Introduction

Multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are inflammatory diseases of the central nervous system (CNS). Although the prevalence of MS is gradually increasing in Japan, it is still low (< 20/100,000) compared with Europe and North America (> 100/100,000)1,2,3. Clinical features are also different between Japanese and Caucasian patients. Disease severity of MS is milder in Japanese compared with Caucasian patients4,5. The prevalence of oligoclonal IgG bands (OCBs) was estimated to be lower in Japanese compared with Caucasian MS patients6,7. On MRI, cerebellar and parietal lesions were less frequently detected in Japanese compared with European MS patients5,8. Moreover, genetic background is also different between these populations. For example, human leukocyte antigen (HLA)-DRB1*15:01 is the strongest genetic susceptibility factor for MS in Europeans, while both HLA-DRB1*15:01 and DRB1*04:05, which is a rare allele in Northern Europeans, are frequent susceptibility alleles in Japanese MS patients9,10,11,12,13. In NMOSD, although the prevalence between Japan and other countries is similar14, the genetic risk factors are thought to be different15. These observations prompted us to establish a biobank for Japanese MS and NMOSD (Japan MS/NMOSD Biobank) to collect clinical information and biological samples, such as DNA, as a part of the Rare Disease Bank at the National Institutes of Biomedical Innovation, Health and Nutrition in Japan16.

Various susceptibility and protective HLA class II alleles have been reported in MS and NMOSD, including in Japanese patients11,13,15,17. HLA genotype-clinical phenotype correlations have been reported, focusing on the major susceptibility alleles in Caucasian (HLA-DRB1*15:01) and Japanese MS patients (HLA-DRB1*15:01 and DRB1*04:05). However, phenotype correlations are unknown in NMOSD. In Caucasian patients with MS, HLA-DRB1*15:01 is associated with younger age at onset, higher white matter lesion volume, greater brain atrophy and impairment of cognitive function10,18, but its association with clinical course, disease severity, and prognosis is controversial19,20,21,22,23. In Japanese patients with MS, HLA-DRB1*15:01 is associated with a higher frequency of cerebrospinal fluid (CSF) IgG abnormality, including OCBs, while DRB1*04:05 is associated with younger age at onset, lower frequency of CSF IgG abnormality, milder disease course and fewer intracortical lesions7,11,13,24. In the present study, we aimed to identify relationships between various susceptibility/protective HLA alleles and clinical manifestations in MS and NMOSD using the newly established Japan MS/NMOSD Biobank data.

