Journal of the Neurological Sciences 348 (2015) 132–135
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Presence of anti-Ro/SSA antibody may be associated with anti-aquaporin-4 antibody positivity in neuromyelitis optica spectrum disorder Jae-Hyun Park a, Jaechun Hwang a, Ju-Hong Min a, Byoung Joon Kim a, Eun-Suk Kang b, Kwang Ho Lee a,⁎ a b
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 23 June 2014 Received in revised form 12 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Neuromyelitis optica spectrum disorder Anti-Ro/SSA antibody Anti-aquaporin-4 antibody Neuromyelitis optica Sjögren's syndrome Non-organ-specific autoantibody
a b s t r a c t Background: Neuromyelitis optica (NMO) is often associated with systemic autoimmune diseases or serological markers of non-organ-specific autoimmunity, and has been most frequently associated with Sjögren's syndrome and anti-Ro/SSA antibody (SSA-Ab) positivity in Asian populations. Objective: We evaluated the clinical significance of anti-Ro/SSA antibody positivity in patients with NMO spectrum disorder (NMOSD). Methods: We retrospectively collected data from 106 consecutive patients with NMOSD and reviewed clinical features and laboratory findings. All patients underwent tests for SSA-Ab and anti-aquaporin-4 antibody (AQP4-Ab) using cell-based indirect immunofluorescence assays. Results: Among 106 patients, 20 (18.9%) were positive for SSA-Ab. Of 48 AQP4-Ab-positive patients, 18 (37.5%) had SSA-Ab. AQP4-Ab seropositivity was 90.0% in patients positive for SSA-Ab, and 32.6% in patients without SSA-Ab (p b 0.001). Presence of SSA-Ab was associated with systemic autoimmune diseases, including Sjögren's syndrome (p b 0.001) and systemic lupus erythematosus (p = 0.003), and with the presence of non-organ-specific autoantibodies such as anti-nuclear antibody and anti-dsDNA antibody in patients with NMOSD, but was not associated with annualized relapse rate or final Expanded Disability Status Scale score independent of AQP4-Ab positivity. Conclusion: We found that the presence of SSA-Ab was highly associated with seropositivity for AQP4-Ab in patients with NMOSD. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neuromyelitis optica (NMO) is a severe idiopathic inflammatory disease of the central nervous system (CNS) that predominantly affects the optic nerves and spinal cord. [1] Antibodies against aquaporin-4 (AQP4), the main water channel in the CNS, are present in 60–80% of NMO patients [2,3]. Anti-AQP4 antibody (AQP4-Ab) distinguishes NMO from other demyelinating disorders and is related to an increased NMO relapse rate [2–6]. The broadened group of disorders associated with AQP4-Ab has been suggested to constitute NMO spectrum disorder (NMOSD) [1]. NMO is strongly associated with other systemic autoimmune diseases, such as Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE), and with the presence of non-organ-specific autoantibodies [7,8]. These associations have recently emerged, which are important for understanding the pathophysiology of NMO [9].
⁎ Corresponding author at: Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135710, Republic of Korea. Tel.: +82 2 3410 3590; fax: +82 2 3410 0052. E-mail address: [email protected] (K.H. Lee).
http://dx.doi.org/10.1016/j.jns.2014.11.020 0022-510X/© 2014 Elsevier B.V. All rights reserved.
In particular, the co-occurrence of NMO and SS has been suggested by several Asian population studies [10–13]. In those studies, many patients had initial neurological manifestations before the development of other features of systemic SS, which may result in an underestimation of NMO associated with SS at the time of initial neurological presentation [7,10]. Anti-Ro/SSA antibody (SSA-Ab) is more frequently detected than anti-La/SSB antibody (SSB-Ab) in patients with NMO or NMOSD, and is associated with recurrent transverse myelitis [7,11]. Therefore, in this study, we used SSA-Ab as a surrogate marker for primary and secondary SS in patients with NMO/NMOSD of optic neuritis and/or longitudinally extensive transverse myelitis (LETM). We elucidated clinical and immunological characteristics of SSA-Ab positivity and negativity in NMO/NMOSD patients, and specifically the frequency of anti-AQP4 antibody positivity. 2. Materials and methods 2.1. Patients We retrospectively collected data from consecutive patients with NMOSD, including NMO, who visited the Neurology Department of
J.-H. Park et al. / Journal of the Neurological Sciences 348 (2015) 132–135
