Investigation of anti-neurofascin and anti-myelin oligodendrocyte glycoprotein antibodies in patients with chronic inflammatory demyelinating polyneuropathy

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a chronic immune-mediated peripheral neuropathy that can affect people of all ages. Walking difficulty, arm and leg weakness, numbness and tingling and tremor can be seen in CIDP. In around 2-18% of patients diagnosed with CIDP, antibodies against paranodal antigens, including Neurofascin 155 (NF155), Neurofascin 186 (NF186), Contactin1 and Caspr1 can be detected in the serum, identifying this subgroup as seropositive CIDP. Treatment approach can be different is this subgroup compared to common CIDP, therefore, it is important to identify these patients. However, antibody tests against the paranodal antigens are not available in Turkey. In this study, we first aimed to establish a live cell-based assay to test the presence of antibodies against NF-155, NF-186 in the serum and cerebrospinal fluid of patients. Then we aimed to test the sera obtained from a nationwide cohort of CIDP patients for these antibodies, as well as anti-MOG antibodies to identify the frequency and characteristics of anti-NF155 antibody positive CIDP in Turkey. The live cell base assay method was applied to live, unfixed cells to preserve the structural integrity of the NF protein. Initially, plasmids containing NF155-EGFP, NF186-EGFP or EGFP (as control) gene was transfected into HeLA cells using a transfection reagent. Then, patient serum or CSF was incubated the transfected cells followed by incubation with a biotin-conjugated secondary antibody. Finally, Alexa fluor-647 conjugated streptavidin was added to detect antibody binding by flow cytometry. Delta and ratio of mean fluorescent intensity (MFI) was calculated by using the data from protein and GFP-only expressing cells. A cut-off value was determined by using sera from healthy controls. We tested sera from 182 CIDP patients. The patients were 41 (21.8) years old on average, with a F:M of 70/104. 147 patients were adult, and 35 patients were pediatric. A total of 11 samples were positive for NF-155-IgG. Among these samples, 6/11 had IgG4 as the dominant isotype, verifying anti-NF155 antibody positivity. Detailed clinical information was present in five patients, that was compatible with the clinical characteristics of seropositive CIDP patients previously reported in the literature. Among seven NF155-IgG4+ patients six were adults and one was a pediatric patient. IgG2 and IgG3 antibodies were also present in addition to IgG4 subtype in three patients. The frequency of anti-NF155 antibody positivity in the Turkish cohort was found to be 3.5%. In the rest of the four patients no IgG positivity was detected and low IgM positivity was detected in three samples. CSF from one NF155 IgG4 positive patient was also analysed and showed the presence of antibodies in the CSF, as well. In another 13 CSF samples tested from CIDP patients one samples had low titers of NF155-IgG, NF186-IgG and MOG-IgG. This patient's serum was negative for these antibodies. Among 12 Guillain-Barre Syndrome (GBS) CSF samples tested, two patients had low titers of NF155-IgG. Among eight MS patients and 22 patients with other neurological disorders, none of the CSF samples tested positive for NF155-IgG or NF186-IgG. In addition, we analysed the presence MOG antibodies in CIDP patients. Serum tests were positive for MOG antibodies in nine patients. Two of the patients were children. Pediatric patients were diagnosed with CIDP and combined central and peripheral demyelination syndrome (CCPD). Adult patients comprised the remaining seven cases, of which two were diagnosed with CCPD and the remaining five with CIDP. In the nine patients with positive serum MOG-IgG, NF155 and NF186 antibodies were negative. Analysis of 16 CSF samples obtained from CIDP and GBS patients revealed that, five patients had low titers of MOG-IgG. Serum MOG-IgG was found to be borderline positive in two of these patients. In the other three patients, serum positivity was not detected. Among patients with both serum and CSF MOG-IgG positivity, one patient had CCPD and the other had CIDP. In conclusion, we have established a live cell-based assay to test anti-NF155 and anti-NF186 antibodies from serum and CSF. We further identified the frequency of these antibodies in a large cohort of Turkish CIDP patients containing adult and pediatric patients, together with their immunological and clinical characteristics. Additionally, we found that MOG-IgG can also be present in the serum and CSF of CIDP patients as well as CCPD patients. Key words: Chronic inflammatory demyelinating polyneuropathy, combined central and peripheral demyelination syndrome, autoantibody, neurofascin, myelin oligodendrocyte glycoprotein