An association analysis of HLA-DQB1 with narcolepsy without cataplexy and idiopathic hypersomnia with/without long sleep time in a Japanese population

Narcolepsy without cataplexy (NA w/o CA) (narcolepsy type 2) is a lifelong disorder characterized by excessive daytime sleepiness and rapid eye movement (REM) sleep abnormalities, but no cataplexy. In the present study, we examined the human leukocyte antigen HLA-DQB1 in 160 Japanese patients with NA w/o CA and 1,418 control subjects. Frequencies of DQB1*06:02 were significantly higher in patients with NA w/o CA compared with controls (allele frequency: 16.6 vs. 7.8%, P=1.1×10−7, odds ratio (OR)=2.36; carrier frequency: 31.3 vs. 14.7%, P=7.6×10−8, OR=2.64). Distributions of HLA-DQB1 alleles other than DQB1*06:02 were compared between NA w/o CA and narcolepsy with cataplexy (NA-CA) to assess whether the genetic backgrounds of the two diseases have similarities. The distribution of the HLA-DQB1 alleles in DQB1*06:02-negative NA w/o CA was significantly different from that in NA-CA (P=5.8×10−7). On the other hand, the patterns of the HLA-DQB1 alleles were similar between DQB1*06:02-positive NA w/o CA and NA-CA. HLA-DQB1 analysis was also performed in 186 Japanese patients with idiopathic hypersomnia (IHS) with/without long sleep time, but no significant associations were observed.