Samsung Medical Center from 2007 to 2011. We identified patients who underwent both SSA-Ab and anti-AQP4 antibody tests, and 106 patients were ultimately included. We used the NMOSD criteria proposed by Wingerchuk et al. [1], and all patients underwent a thorough clinical history taking, neurological examinations, and routine laboratory tests. We reviewed demographic features, clinical features, disability as measured by the Expanded Disability Status Scale (EDSS), and the results of serological tests, including those for antinuclear antibody (ANA), SSA-Ab, SSB-Ab, and anti-double stranded DNA (anti-dsDNA) antibody. To evaluate the prognostic value of the SSA-Ab, we reviewed the initial SSA-Ab results obtained at our hospital. We used laboratory data obtained within 1 month of the onset of a recurrent clinical attack. Diagnoses of SS and SLE were based on American-European criteria for SS [14] and updated classification criteria for SLE [15,16]. The Institutional Review Board of Samsung Medical Center approved this study, and all patients provided informed consent to participate in this study. 2.2. Anti-AQP4 antibody assay Serum samples were obtained from patients within 1 month of attack onset. The test for AQP4-Ab was performed either immediately after serum sampling or after storage at − 70 °C. AQP4-Ab was measured using a commercially available cell-based immunofluorescence assay kit from EUROIMMUN AG (Lubeck, Germany) according to the manufacturer's instructions [17]. 2.3. Statistical analyses We analyzed the clinical and laboratory differences between the SSA-Ab-positive and -negative groups using Pearson's χ [2] test or Fisher's exact test for categorical variables. For continuous variables, t-tests or Mann–Whitney U tests were used as appropriate. Data were processed using the PASW Statistics 18 (SPSS Inc., Chicago, IL, USA) software package. A p-value b 0.05 was considered statistically significant. 3. Results Among the 106 NMOSD patients, 20 (18.9%) were seropositive for SSA-Ab and 86 (81.1%) were seronegative. A comparison of the demographics and clinical characteristics of SSA-Ab-positive and -negative groups is shown in Tables 1 and 2. Females were more frequently SSA-Ab positive than negative (18/20 vs. 53/86, p = 0.015) and AQP4Ab positive than negative (40/46 vs. 31/60, p b 0.001). There were no significant differences in clinical features, disease duration, attack number, or annualized relapse rate between the SSA-Ab-positive and -negative groups. EDSS score at last observation was higher in the SSA-Ab-positive than it was in the -negative group (median: 3.5 vs. 2, p = 0.004). Considering the influence of AQP4-Ab on clinical course in patients with SSA-Ab, we compared EDSS scores at the last observation between SSA-Ab-positive and -negative subgroups of the AQP4-Abpositive group (Table 2). There were no significant difference in EDSS score (at last observation) between the SSA-Ab-positive and -negative subgroups (median: 3 vs. 4, p = 0.273) of the AQP4-Ab-positive group. Eighteen of 20 patients (90.0%) positive for SSA-Ab also exhibited AQP4-Ab positivity, while 28 (32.6%) patients negative for SSA-Ab were AQP4-Ab-positive (p b 0.001). Table 3 shows the comparison of AQP4-Ab positivity between SSA-Ab-positive and -negative groups with characteristic clinical syndrome or features of NMOSD. In patients presenting with overall myelitis (including recurrent myelitis, LETM, or a combination of myelitis and optic neuritis), with LETM specifically, or with recurrent/bilateral optic neuritis, the frequency of AQP4-Ab positivity was significantly higher in the SSA-Ab-positive group than in the -negative group (p-values for myelitis group b 0.001, for LETM b 0.001, and for recurrent/bilateral optic neuritis = 0.001). Seventeen NMOSD patients positive for SSA-Ab had recurrent episodes involving
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Table 1 Demographic, clinical and serological features of patients with NMOSD based on SSA positivity.
Age at onset, years Male:female, n Follow-up duration, years Neurological manifestation Optic neuritis, n (%) Recurrent or bilateral optic neuritis, n (%) Myelitis, n (%) LETM, n (%) Recurrent LETM, n (%) Optic-spinal multiple sclerosis, n (%) Disease duration, years Number of clinical attacks Annualized relapse rate Time to immunosuppressive therapy, years Final EDSS score Anti-AQP4 antibody, n (%) Met Sjögren's syndrome criteria, n (%) Met SLE criteria, n (%) Anti-La/SSB antibody, n (%) Anti-nuclear antibody, n (%) Anti-dsDNA antibody, n (%) Met 2006 NMO criteria, n (%) Titer of anti-Ro/SSA antibody
SSA-Ab positive (n = 20)
SSA-Ab negative (n = 86)
p-Value
39.1 ± 13.0 2:18 2.3 (0.6–6.9)
37.5 ± 12.9 33:53 1.5 (0.5–4.5)
0.616 0.015 0.208
12 (60.0) 7 (35.0)
53 (61.6) 40 (46.5)
0.893 0.351
18 (90.0) 15 (75.0) 10 (50.0) 0 (0)
65 (75.6) 50 (58.1) 27 (31.8) 4 (4.7)
0.159 0.163 0.125 0.326
3.1 (1.4–7.6) 4 (2–7) 1.16 (0.68–1.87) 1.34 (0.62–7.06)
2.9 (1.2–9.2) 3 (2–5) 1.02 (0.57–2.13) 2.4 (0.9–7.8)
0.691 0.364 0.920 0.438
3.5 (2–8) 18 (90.0) 14 (70.0)
2 (1–5) 28 (32.6) 7 (8.1)
0.004 b0.001 b0.001
2 (10.0) 9 (45.0) 19 (95.0) 4/17 (23.5) 10 (50.0) 420 (180–1060)
0 (0) 0 (0) 33 (38.4) 2/73 (2.7) 28 (32.6) –
0.003 b0.001 b0.001 0.002 0.143 –
Continuous variables are mean ± standard deviation (SD) or median (interquartile range). LETM, longitudinally extensive transverse myelitis; EDSS, Expanded Disability Status Scale; AQP4, aquaporin-4; SLE, systemic lupus erythematosus; SSA-Ab, anti-Ro/SSA antibody; dsDNA, double-stranded DNA.
