Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers

Abstract

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

Keywords: BET1; HNRNPDL; NEFM and PHGDH; Von Willebrand factor a domain containing 1 protein; neuromyopathy.

FIGURE 1

Proteomic studies on VWA1‐patient derived blood samples. (A) Results of GTex‐based in silico analysis of VWA1 expression/transcript abundance in different tissues and cellular populations. (B) VWA1 protein abundances presented as ratios with respect to GAPDH level in in house generated spectral libraries (protein catalogues) of fibroblasts and skeletal muscle. (C) Schematic representation of the applied workflow to study the proteomic signatures of white blood cells and plasma from the same blood sample. (D) Abundance plot for proteomic profiling data obtained on white blood cells showing the dynamic range of all identified proteins. This is based on their relative quantification of the three highest abundant peptides for each protein, allowing protein comparison within an experiment. All identified proteins of the control (black) are sorted with decreasing abundance while the patient (red) was plotted in the same order to directly compare the different abundances. All identified proteins cover a dynamic range of eight orders of magnitude. (E) Volcano plot for proteomic findings obtained in white blood cells highlighting statistically significant increased proteins (green dots) as well as decreased proteins (red dots). Fc, fold change. Four proteins of particular neuromuscular relevance are highlighted. (F) Results of GO‐Term based in silico studies of proteomic findings in white blood cells show that increased proteins impact on intracellular protein transport, membrane fusion, protein ubiquitination and ribosome disassembly. Cellular compartments affected by increased protein abundances include membranes of the ER‐Golgi network as well as SNARE complex, extracellular exosomes and cytoplasmic vesicles (upper panel). Decreased proteins impact on immune response along with antigen processing and presentation and also affect the ER‐Golgi network (lower panel). (G) Abundance plot for proteomic profiling data obtained on plasma showing the dynamic range of all identified proteins. This is based on their relative quantification of the three highest abundant peptides for each protein, allowing protein comparison within an experiment. All identified proteins of the control (black) are sorted with decreasing abundance while the patient (red) was plotted in the same order to directly compare the different abundances. All identified proteins cover a dynamic range of eight orders of magnitude. (H) Volcano plot for proteomic findings obtained in plasma highlighting statistically significant increased proteins (green dots) as well as decreased proteins (red dots). (I) GO‐Term based in silico studies showed that increased proteins are indicative for positive regulation of cell death, oxidative stress burden (nitric oxide transport, response to hydrogen peroxide and cellular oxidant detoxification) and inflammatory response and impact on extracellular regions and endocytic vesicles as cellular compartments whereas decreased proteins are also indicative for immune response and affect the immunoglobulin complex and the extracellular space. Full names of proteins depicted in this figure are listed in Table 2 and Table S1.

FIGURE 2

Immunofluorescence findings in VWA1‐patient derived muscle biopsies. (A) Immunofluorescence studies of BET1 (green) showed a sarcoplasmic increase accompanied by the presence of focal dot‐like structures in patient‐derived muscle cells compared to muscle cells derived from a control case. Spectin (SPEC) staining (red) visualizes the sarcolemma. (B) Immunostaining of HNRNPDL also revealed a sarcoplasmic increase accompanied by the presence of dot‐like immunoreactive structures in patient‐derived muscle cells compared to control cells. Spectin (SPEC) staining (red) visualizes the sarcolemma. (C) VWA1‐mutant muscle cells show a generalized sarcoplasmic increase with the presence of focal accumulations of PHGDH (green) in comparison to non‐mutant muscle cells. Spectin (SPEC) staining (red) visualizes the sarcolemma. (D) Immunofluorescence‐based studies of NEFM revealed a sarcoplasmic increase only in few myofibres in addition to a considerable increase in extra muscular cells localized within the extracellular matrix in VWA1‐mutant muscle compared to controls. One representative control biopsy is shown for the different staining studies. Spectrin (SPEC) staining (red) visualizes the sarcolemma. DAPI staining visualizes nuclei. Scale bar 50 μm.

FIGURE 3

Immunohistochemistry findings in a VWA1‐patient derived muscle biopsy. (A) Immunostaining of CD20 revealed cells in capillaries and vessels within the muscle tissue in addition to reactivity in few circulating cells. (B) Increased immunoreactivity of MUM1⁺ cells in muscular capillaries and vessels in addition to immunoreactivity in few circulating cells. (C) CD138 immunostaining showed a reactivity of singular cells in muscular capillaries and vessels as well as in few circulating cells. (D) CRP staining revealed a pronounced immunoreactivity in the thickened connective tissue in addition to immunoreactivity in big, foamy, rounded cells (most likely macrophages).