Results

Comparison of demographic features between MS and NMOSD

We enrolled 739 patients and collected 731 DNA, 528 plasma and 566 serum samples. Overall, 19, 8, and 1 patients were excluded from the analyses because of a lack of clinical information, a lack of DNA samples, and double registration, respectively. Clinical information and genotypes of HLA-DRB1 and -DPB1 alleles were available from 528 MS and 183 NMO/NMOSD patients based on the 2010 revised McDonald criteria25 and NMO and NMOSD criteria advocated by Wingerchuk in 2006 and 200726,27, respectively. Of 183 NMO/NMOSD patients enrolled, 165 patients fulfilled the international consensus diagnostic criteria of NMOSD published in 201528. In the context of the current clinical situation, we conducted the following analyses of NMOSD using data from these 165 patients. MS patients included 438 relapsing–remitting MS (RRMS), 70 secondary progressive MS (SPMS), and 19 primary progressive MS (PPMS) cases. Comparing clinical and laboratory information between MS and NMOSD (Table 1), MS patients were younger at onset and at registration compared with NMOSD patients (both p < 0.001). The female to male ratio in NMOSD was higher than in MS (p < 0.001). Disease duration was longer in MS than in NMOSD patients (p = 0.004). There was no significant difference in the total number of relapses between the two groups, but annualized relapse rate (ARR) was higher in NMOSD than in MS (p = 0.001). NMOSD patients experienced optic neuritis more frequently than MS patients, while MS patients had higher frequencies of cerebrum, cerebellum, and brainstem involvement than NMOSD patients. Myelitis, especially episodes of transverse myelitis and the presence of longitudinally extensive spinal cord lesions (LESCLs), was more frequent in NMOSD compared with MS (p = 0.031, p < 0.001 and p < 0.001, respectively). NMOSD patients had higher Kurtzke’s Expanded Disability Status Scale (EDSS) scores29 and progression index (PI) values than MS patients (both p < 0.001). Of the seven functional system (FS) scores29, visual, pyramidal, sensory, and bowel and bladder FS scores were significantly higher in NMOSD than in MS, while brainstem, cerebellar and cerebral FS scores were significantly higher in MS than in NMOSD. Patients with MS more frequently had CSF IgG abnormalities including OCB-positivity and/or high IgG index (> 0.658)30 than those with NMOSD (p < 0.001). Anti-aquaporin 4 (AQP4) antibody was positive in 95.1% of NMOSD patients. In this study, four cases with anti-AQP4 antibody were diagnosed as MS. These diagnoses were ultimately made by each doctor-in-charge based on the patients’ clinical and MRI characteristics and clinical courses, and they were treated with interferon-β (IFN-β). Excluding these cases from the MS group did not significantly change our findings. Half of the MS patients and one-quarter of the NMOSD patients showed a benign course. Co-existing autoimmune diseases and/or autoantibodies, especially Sjögren syndrome, anti-nuclear antibody (ANA), and anti-SS-A/SS-B antibodies, were more frequent in NMOSD patients compared with MS patients (all p < 0.001), while frequencies of co-existing thyroid disease and/or thyroid-related antibodies did not differ between the two groups. Almost all NMOSD patients in the cohort were treated with oral corticosteroids, and two thirds with immunosuppressant drugs. More than 85% of MS patients were treated with disease-modifying drugs (DMDs). Twenty-seven NMOSD patients were treated with IFN-β. Out of the 23 patients for whom data on IFN-β efficacy was obtained, IFN-β was not effective in 18 (78.3%). The other five patients who showed effective treatment responses to IFN-β were additionally treated with corticosteroids.

Table 1 Clinical and laboratory characteristics of Japanese patients with MS and NMOSD.
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Comparison of the demographic and clinical features of MS patients between northern and southern Japan

We previously reported a difference in the clinical characteristics of MS patients between northern and southern Japan1,13; therefore, MS patients were stratified by their resident area into two groups (northern and southern Japan) at a latitude of 37° North. Of 528 MS patients, 125 and 371 patients lived in northern and southern Japan, respectively (residence information was missing for 32 patients). As shown in Supplementary Table S1, northern MS patients had lower numbers of relapses (p < 0.001), annualized relapse rate (p = 0.001), and FS score of cerebellar function (p = 0.050), but they experienced cerebrum relapses more frequently than southern MS patients (p = 0.005). Although disease disability and severity did not differ between northern and southern MS patients, patients with a benign disease course (EDSS ≤ 3 at 10 or more years of disease duration) were more frequently observed in southern Japan than in northern Japan (p = 0.027). Patients with a high IgG index were more frequent in northern Japan than in southern Japan (p = 0.039), but the OCB positivity rate was similar between groups. Patients who experienced transverse myelitis and autoantibody positivity, especially for ANA and anti-SS-A and/or -SS-B antibodies, were more frequent in southern Japan than in northern Japan (p = 0.007, 0.001, 0.001 and 0.026, respectively). Although a small number of PPMS patients were registered only in southern Japan, the above-mentioned differences were similar if these 19 PPMS patients were excluded (data not shown).

Frequencies of the HLA-DRB1 and -DPB1 alleles

As shown in Tables 2 and 3, the carrier frequencies of the HLA-DRB1*04:05, DRB1*15:01, and DPB1*03:01 alleles were higher in MS patients than in healthy controls (corrected p [pcorr] < 0.001, pcorr < 0.001 and pcorr = 0.006, respectively). Those of HLA-DRB1*01:01, DRB1*09:01, DRB1*13:02 and DPB1*04:01 were lower in MS patients than in healthy controls (pcorr = 0.001, pcorr < 0.001, pcorr < 0.001 and pcorr = 0.006, respectively). In NMOSD patients, the carrier frequencies of HLA-DRB1*08:02 and DPB1*05:01 were higher and those of HLA-DRB1*09:01 were lower than in healthy controls (pcorr = 0.008, pcorr < 0.001 and pcorr < 0.001, respectively).