the CNS, and only three patients positive for SSA-Ab presented with a first-ever attack when they were seen initially at our hospital. We additionally evaluated clinical features and autoantibody profiles according to the presence of AQP4-Ab. The presence of AQP4-Ab was associated with recurrent LETM (21/46 vs. 16/60, p = 0.034) but not with recurrent or bilateral optic neuritis (21/46 vs. 26/60, p = 0.812). The presence of AQP4-Ab was related with that of non-organspecific autoantibodies, namely SSA-Ab (18/46 vs. 2/60, p b 0.001), SSB-Ab (8/46 vs. 1/60, p = 0.004), ANA (37/46 vs. 15/60, p b 0.001), and anti-dsDNA antibody (5/39 vs. 1/51, p = 0.041). Fourteen (70.0%) SSA-Ab-positive patients and 7 (8.1%) who were SSA-Ab-negative met the criteria for SS (p b 0.001). Among the 21 patients who ultimately fulfilled the criteria for SS, none were diagnosed
Table 2 Comparison of demographic and clinical features between anti-Ro/SSA-positive and -negative groups among anti-AQP4-Ab positive NMOSD patients. AQP4-Ab positive (n = 46)
Age at onset, years Male:female, n Follow-up duration, years Disease duration, years Number of clinical attacks Annualized relapse rate Final EDSS score Met 2006 NMO criteria, n (%)
SSA-Ab positive (n = 18)
SSA-Ab negative (n = 28)
p-Value
38.9 ± 13.4 2:16 2.3 (0.4–7.5) 3.1 (1.2–8.4) 4 (2–8) 1.35 (0.74–1.90) 3 (2–8) 10 (55.6)
34.3 ± 13.8 4:24 3.5 (0.7–9.3) 6.1 (1.6–11.2) 4 (3–7) 0.96 (0.58–2.10) 4 (1–5) 21 (75.0)
0.270 0.755 0.485 0.209 0.754 0.471 0.273 0.170
Continuous variables are mean ± standard deviation (SD) or median (interquartile range). EDSS, Expanded Disability Status Scale; SSA-Ab, anti-Ro/SSA antibody.
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Table 3 Frequency of AQP4-Ab positivity in patients with characteristic clinical syndrome or features of NMOSD. AQP4-Ab positivity Clinical syndrome or features of NMOSD
N
SSA-Ab positive
SSA-Ab negative
p-Value
NMO, n Myelitis, n LETM, total, n Recurrent LETM, n Recurrent or bilateral optic neuritis, n
38 83 65 37 47
10/10 16/18 14/15 9/10 7/7
21/28 26/65 19/50 12/27 14/40
0.156 b0.001 b0.001 0.013 0.001
LETM, longitudinally extensive transverse myelitis; SSA-Ab, anti-Ro/SSA antibody.
with SS before having developed NMOSD symptoms. Three patients had preceding systemic SS symptoms, such as dry eye, dry mouth, and arthritis before their initial neurological presentation. However, the remaining 18 patients did not experience SS symptoms. SS was diagnosed in 5 patients at the initial evaluation for NMOSD, and 16 were diagnosed with SS at NMOSD follow-up. Anti-dsDNA antibodies, which are highly specific serological markers for SLE, were observed in 4/17 (23.5%) patients positive for SSA-Ab, and in 2/73 (2.7%) negative for SSA-Ab (p = 0.002). Two of 20 SSA-Ab-positive patients (10.0%) met the criteria for SLE and SS. No patient satisfied the diagnostic criteria for SLE in the SSA-Ab-negative group. SSB-Ab was observed in 9 (45.0%) patients in the SSA-Abpositive group, but none in the negative group (p b 0.001). Nineteen of 20 (95.0%) SSA-Ab-positive patients and 33 (38.4%) SSA-Abnegative patients were positive for ANA (p b 0.001). The frequency of patients who met the NMO diagnostic criteria did not differ between the two groups (anti-Ro/SSA-positive vs. -negative group: 50.0% vs. 32.6%, respectively, p = 0.143). 4. Discussion The main findings of this study were that NMOSD patients were often seropositive for SSA-Ab, and that SSA-Ab positivity was highly associated with the presence of AQP4-Ab. SSA-Ab positivity was also closely related to systemic autoimmune diseases, including SS and SLE, and to positivity for non-organ-specific autoantibodies, such as ANA and anti-dsDNA, in patients with NMOSD. The presence of SSA-Ab was not associated with clinical manifestation of the disease or relapse rate. The final EDSS score was higher in the SSA-Ab-positive group, but no significant difference was observed in the final EDSS score between the SSA-Ab-positive and -negative subgroups of the AQP4-Ab-positive group. AQP4-Ab positivity was associated with positivity for other non-organ-specific autoantibodies, including SSA-Ab, SSB-Ab, ANA, and anti-dsDNA, and was also related with recurrent LETM. The frequency of AQP4-Ab positivity was significantly higher in the SSA-Ab-positive than in the -negative group in overall patients and in patients with characteristic clinical syndrome or features of NMOSD. These findings may indicate that NMOSD patients with AQP4-Ab may have