Detection of aquaporin‐4 antibodies for patients with CNS inflammatory demyelinating diseases other than typical MS in Lithuania

Abstract Objectives Neuromyelitis optica (NMO) is frequently associated with aquaporin‐4 autoantibodies (AQP4‐Ab); however, studies of NMO in Lithuania are lacking. Therefore, the main objective of our study is to assess positivity for AQP4‐Ab in patients presenting with inflammatory demyelinating central nervous system (CNS) diseases other than typical multiple sclerosis (MS) in Lithuania. Materials and methods Data were collected from the two largest University hospitals in Lithuania. During the study period, there were 121 newly diagnosed typical MS cases, which were included in the MS registry database. After excluding these typical MS cases, we analyzed the remaining 29 cases of other CNS inflammatory demyelinating diseases, including atypical MS (n = 14), acute transverse myelitis, TM (n = 8), acute disseminated encephalomyelitis, ADEM (n = 3), clinically isolated syndrome, CIS (n = 2), atypical optic neuritis, ON (n = 1), and NMO (n = 1). We assessed positivity for AQP4‐Ab for the 29 patients and evaluated clinical, laboratory, and instrumental differences between AQP4‐Ab seropositive and AQP4‐Ab seronegative patient groups. Results AQP4‐Ab test was positive for three (10.3%) patients in our study, with initial diagnoses of atypical MS (n = 2) and ADEM (n = 1). One study patient was AQP4‐Ab negative despite being previously clinically diagnosed with NMO. There were no significant clinical, laboratory, or instrumental differences between the groups of AQP4‐Ab positive (3 [10.3%]) and negative (26 [89.7%]) patients. Conclusions AQP4‐Ab test was positive for one‐tenth of patients with CNS inflammatory demyelinating diseases other than typical MS in our study. AQP4‐Ab testing is highly recommended for patients presenting with not only TM and ON but also an atypical course of MS and ADEM.


| INTRODUC TI ON
Neuromyelitis optica (NMO)-autoimmune inflammatory central nervous system (CNS) disease characterized by severe attacks of optic neuritis (ON) and longitudinally extensive transverse myelitis (LETM; Wingerchuk et al., 2015). A report by Antoine Portal , the first physician to king Louis XVIII, represents probably the first account of visual loss in a patient with spinal cord inflammation but no brain pathology in the Western literature (Jarius & Wildemann, 2012). French term neuro-myélite optique aiguë was first used by Eugène Devic  in a paper communicated on the occasion of the Congrès Français de Médecine in Lyon in 1894, where he denoted a novel syndrome characterized by acute myelitis and optic neuritis (Jarius & Wildemann, 2013).
In recent years, NMO has raised enormous interest among scientists and clinical neurologists, fueled by the detection of a highly specific serum immunoglobulin G autoantibody targeting the astrocytic water channel aquaporin-4 (AQP4) by Dr. Lennon and colleagues in 2004 (Jarius & Wildemann, 2013;Lennon et al., 2004;Lennon, Kryzer, Pittock, Verkman, & Hinson, 2005). This discovery has made clear that in most cases NMO is not a subform of multiple sclerosis (MS), but an autoimmune condition with an immunopathogenesis distinct from that of MS despite considerable overlap in clinical presentation and paraclinical findings . In 2007, the term NMO spectrum disorders (NMOSD) was introduced to include AQP4-Ab seropositive patients with limited or inaugural forms of NMO: first-attack LETM, recurrent or bilateral ON (Wingerchuk, Lennon, Lucchinetti, Pittock, & Weinshenker, 2007). The term also encompasses the cerebral, diencephalic, and brainstem lesions that occur in a minority of patients with otherwise typical NMO (Wingerchuk et al., 2015).
The percentage of NMO in Asia and the West Indies was known to be almost 50% of CNS demyelinating disorders (Kowarik et al., 2014). NMO was considered to be a rare disorder in Caucasians; however, this view was based on few studies with small patient populations from tertiary hospitals (Wu, Zhang, & Carroll, 2008).
The prevalence of AQP4-Ab positive patients is unknown in Lithuania. Therefore, the main objective of our study is to assess positivity for AQP4-Ab in patients presenting with demyelinating inflammatory CNS diseases other than typical MS in Lithuania (an atypical course of MS; acute transverse myelitis [TM]; severe, atypical ON; NMO; acute disseminated encephalomyelitis [ADEM]; and clinically isolated syndrome [CIS]). The secondary objectives of this study are to evaluate clinical, laboratory, and instrumental differences between AQP4-Ab seropositive and AQP4-Ab seronegative patient groups. To our knowledge, this is the first study to assess the frequency of AQP4-Ab in patients with demyelinating CNS diseases other than typical MS in Lithuania.

