Narcolepsy risk loci outline role of T cell autoimmunity and infectious triggers in narcolepsy

Abstract

Narcolepsy type 1 (NT1) is caused by a loss of hypocretin/orexin transmission. Risk factors include pandemic 2009 H1N1 influenza A infection and immunization with Pandemrix®. Here, we dissect disease mechanisms and interactions with environmental triggers in a multi-ethnic sample of 6,073 cases and 84,856 controls. We fine-mapped GWAS signals within HLA (DQ0602, DQB1*03:01 and DPB1*04:02) and discovered seven novel associations (CD207, NAB1, IKZF4-ERBB3, CTSC, DENND1B, SIRPG, PRF1). Significant signals at TRA and DQB1*06:02 loci were found in 245 vaccination-related cases, who also shared polygenic risk. T cell receptor associations in NT1 modulated TRAJ*24, TRAJ*28 and TRBV*4-2 chain-usage. Partitioned heritability and immune cell enrichment analyses found genetic signals to be driven by dendritic and helper T cells. Lastly comorbidity analysis using data from FinnGen, suggests shared effects between NT1 and other autoimmune diseases. NT1 genetic variants shape autoimmunity and response to environmental triggers, including influenza A infection and immunization with Pandemrix®.

Fig. 1. Multi-ethnic genetic analysis of NT1.

A Multi-ethnic analysis conducted in 6073 cases and 84,856 controls reveals significant associations in 13 loci in addition to HLA. The x-axis shows genomic location by chromosome and the y-axis shows –log₁₀ P-values. The red horizontal line indicates the genome-wide significant P-value threshold of 5 × 10−⁸. P-values larger than 10⁻⁷⁵ were set to 10⁻⁷⁵ (HLA locus has many SNPs with P-value < 10⁻²¹⁶). Variants are shared at individual level with known autoimmune traits, with notable exception at variants within the TRA and TRB loci and variants within CD207 and INFAR1 (see Supplementary Data 1). Raw P-values are reported using two-sided fixed-effects meta-analysis. Multiple testing correction has been done at genome-wide level so that variants with nominal P-value under 5e-8 were considered statistically significant. B Associated variants are located on chromosome positions that have active eQTLs in blood samples, as evidenced by an analysis using GARFIELD. Enrichment analysis has been binned by P-value threshold and raw two-sided P-values are reported. C When using stratified LD score regression, association within individual blood cell types implicate NK cells, CD4+ T and CD8+ T cells. Statistically significant enrichment is marked with a line corresponding to enrichment P-value = 0.05 (dashed line) and FDR corrected P-value = 0.05 (dotted line). Raw two-sided P-values are reported and we have show significance also by false-discovery rate of 0.05 (dotted line). D Global enrichment is seen with autoimmune traits in general using variants that were genome-wide significant. Raw two-sided P-values from hypergeometric test are reported. Fig. 2A, B, E: Raw P-values are reported using two-sided fixed-effects meta-analysis. E Overall enrichment was seen with MS and SLE using LDSC. F PheWAS with narcolepsy risk variants showed association across different autoimmune traits. positive beta is depicted with square and negative with triangle. For details, see methods.

Fig. 2. TRA rs1154155 is associated with multiple functional SNPs affecting TRAJ24 and TRAJ28; TRB lead variant affects TRBV 4-2 usage.

A NT1 association with rs1154155 within the T-cell receptor alpha chain (TRA) locus. B NT1 association with rs7458379 within the T-cell receptor beta chain (TRB) locus. C The NT1 association within the TRA locus spans a region containing ~30 TRAJ genes that contains 4 SNPs in almost perfect LD (rs1154155, rs1483979, rs3764159, rs3764160) over a 18 kb region. D Exemplary TCR receptor structure of a TCR containing TCR J24. rs1483979 encodes a leucine (J24*02) to phenylalanine (J24*01) substitution (F8L) within the J24 segment. The substitution is projected within the Complementary Determining Region (CDR) 3 of the corresponding family of receptors, in an area anticipated to interact directly with the peptide presented by HLA. E Usage of TRAJ28*01 in 895 individuals shows similar association with NT1 lead variant rs1154155, with posterior probability of 0.958 between NT1 and increased TRAJ28 usage. F T-cell receptor sequencing in CD4+ T memory cells in 60 NT1 patients and 42 matched controls confirmed the effect of rs1483979 on usage of TRAJ28*01 with similar effect seen in the NT1 cases. It also shows a decreasing effect of rs1483979C on TRAJ24 expression, with a ratio of 24 F/L (associated allele over non-associated allele in ~0.4 in heterozygous subjects, see Supplementary Fig. 5). The center of the boxplot corresponds to the median corresponding to 50th percentile, the box indicates the upper and lower bounds of the interquartile range corresponding to 25th and 75th percentile, and the min and max values correspond to the plus/minus two interquartile ranges.

Fig. 3. Postulated potential disease mechanisms in autoimmune narcolepsy.

(1) Peripheral response: Influenza virions or vaccine protein debris are ingested by DCs facilitated by CD207; flu proteins are processed by cathepsins CTSH and CTSC for presentation by HLA molecules to specific TCRα-bearing CD4+ cells, initiating an immunological synapse and responses to influenza. Presentation by DC is modulated by IFNAR1 in the context of influenza infection and the type 1 INF response. Cross presentation of influenza antigens processed via the MHC class I pathway in concert with TNFSF4 in DCs is necessary to activate CD8+ cells that mature into cytotoxic lymphocytes (CTLs), initiating cell killing of viron infected cells. Activated Th1 CD4+ cells produce cytokines such as IFNγ and IL-2, which augment cytotoxic activity of CTLs via perforin (PRF1). SIRPG on activated T cells may also promote cell-to-cell adhesion and proliferation in this response. (2) CNS autoimmunity: Activated and primed specific CD4+ cells migrate to the CNS, where they interact with microglia and resident DCs via DQ0602 bound to an influenza-mimic autoimmune-epitope (derived from hypocretin cells), initiating a secondary memory response. Hypocretin cell proteins are processed by cathepsins CTSH and CTSC for presentation by DQ0602 to specific TCRα-bearing CD4+ cells, initiating an immunological synapse and autoimmune response. Chain usage for TRAJ24-2, TRAJ28 and TRBV4-2 is associated with NT1 risk and may be crucial for autoantigen recognition. Further, cross presentation by resident DCs and microglial cells activates specific CD8+ cells via MHC class I binding of another HCRT neuron-derived peptide. These primed cytotoxic CD8+ cells then kill HCRT neurons after recognizing MHC class I (such as A*11:01, associated with NT1 independently of DQ0602) bound, cognate HCRT neuron-derived peptide, may be derived from RFX4 or LHX9, on hypocretin neurons. SIRPG1 on DCs, microglia or activated T cells may also promote cell-to-cell adhesion and proliferation in this response.