other non-organ-specific autoantibodies, and may consequently experience recurrent attacks. Pittock and colleagues reported that 10.5% of 153 American NMOSD patients and 44.4% of 18 French patients were SSA-Ab positive [7]. In another study of 106 Korean patients with AQP4-Ab, 33% had non-organspecific autoantibodies, most commonly ANA and SSA-Ab [18]. In this study, the frequency of SSA-Ab was 18.9% in all NMOSD patients and 39.1% in NMOSD patients with AQP4-Ab, which is in agreement with the results of previous reports. The mechanisms underlying the coassociation of systemic humoral autoimmune diseases/non-organ– specific autoantibodies and AQP4-Ab in patients with NMOSD presenting with myelitis and optic neuritis remain to be elucidated. AQP4-Ab is considered to be associated with pathogenicity of NMO; however, several findings indicate that AQP4-Ab alone may not cause
CNS lesions. First, AQP4-Ab was detected during remission in some patients with NMO [19], and many years before the onset of NMO in other patients [20]. Second, in animal models of NMO, passive antibody transfer to rodents had no effect unless disruption of the blood–brain barrier or brain inflammation was induced [21]. Systemic inflammatory factors such as autoantibodies or other inflammatory mechanisms may contribute to the disruption of the blood–brain barrier, or create the inflammatory environment required for AQP4-Ab induction. SSA-Ab may be associated with disruption of the blood–brain barrier. SSA-antigen is present in endothelial cells and SSA-Ab is thought to cause endothelial damage [22], allowing AQP4-Ab to gain access to aquaporin-4, the target antigen of AQP4-Ab, after disruption of the blood–brain barrier. In SS, other possible manifestations of CNS lesions include small-vessel vasculitis and antiphospholipid syndrome [22,23]. In this study, 17 of 20 SSA-Ab-positive NMOSD patients had recurrent episodes of CNS involvement, similar to a previous report demonstrating that SSA-Ab was associated with recurrent transverse myelitis [24]. In this study, two SSA-Ab-positive patients did not initially show AQP4-Ab positivity. One developed AQP4-Ab with relapses during follow-up and the other did not undergo further AQP4-Ab testing. Both received immunosuppressive therapy at the time of initial blood sampling. Fluctuation in levels of serum autoantibodies (e.g. anti-ds DNA, and antibodies to Ro/SSA or AQP4) and positive or negative seroconversion have been reported previously in patients with autoimmune diseases [25–27]. Therefore serial follow-up of the AQP4-Ab assays would be helpful to detect AQP4-Ab if the initial test was negative. The sensitivity of the AQP4-Ab assays can affect the detection of AQP4-Ab in those patients. We used cell-based immunofluorescence assays that are as sensitive as in-house fluorescence-activated cell sorting assays and more sensitive than indirect immunofluorescence assays and enzyme-linked immunosorbent assays [27,28]. This study had several limitations. The study's results should be interpreted with caution due to its retrospective design in a tertiary medical center. Further prospective large studies are needed to elucidate the clinical impact of SSA-Ab and the association of systemic autoimmune diseases with NMO. In conclusion, we suggest that determination of SSA-Ab positivity is useful in clinical practice for patients with NMOSD. Moreover, this study can help clarify the complex relationship between NMO and other systemic autoimmune diseases. Conflicts of interest None to be declared. Acknowledgments This study was supported by a grant from the Korea Healthcare Technology R&D project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (grant number A080588-28). References [1] Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG. The spectrum of neuromyelitis optica. Lancet Neurol 2007;6:805–15. [2] Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202: 473–7. [3] Lennon VA, Wingerchuk DM, Kryzer TJ, Pittock SJ, Lucchinetti CF, Fujihara K, et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364:2106–12. [4] Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–9. [5] Jarius S, Franciotta D, Bergamaschi R, Wright H, Littleton E, Palace J, et al. NMO-IgG in the diagnosis of neuromyelitis optica. Neurology 2007;68:1076–7. [6] Weinshenker BG, Wingerchuk DM, Vuklisic S, Linbo L, Pittock SJ, Lucchinetti CF, et al. Neuromyelitis optica IgG predicts relapse after longitudinally extensive transverse myelitis. Ann Neurol 2006;59:566–9.