Idiopathic hypersomnia (IHS) is a sleep disorder of presumed central nervous system origin that is associated with excessive daytime sleepiness consisting of prolonged non-REM sleep episodes. Daytime naps of IHS patients tend to be longer and less refreshing than those of NA-CA patients. IHS is a rare disease, representing 8:10 to 1:10 patients with NA-CA. This suggests that the prevalence of IHS approximates 0.005%. 18 The ICSD-2 describes two clinical forms of IHS by the difference in nocturnal sleep time: IHS with long sleep time (IHS-LST) and IHS without long sleep time (IHS w/o LST). The nocturnal sleep time of IHS-LST is prolonged to at least 10 h, while that of IHS w/o LST is either normal or slightly prolonged (less than 10 h). CSF orexin A levels in IHS are normal. 6 The cause and pathogenesis of IHS remain largely unknown. NA w/o CA and IHS w/o LST have several common characteristics except for REM-related symptoms. Distinguishing NA w/o CA and IHS w/o LST is impossible without the multiple sleep latency test to identify sleep-onset REM periods. According to the ICSD-2, the diagnosis is based on the number of sleep-onset REM periods, two or more in the former and less than two in the latter. In the present study, we tested whether HLA-DQB1 alleles have an influence on susceptibility to IHS w/o LST and IHS-LST.
A total of 346 Japanese patients and 1,418 Japanese healthy controls were included in this study. NA w/o CA, IHS w/o LST and IHS-LST were diagnosed according to the ICSD-2 criteria. The patient groups consisted of NA w/o CA (n = 160), IHS w/o LST (n = 118) and IHS-LST (n = 68). We utilized HLA data of healthy individuals, who have been previously studied for disease association analyses. 17,19,20 In addition, to assess genetic similarities between the above hypersomnia disorders and NA-CA, HLA-DQB1 data from 664 patients with NA-CA were utilized. 17 All of the patients and controls were mainland Japanese and gave written informed consent. This study was approved by the local institutional review boards at participating institutions. Typing for the HLA-DQB1 locus was performed by a Luminex Multi-Analyte Profiling system (xMAP) with WAKFlow HLA typing kits (Wakunaga Pharmaceutical, Wakunaga, Hiroshima, Japan). Comparisons of frequencies were performed using the Chi-square test or Fisher's Exact test as appropriate. To account for multiple testing, the significance level was adjusted by the number of HLA alleles with allele frequencies no less than 0.5% in controls (12 for HLA-DQB1 alleles). The significance level was set to be Po 4.2 × 10 − 3 (0.05/12). If any of the four cells was zero, the Woolf-Haldane correction was applied (adding 0.5 to all cells). An association analysis controlling for the effects of DQB1*06:02 was needed to test the other HLA-DQB1 alleles. Specifically, the analysis was performed using counts of the other HLA-DQB1 alleles remaining after excluding allele counts of DQB1*06:02 from both cases and controls, which is the relative predispositional effects method. 21 Briefly, frequencies and ORs were calculated for the alleles carried by the non-DQB1*06:02 chromosomes. To determine whether there was a different allelic distribution between two groups, the overall frequency distribution of HLA-DQB1 alleles in one group was compared with the distribution in another group by using the global Chi-square test with 12 degrees-of-freedom.
Next, we tested whether HLA-DQB1 allele distributions (except for DQB1*06:02) in DQB1*06:02-positive and -negative NA w/o CA patients showed similarities with those of NA-CA patients ( Table 2 and Supplementary Figure 1). The allele distribution of DQB1*06:02-negative NA w/o CA was significantly different from that of NA-CA (P = 5.8 × 10 − 7 ). On the other hand, no significant difference was observed in the distribution between DQB1*06:02positive NA w/o CA and NA-CA (P = 0.97). When we focused on HLA-DQB1 alleles with frequencies no less than 5% in DQB1*06:02positive NA w/o CA, all the ORs of the alleles were found in the same direction as those of NA-CA (Table 2). These results suggest that a common etiological pathway might exist for DQB1*06:02positive NA w/o CA and NA-CA, whereas DQB1*06:02-negative NA w/o CA has a different etiological pathway from NA-CA. Orexin A in the CSF of typical NA-CA patients is known to be reduced or undetectable. 5,6 A minority of NA w/o CA patients have low concentrations of CSF orexin A levels, although a majority of NA w/o CA patients have normal concentrations; 6,10 thus, an etiologic heterogeneity in NA w/o CA is suggested. In addition, most of the NA w/o CA patients with low CSF orexin A are DQB1*06:02positive, as in NA-CA. 6,10 In contrast, DQB1*06:02 positivity of patients with normal CSF orexin A is not higher than that seen in the general population. 10 Table 3 and Supplementary Table 2, respectively. DQB1*06:02, known to be associated with NA-CA and NA w/o CA, was not associated with IHS w/o LST or IHS-LST. Although DQB1*05:02 (P = 6.3 × 10 − 3 ) and DQB1*03:01 (P = 0.04) showed nominally significant associations with IHS-LST, there were no HLA-DQB1 alleles that reached the threshold after correction for multiple comparisons. The similarity of HLA-DQB1 allele distribution between NA-CA and IHS w/o LST or IHS-LST was tested after controlling for the effects of DQB1*06:02, and significant differences were found: for IHS w/o LST: P = 2.1 × 10 − 4 and for IHS-LST: P = 2.2 × 10 − 8 . Taken together, these results indicate that IHS w/o LST and IHS-LST are caused by different etiological genetic factors than those that give rise to NA-CA.
The International Classification of Sleep Disorders was recently revised for the 3rd Edition (ICSD-3). When we had recruited patient samples, the 2nd edition (ICSD-2) was used. Main differences between the ICSD-2 and ICSD-3 regarding NA w/o CA, IHS w/o LST and IHS-LST are as follows. The terminology has been changed from 'narcolepsy without cataplexy (NA w/o CA)' to 'narcolepsy type 2'. The concept of NA w/o CA and narcolepsy type 2 is almost the same. IHS w/o LST and IHS-LST were unified to 'idiopathic hypersomnia (IHS)' regardless of the extension of the sleep time. Therefore, we combined our IHS w/o LST and IHS-LST data to analyze the HLA-DQB1 data as IHS (Supplementary Tables  3 and 4). As a result, no significant association was observed between each HLA-DQB1 allele and IHS. Distribution of HLA-DQB1 The overall frequency distribution of HLA-DQB1 alleles in NA-CA group was compared with that in another group using the global Chi-square test. Analysis of HLA-DQB1 association with narcolepsy without cataplexy T Miyagawa et al alleles in IHS was also significantly different from that of NA-CA, even after the effect of DQB1-06:02 was controlled (P = 8.6 × 10 − 9 ). To conclude, the association of DQB1*06:02 in NA w/o CA was confirmed. Our results also suggested an immunological pathogenesis of DQB1*06:02-positive NA w/o CA, which is similar to that of NA-CA. DQB1*06:02-negative NA w/o CA, IHS w/o LST and IHS-LST may have a different etiology, which is not well understood.

HGV DATABASE
The relevant data from this Data Report are hosted at the Human Genome Variation Database at http://dx.doi.org/10.6084/m9. fig  share.hgv.688.