CAA ATC TTG Cag aca gga gtg gga cag agg gag gag gca gct gca gtc ctt ccc caa act gcg cac agc acc tca ccc atg cgc tca gtg ctg ctc act ctg gtg gcc ctg gca ggt gtg gct ggg ctg ctg gtg gct ctg gct gtg gct ctg tgt gtg cgg cag cat gcg cgg cag caa gac aag gag cgc ctg gca gcc ctg ggg cct gag ggg gcc cat ggt gac act acc ttt gag tac cag gac ctg tgc cgc cag cac atg gcc acg aag tcc ttg ttc aac cgg gca gag ggt cca ccg gag cct tca cgg gtg agc agt gtg tcc tcc caG TTC AGC GAC GCA GCC CAG GCc agc ccc agc tcc cac agc agc acc ccg tcc tgg tgc gag gag ccg gcc caa gcc aac atg gac atc tcc acg gga cac atg att ctg gca tac atg gag gat cac ctg cgg aac cgg gac cgc ctt gcc aag gag tgg cag gcc ctc tgt gcc tac caa gca gag cca aac acc tgt gcc acc gcg cag ggg gag ggc aac atc aaa aag aac cgg cat cct gac TTC CTG CCC TAT GAC CAT GCC 
Partial amino acid sequence (aa 545–749) 
T G L Q I L Q T G V G Q R E E A A A V L P Q T A H S T S P M R S V L L T L V A L A G V A G L L V A L A V A L C V R Q H A R Q Q D K E R L A A L G P E G A H G D T T F E Y Q D L C R Q H M A T K S L F N R A E G P P E P SR V S S V S S Q F S D A A Q A S P S S H S S T P S W C E E P A Q A N M D I S T G H M I L A Y M E D H L R N R D R L A K E W Q A L C A Y Q A E P N T C A T A Q G E G N I K K N R H P D F L P Y D H A 
*

Primer sequences are shown in capital letters, top to bottom primers IA-2-F, IA-2-R, IA-2-R2; the spliced exon 13 sequence is shown in bold, and the spliced exon 14 sequence is shown in bold and italics (this sequence also contains primer IA-2-R).

Bolded letters represent alternatively spliced residues from exon 13 (aa 557–629), and bolded and italicized letters represent alternatively spliced residues from exon 14 (aa 653–695).

This study was supported by a Career Development Award from the Juvenile Diabetes Foundation International (A.P.) (JDFI CDA no. 296117), research grants from the American Diabetes Association and the National Institutes of Health (DK-32083, AI-44456), and the Diabetes Research Institute Foundation.

We thank Carol K. Petito and Inna Logvisnki of the University of Miami Brain and Fetal Tissue Bank (a joint effort with the University of Maryland Brain and Tissue Banks through National Institute of Child Health and Human Development contract no. NO1-HD-8-3284) and Topaz Kirlew and the Diabetes Research Institute Cell Transplant Center Team for providing tissues for this study. We are indebted to Dr. Shannon Gleason at Bayer and Drs. Michele Solimena and Ronald Dirkx at Yale University for kindly providing rabbit antibodies against the ICA512 (IA-2) molecule. We also thank Mr. David Stenger for assisting in the editing of the manuscript.

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Address correspondence and reprint requests to Alberto Pugliese, Immunogenetics Program, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Ave., Miami, FL 33136 USA. E-mail: apuglies@med.miami.edu.

Received for publication 28 July 2000 and accepted in revised form 29 December 2000.