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Presence of anti-Ro/SSA antibody may be associated with anti-aquaporin-4 antibody positivity in neuromyelitis optica spectrum disorder Jae-Hyun Park a, Jaechun Hwang a, Ju-Hong Min a, Byoung Joon Kim a, Eun-Suk Kang b, Kwang Ho Lee a,⁎ a b
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 23 June 2014 Received in revised form 12 November 2014 Accepted 14 November 2014 Available online 20 November 2014 Keywords: Neuromyelitis optica spectrum disorder Anti-Ro/SSA antibody Anti-aquaporin-4 antibody Neuromyelitis optica Sjögren's syndrome Non-organ-specific autoantibody
a b s t r a c t Background: Neuromyelitis optica (NMO) is often associated with systemic autoimmune diseases or serological markers of non-organ-specific autoimmunity, and has been most frequently associated with Sjögren's syndrome and anti-Ro/SSA antibody (SSA-Ab) positivity in Asian populations. Objective: We evaluated the clinical significance of anti-Ro/SSA antibody positivity in patients with NMO spectrum disorder (NMOSD). Methods: We retrospectively collected data from 106 consecutive patients with NMOSD and reviewed clinical features and laboratory findings. All patients underwent tests for SSA-Ab and anti-aquaporin-4 antibody (AQP4-Ab) using cell-based indirect immunofluorescence assays. Results: Among 106 patients, 20 (18.9%) were positive for SSA-Ab. Of 48 AQP4-Ab-positive patients, 18 (37.5%) had SSA-Ab. AQP4-Ab seropositivity was 90.0% in patients positive for SSA-Ab, and 32.6% in patients without SSA-Ab (p b 0.001). Presence of SSA-Ab was associated with systemic autoimmune diseases, including Sjögren's syndrome (p b 0.001) and systemic lupus erythematosus (p = 0.003), and with the presence of non-organ-specific autoantibodies such as anti-nuclear antibody and anti-dsDNA antibody in patients with NMOSD, but was not associated with annualized relapse rate or final Expanded Disability Status Scale score independent of AQP4-Ab positivity. Conclusion: We found that the presence of SSA-Ab was highly associated with seropositivity for AQP4-Ab in patients with NMOSD. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neuromyelitis optica (NMO) is a severe idiopathic inflammatory disease of the central nervous system (CNS) that predominantly affects the optic nerves and spinal cord. [1] Antibodies against aquaporin-4 (AQP4), the main water channel in the CNS, are present in 60–80% of NMO patients [2,3]. Anti-AQP4 antibody (AQP4-Ab) distinguishes NMO from other demyelinating disorders and is related to an increased NMO relapse rate [2–6]. The broadened group of disorders associated with AQP4-Ab has been suggested to constitute NMO spectrum disorder (NMOSD) [1]. NMO is strongly associated with other systemic autoimmune diseases, such as Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE), and with the presence of non-organ-specific autoantibodies [7,8]. These associations have recently emerged, which are important for understanding the pathophysiology of NMO [9].
⁎ Corresponding author at: Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135710, Republic of Korea. Tel.: +82 2 3410 3590; fax: +82 2 3410 0052. E-mail address: [email protected] (K.H. Lee).
http://dx.doi.org/10.1016/j.jns.2014.11.020 0022-510X/© 2014 Elsevier B.V. All rights reserved.
In particular, the co-occurrence of NMO and SS has been suggested by several Asian population studies [10–13]. In those studies, many patients had initial neurological manifestations before the development of other features of systemic SS, which may result in an underestimation of NMO associated with SS at the time of initial neurological presentation [7,10]. Anti-Ro/SSA antibody (SSA-Ab) is more frequently detected than anti-La/SSB antibody (SSB-Ab) in patients with NMO or NMOSD, and is associated with recurrent transverse myelitis [7,11]. Therefore, in this study, we used SSA-Ab as a surrogate marker for primary and secondary SS in patients with NMO/NMOSD of optic neuritis and/or longitudinally extensive transverse myelitis (LETM). We elucidated clinical and immunological characteristics of SSA-Ab positivity and negativity in NMO/NMOSD patients, and specifically the frequency of anti-AQP4 antibody positivity. 2. Materials and methods 2.1. Patients We retrospectively collected data from consecutive patients with NMOSD, including NMO, who visited the Neurology Department of
J.-H. Park et al. / Journal of the Neurological Sciences 348 (2015) 132–135
