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Relationship between T-cell receptor α gene polymorphisms and symptomatic differences in patients with narcolepsy type 1

Supplemental Digital Content is available in the text


Introduction
In the last decades, significant progress has been made in the fields of immunotherapy and immunoprecision medicine. [1,2] Due to the advanced genome sequencing method and detailed genetic data, immune therapy and monitoring based on precision medicine have been successfully applied to several diseases including cancers, infectious diseases, allergies, and asthma. [3][4][5][6] The T-cell receptor (TCR) was suggested to play a crucial role in immunity. The clinical trials involving TCR-engineered T-cell immune therapy have shown some clinical responses. [2] Narcolepsy type 1 (NT1) is a chronic immune-mediated disease caused by a hypocretin-1 deficiency, [7][8][9] which is associated with the autoimmune process targets a peptide unique to hypocretin-producing neurons via specific human leucocyte antigen (HLA)-peptide-TCR interactions. [10] Recently, hypocretin-specific T cells were detected in the blood and cerebrospinal fluid of patients with narcolepsy, [11] which provided evidence that T cells play an important role in the onset of narcolepsy.
The gene encodes the TCR, which plays a prominent role in antigen presentation and T-cell activation, is T-cell receptor a (TRA) gene, which is a strong candidate gene for narcolepsy susceptibility. Several genome-wide association studies (GWASs) have revealed that the large TRA locus encoding the a-chain of the TCR showed strong association with the risk for narcolepsy, [10,12,13] independent of the HLA, across Caucasians, Asians, and African Americans. [10,[12][13][14] (less than 25%) suffer from all the symptoms. [15] Two or three symptoms occur in approximately 30% of narcoleptic patients. [16] EDS is the most common presenting and disabling symptom and is required for the diagnosis of narcolepsy. [15,17] Cataplexy is the most specific symptom of NT1. [18] The estimated prevalence of hypnagogic hallucinations ranges from 30% to 80% of patients and that of sleep paralysis ranges from 25% to 50%. [18,19] The presence of different narcolepsy symptoms is associated with different qualities of life and psychological impairments. [20] Thus, reducing the symptoms of narcolepsy is one of the important aims of treatment, including the possible immunotherapy in the future.
Despite the importance of TRA in narcolepsy risk and in the field of immunoprecision medicine, the role of TRA haplotype polymorphisms in the symptomatic diversity of narcolepsy remains unclear since it has not been extensively investigated. Therefore, in this retrospective study, we aimed to investigate the association between the single nucleotide polymorphisms (SNPs) and haplotypetagging polymorphisms of TRA and the symptom diversity of narcolepsy in a Chinese population.

Ethical approval
The research protocols were approved by the Institutional Review Board Panel on Medical Human Subjects at the Peking University People's Hospital. Informed consent was obtained from all subjects.

Study population
We performed a study consisting of 1062 patients with NT1. Briefly, the patients were recruited from the Sleep Laboratory of the Peking University People's Hospital, a unit which evaluates patients with sleep disorders and receives referrals from all over China. The medical record of all the patients with narcolepsy was reviewed and rediagnosed by clinical experts to ensure that they all met the diagnostic criteria for NT1 according to the third edition of the International Classification of Sleep Disorders-3. [21] These criteria include (A and B must be met): A. The patient has been experiencing daily periods of irresistible need to sleep or daytime lapses into sleep occurring for at least 3 months; B. The presence of one (or both) of the following: (1) Cataplexy and a mean sleep latency of 8 min and two or more sleep-onset rapid eyes movement -sleep periods (SOREMPs) on a multiple sleep latency testing (MSLT) performed according to standard techniques. A SOREMP (within 15 min of sleep onset) on the preceding nocturnal polysomnography may replace one of the SOREMPs on the MSLT; (2) Cerebral spinal fluid hypocretin-1 concentration, measured by immunoreactivity, is either 110 pg/mL or 1/3 of mean values obtained in normal subjects with the same standardized assay. Patients were all Chinese, and most of them were Han ethnicity (95.2%). Clinical data included presence or absence of cataplexy, sleepiness, sleep paralysis, hypnogogic hallucination, disturbed nocturnal sleep, and the degree of impairment of life quality. Trained interviewers used structured questionnaires to collect information on demographic variables, medical history, and medications. Genotyping, quality control, and genetic loci selection SNP genotyping and quality control have been described in detail elsewhere. [22] In brief, samples were genotyped and analyzed using the Affymetrix Axiom CHB array and Affymetrix Genotyping Console (Affymetrix, lnc., Cambridge, Massachusett, USA). Totally, 13 SNPs of the TRA gene, which were previously discovered narcolepsy risk loci from GWAS studies published before September, 2018 or discovered in the GWAS study using our own data, including rs227000, rs1258667, rs227017, rs8016421, rs1154153, rs1154155, rs1154158, rs1263638, rs1263640, rs7153643, rs1263642, rs1263645, and rs1263647 were genotyped (the TRA polymorphisms of healthy controls are shown in Supplementary Table 2, http://links.lww.com/CM9/A68). All samples used in the current study achieved a genotype call rate of >99%. Totally, 903 of the 1062 narcolepsy cases were included for further analysis after quality control.

Statistical analysis
We used the Chi-square test to assess whether the SNPs were in Hardy-Weinberg equilibrium and to determine differences in genotype frequencies between patients with narcolepsy. Statistical significance was set at a twosided P-value of less than 0.05. We defined statistical significance using the Bonferroni correction and set the experiment-wide association significance threshold at 5.6 Â 10 -3 for single-variant analysis (0.05/9 variants tested). For the SNPs in linkage disequilibrium (LD), only one SNP was included in the single-variant analysis. Analyses of LD between SNPs of TR were performed to identify the existing haplotype blocks. The association of the TRA haplotypes with the clinical features of narcolepsy was evaluated using a logistic regression model. Odds ratio (OR), 95% confidence interval (CI), and P-value were calculated. We used Haploview (version 4.2, Broad Institute, USA) to calculate the LD of selected SNPs. The subsequent statistical analyses were performed using R statistical software (version 3.5.0, R Development Core Team, The New Zealand).

