Immune response profiling identifies autoantibodies specific to Moyamoya patients

Moyamoya Disease (MMD) is a rare cerebrovascular disorder that involves progressive bilateral stenosis/occlusion of the supraclinoid internal carotid arteries (ICAs) as well as the proximal anterior and middle cerebral arteries (ACAs and MCAs) [1, 2]. MMD is also characterized by the development of abnormal, fragile collateral blood vessels, usually from enlarged and proliferated lenticulostriate arteries [3]. Early diagnosis is difficult and is usually made angiographically after clinical presentation of cerebral ischemia (stroke or TIA) or hemorrhage, often well after damage to the brain has occurred. The etiology of MMD is currently unknown. Approximately 10–12% of MMD cases are clearly familial with an autosomal dominant inheritance pattern, and several MMD-associated loci have been identified, including 3p24.2-p26, 6q25, 8q23, 12p12, and 17q25.3 [1, 4]. Mutations in individual genes have also been described, including TGFβ1 and PDGFRB [5], BRCC3 [6], and RNF213 [1, 4, 7]. There is also some evidence for active vascular remodeling in MMD collaterals, including proliferation of the smooth muscle cell layer in the intima of MMD vessels [1, 4]. The expression levels of several proteins with known roles in angiogenesis have been described, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-β) [1, 4]. There are also reports of increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the cerebral spinal fluid (CSF) of MMD patients [8].

MMD is often described as Moyamoya Syndrome (MMS) when it is associated with other disorders, such as Majewski Osteodysplastic Primordial Dwarfism Type II (MOPD II), Down syndrome, Seckel syndrome, Neurofibromatosis and Graves’s disease, a known autoimmune disorder [9–11]. Although MMD is not traditionally considered an autoimmune disease, several groups have reported elevated levels of circulating autoantibodies (autoAbs), including anti-cardiolipin [12], anti-thyroid [13, 14] and anti-α-fodrin [15]. In addition, a SEREX profile of CSF obtained from MMD patients identified 5 proteins against which autoAbs were expressed [16].

To comprehensively characterize the serum autoAb repertoire in MMD patients, our unbiased approach used a commercially-available high-density protein microarray platform to identify circulating serum autoAbs. These arrays have been useful for profiling disease-relevant autoAbs and have led to the identification of novel autoAbs in chronic renal [17], rheumatologic and autoimmune diseases [18]. Our study identified a number of MMD-associated autoAbs and the biological pathways and networks associated with them.

We report the first comprehensive analysis of the presence of autoAbs in sera of MMD patients. Using high density autoantibody arrays we identified 165 autoAbs associated with MMD as compared to control sera. We further identified a subset of 6 candidate MMD-specific autoAbs, using the novel bioinformatics tool fitSNPs, which were validated by indirect custom-designed ELISAs in a larger cohort of MMD patients (n = 46) compared to CVD control patients (n = 22). These novel autoAbs have not been previously associated with MMD and their precise relationship to MMD remains unknown. The mechanisms leading to autoantibody generation are not yet well described, although several theories exist. These include immune responses to altered protein post-translational modifications, protein mutations and increased levels of protein expression, either as a result of aberrant over-expression or as a consequence of release following cellular reorganization or damage. Ingenuity Pathway analysis, however, revealed that specific pathways were over-represented by the expressed reactive antigens, including Cell-to-Cell Signaling and Interaction, Hematological Disease, and Immunological Disease (Figure 3). These data make biological sense given the nature of MMD, as it is associated with active angiogenesis, smooth muscle cell proliferation and tissue remodeling [1]. However, only 21 autoAbs against antigens with previously described functions in angiogenesis, vascular remodeling/extracellular matrix deposition/, and vascular stability were differentially expressed. These include endoglin (ENG), angiomotin (AMOT) and fibroblast growth factor receptor 1 (FGFR1), in addition to the 4 in the subset of 6 proteins identified by fitSNP (Table 3). Given that MMD is considered a vascular disease, this represents a small percent of the total antigens identified in this paper. There are several possible explanations for this: firstly, the antigens identified in MMD may have as yet undefined roles in angiogenesis, are thus not annotated as such and were not identified in our analysis. This is certainly the case for RNF213, which is currently not present on the array and has only very recently been implicated in MMD and angiogenesis [7]; secondly, the Protein Microarray v4.0 arrays do not contain the full complement of expressed proteins, nor are these angiogenesis-specific arrays; finally, and perhaps most intriguingly, is the fact that these antigens are significantly expressed both pre- and post-surgery. If MMD was a disorder affecting only the cerebrovasculature, we would not only expect more ‘angiogenic’ autoAb expression pre-surgery but for there to be a significant decrease in these same autoAb levels following bypass when the patients have been treated. That autoAb levels remain high both pre- and post-bypass surgery (ie, after the cerebral flow had been re-established) may suggest some novel aspect of the basic disease biology.

