This study is, to our knowledge, the first to report on the frequency of autoantibodies recognizing the AQP4 water channel in patients with NMO and to evaluate its specificity when compared to an adequate control group of different patient populations and healthy individuals. Based on the seminal observations of Lennon and colleagues suggesting AQP4 as the target antigen in NMO [7
], we cloned and expressed human AQP4 to establish a quantitative radioimmunoprecipitation assay. This novel approach allows standardized high-throughput analysis. In our series of patients with NMO and related disorders, we show that antibodies to the AQP4 water channel are present in 63% of patients with NMO or at high risk of the disease, but are virtually absent in patients with MS and other inflammatory and noninflammatory neurological diseases (5/217), as well as in patients with rheumatological diseases and in healthy controls (0/74). A methodological limitation here is the moderate interassay variation coefficient in this type of assay, although this can be overcome because of the very low intra-assay variation coefficient and the fact that a high number of samples can be measured simultaneously.
Recently, the general presence of a brain-specific autoantibody, designated NMO-IgG, was described in NMO and determined in patients by indirect immunofluorescence [6
]. This method of testing for NMO-IgG entails laborious and cost-intensive preabsorption of patient sera, employing blocking procedures in order to avoid unspecific binding and false-positive results. Our detection assay for the specific antibody AQP4 provides observer-independent. quantitative data, and is therefore easily reproducible. It remains to be shown whether all NMO-IgG-positive patients reported also reveal reactivity against the AQP4 water channel.
In our study, antibodies to the AQP4 water channel were sufficiently specific for NMO to allow differentiation from MS and other inflammatory neurological diseases. This finding is of clinical relevance, as diagnosis of NMO based solely on clinical, neuroradiological, and CSF findings may be difficult or impossible. One question that has yet to be answered satisfactorily is whether testing for AQP4 antibodies allows diagnosis of NMO after the first event (i.e., first optic neuritis or myelitis). Weinshenker et al. [9
] recently demonstrated that NMO-IgG seropositivity at the initial presentation of LETM predicts relapse of myelitis or development of optic neuritis. All six patients in our series with isolated LETM were positive for antibodies to AQP4, a finding that supports the hypothesis that LETM is an inaugural or limited form of NMO [9
]. We did not, however, detect the antibody in patients with less extensive lesions, i.e., myelitis involving fewer than three segments.
Finally, our findings suggest that testing for AQP4 antibodies not only enables a reliable distinction to be made between NMO and MS, but also facilitates differential diagnosis concerning other autoimmune diseases affecting the CNS. This ability to distinguish is particularly important when patients presenting with myelitis have concomitant serological findings suggesting systemic autoimmunity. Positive testing for autoantibodies (ANA, SS-A, SS-B) has been reported in 38%–75% of patients with NMO [12
], which poses diagnostic challenges to clinicians in many cases. The fact that sera positive for AQP4 antibodies did not show reactivity in testing for the presence of autoantibodies against three unrelated antigens using the same assay system largely rules out nonspecific binding as a cause for anti-AQP4 positivity, and argues for the reliability and specificity of our assay. The negative results in the rheumatological disease group also argue against nonspecific binding of patients' sera to AQP4. Further studies will have to address the question of whether AQP4 is the only target antigen in NMO, and whether antibodies to AQP4 are pathogenic in NMO or a mere epiphenomenon of the disease.