Samsung Medical Center from 2007 to 2011. We identified patients who underwent both SSA-Ab and anti-AQP4 antibody tests, and 106 patients were ultimately included. We used the NMOSD criteria proposed by Wingerchuk et al. [1], and all patients underwent a thorough clinical history taking, neurological examinations, and routine laboratory tests. We reviewed demographic features, clinical features, disability as measured by the Expanded Disability Status Scale (EDSS), and the results of serological tests, including those for antinuclear antibody (ANA), SSA-Ab, SSB-Ab, and anti-double stranded DNA (anti-dsDNA) antibody. To evaluate the prognostic value of the SSA-Ab, we reviewed the initial SSA-Ab results obtained at our hospital. We used laboratory data obtained within 1 month of the onset of a recurrent clinical attack. Diagnoses of SS and SLE were based on American-European criteria for SS [14] and updated classification criteria for SLE [15,16]. The Institutional Review Board of Samsung Medical Center approved this study, and all patients provided informed consent to participate in this study. 2.2. Anti-AQP4 antibody assay Serum samples were obtained from patients within 1 month of attack onset. The test for AQP4-Ab was performed either immediately after serum sampling or after storage at − 70 °C. AQP4-Ab was measured using a commercially available cell-based immunofluorescence assay kit from EUROIMMUN AG (Lubeck, Germany) according to the manufacturer's instructions [17]. 2.3. Statistical analyses We analyzed the clinical and laboratory differences between the SSA-Ab-positive and -negative groups using Pearson's χ [2] test or Fisher's exact test for categorical variables. For continuous variables, t-tests or Mann–Whitney U tests were used as appropriate. Data were processed using the PASW Statistics 18 (SPSS Inc., Chicago, IL, USA) software package. A p-value b 0.05 was considered statistically significant. 3. Results Among the 106 NMOSD patients, 20 (18.9%) were seropositive for SSA-Ab and 86 (81.1%) were seronegative. A comparison of the demographics and clinical characteristics of SSA-Ab-positive and -negative groups is shown in Tables 1 and 2. Females were more frequently SSA-Ab positive than negative (18/20 vs. 53/86, p = 0.015) and AQP4Ab positive than negative (40/46 vs. 31/60, p b 0.001). There were no significant differences in clinical features, disease duration, attack number, or annualized relapse rate between the SSA-Ab-positive and -negative groups. EDSS score at last observation was higher in the SSA-Ab-positive than it was in the -negative group (median: 3.5 vs. 2, p = 0.004). Considering the influence of AQP4-Ab on clinical course in patients with SSA-Ab, we compared EDSS scores at the last observation between SSA-Ab-positive and -negative subgroups of the AQP4-Abpositive group (Table 2). There were no significant difference in EDSS score (at last observation) between the SSA-Ab-positive and -negative subgroups (median: 3 vs. 4, p = 0.273) of the AQP4-Ab-positive group. Eighteen of 20 patients (90.0%) positive for SSA-Ab also exhibited AQP4-Ab positivity, while 28 (32.6%) patients negative for SSA-Ab were AQP4-Ab-positive (p b 0.001). Table 3 shows the comparison of AQP4-Ab positivity between SSA-Ab-positive and -negative groups with characteristic clinical syndrome or features of NMOSD. In patients presenting with overall myelitis (including recurrent myelitis, LETM, or a combination of myelitis and optic neuritis), with LETM specifically, or with recurrent/bilateral optic neuritis, the frequency of AQP4-Ab positivity was significantly higher in the SSA-Ab-positive group than in the -negative group (p-values for myelitis group b 0.001, for LETM b 0.001, and for recurrent/bilateral optic neuritis = 0.001). Seventeen NMOSD patients positive for SSA-Ab had recurrent episodes involving
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Table 1 Demographic, clinical and serological features of patients with NMOSD based on SSA positivity.
Age at onset, years Male:female, n Follow-up duration, years Neurological manifestation Optic neuritis, n (%) Recurrent or bilateral optic neuritis, n (%) Myelitis, n (%) LETM, n (%) Recurrent LETM, n (%) Optic-spinal multiple sclerosis, n (%) Disease duration, years Number of clinical attacks Annualized relapse rate Time to immunosuppressive therapy, years Final EDSS score Anti-AQP4 antibody, n (%) Met Sjögren's syndrome criteria, n (%) Met SLE criteria, n (%) Anti-La/SSB antibody, n (%) Anti-nuclear antibody, n (%) Anti-dsDNA antibody, n (%) Met 2006 NMO criteria, n (%) Titer of anti-Ro/SSA antibody
SSA-Ab positive (n = 20)
SSA-Ab negative (n = 86)
p-Value
39.1 ± 13.0 2:18 2.3 (0.6–6.9)
37.5 ± 12.9 33:53 1.5 (0.5–4.5)
0.616 0.015 0.208
12 (60.0) 7 (35.0)
53 (61.6) 40 (46.5)
0.893 0.351
18 (90.0) 15 (75.0) 10 (50.0) 0 (0)
65 (75.6) 50 (58.1) 27 (31.8) 4 (4.7)
0.159 0.163 0.125 0.326
3.1 (1.4–7.6) 4 (2–7) 1.16 (0.68–1.87) 1.34 (0.62–7.06)
2.9 (1.2–9.2) 3 (2–5) 1.02 (0.57–2.13) 2.4 (0.9–7.8)
0.691 0.364 0.920 0.438
3.5 (2–8) 18 (90.0) 14 (70.0)
2 (1–5) 28 (32.6) 7 (8.1)
0.004 b0.001 b0.001
2 (10.0) 9 (45.0) 19 (95.0) 4/17 (23.5) 10 (50.0) 420 (180–1060)
0 (0) 0 (0) 33 (38.4) 2/73 (2.7) 28 (32.6) –
0.003 b0.001 b0.001 0.002 0.143 –
Continuous variables are mean ± standard deviation (SD) or median (interquartile range). LETM, longitudinally extensive transverse myelitis; EDSS, Expanded Disability Status Scale; AQP4, aquaporin-4; SLE, systemic lupus erythematosus; SSA-Ab, anti-Ro/SSA antibody; dsDNA, double-stranded DNA.