Table 2 Carrier frequencies of HLA-DRB1 in patients with MS and NMOSD and in healthy controls.
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Table 3 Carrier frequencies of HLA-DPB1 in patients with MS and NMOSD and in healthy controls.
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Comparison of clinical and laboratory characteristics between risk HLA-allele-positive and -negative patients

The main findings of the HLA genotype-clinical phenotype correlations are summarized in Tables 4 and 5.

Table 4 Summary of HLA genotype-clinical phenotype correlations in MS.
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Table 5 Summary of HLA genotype-clinical phenotype correlations in NMOSD.
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MS patients

Concerning the susceptibility alleles, MS patients with HLA-DRB1*04:05 were younger at disease onset (p = 0.017), had lower frequencies of CSF IgG abnormalities, including presence of OCBs and high IgG index (all p < 0.001), and higher visual FS scores (p = 0.001), and tended to have optic nerve involvement (optic neuritis) more frequently than patients without DRB1*04:05 (p = 0.052) (Table 6). Because disease duration differed between the two groups, the frequency of patients who experienced relapses with optic neuritis and FS scores (≥ 1) of visual function were compared, adjusting for disease duration. The frequencies of patients who experienced optic neuritis tended to be higher in patients with HLA-DRB1*04:05 than those without the allele, but this did not reach statistical significance (odds ratio [OR] 1.33 [95% confidence interval [CI] 0.92–1.94], p = 0.132). Regarding visual function, HLA-DRB1*04:05 was an independent risk factor for functional disability of the optic nerve even after adjusting for disease duration (OR 2.30 [95% CI 1.29–4.10], p = 0.005).

Table 6 Comparison of the clinical characteristics between MS patients with and without HLA-DRB1*04:05.
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We previously reported that the effect of HLA-DRB1*04:05 on MS patients differed between northern and southern Japan13; therefore, we additionally compared MS patients from northern and southern Japan with and without HLA-DRB1*04:05 (Supplementary Tables S2, S3). CSF IgG abnormality was less frequent in HLA-DRB1*04:05-positive than -negative patients from northern or southern Japan (p = 0.005 and p < 0.001, respectively) similar to that for all of Japan. In southern Japan, MS patients with HLA-DRB1*04:05 were younger at disease onset (p = 0.009) with higher visual FS scores (p = 0.003) and tended to have optic nerve involvement (optic neuritis) more frequently than patients without DRB1*04:05 (p = 0.090). In northern Japan, HLA-DRB1*04:05-positive patients had higher pyramidal FS scores (p = 0.029) and experienced fingolimod treatment more frequently than HLA-DRB1*04:05-negative patients (p = 0.006). Moreover, when excluding PPMS, as in a previous study13, PI and MS severity scores (MSSS)31 were lower in southern MS patients with HLA-DRB1*04:05 than in those without the allele (p = 0.22 and 0.050, respectively) (Supplementary Table S4). When we compared the clinical characteristics of MS patients with HLA-DRB1*04:05 (excluding PPMS) between northern and southern Japan, northern patients had a lower number of relapses (p < 0.001) and annualized relapse rate (p = 0.006) but higher PI (p = 0.018) and MSSS (p = 0.008). Patients who experienced transverse myelitis and who had ANA were more frequent in southern Japan than in northern Japan (p = 0.023 and 0.012, respectively). Southern MS patients with HLA-DRB1*04:05 were less frequently treated with DMD, especially with fingolimod, compared with northern patients (p = 0.018 and 0.009, respectively) (Supplementary Table S5).

HLA-DRB1*15:01-positive MS patients had CSF IgG abnormalities more commonly than DRB1*15:01-negative patients (p = 0.036) (Table 7) and they experienced attacks in the optic nerve (optic neuritis) and cerebellum more frequently (p = 0.044 and 0.031, respectively), and also tended to have a higher FS score of visual function (p = 0.060) compared with those without the allele. FS scores of cerebellar function were similar between these two groups. FS scores of pyramidal function and PI were significantly higher in DRB1*15:01-positive patients than in DRB1*15:01-negative patients (p = 0.027 and 0.031, respectively), although EDSS scores and MSSS were not significantly different between them. MS patients with DRB1*15:01 had anti-SS-A and/or -SS-B antibodies more frequently than those without (p = 0.007). HLA-DPB1*03:01 carriers had higher FS scores of brainstem and cerebellar function than non-carriers (p = 0.035 and 0.004, respectively) (Supplementary Table S6).

