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Characterization of the binding pattern of human aquaporin-4 autoantibodies in patients with neuromyelitis optica spectrum disorders.

Tuller F, Holzer H, Schanda K, Aboulenein-Djamshidian F, Höftberger R, Khalil M, Seifert-Held T, Leutmezer F, Berger T, Reindl M - J Neuroinflammation (2016)

Bottom Line: These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study.Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples.Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.

View Article: PubMed Central - PubMed

Affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.

ABSTRACT

Background: The discovery of a highly specific antibody against the aquaporin-4 (AQP4) water channel (AQP4-IgG) unified the spectrum of neuromyelitis optica spectrum disorders (NMOSD), which are considered to be antibody-mediated autoimmune diseases. The AQP4 water channel is located on astrocytic end-feet processes and consists of six transmembrane helical domains forming three extracellular loops A, C, and E in which defined amino acids were already proven to be critical for AQP4-IgG binding. However, the clinical relevance of these findings is unclear. Therefore, we have characterized the epitope specificity of AQP4-IgG-positive NMOSD patients.

Methods: We established a cell-based flow cytometry assay for the quantitative detection of AQP4-IgG-positive serum samples. Human embryonic kidney (HEK) cells were transiently transfected with an EmGFP-tagged AQP4-M23, AQP4-M1, or six AQP4-M23 extracellular loop mutants including two mutations in loop A (serial AA substitution, insertion of a myc-tag), two in loop C (N153Q, insertion of a myc-tag), and two in loop E (H230G, insertion of a myc-tag). Fourty-seven baseline and 49 follow-up serum samples and six paired cerebrospinal fluid (CSF) baseline samples of 47 AQP4-IgG-positive Austrian NMOSD patients were then tested for their binding capability to AQP4-M1 and AQP4-M23 isoforms and these six extracellular loop mutants.

Results: Overall, we could identify two broad patterns of antibody recognition based on differential sensitivity to mutations in extracellular loop A. Pattern A was characterized by reduced binding to the two mutations in loop A, whereas pattern B had only partial or no reduced binding to these mutations. These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study. Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples. Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.

Conclusions: Our study demonstrates that AQP4-IgG in sera of NMOSD patients show distinct patterns of antibody recognition. The clinical and diagnostic relevance of these findings have to be addressed in prospective studies.

No MeSH data available.


Related in: MedlinePlus

AQP4-M23 binding ratios in seropositive NMOSD patients and seronegative control groups. AQP4-IgGs were exclusively detected in serum samples of patients with NMOSD, but not in MS patients, patients with OND or HC. The cutoff value of 1.323 is indicated by a dashed horizontal line. Medians are indicated by horizontal bars. Binding ratios were compared by using a non-parametric test (Kruskal-Wallis Test) revealing p < 0.0001. NMOSD neuromyelitis optica spectrum disorders, MS multiple sclerosis, OND other neurological diseases, HC healthy controls
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Fig2: AQP4-M23 binding ratios in seropositive NMOSD patients and seronegative control groups. AQP4-IgGs were exclusively detected in serum samples of patients with NMOSD, but not in MS patients, patients with OND or HC. The cutoff value of 1.323 is indicated by a dashed horizontal line. Medians are indicated by horizontal bars. Binding ratios were compared by using a non-parametric test (Kruskal-Wallis Test) revealing p < 0.0001. NMOSD neuromyelitis optica spectrum disorders, MS multiple sclerosis, OND other neurological diseases, HC healthy controls

Mentions: We established a cell-based flow cytometry assay as detection method for AQP4-antibodies and screened 47 AQP4-IgG positive baseline serum samples of 47 NMOSD patients and 197 AQP4-IgG negative control serum samples for their binding ratio against the AQP4-M23 isoform expressed on HEK-293A cells (Fig. 2). HEK-293A cells with the fluorescence intensity of 104–105 were gated to determine the binding ratio to the transfected cell population and to ensure a high and comparable expression level between the transfectants (Additional file 2). A cutoff value (1.323) was calculated by the binding ratio of our seronegative control groups using ROC analysis. Serum positivity of AQP4 autoantibodies was confirmed in all NMOSD samples whereas none of the controls was seropositive, yielding an assay sensitivity and specificity of 100 % (95 % CI 92–100 % and 95 % CI 98–100 %, respectively).Fig. 2


Characterization of the binding pattern of human aquaporin-4 autoantibodies in patients with neuromyelitis optica spectrum disorders.

