title: Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.
abstract: Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 translation initiation signals; b) AQP4 isoforms (M1 and M23) and fluorescent tag position; c) NMO serum concentration and AQP4 degradation. Our results demonstrate that when using AQP4-M1, the nucleotide in position -3 of the AUG greatly affects the AQP4-M1/M23 protein ratio, NMO-IgG binding, and consequently test sensitivity. Test sensitivity was highest with M23 expressing cells (97.5%) and only 27.5% with AQP4-M1. The fluorescent tag added to the N-terminus of AQP4-M23 considerably affected the NMO-IgG binding, and test sensitivity, due to disruption of AQP4 suprastructures. Furthermore, sera used at high concentration resulted in AQP4 degradation which affected test sensitivity. To further evaluate the reliability of the M23 based CBA test, samples of one NMO patient collected during about 2 years clinical follow-up were tested. The results of serum titer correlated with disease activity and treatment response. In conclusion, we provide a molecular explanation for the contrasting CBA test data reported and suggest the use of M23 with a C-terminus fluorescent tag as the proper test for NMO diagnosis.
authors.affiliation: Department of Bioscience, Biotechnologies and Biopharmaceutic and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pisani
authors.forename: Angelo
authors.initials: A
authors.lastname: Sparaneo
authors.forename: Carla
authors.initials: C
authors.lastname: Tortorella
authors.forename: Maddalena
authors.initials: M
authors.lastname: Ruggieri
authors.forename: Maria
authors.initials: M
authors.lastname: Trojano
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Mola
authors.forename: Grazia Paola
authors.initials: GP
authors.lastname: Nicchia
authors.forename: Antonio
authors.initials: A
authors.lastname: Frigeri
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
mesh_terms: D000918:Antibody Specificity; D051401:Aquaporin 4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology*; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008297:Male; D009471:Neuromyelitis Optica / Q000097:blood* / Q000235:genetics / Q000276:immunology*; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D001323:Autoantibodies; D020033:Protein Isoforms