Table 7 Comparison of the clinical characteristics between MS patients with and without HLA-DRB1*15:01.
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With respect to resistance alleles, MS patients with the HLA-DRB1*01:01 allele had a lower female to male ratio than those without (p = 0.006) (Supplementary Table S7). HLA-DRB1*01:01 carriers had spinal cord involvement less frequently and had a lower FS score of sensory function than non-carriers (p = 0.022 and 0.048, respectively). HLA-DRB1*09:01-positive MS patients had lower ARR and FS scores of bowel and bladder function and had brainstem involvement less frequently than DRB1*09:01-negative patients (p = 0.047, 0.015 and 0.005, respectively) (Supplementary Table S8). EDSS scores, PI and MSSS were not different between these two groups. MS patients with HLA-DRB1*13:02 tended to have brainstem and spinal cord involvement less frequently than those without the allele (p = 0.055 and 0.058, respectively). Moreover, HLA-DRB1*13:02-carriers had a lower frequency of OCB-positivity than non-carriers (p = 0.002) (Supplementary Table S9). MS patients with the HLA-DPB1*04:01 allele had OCBs less frequently and higher frequencies of LESCL compared with those without the allele (p = 0.004 and 0.043, respectively) (Supplementary Table S10).

NMOSD patients

With respect to susceptibility alleles, NMOSD patients with the HLA-DRB1*08:02 allele had lower frequencies of OCB-positivity compared with those without the allele (p = 0.012) (Supplementary Table S11). Moreover, HLA-DRB1*08:02 carriers were more prone to having autoimmune diseases and/or autoantibodies, especially anti-SS-A and/or -SS-B antibodies, compared with non-carriers (p = 0.033 and 0.035, respectively). NMOSD patients with HLA-DPB1*05:01 had longer disease durations and a larger number of relapses compared with those without (p = 0.016 and 0.001, respectively) (Supplementary Table S12), while the ARR was comparable between them. Moreover, they tended to have more frequent involvement of the cerebrum and cerebellum compared with those without (p = 0.090 and 0.077, respectively). PI was lower in patients with the HLA-DPB1*05:01 allele compared with those without (p = 0.034), although EDSS scores were not statistically different. They had Sjögren syndrome and anti-SS-A and/or -SS-B antibodies more often than DPB1*05:01-negative patients (p = 0.044 and 0.028, respectively). With respect to protective alleles, HLA-DRB1*09:01-positive NMOSD patients had co-existing autoimmune diseases, especially Sjögren syndrome, more frequently than DRB1*09:01-netagive patients (p = 0.030 and 0.034, respectively) (Supplementary Table S13).

Factors associated with disability

Finally, we evaluated which clinical parameters, laboratory findings and HLA subtypes were associated with disability. In RRMS, we focused on HLA-DRB1*04:05 and DRB1*15:01 as genetic factors. Multivariable models showed that old age at onset, long disease duration, many relapses, and being a carrier of the HLA-DRB1*15:01 allele were independent risk factors for high EDSS scores (Table 8). For NMOSD, old age at onset, long disease duration and many relapses were risk factors for high EDSS scores, but episodes of bilateral optic neuritis, experience of LESCL and harbouring the HLA-DPB1*05:01 allele were not (Supplementary Table S14).

Table 8 Clinical and genetic factors associated with EDSS scores in RRMS.
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Discussion

We used the Japan MS/NMOSD Biobank and the largest cohort of Japanese MS and NMOSD patients studied to date to show that HLA-DRB1*04:05, DRB1*15:01 and DPB1*03:01 are susceptibility alleles, while HLA-DRB1*01:01, DRB1*09:01, DRB1*13:02 and DPB1*04:01 are protective alleles for MS, which is consistent with previous Japanese studies on MS11,13. For NMOSD, HLA-DRB1*08:02 and DPB1*05:01 are susceptibility alleles and DRB1*09:01 is a protective allele. This is a novel finding for HLA-DRB1*08:02 and confirmatory for DPB1*05:01 and DRB1*09:0115. Importantly, we discovered new significant HLA genotype-clinical phenotype correlations in MS and NMOSD.