| Participants
Patients were selected from the Departments of Neurology, MS centers of the two largest university hospitals in Lithuania (2,944,459 inhabitant population, 2014) Twenty-nine patients over 18 years of age who presented with demyelinating CNS diseases other than typical MS were included in the study. We assessed positivity for AQP4-Ab in patients presenting with an atypical course of MS that did not fulfill the 2010 McDonald MRI Criteria for lesion dissemination in time and space (Polman et al., 2011), acute TM, severe, atypical ON, NMO, ADEM, and clinically isolated syndrome (CIS). We evaluated clinical, laboratory, and instrumental differences between AQP4-Ab seropositive and AQP4-Ab negative patient groups.
NMO diagnosis was made according to the revised diagnostic criteria of Wingerchuk, Lennon, Pittock, Lucchinetti, and Weinshenker (2006) After this study, clinical diagnoses were revised and some patients were diagnosed with NMO or NMOSD (Table 1)  The study was approved by the Lithuanian Bioethics Committee on January 27, 2011 (No. L-12-01/2), and all patients provided written informed consent. Samples were stored up to 10 days at temperatures between +2°C and +8°C.

| Statistical analyses
Continuous variables were expressed as mean ± SD and/or median

| Characteristics of study population
Twenty-nine patients were included in the study: seven (24.1%) males and 22 (75.9%) females 18 years of age or older (mean age was 41.3 ± 12.5 years, from 22 to 64 years). At study entry, the largest proportions of patients were diagnosed with atypical MS-14 (48.3%) and TM-8 (27.6%; Figure 1). One patient's initial diagnosis was NMO (the diagnosis was based on Wingerchuk's clinical 2006 criteria: two absolute criteria were positive and two out of three supportive criteria were positive; serum testing for AQP4-Ab was not available before this study).

| Comparison between AQP4-Ab seropositive and AQP4-Ab negative patient groups
The duration of the disease in the AQP4-Ab seropositive and AQP4-Ab seronegative group was 55.0 ± 57.9 months and 48.6 ± 61.3 months, respectively, when the AQP4-Ab testing was performed. There were no significant clinical, laboratory, or instrumental differences between the groups of AQP4-Ab positive (

