Novel Autoantibodies in Idiopathic Small Fiber Neuropathy

Abstract

Objective: Small fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high-throughput protein microarray platform that captures autoantibodies expressed in the native conformational state.

Methods: Sera from 58 SFN patients and 20 age- and gender-matched healthy controls (HCs) were screened against >1,600 immune-related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts.

Results: Nine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC = 2.99 and 3.07, respectively, p = 0.003, q = 0.076), DBNL (FC = 2.11 and 2.16, respectively, p = 0.009, q < 0.003), and KRT8 (FC = 1.65 and 1.70, respectively, p = 0.043, q < 0.003). Further subgroup analysis into iSFN and SFN by secondary causes (secondary SFN) in the main cohort showed that MX1 is higher in iSFN compared to secondary SFN (FC = 1.61 vs 0.106, p = 0.009).

Interpretation: Novel autoantibodies MX1, DBNL, and KRT8 are found in iSFN. MX1 may allow diagnostic subtyping of iSFN patients. ANN NEUROL 2022;91:66-77.

FIGURE 1

Sengenics Immunome KREX Protein Array platform. The biotin carboxyl carrier protein (BCCP) folding marker acts as a marker for correctly folded proteins. The unique BCCP folding marker conserves the native protein conformation. Proteins are immobilized on the array only when they are properly folded and biotinylated on the BCCP folding marker. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 2

Flowchart of recruitment, proteomics testing, and data analysis of small fiber neuropathy (SFN) patients. Fifty‐nine patients were recruited to the main cohort. One patient was excluded due to failure in meeting diagnostic criteria, and the remaining samples (n = 58) were analyzed and compared with 20 healthy controls who were age‐ and gender‐matched. Thirty‐six patients were recruited to the validation cohort and were run independently and compared with 20 healthy controls. Three samples were excluded due to failure in quality control (QC) or normalization (n = 33). Bioinformatic analysis was performed to identify reproducible autoantigens. NCS = nerve conduction studies. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 3

Global differences among the groups and autoantibodies with significantly altered serum levels between idiopathic small fiber neuropathy (iSFN; n = 34 for main cohort, n = 18 for validation cohort), secondary SFN (sSFN; n = 24 for main cohort, n = 15 for validation cohort), and healthy controls (HCs; n = 20). (A) Partial least squares discriminant analysis plots showing separability of subjects based on the overall molecular profiles between the iSFN, sSFN, and HC groups. Positive separation is seen between SFN and HCs in both main and validation cohorts. (B) Heatmap visualization of autoantibodies reproducibly altered between SFN, iSFN, and HCs of the main cohort and the validation cohort. [Color figure can be viewed at www.annalsofneurology.org]

FIGURE 4

Nine proteins were significantly elevated in small fiber neuropathy (SFN) patients and idiopathic SFN (iSFN) compared to healthy controls (HC). (A, B) Common proteins between the main and validation cohorts with significant log2 fold change (FC) were identified. (C) Scatterplot analysis shows 9 proteins that were reproducibly altered in SFN in both cohorts with statistical significance. MX1, DBNL, and KRT8 showed the largest FCs in both cohorts. (D) In the iSFN analysis, MX1 showed the highest FC, but did not meet statistical significance in the validation cohort. DBNL and KRT8 showed the next highest FCs, which reached significance in both cohorts. p < 0.05 is considered to be significant, whereas q < 0.1 is considered to be significant in the validation cohort. [Color figure can be viewed at www.annalsofneurology.org]