In our MS cohort, the susceptibility alleles, DRB1*15:01, and DPB1*03:01, were associated overall with worsening functional disability scores. The finding that HLA-DRB1*15:01 increased the involvement of the optic nerve (optic neuritis) and cerebellum and worsened visual and pyramidal FS scores, resulting in greater PI, is a new finding in Asians and is compatible with several reports in Caucasians showing that DRB1*15:01 is associated with greater MSSS and more severe cognitive impairment18,20. The relationship of HLA-DRB1*04:05 to clinical manifestations has not been reported in Caucasians, primarily because this allele is rare in most European descendants32,33. MSSS was lower in southern MS patients with HLA-DRB1*04:05 than in those without the allele when excluding PPMS in this study, which is in accord with a previous finding that HLA-DRB1*04:05 was associated with a milder disease course as evaluated by EDSS scores and PI in Japanese MS patients, particularly from southern Japan11,13. Unexpectedly, we also found an association of this allele with increased tendency of involvement of the optic nerve (optic neuritis) and worsening of visual FS scores. Although these results appear contradictory, the fact that motor disability involving pyramidal and cerebellar functions is more contributory to EDSS scores than other systems, such as visual function, may explain this discrepancy. A comparison of the clinical characteristics of MS patients with HLA-DRB1*04:05 between northern and southern Japan showed that southern patients had a milder disease course and atypical presentation including episodes of transverse myelitis and having ANA, which may result in the lower frequency of DMD treatment. Given that they share HLA-DRB1*04:05 as a common genetic background, environmental factors including residential latitude may contribute in part to these differences in the clinical manifestations. In Caucasians, HLA-DRB1*15:01 has repeatedly been shown to be associated with younger age at onset in MS patients10,18,34, while in Japanese, HLA-DRB1*04:05 is associated with a younger age at onset11,13, which was confirmed in the present study. Thus, the contribution of HLA alleles to a younger age at onset could vary according to race. The association of HLA-DPB1*03:01 with an increase in brainstem and cerebellar FS scores is a novel finding, which further supports a contribution of the susceptibility alleles to aggravation of disease severity in Japanese MS patients.

In contrast, the resistance alleles, HLA-DRB1*01:01, DRB1*09:01 and DRB1*13:02, were associated with decreased functional disability in the present cohort. HLA-DRB1*01:01 decreased involvement of the spinal cord and sensory FS scores, DRB1*09:01 decreased ARR, involvement of the brainstem and bowel and bladder FS scores, and DRB1:13:02 decreased involvement of the spinal cord and brainstem. HLA-DRB1*01 was reported to decrease PI and the frequency of malignant subtype and to extend the time to reach an EDSS score of 6 in Caucasian patients35, which is in line with our findings. Another resistance allele, HLA-DPB1*04:01, was reported to be associated with a reduced MSSS under corticosteroid or azathioprine in Brazilian patients36. This was not obvious in the present study, possibly because of the low frequency of HLA-DPB1*04:01 in this cohort. Collectively, these resistance alleles are indicated to decrease disease susceptibility and to decrease involvement of relevant CNS sites in different races.

We examined CSF IgG in our cohort and found that the resistance alleles, HLA-DRB1*13:02 and DPB1*04:01, decreased OCBs while one of the susceptibility alleles, DRB1*15:01, increased, and another, DRB1*04:05, decreased CSF IgG abnormalities. Potentiation of CSF IgG abnormalities by DRB1*15:01 was reported in both Caucasians9,37,38 and Japanese patients with MS11,13,39, while mitigation of CSF IgG abnormalities by DRB1*04:05 has been repeatedly reported in Japanese patients with MS7,11,13,39. Collectively, even for susceptibility alleles, their influence on CSF IgG abnormalities, including OCBs, appears to be heterogeneous. The association of two resistance alleles, HLA-DRB1*13:02 and DPB1*04:01, with lower OCB frequency is a novel finding, which should be confirmed by a large scale study. HLA-DRB1*13:02 is a protective allele for systemic and organ-specific autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, and autoimmune hepatitis40. Therefore, a person with HLA-DRB1*13:02 may have resistance to producing autoreactive immunoglobulins. Only a few studies of Caucasians reported such associations of distinct HLA alleles with CSF IgG abnormalities, which may be partly accounted for by the extremely high overall prevalence of OCBs (> 90%)9,41 that obscures the effects of HLA alleles. Thus, investigation of a Japanese MS cohort, in which the positivity rate of OCBs is not very high (around 60%)6,7, for any association of HLA alleles with CSF IgG abnormalities is warranted.