the CNS, and only three patients positive for SSA-Ab presented with a first-ever attack when they were seen initially at our hospital. We additionally evaluated clinical features and autoantibody profiles according to the presence of AQP4-Ab. The presence of AQP4-Ab was associated with recurrent LETM (21/46 vs. 16/60, p = 0.034) but not with recurrent or bilateral optic neuritis (21/46 vs. 26/60, p = 0.812). The presence of AQP4-Ab was related with that of non-organspecific autoantibodies, namely SSA-Ab (18/46 vs. 2/60, p b 0.001), SSB-Ab (8/46 vs. 1/60, p = 0.004), ANA (37/46 vs. 15/60, p b 0.001), and anti-dsDNA antibody (5/39 vs. 1/51, p = 0.041). Fourteen (70.0%) SSA-Ab-positive patients and 7 (8.1%) who were SSA-Ab-negative met the criteria for SS (p b 0.001). Among the 21 patients who ultimately fulfilled the criteria for SS, none were diagnosed
Table 2 Comparison of demographic and clinical features between anti-Ro/SSA-positive and -negative groups among anti-AQP4-Ab positive NMOSD patients. AQP4-Ab positive (n = 46)
Age at onset, years Male:female, n Follow-up duration, years Disease duration, years Number of clinical attacks Annualized relapse rate Final EDSS score Met 2006 NMO criteria, n (%)
SSA-Ab positive (n = 18)
SSA-Ab negative (n = 28)
p-Value
38.9 ± 13.4 2:16 2.3 (0.4–7.5) 3.1 (1.2–8.4) 4 (2–8) 1.35 (0.74–1.90) 3 (2–8) 10 (55.6)
34.3 ± 13.8 4:24 3.5 (0.7–9.3) 6.1 (1.6–11.2) 4 (3–7) 0.96 (0.58–2.10) 4 (1–5) 21 (75.0)
0.270 0.755 0.485 0.209 0.754 0.471 0.273 0.170
Continuous variables are mean ± standard deviation (SD) or median (interquartile range). EDSS, Expanded Disability Status Scale; SSA-Ab, anti-Ro/SSA antibody.
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Table 3 Frequency of AQP4-Ab positivity in patients with characteristic clinical syndrome or features of NMOSD. AQP4-Ab positivity Clinical syndrome or features of NMOSD
N
SSA-Ab positive
SSA-Ab negative
p-Value
NMO, n Myelitis, n LETM, total, n Recurrent LETM, n Recurrent or bilateral optic neuritis, n
38 83 65 37 47
10/10 16/18 14/15 9/10 7/7
21/28 26/65 19/50 12/27 14/40
0.156 b0.001 b0.001 0.013 0.001
LETM, longitudinally extensive transverse myelitis; SSA-Ab, anti-Ro/SSA antibody.
with SS before having developed NMOSD symptoms. Three patients had preceding systemic SS symptoms, such as dry eye, dry mouth, and arthritis before their initial neurological presentation. However, the remaining 18 patients did not experience SS symptoms. SS was diagnosed in 5 patients at the initial evaluation for NMOSD, and 16 were diagnosed with SS at NMOSD follow-up. Anti-dsDNA antibodies, which are highly specific serological markers for SLE, were observed in 4/17 (23.5%) patients positive for SSA-Ab, and in 2/73 (2.7%) negative for SSA-Ab (p = 0.002). Two of 20 SSA-Ab-positive patients (10.0%) met the criteria for SLE and SS. No patient satisfied the diagnostic criteria for SLE in the SSA-Ab-negative group. SSB-Ab was observed in 9 (45.0%) patients in the SSA-Abpositive group, but none in the negative group (p b 0.001). Nineteen of 20 (95.0%) SSA-Ab-positive patients and 33 (38.4%) SSA-Abnegative patients were positive for ANA (p b 0.001). The frequency of patients who met the NMO diagnostic criteria did not differ between the two groups (anti-Ro/SSA-positive vs. -negative group: 50.0% vs. 32.6%, respectively, p = 0.143). 4. Discussion The main findings of this study were that NMOSD patients were often seropositive for SSA-Ab, and that SSA-Ab positivity was highly associated with the presence of AQP4-Ab. SSA-Ab positivity was also closely related to systemic autoimmune diseases, including SS and SLE, and to positivity for non-organ-specific autoantibodies, such as ANA and anti-dsDNA, in patients with NMOSD. The presence of SSA-Ab was not associated with clinical manifestation of the disease or relapse rate. The final EDSS score was higher in the SSA-Ab-positive group, but no significant difference was observed in the final EDSS score between the SSA-Ab-positive and -negative subgroups of the AQP4-Ab-positive group. AQP4-Ab positivity was associated with positivity for other non-organ-specific autoantibodies, including SSA-Ab, SSB-Ab, ANA, and anti-dsDNA, and was also related with recurrent LETM. The frequency of AQP4-Ab positivity was significantly higher in the SSA-Ab-positive than in the -negative group in overall patients and in patients with characteristic clinical syndrome or features of NMOSD. These findings may indicate that NMOSD patients with AQP4-Ab may have other non-organ-specific autoantibodies, and