Demographic and clinical characteristics
The demographic and clinical data of the patients with narcolepsy are presented in Table 1. Among these patients, 860 (95.2%) were of Chinese Han ethnicity, 285 (31.6%) were women, 618 (68.4%) were men, and 18 (2.0%) had a family history of narcolepsy.

Association between SNPs of TRA and clinical features of narcolepsy
According to the Chi-square test, we examined the association between the individual genetic variant and symptoms of narcolepsy, After the Bonferroni correction, only the association of rs7153643 with visual hallucination was statistically significant. The association between each individual SNP and the symptoms of narcolepsy is summarized in Table 2.

Construction of TRA haplotypes
In addition to the association between SNPs and symptomatic diversity of narcolepsy, we observed that the SNPs in TRA were in LD with each other, forming several haplotypes. Considering the SNPs in TRA gene were in LD, we constructed TRA haplotype in order to explore the association between haplotype block and symptoms of patients with narcolepsy in subsequent analysis.

Distribution of TRA haplotypes in patients with narcolepsy with different symptoms
Our data indicated that the polymorphisms of three haplotype blocks, formed by rs227017, rs1154153, and rs1154158; rs1263638 and rs1263640; and rs1263645 and rs12636470, were associated with auditory hallucination in patients with narcolepsy. As shown in Table 3, the higher TRA SNPs haplotype frequency of CT (OR = 1.236, 95% CI 1.012-1.511, P = 0.038) and TG (OR = 1.235, 95% CI 1.012-1.507, P = 0.038) were associated with an increased risk for auditory hallucination. The ATG haplotype (OR = 0.775, 95% CI 0.609-0.987, P = 0.039) and CA haplotype (OR = 0.810, 95% CI 0.663-0.988, P = 0.038); however, were more frequently observed in patients with narcolepsy who did not experience auditory hallucination. Haplotypes with a frequency of <1% were excluded from the analysis. Therefore, the three haplotype blocks were significantly associated with auditory hallucination in patients with narcolepsy.

Discussion
The present study reports two main findings. First, we identified the main haplotype blocks among patients with narcolepsy in the Chinese population. Second, we demonstrated that the different distributions of haplotypes of the TRA were significantly associated with auditory hallucination in patients with NT1.
The major strengths of the current study are as follows: (1) Our study comprised a large number of Chinese patients with phenotype and genotype data consistently collected using the same procedure for years; (2) We identified the distribution of haplotypes in the TRA, without ambiguity, in a Chinese population with narcolepsy; and (3) We extensively examined the impact of TRA on the symptomatic diversity of narcolepsy.
A previous study conducted in both Europeans and Asians [12] (only Japanese and Koreans) participants also constructed the haplotype blocks of TRA; however, the haplotype blocks of TRA that we constructed were different from those reported in that study.
A possible explanation is that the patients with narcolepsy in our study are all Chinese, and the Chinese population may have a distinctive genetic structure and distinctive TRA haplotypes.
TRA has been identified as one of the narcolepsy-risk genes in international SNP-based GWASs. [10,12,23] Some identified variants of TRA can influence the function of the immune system and are relevant to the onset of narcolepsy. [13,24] Previous studies indicated that the polymorphisms of TRA are associated with the age-atonset of narcolepsy, [13,22] and found a different distribution of TRA haplotypes between patients with narcolepsy and controls. [12] Through the haplotype analysis, we found that the haplotype polymorphisms of the TRA were also associated with the symptoms among patients with narcolepsy. Our results confirmed the important role of TRA in the phenotypes of narcolepsy, and indicated that symptomatic sub-types of narcolepsy may exist. This will contribute to the redefinition of the clinical sub-types of narcolepsy based on the concept of precision medicine. The Our results indicate that the patterns of haplotype in TRA (haplotypes TG and CT) is closely associated with auditory hallucination. The underlying pathophysiology of auditory hallucination remains elusive, but several studies proposed that the primary auditory cortex (A1) may be involved in the generation of auditory hallucinations. [25][26][27] Serotonin 2A receptor (5-HT2AR) activation may play a crucial role in causing hallucinations during sleep paralysis. [28] It has been estimated that although the core underlying pathologic mechanism in narcolepsy is the loss of large quantities of hypocretin (orexin) neurons in the hypothalamus, narcolepsy also considerably affects endocrine, metabolism, and mood regulation systems. [29] Our results indicate that TRA polymorphisms may be associated with narcolepsy sub-types with different underlying pathogenesis and mechanisms, which may affect different parts of the brain and present different symptoms. The results also indicate that the diversity of symptoms of narcolepsy caused by TCR diversity is closely associated with different immune processes. Although the underlying mechanism is not yet clear, these results may help to elucidate possible underlying mechanisms in narcolepsy.
Some limitations and shortcomings exist in our study. First, our study only explored the effect of genetic factors for the symptomatic differences of narcolepsy. We did not investigate the effects of non-genetic factors. Second, the polymorphisms discussed in this study were limited in number due to the limited tagging SNPs. Thus, a more comprehensive tagging SNP-based approach, which relies  The present study identified the main haplotype blocks among patients with narcolepsy in a Chinese population and demonstrated that the different distributions of haplotypes of the TRA were significantly associated with the symptomatic differences in narcolepsy. These results may help in defining different sub-types of narcolepsy and  treating narcolepsy symptoms using immunotherapy based on the concept of precision medicine.