Multivariable analysis also revealed HLA-DRB1*15:01 to be an independent risk factor for high EDSS scores in addition to old age at onset, long disease duration, and many relapses. Older age at onset, male sex, and high relapse rate especially in the early disease course, are well known poor prognostic factors in Caucasians42,43,44,45, but the natural history of MS in Asia is less well studied. We previously assessed the association between age at onset and disease severity using MSSS13. Old age at onset is thought to be an independent risk factor for increased disease severity and disability in both Caucasians and Japanese. In contrast to previous studies of Caucasian MS patients42,43,44, this and our previous studies did not show an effect of sex on disease severity or disability in Japanese MS patients13. Therefore, male sex can be a poor prognostic factor for Caucasians but not Japanese, although this should be confirmed in a large study, such as a nation-wide survey. The effect of the total number of relapses on prognosis is controversial46, but relapse frequency up to year 5 was reported to be predictive in some studies44,47. The median disease duration in RRMS patients was 8 years in our study, which may reflect relatively early disease activity.

In NMOSD, no association between any HLA allele and clinical manifestations has been found. The present NMOSD patients had highly comparable demographic and clinical features, including sex ratio, age of onset, and frequency of OCB-positivity with previous Japanese clinical studies48,49. One of the susceptibility alleles, HLA-DPB1*05:01, was associated with lower PI compared with HLA-DPB1*05:01-negative patients, even though EDSS scores did not differ between them. Because the disability of NMOSD patients is mainly caused by acute attacks, and a progressive disease course is uncommon in NMOSD compared with MS50,51,52, the PI decreases as the disease duration increases, particularly when treatments such as corticosteroids reduce relapses53. Indeed, NMOSD patients with DPB1*05:01 had a significantly longer disease duration than those without the allele in our cohort, which confirms this idea. Multivariable analyses also supported the idea that the HLA-DPB1*05:01 allele has no significant impact on disability.

We found that the susceptibility alleles, HLA-DRB1*08:02 and DPB1*05:01, increased the frequencies of concomitant autoimmune diseases and autoantibodies such as Sjögren syndrome and anti-SS-A/SS-B antibodies, while the resistance allele, DRB1*09:01, was also associated with increased frequencies of co-existing autoimmune diseases, such as Sjögren syndrome. Thus, how associations of either the susceptibility or protective alleles with autoimmune traits modulate NMOSD susceptibility or resistance through autoimmune backgrounds remain elusive.

In our multivariable analyses, while higher age at onset, longer disease duration, and more relapses were independent risks for disability, the susceptibility allele, HLA-DPB1*05:01, was not an independent risk factor for disability, in contrast to MS. HLA-DPB1*05:01 could increase the susceptibility to NMOSD but may not confer disability. Associations between older age at onset and higher EDSS scores and between death and high ARR have been reported in European and Asian cohorts52,54,55,56. Collectively, older age at onset and relapse number are regarded as risk factors for disability, underscoring the importance of early introduction of efficacious preventative therapies in elderly-onset NMOSD patients. Although chronic progression is not considered to contribute to NMOSD, the longer disease duration was also an independent risk for disability in our NMOSD patients. We have reported a persistent increase of serum neurofilament light chain in the remission phase in a fraction of NMOSD patients, indicating continuous damage to axons following acute relapses57. Long term follow-up studies are required to elucidate whether, and if so how, a longer disease duration influences disability in NMOSD.

