Narcolepsy-cataplexy, characterized by sleepiness and rapid onset into REM sleep, affects 1 in 2,000 individuals1,2. Narcolepsy was first shown to be tightly associated with HLA-DR23, and later sublocalized to DQB1*06024. Following studies in dogs5 and mice6, a 95% loss of hypocretin-producing cells in human postmortem hypothalami was reported7,8, Using Genome Wide Association (GWA) in Caucasians with replication in three ethnic groups, we found association with polymorphisms in the T-Cell receptor alpha (TCRA) locus, with highest significance at rs1154155 (average allelic odds ratio 1.69, genotype odds ratios 1.94 and 2.55, p 0.8 with rs1154155 are known to exist from HapMap data. This SNP is located 176bp 3′ to TRAJ10, a J segment without known coding polymorphisms. Genotype analysis suggested a dosage effect (CC vs. AA MH OR=2.55, 95% CI 1.92-3.38; AC vs. AA MH OR=1.94, 95% CI 1.68-2.25) (Table 3). Population attributable risks16 for TCRA rs1154155C in Caucasians and Asians were 20% and 42%, respectively. The increased frequency of rs1154155C in Asians likely contributes to the reported increased prevalence in Japan1 despite lower DQB1*0602 frequency4. Our identified TCRA rs1154155C polymorphism showed no interaction with the nominally significant TCRB rs17231T polymorphism of the GWA data (OR interaction=1.0). In our much larger sample, we also did not replicate a previously published rs5770917 association in Japanese narcolepsy (Table 1), suggesting an ethnic specific effect17. Further, interactions between rs5770917 and rs1154155 were non-significant in Caucasians, Asians, and African Americans (OR interaction=1.0 in all samples). The TCRA locus encodes the α . -chain of the TCRαβ-heterodimer, a protein expressed by T lymphocytes18. The T-cell receptor is a unique protein which interacts with both HLA class I (CD8 in cytotoxic T-cells) and HLA Class II (CD4 in helper T-cells), including the DQαβ heterodimer denoted DQ0602, encoded by DQB1*0602 and the closely linked DQA1*0102 allele. The TCRA locus, like the TCRB and the Immunoglobulin variable heavy and light chain loci, is unusual in undergoing somatic cell recombination. TCRA and TCRB recombination occur in the thymus, resulting, after deletion of auto reactive clones and positive selection, in the generation of T-cell clones with unique TCRA and TCRB recombined loci. In the TCRA locus, recombination occurs between the 5′ area of one of the 46 functional Variable (V) segments19and the 3′ area of one of the 49 functional J segments20,21,22, with additional amino acid junctional diversity generated by N- and P-additions in the V-J border region. In the TCRB locus, diversity is even more complex and generated by recombination of 48V, 2D and 13J segments22. This mechanism produces a diverse repertoire of distinct TCRαβ idiotype bearing T-cells21, which can be called upon to recognize antigens presented by HLA class I or class II molecules23. Unlike most other autoimmune diseases9, narcolepsy is almost completely associated with a single HLA allele, DQB1*0602, across Caucasians, Asians and African Americans4. Considering the tight DQB1*0602 association in narcolepsy, it is logical to hypothesize that the DQB0602 heterodimer should interact with a specific TCRαβ receptor subtype whose occurrence is marked by rs1154155C, and less strongly by rs17231T at both TCR loci. This TCR idiotype would bear specific VJα and VDJβ recombinants, with recognition of a peptide that also binds DQ0602, mediating further immune reaction leading to the destruction of hypocretin-producing cells. Precisely how a J segment region polymorphism such as rs1154155C could increase the risk of occurrence of this narcolepsy associated T-cell clone is unknown, but could involve non-random VJα choices in recombination21, as previously reported. Similarly, a polymorphism in the TCRB V region could influence VDJ recombination for the complementary TCRβ chain. Less probably, the TCR-DQ association could also occur without the need for peptide binding, through superantigen-like bridging of TCR and DQ, although most known superantigens interact with TCRβ rather than TCR α . chains24. Further, superantigen bridging typically results in stimulation of large systemic lymphocyte populations carrying specific TCRB segments such as that seen in toxic shock syndrome. Surprisingly, of over 10 HLA associated autoimmune diseases that have been subjected to genome-wide analyses and candidate gene studies, none has shown consistent association with either TCR locus25. Further studies of the TCR loci in narcolepsy may for the first time reveal a role for a specific TCR receptor idiotype in the pathophysiology of an autoimmune disorder. Methods Cases and Controls Narcolepsy patients were selected as described, 98% of whom are predicted to be hypocretin deficient. The initial Caucasian sample was comprised of 807 cases and 1074 controls of mixed European ancestry; 415 cases and 753 controls were recruited from the US and Canada; 392 cases and 321 controls were recruited from European centers. The Caucasian replication sample contained 718 individuals of whom 542 were recruited from the US and Canada (259 cases, 283 controls), and 176 from Europe (104 cases 72 controls). The Asian sample included 866 Japanese (433 cases, 433 controls) and 300 Koreans (128 cases, 172 controls). Finally, 277 African Americans were studied (133 cases, 144 controls). All subjects had given written informed consent approval. HLA-DQB1*0602 typing The presence or absence of DQB1*0602 was determined using DQB1 exon 2 sequence-specific primers (see Supplementary Table 2). These primers amplify DQB1*0602 and a few exceptionally rare DQB1*06 alleles (allele frequency<0.5%) as a 218 bp PCR product. The assay includes a DRB1 internal positive control. Analysis of Affymetrix Data We obtained Cel file data for all samples and performed genotyping using the birdseed-dev algorithm for Affy 6.0 (Affymetrix Power Tools \apt-1.8.5) (1544 samples) (http://www.affymetrix.com/products/software/specific/birdseed_algorithm.affx), and BRLMM for Affy 500K array set chips (337 samples) (http://www.affymetrix.com/support/technical/whitepapers/brlmm_whitepaper.pdf). In each genotype-calling group, individual chips with outlier low call rates (typically <97%) or high heterozygosity were excluded from further analysis. For each Birdseed calling run, SNPs with call rates <0.9, or Hardy Weinberg P<0.01 in controls were excluded. A total of 549,596 SNPs passed all quality control filters and were included in the final analysis. Genotype data was maintained in our database (Progeny Lab 7, http://www.progenygenetics.com), and analyses were performed using the PLINK software package (v1.04 26/Aug/2008, http://pngu.mgh.harvard.edu/purcell/plink/ 14). Interaction studies were performed in the initial set and in replication sets (cases and controls) using Plink epistasis, which performs a logistic regression including main genotype effects plus an interaction term. Supplementary Material 1 2
