Immune response profiling identifies autoantibodies specific to Moyamoya patients

Abstract

Background: Moyamoya Disease is a rare, devastating cerebrovascular disorder characterized by stenosis/occlusion of supraclinoid internal carotid arteries and development of fragile collateral vessels. Moyamoya Disease is typically diagnosed by angiography after clinical presentation of cerebral hemorrhage or ischemia. Despite unclear etiology, previous reports suggest there may be an immunological component.

Methods: To explore the role of autoimmunity in moyamoya disease, we used high-density protein arrays to profile IgG autoantibodies from the sera of angiographically-diagnosed Moyamoya Disease patients and compared these to healthy controls. Protein array data analysis followed by bioinformatics analysis yielded a number of auto-antibodies which were further validated by ELISA for an independent group of MMD patients (n = 59) and control patients with other cerebrovascular diseases including carotid occlusion, carotid stenosis and arteriovenous malformation.

Results: We identified 165 significantly (p < 0.05) elevated autoantibodies in Moyamoya Disease, including those against CAMK2A, CD79A and EFNA3. Pathway analysis associated these autoantibodies with post-translational modification, neurological disease, inflammatory response, and DNA damage repair and maintenance. Using the novel functional interpolating single-nucleotide polymorphisms bioinformatics approach, we identified 6 Moyamoya Disease-associated autoantibodies against APP, GPS1, STRA13, CTNNB1, ROR1 and EDIL3. The expression of these 6 autoantibodies was validated by custom-designed reverse ELISAs for an independent group of Moyamoya Disease patients compared to patients with other cerebrovascular diseases.

Conclusions: We report the first high-throughput analysis of autoantibodies in Moyamoya Disease, the results of which may provide valuable insight into the immune-related pathology of Moyamoya Disease and may potentially advance diagnostic clinical tools.

Figure 1

Increased levels of specific autoAbs were observed in MMD patients compared to healthy controls by protein array. Targets included (A) calcium/calmodulin-dependent protein kinase II alpha (CAMK2A); (B) B-cell antigen receptor complex-associated protein alpha-chain (CD79A); (C) G protein pathway suppressor 1 (GPS1); (D) ephrin-A3 (EFNA3); (E) Na+/K + transporting ATPase interacting 4 (NKAIN4); (F) Transmembrane Protein 32 (TMEM32). RFU = relative fluorescence units.

Figure 2

Validation of autoAb expression by indirect ELISA. Six MMD-specific proteins identified through a fitSNPs approach were analyzed by indirect ELISA for IgG levels against these antigens in MMD sera. All antigens were found to be significantly increased in MMD (n = 46) compared to CVD controls (n = 22): (A) STRA13 (p = 0.01), (B) APP (p = 0.01), (C) CTNNB1 (p = 0.02), and (D) GPS1 (p = 0.01), (E) ROR1 (p = 0.04), and (F) EDIL3 (p = 0.02) (OD@405 nm = optical density at 405 nm) (G) A ROC curve built on a logistic regression model using 6 autoAbs ELISA data (Area Under Curve = 0.76) demonstrates a 74% sensitivity, 77% specificity, 87% PPV, 59% NPV and overall 75% accuracy set of all MMD samples tested.

Figure 3

Association of MMD-specific autoAbs with a Cell-to-Cell Signaling and Interaction, Hematological Disease, and Immunological Disease IPA network. The autoAbs validated in this study are encircled in bold red.