In terms of disability in NMOSD patients, our cohort showed relatively mild disability compared with the clinical course of NMOSD patients described by Wingerchuk et al.27. However, NMOSD patients without LESCL were reported to have a milder disease course than those with LESCL58. Because patients without LESCL occupied 57.4% of NMOSD patients in our cohort, overall disability in our NMOSD cases appeared to be mild. Moreover, a recent study showed that Japanese NMOSD patients with AQP4-IgG had a lower risk of relapse, especially transverse myelitis and optic neuritis attacks, compared with Caucasian patients52. This might explain why our cohort had lower EDSS scores and lower frequencies of LESCL than expected values based on the results of Caucasian NMOSD patients. In addition, more than 95% of NMOSD patients received prednisolone treatment, which can decrease disease severity by preventing relapses53. Therefore, we think that our cohort showed normal attributes for Japanese NMOSD patients.

In this study, we compared the demographic and clinical features of MS and NMOSD patients and assessed their distinct genetic backgrounds and the differential effects of HLA class II alleles on clinical features between these two diseases. Although MS and NMOSD are distinct diseases, both are inflammatory diseases affecting CNS tissues and their clinical findings sometimes overlap. Moreover, it is occasionally difficult to distinguish between these two diseases, particularly for cases of seronegative NMOSD in clinical settings. Thus, we consider that the collection of MS and NMOSD information and biosamples by the Japan MS/NMOSD biobank will be very useful for the characterization of clinical and laboratory findings to differentiate between the two conditions. By describing the differences in clinical and laboratory tests between MS and NMOSD concurrently, the differential aspects of these two diseases will become clearer.

There are several limitations in our study. First, susceptibility HLA alleles for MS and NMOSD, other than HLA-DRB1 and -DPB1, were recently reported for Japanese patients using next-generation sequencing17. These include HLA-B*39:01 and HLA-B*15:01 for MS and HLA-DQA1*05:03 for NMOSD17. We genotyped only two classical HLA class II genes (DRB1 and DPB1) using sequence-specific oligonucleotide hybridization. By applying new methodology, such as next-generation sequencing, to this newly established Japan MS/NMOSD Biobank of samples, new risk genes of MS or NMOSD could be identified in the future. Second, healthy controls were not included in the MS/NMOSD Biobank; therefore, we used data of healthy controls collected by the Japan Multiple Sclerosis Genetics Consortium13. Therefore, similar risk and resistance alleles to those in the previous study may tend to be obtained; however, the HLA genotype-clinical phenotype correlations were newly discovered in the present study. Third, in this MS/NMOSD biobank, the number of enrolled MS patients from southern Japan were approximately 3 times higher than that from northern Japan despite the higher prevalence rate of MS in northern Japan59. However, the general population of southern Japan is about 7 times higher than that of northern Japan. Therefore, although potential enrolment bias cannot be completely excluded, we do not think that the 3:1 ratio of enrolled MS patients between southern and northern Japan distorted the results of the present study. Fourth, as we did not systematically examine anti-myelin oligodendrocyte glycoprotein (MOG) antibodies, some patients with anti-MOG antibodies may be included in our MS cohort. However, most adult patients with typical MS, including Japanese, are negative for anti-MOG antibodies60,61; therefore, possible contamination of MOG antibody disease would not severely distort our results.

In conclusion, based on the Japan MS/NMOSD Biobank data, we show HLA genotype-clinical phenotype correlations concerning the confirmed and newly identified susceptibility and resistance DRB1 and DPB1 genes in Japanese patients with MS or NMOSD. The susceptibility alleles were mostly associated with worsening of disease severity, whereas the resistance alleles were associated with amelioration of the disease in MS. By contrast, such genotype–phenotype correlations were not obvious in NMOSD. Together these findings indicate that both susceptibility and resistance alleles can influence the clinical manifestations in MS but not in NMOSD.

