Original Article Genetic Control of Alternative Splicing in the TAP2 Gene Possible Implication in the Genetics of Type 1 Diabetes Hui-Qi Qu, 1 Yang Lu, 1 Luc Marchand, 1 Franc ¸ois Bacot, 1 Rosalie Fre ´ chette, 1 Marie-Catherine Tessier, 1 Alexandre Montpetit, 2 and Constantin Polychronakos 1,3 The transporter 2, ATP-binding cassette, subfamily B (TAP2) is involved in the transport of antigenic peptides to HLA molecules. Coding TAP2 polymorphisms shows a strong association with type 1 diabetes, but it is not clear whether this association may be entirely due to linkage disequilibrium with HLA DR and DQ. Functionally, rat Tap2 nonsynonymous single-nucleotide polymorphisms (nsSNPs) confer differential selectivity for antigenic pep- tides, but this was not shown to be the case for human TAP2 nsSNPs. In the human, differential peptide selectivity is rather conferred by two splicing isoforms with alterna- tive carboxy terminals. Here, we tested the hypothesis that alleles at the coding SNPs favor different splicing isoforms, thus determining peptide selectivity indirectly. This may be the basis for independent contribution to the type 1 diabetes association. In RNA from heterozygous lympho- blastoid lines, we measured the relative abundance of each SNP haplotype in each isoform. In isoform NM_000544, the G (Ala) allele at 665 Thr>Ala (rs241447) is more than twice as abundant as A (Thr) (GA  2.2  0.4, P  1.5  10 4 ), while isoform NM_018833 is derived almost exclu- sively from chromosomes carrying A (AG  18.1  5.6, P  2.04  10 7 ). In 889 Canadian children with type 1 diabe- tes, differential transmission of parental TAP2 alleles persisted (P  0.011) when analysis was confined to chro- mosomes carrying only DQ*02 alleles, which mark a con- served DR-DQ haplotype, thus eliminating most of the variation at DR-DQ. Thus, we present evidence of TAP2 association with type 1 diabetes that is independent of HLA DR-DQ and describe a plausible functional mechanism based on allele dependence of splicing into isoforms known to have differential peptide selectivities. Diabetes 56: 270 –275, 2007 G enetic susceptibility to the autoimmune -cell destruction that causes type 1 diabetes is a complex trait, 50% of which is explained by the HLA locus (1). Most of the linkage to the HLA locus is accounted for by association with coding polymorphisms in the class II genes, specifically DR and DQ (2– 4), but HLA class I genes (HLA-A, -B, and -C) may also contribute (5,6) and is becoming increasingly appar- ent that non-HLA genes within the class II locus play a role in type 1 diabetes susceptibility (7,8). Among the latter, the transporter 2, ATP-binding cassette, subfamily B (TAP2) gene is one of the most studied. Nonsynonymous single- nucleotide polymorphisms (nsSNPs) on TAP2 are strongly associated with type 1 diabetes (7,9 –12). However, TAP2 is located only 156 kb telomeric to DQB1, and, because of strong linkage disequilibrium (LD) with DR and DQ, it is not easy to determine what part of the association, if any, is independent of DR and DQ. Published studies suggest independent contribution (7), but conclusive evidence is lacking. Functionally, the TAP1 and TAP2 genes, being involved in antigen presentation, are excellent candidates for asso- ciation with autoimmune disease. The two gene products form a heterodimer that transports antigenic peptides from the cytoplasm into the endoplasmic reticulum (13) and is essential for loading antigen on HLA class I protein on the cell surface (14,15). Moreover, as TAP2 protein comes in direct contact with antigenic peptides, the pos- sibility exists that its coding polymorphisms confer anti- gen selectivity, which complements that of the HLA molecules. This is consistent with an apparent excess of nsSNPs (four high-frequency nsSNPs, including 687 GlnTer that creates a stop codon truncating the carboxyl terminal by 17 amino acids). Like HLA (16,17), overabun- dance of coding TAP2 polymorphisms may reflect strong recent positive selection favoring heterozygosity and spec- ificity for emerging pathogens. In the rat, strong effects on peptide selectivity by coding TAP2 polymorphisms between strains have indeed been unequivocally demonstrated (18). Although a similar effect by human polymorphisms has been claimed (19), two thorough studies failed to find any evidence of it (20,21). Interestingly, marked differences in antigenic selectivity have been reported (22) between the two known splicing isoforms of TAP2 (Fig. 1). The first 10 exons are common to the two isoforms (NM_000544 and NM_018833), which differ by having a completely distinct 11th exon and, consequently, distinct carboxyl terminal ends and 3' un- From the 1 Endocrine Genetics Laboratory, The McGill University Health Center (Montreal Children’s Hospital), Montre ´ al, Quebec, Canada; the 2 McGill University and Genome Quebec Innovation Centre, Montre ´ al, Quebec, Can- ada; and the 3 Department of Pediatrics, The McGill University Health Center (Montreal Children’s Hospital), Montre ´ al, Quebec, Canada. Address correspondence and reprint requests to Constantin Polychronakos, MD, The McGill University Health Center (Montreal Children’s Hospital), 2300 Tupper, Montre ´ al, QC, Canada, H3H 1P3. E-mail: constantin.polychronakos@ mcgill.ca. Received for publication 25 June 2006 and accepted in revised form 9 October 2006. H.-Q.Q. and Y.L. contributed equally to this work. Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. AI, allelic imbalance; gDNA, genomic DNA; LD, linkage disequilibrium; nsSNP, nonsynonymous single-nucleotide polymorphism; SNP, single-nucle- otide polymorphism. DOI: 10.2337/db06-0865 © 2007 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 270 DIABETES, VOL. 56, JANUARY 2007