Tuller F, Holzer H, Schanda K, Aboulenein-Djamshidian F, Höftberger R, Khalil M, Seifert-Held T, Leutmezer F, Berger T, Reindl M - J Neuroinflammation (2016)

AQP4-M23 binding ratios in seropositive NMOSD patients and seronegative control groups. AQP4-IgGs were exclusively detected in serum samples of patients with NMOSD, but not in MS patients, patients with OND or HC. The cutoff value of 1.323 is indicated by a dashed horizontal line. Medians are indicated by horizontal bars. Binding ratios were compared by using a non-parametric test (Kruskal-Wallis Test) revealing p < 0.0001. NMOSD neuromyelitis optica spectrum disorders, MS multiple sclerosis, OND other neurological diseases, HC healthy controls
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4930584&req=5

Fig2: AQP4-M23 binding ratios in seropositive NMOSD patients and seronegative control groups. AQP4-IgGs were exclusively detected in serum samples of patients with NMOSD, but not in MS patients, patients with OND or HC. The cutoff value of 1.323 is indicated by a dashed horizontal line. Medians are indicated by horizontal bars. Binding ratios were compared by using a non-parametric test (Kruskal-Wallis Test) revealing p < 0.0001. NMOSD neuromyelitis optica spectrum disorders, MS multiple sclerosis, OND other neurological diseases, HC healthy controls
Mentions: We established a cell-based flow cytometry assay as detection method for AQP4-antibodies and screened 47 AQP4-IgG positive baseline serum samples of 47 NMOSD patients and 197 AQP4-IgG negative control serum samples for their binding ratio against the AQP4-M23 isoform expressed on HEK-293A cells (Fig. 2). HEK-293A cells with the fluorescence intensity of 104–105 were gated to determine the binding ratio to the transfected cell population and to ensure a high and comparable expression level between the transfectants (Additional file 2). A cutoff value (1.323) was calculated by the binding ratio of our seronegative control groups using ROC analysis. Serum positivity of AQP4 autoantibodies was confirmed in all NMOSD samples whereas none of the controls was seropositive, yielding an assay sensitivity and specificity of 100 % (95 % CI 92–100 % and 95 % CI 98–100 %, respectively).Fig. 2

Bottom Line: These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study.Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples.Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.

View Article: PubMed Central - PubMed

Affiliation: Clinical Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria.

ABSTRACT

Background: The discovery of a highly specific antibody against the aquaporin-4 (AQP4) water channel (AQP4-IgG) unified the spectrum of neuromyelitis optica spectrum disorders (NMOSD), which are considered to be antibody-mediated autoimmune diseases. The AQP4 water channel is located on astrocytic end-feet processes and consists of six transmembrane helical domains forming three extracellular loops A, C, and E in which defined amino acids were already proven to be critical for AQP4-IgG binding. However, the clinical relevance of these findings is unclear. Therefore, we have characterized the epitope specificity of AQP4-IgG-positive NMOSD patients.

Methods: We established a cell-based flow cytometry assay for the quantitative detection of AQP4-IgG-positive serum samples. Human embryonic kidney (HEK) cells were transiently transfected with an EmGFP-tagged AQP4-M23, AQP4-M1, or six AQP4-M23 extracellular loop mutants including two mutations in loop A (serial AA substitution, insertion of a myc-tag), two in loop C (N153Q, insertion of a myc-tag), and two in loop E (H230G, insertion of a myc-tag). Fourty-seven baseline and 49 follow-up serum samples and six paired cerebrospinal fluid (CSF) baseline samples of 47 AQP4-IgG-positive Austrian NMOSD patients were then tested for their binding capability to AQP4-M1 and AQP4-M23 isoforms and these six extracellular loop mutants.

Results: Overall, we could identify two broad patterns of antibody recognition based on differential sensitivity to mutations in extracellular loop A. Pattern A was characterized by reduced binding to the two mutations in loop A, whereas pattern B had only partial or no reduced binding to these mutations. These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study. Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples. Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF.

Conclusions: Our study demonstrates that AQP4-IgG in sera of NMOSD patients show distinct patterns of antibody recognition. The clinical and diagnostic relevance of these findings have to be addressed in prospective studies.

No MeSH data available.


Related in: MedlinePlus