Patients and methods

Participants

The MS/NMOSD Biobank of Japan was established in 2013 as a part of the Rare Disease Bank at the National Institute of Biomedical Innovation, Health and Nutrition in Japan16. Japanese patients with MS and NMOSD, consenting to be registered in this biobank, were recruited from 20 institutes in Japan. MS was diagnosed using the 2010 revised McDonald criteria25. The diagnoses of NMO and NMOSD were based on the criteria advocated by Wingerchuk in 2006 and 200726,27. Diagnoses were recorded at the time informed consent was provided by the participants. However, given that the NMOSD criteria updated in 2015 are now widely used and that the items of NMOSD proposed in 2007 are relatively complicated, we limited our analyses to the 165 NMOSD patients who fulfilled the criteria of Wingerchuk 201528 based on the data collected. Participants’ demographic data, clinical information and biological samples (sera, plasma and DNA) were also collected. The biological samples were taken between December 2002 and March 2017. Among 739 patients enrolled, samples from 628 patients were obtained within 1 year after written informed consent was obtained and previously stocked samples from 105 patients were provided from each institute to the Japan MS/NMOSD biobank (6 patients lacked sampling date information). The collected demographic data and clinical information were as follows: gender, age at onset and registration, date and residence at clinical evaluation, EDSS scores29, FS scores29, number of relapses, relapse sites (based on clinical topography), presence of anti-AQP4 antibodies, particular MRI findings, such as a large brain lesion (> 3 cm) or a LESCL, presence of OCBs and IgG index, presence of co-existing autoimmune diseases and autoantibodies, and therapy conditions, including whether patients had been exposed to DMDs, corticosteroids or other immunosuppressant agents. PI (EDSS score/disease duration) and ARR (number of relapses/disease duration) were calculated. For MS, disease course (RRMS, SPMS or PPMS) and the MSSS31 were also assessed. Based on previous studies, “benign course” was defined following the most popular definitions, in which the EDSS cut-off point was ≤ 2.0 or 3.0 after disease duration of at least 10 years43,62. In the subanalysis, participants were stratified by their resident area into two groups (northern and southern Japan) at a latitude of 37° North, as previously described1. Ethical committees of National Institutes of Biomedical Innovation, Health and Nutrition, Kyushu University, National Hospital Organization Hokkaido Medical Center, Saitama Medical Center, National Hospital Organization Utano National Hospital, Ehime University Graduate School of Medicine, Yamaguchi University Graduate School of Medicine, Tokyo Medical and Dental University, Kanazawa Medical University, Kindai University, Iwate Medical University, Niigata University, Tokyo Women's Medical University Yachiyo Medical Center, Sapporo Medical University School of Medicine, Kitasato University School of Medicine, Osaka University Graduate School of Medicine, Hiroshima University, National Hospital Organization Omuta National Hospital, The University of Tokyo, Tokyo Women’s Medical University, National Hospital Organization Nagasaki Kawatana Medical Center and University of Occupational and Environmental Health School of Medicine reviewed and approved this study. This study was conducted according to the World Medical Association Declaration of Helsinki. All participants provided written informed consent between September 2013 and June 2017.

Genotyping

The participants’ genotypes of HLA-DRB1 and -DPB1 alleles were determined by the sequence-specific oligonucleotide probe method (Luminex method) using DNA samples at the National Institutes of Biomedical Innovation, Health and Nutrition and the HLA Laboratory (Kyoto, Japan), as described previously16,63. The carrier frequencies of Japanese healthy controls from a Japan Multiple Sclerosis Genetics Consortium study were used13.

Statistical analysis

To compare the demographic characteristics and clinical information between two groups, Fisher’s exact test was used for categorical data, whereas the Wilcoxon rank sum test was used for continuous data. To identify the susceptibility and protective alleles of HLA-DRB1 and -DPB1, the phenotypic frequencies of these alleles were compared using Fisher’s exact test. Uncorrected p values (puncorr) were multiplied by the number of comparisons (Bonferroni-Dunn’s correction) to calculate pcorr values. To calculate the risk of optic neuritis and FS scores (≥ 1) of visual function for MS patients with HLA-DRB1*04:05, logistic regression analyses were used with adjustment for disease duration. When comparing the proportions of disease course between groups, a likelihood ratio chi-square test was employed as it includes more than two categories. To identify independent factors that correlate with disease disability, EDSS scores were rank-normalized and multivariable linear regression analyses were performed. All analyses were carried out using JMP version 14.1.0 (SAS Institute, Cary, NC, USA). Statistical significance was set at p < 0.05.