may consequently experience recurrent attacks. Pittock and colleagues reported that 10.5% of 153 American NMOSD patients and 44.4% of 18 French patients were SSA-Ab positive [7]. In another study of 106 Korean patients with AQP4-Ab, 33% had non-organspecific autoantibodies, most commonly ANA and SSA-Ab [18]. In this study, the frequency of SSA-Ab was 18.9% in all NMOSD patients and 39.1% in NMOSD patients with AQP4-Ab, which is in agreement with the results of previous reports. The mechanisms underlying the coassociation of systemic humoral autoimmune diseases/non-organ– specific autoantibodies and AQP4-Ab in patients with NMOSD presenting with myelitis and optic neuritis remain to be elucidated. AQP4-Ab is considered to be associated with pathogenicity of NMO; however, several findings indicate that AQP4-Ab alone may not cause
CNS lesions. First, AQP4-Ab was detected during remission in some patients with NMO [19], and many years before the onset of NMO in other patients [20]. Second, in animal models of NMO, passive antibody transfer to rodents had no effect unless disruption of the blood–brain barrier or brain inflammation was induced [21]. Systemic inflammatory factors such as autoantibodies or other inflammatory mechanisms may contribute to the disruption of the blood–brain barrier, or create the inflammatory environment required for AQP4-Ab induction. SSA-Ab may be associated with disruption of the blood–brain barrier. SSA-antigen is present in endothelial cells and SSA-Ab is thought to cause endothelial damage [22], allowing AQP4-Ab to gain access to aquaporin-4, the target antigen of AQP4-Ab, after disruption of the blood–brain barrier. In SS, other possible manifestations of CNS lesions include small-vessel vasculitis and antiphospholipid syndrome [22,23]. In this study, 17 of 20 SSA-Ab-positive NMOSD patients had recurrent episodes of CNS involvement, similar to a previous report demonstrating that SSA-Ab was associated with recurrent transverse myelitis [24]. In this study, two SSA-Ab-positive patients did not initially show AQP4-Ab positivity. One developed AQP4-Ab with relapses during follow-up and the other did not undergo further AQP4-Ab testing. Both received immunosuppressive therapy at the time of initial blood sampling. Fluctuation in levels of serum autoantibodies (e.g. anti-ds DNA, and antibodies to Ro/SSA or AQP4) and positive or negative seroconversion have been reported previously in patients with autoimmune diseases [25–27]. Therefore serial follow-up of the AQP4-Ab assays would be helpful to detect AQP4-Ab if the initial test was negative. The sensitivity of the AQP4-Ab assays can affect the detection of AQP4-Ab in those patients. We used cell-based immunofluorescence assays that are as sensitive as in-house fluorescence-activated cell sorting assays and more sensitive than indirect immunofluorescence assays and enzyme-linked immunosorbent assays [27,28]. This study had several limitations. The study's results should be interpreted with caution due to its retrospective design in a tertiary medical center. Further prospective large studies are needed to elucidate the clinical impact of SSA-Ab and the association of systemic autoimmune diseases with NMO. In conclusion, we suggest that determination of SSA-Ab positivity is useful in clinical practice for patients with NMOSD. Moreover, this study can help clarify the complex relationship between NMO and other systemic autoimmune diseases. Conflicts of interest None to be declared. Acknowledgments This study was supported by a grant from the Korea Healthcare Technology R&D project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (grant number A080588-28). References [1] Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG. The spectrum of neuromyelitis optica. Lancet Neurol 2007;6:805–15. [2] Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005;202: 473–7. [3] Lennon VA, Wingerchuk DM, Kryzer TJ, Pittock SJ, Lucchinetti CF, Fujihara K, et al. A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004;364:2106–12. [4] Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006;66:1485–9. [5] Jarius S, Franciotta D, Bergamaschi R, Wright H, Littleton E, Palace J, et al. NMO-IgG in the diagnosis of neuromyelitis optica. Neurology 2007;68:1076–7. [6] Weinshenker BG, Wingerchuk DM, Vuklisic S, Linbo L, Pittock SJ, Lucchinetti CF, et al. Neuromyelitis optica IgG predicts relapse after longitudinally extensive transverse myelitis. Ann Neurol 2006;59:566–9.
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