Purpose: To utilize a high content protein array to generate an immune response biomarker profile (IRBP) to identify autoantibodies and/or proteins that could be biomarkers for hypertrophic cardiomyopathy
Methods: It is generally believed that a patient’s own aberrantly-expressed proteins can trigger an immune response. The molecular mechanisms underlying this phenomenon still remain unclear, but based on this principle, all of the proteins present on a human proteome array can be assayed for their immunogenic activity by performing plasma profiling assays. Autoantibodies in diseased versus non diseased plasma can be identified and used as serological markers of disease. In addition, disease-associated autoantibodies may target proteins that are mutated, modified or aberrantly expressed and so could also be considered immunologic reporters to help uncover biology of the disease. Lastly, the proteins associated with the autoantibodies can be examined for differential expression. As such, the ProtoArray™ human protein microarray from ThermoFisher was assayed with healthy volunteer plasma and diseased state plasma from hypertrophic cardiomyopathy patients. The ProtoArray™ is a high-content, functional protein microarray containing 9375 human proteins printed on a glass slide. Each human open reading frame is expressed as an N-terminal GST fusion protein using a baculovirus expression system. The slides were sent to ThermoFisher for image rendering (Figure 1) followed by image and file indexing resulting in a final raw results data file.
Two different methods of data analysis were explored. First, the ThermoFisher free-ware, Prospector Analyzer, was used to compare data across healthy volunteer and diseased state samples using Z-factor, Z-score and CI P-values. Second, Ingenuity Pathway Analysis (IPA) was completed to investigate the differential expression of related proteins which could point to important cellular pathways relevant to hypertrophic cardiomyopathy.
Results: For the Prospector Analyzer analysis, the three scores were considered collectively resulting in 14 top hits for autoantibody expression (Figure 2). Of these 14 hits, two proteins (GAK and FKBP5) were selected for quantitation via ELISA. GAK is associated with focal adhesions and FKBP5 plays a role in immunoregulation and is involved in protein folding and trafficking. These cellular activities were of interest based on disease presentation. GAK and FKBP5 were quantitated via ELISA in healthy volunteer and diseased state patient plasma. Differences in autoantibody expression did not correlate with difference in protein expression for these GAK and FKBP5.
For the IPA, protein hits in healthy volunteer and diseased state patient samples with p<0.05 were included. This corresponded to approximately 900 proteins. Twenty-nine pathways were identified as significant (p<0.05). The two top pathways were B cell receptor signaling (Figure 3) and primary immunodeficiency signaling, which included 20 enriched molecules and 11 enriched molecules, respectively. These two pathways have 3 overlapping enriched molecules which may be of particular interest to examine further. Signals propagated through the B cell antigen receptor (BCR) are crucial to the development, survival and activation of B lymphocytes. These signals also play a central role in the removal of potentially self-reactive B lymphocytes.
Primary immunodeficiency (PI) diseases are a genetically heterogeneous group of disorders that affect distinct components of the innate and adaptive immune system. These include neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, and T and B lymphocytes. These disorders of the immune system cause increased susceptibility to infection, autoimmune disease, and malignancy.
Conclusion: In summary, autoantibodies against proteins that result from abnormal gene expression and gene mutation in hypertrophic cardiomyopathy patients could be plasma biomarkers. The two proteins tested here with differential autoantibody expression in diseased state patient plasma did not show differential protein expression. The IPA identified related proteins in the B cell receptor signaling and primary immunodeficiency pathways which will be examined further.