| D ISCUSS I ON
In North America, Australia, and Europe NMO patients represent a small fraction (1%-2%) of Caucasians with inflammatory white matter diseases (Kowarik et al., 2014). As about 70%-80% of NMO cases are associated with aquaporin-4 antibodies, AQP4-Ab testing is an essential tool for NMO diagnosis and consideration of treatment options, especially long-term immunosuppression (Sellner et al., 2010).
It was also suggested that NMO spectrum should be broadened to include AQP4-Ab positive patients with monophasic or more limited phenotypes (Sato et al., 2013).
AQP4-Ab test was positive for three (10.3%) patients with demyelinating CNS diseases other than typical MS in our study. In a previous study, Korean patients with inflammatory demyelinating CNS diseases (including MS patients) were tested for AQP4-Ab, and 106 (out of 388) were found to be positive (Kim, Kim, Li, Jung, & Kim, 2012). In contrast, it has been reported that NMO is rare among ON patients in the population of southern Finland; of the 300 patients with suspected ON, only three patients (1.6%) were found to be positive for AQP4-Ab (Siuko et al., 2014). Further, AQP4-Ab is relatively rare among patients with acute monosymptomatic ON: AQP4-Ab were detected in only eight (5.8%) out of 139 patients from European countries and Turkey who presented with acute monosymptomatic ON (Jarius, Frederikson, et al., 2010). In this same study, all the 32 MS patients were tested negative for AQP4-Ab (Jarius, Frederikson, et al., 2010). We strongly believe that the implementation of AQP4-Ab testing could greatly improve NMO and NMOSD diagnosis in Lithuania.  One NMO patient in the present study was AQP4-Ab seronegative.
About 12% to 30% of patients with NMO or NMOSD remain AQP4-Ab negative (Marignier et al., 2013). Some of our AQP4-Ab negative patients may be positive for antibodies to myelin oligodendrocyte glycoprotein (anti-MOG). It was previously reported that antibodies to MOG were detected in some AQP4-Ab negative patients manifesting clinical and neuroimaging signs of NMO or NMOSD (Zamvil & Slavin, 2015). Furthermore, the results of one observational study showed that serum peptide reactivities may also have the potential to distinguish between both NMOSD subgroups and MS (Metz et al., 2016).
Oligoclonal bands (OCB) were detected in the CSF for the two AQP-4 positive patients in our study. Two studies in which the OCB were analyzed, but OCB were not the primary goal, showed that OCB  (Thompson, 2005).
All patients receiving DMTs were initially diagnosed with atypical MS in the present study. Two AQP4-Ab positive patients, who were initially diagnosed with atypical MS, were also on DMTs (one received glatiramer acetate, other-IFN-beta); however, their disability progressed. The diagnoses were revised after the AQP4-Ab testing, these patients were switched to azathioprine, and the course of the disease stabilized. Therefore the present study illustrates the importance of AQP4-Ab testing. The detection of AQP4-Ab is substantial, because it justifies consideration of longterm immunosuppression, while DMTs have been reported to be inefficacious or even harmful when used for the NMO treatment (Kowarik et al., 2014).
The time between the onset of clinical myelitis symptoms and spinal cord MRI for patients in the present study was 30 days or less (for the first myelitis episodes); however, we did not perform spinal cord MRI for our patients during all repeated spinal cord attacks.
The timing of MRI in the evolution of NMOSD may influence the length of the imaged lesion: early imaging may miss a long lesion, and late imaging may reveal discontinuous or short lesions or no lesions; therefore, it is suggested that short spinal cord lesion does not exclude the diagnosis of NMOSD (Flanagan et al., 2015). However, almost half (44.8%) of the patients in our study had ≥1 spinal cord MRI lesions extending ≥3 VS. Even though LETM was reported to be the most specific radiological finding supporting NMO diagnosis in adult patients (Wingerchuk et al., 2006), short TM is not uncommon in NMOSD and does not exclude NMOSD diagnosis (Flanagan et al., 2015) and decision to perform AQP4-Ab testing. Brain MRI has also an increasingly important role in the differential diagnosis of NMO and NMOSD, particularly from MS, as differentiating these conditions is of prime importance because early initiation of immunosuppressive therapy is the key to preventing attack-related disability in NMOSD (Kowarik et al., 2014), as shown in the case reports in our study.
We acknowledge the limitations of the study: small sample size and possible referral bias as our hospitals are tertiary referral centers.
We hope to address these issues in the future studies by using larger sample sizes and including more patients from regional hospitals.
In conclusion, AQP4-Ab test was positive for one-tenth of patients presenting with inflammatory demyelinating CNS diseases other than typical MS in our study. There were no significant clinical, laboratory, or instrumental differences between the groups of AQP4-Ab positive and negative patients. AQP4-Ab testing is highly recommended for patients presenting with not only TM and ON but also an atypical course of MS and ADEM.

CO N FLI C T O F I NTE R E S T S
The authors state no conflict of interests.