Of particular interest are the roles of cell cycle, DNA maintenance and DNA damage response/repair (DDR) pathways. Two highly expressed MMD-associated autoAbs identified in this array with fitSNP and validated by ELISA include those generated against COP9 signalosome complex subunit 1 (GPS1 or CSN1) and stimulated by retinoic acid 13 (STRA13), both of which have well-described functions in DDR. GPS1 is a component of the COP9 signalosome (CSN) complex whose function is to modulate cullin neddylation, which in turn regulates cullin-RING ubiquitin ligase (CRL) activity and the ubiquitination of proteins involved in DDR [49]. The CSN also forms complexes with CRLs containing cullin4 in order to function in DDR, cell cycle regulation and chromatin remodeling [49]. STRA13 (also known as CENP-X or MHF2) is the DNA-binding component of the Fanconi Anemia (FA) core protein complex, through its association with MHF1 and FANCM proteins [50, 51]. The FA complex plays a critical role in DDR, and is specifically recruited to stalled DNA replication forks occurring as a result of blockage or damage [53]. Two other validated MMD-specific proteins, Amyloid beta A4 protein (APP) and β-catenin, also have roles in DDR and DNA maintenance. APP, a protein most widely studied for its role in Alzheimer disease, has recently been identified as a component of the TIP60 DNA repair complex, a complex that modifies histone acetylation at DNA double strand breaks [40, 41]. β-catenin has a key role in the activation of canonical Wnt signaling and has also recently been implicated in chromosomal instability; specifically, Aoki et al. describe defects in β-catenin leading to activation of Wnt signaling and dysregulation of the cell cycle and chromosomal instability [43]. That DDR and DNA maintenance may have a central role is also supported by the identification in the discovery phase of approximately 20 autoAbs, the antigens against which have known roles in DDR and DNA maintenance, including SWSAP1, CSNK1D, MCM10, NUDT1 and UBE2W.

Further evidence that both MMD/MMS may involve the critical DDR comes from the recent discovery of a novel Xq28 deletion in 3 unrelated hereditary MMS families [6]. This deletion results in the loss of BRCC3, an E3 ubiquitin ligase that is a subunit of the BRCA1-BRCA2-containing complex (BRCC), and deletion of this protein in zebrafish results in defects in angiogenesis [6]. The BRCC complex functions in DDR by stabilizing the BRCA1 protein at DNA break points [54]. Intriguingly, both BRCA1 and BRCA2 are also a part of the FA complex of DDR proteins as described above [53, 55]. In addition, MOPD II and Seckel syndrome, diseases strongly associated with moyamoya angiopathy, are sometimes caused by mutations in the pericentrin gene [9, 10, 56]. Pericentrin is an essential component of the centrosome which mediates, among others, cell cycle progression, spindle organization and orientation, cilia formation and DDR [57].

Given that radiation exposure to the brain is one of the leading environmental causes of moyamoya angiopathy [58], DDR and DNA maintenance machinery may well be compromised in MMD patients. While further research is required to determine if this is the case, precisely how this leads to a complex disease and syndrome remains purely speculative. It is important to note that while defects in DDR, chromosomal instability and cell cycle disregulation often lead to cancer, there is no evidence of increased cancer rates in MMD patients, aside from an association with NF1 [11]. DNA replication and cellular response to DNA damage involve a vast complex of proteins and protein–protein interactions. Given the clinical heterogeneity of MMD and MMS, it would not be unfounded to expect the disease to involve a spectrum of one to several mutations in multiple genes, as is the case with FA patients [53].

The over-representation of antigens pertaining to antigen presentation, immune cell trafficking and inflammatory response may also suggest a significant autoimmune component for MMD. STRA13, one of the validated MMD-specific antigens, also plays an essential role in the stabilization of the outer kinetocore, a protein complex that is essential for correct attachment, segregation and movement of chromosomes during cell division [52]. Reports of the expression of autoAbs to other centromeric proteins of the kinetocore are also associated with a number of autoimmune diseases in humans including rheumatoid arthritis and limited cutaneous sclerosis or CREST syndrome [59]. Therefore, MMD may be related in part to an autoimmune condition.

While the presence of autoAbs may not yet be of diagnostic value in MMD, this prospect may be advanced by validating additional autoAbs and using a larger cohort of patients. Our study demonstrates a novel role for the autoimmune response in MMD and may provide insight into the mechanisms of the disease, including such key cellular processes as cell cycle and DNA repair and maintenance. Future studies are required to further elucidate the clinical relevance of these autoAbs in MMD. There are currently no in vitro models of MMD, but recent advances in disease-specific induced pluripotent stem cells (iPSCs) may show some potential as an in vitro model of this complex and rare disease.

Minnie M Sarwal and Gary K Steinberg are Joint Senior Authors.