Background
Methods
Patients and samples processing
Case | Sex | aIgIsotype stage | |
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1-MM | F | IgA k | II A |
2-MM | M | IgG k | II A |
3-MM | M | IgM k/IgA k | II A |
4-MM | M | IgG \(\lambda\) | III A |
5-MM | M | IgG k | III A |
6-MM | M | IgG k | III A |
7-MM | M | micromolecular k | III A |
8-MM | F | IgG k | III A |
9-MM | M | IgA \(\lambda\) | III A |
10-MM | M | micromolecular k | III A |
1-MGUS | F | IgG \(\lambda\) | |
2-MGUS | M | IgG \(\lambda\) | |
3-MGUS | M | IgG \(\lambda\) | |
4-MGUS | M | IgG k | |
5-MGUS | M | IgG k | |
6-MGUS | M | IgA k | |
7-MGUS | M | IgA k | |
8-MGUS | F | IgG \(\lambda\) / IgA \(\lambda\) |
RNA isolation and label protocol
Scan protocol and data processing
Data approximation via nonnegative matrix factorization
NMF based extraction approach and post-processing phase
Considerations on the NMF-based approach
Results
Preliminary microarray data analysis
Microarray dimensionality reduction
Function analysis
Functional analysis of common genes
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The Prok-1 and GNRHR genes are present in the pathways: GPCR ligand binding, signal transduction and class A/1 (rhodopsin-like receptor).
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The AHSG and B2M genes are present in VEGF and VEGFR signaling network, Arf6 downstream pathway, Sphingosine 1-phosphate (S1P) pathway, proteoglycan syndecan-mediated signaling events, Nectin adhesion pathway, EGFR-dependent signaling events endothelin, and endothelins.
Functional analysis of uncommon genes
Gene symbol | Pathway name | Gene symbol | Pathway name |
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TNF | T cell receptor signaling pathway, osteoclast differentiation, natural killer cell mediated cytotoxicity, Fc epsilon RI signaling pathway, TGF-beta signaling pathway, amyotrophic lateral sclerosis (ALS), malaria | FYN | T cell receptor signaling pathway, osteoclast differentiation, natural killer cell mediated cytotoxicity, Fc epsilon RI signaling pathway, axon guidance, focal adhesion |
PPP3CB | T cell receptor signaling pathway, osteoclast differentiation, natural killer cell mediated cytotoxicity, axon guidance, amyotrophic lateral sclerosis (ALS) | NFATC1 | T cell receptor signaling pathway, osteoclast differentiation, natural killer cell mediated cytotoxicity, axon guidance |
LCP2 | T-cell receptor signaling pathway, osteoclast differentiation, natural Killer cell mediated cytoxicity, Fc epsilon RI signaling pathway | EGF | Pathway in cancer, focal adhesion, endometrial cancer |
HGF | Pathway in cancer, malaria, focal adhesion | TGFB3 | Pathway in cancer, malaria, TGF-beta signaling pathway |
LAT | T-cell receptor signaling pathway, natural Killer cell mediated cytoxicity, Fc epsilon RI signaling pathway | CASP9 | Pathways in cancer, amyotrophic lateral sclerosis (ALS), endometrial cancer |
COMP | TGF-beta signaling pathway, focal adhesion, malaria | GAB2 | Osteoclast differentiation, Fc epsilon RI signaling pathway |
CRK | Pathway in cancer, focal adhesion | APC2 | Pathway in cancer, endometrial cancer |
BMP4 | Pathway in cancer, TGF-beta signaling pathway | SPI1 | Pathway in cancer, osteoclast differentiation |
PDPK1 | Focal adhesion, endometrial cancer | FCGR3B | Osteoclast differentiation, natural killer cell mediated cytoxicity |
CD3E | T cell receptor signaling pathway | CD28 | T cell receptor signaling pathway |
CSF2RA | Pathways in cancer | SLIT2 | Axon guidance |
ID2 | TGF-beta signaling pathway | E2F5 | TGF-beta signaling pathway |
PTPN11 | Cytotoxicity mediated by killer cells | FZD2 | Pathways in cancer |
ACTN1 | Focal adhesion | CFL1 | Axon guide |
RHOD | Axon guide | MAP3K5 | Amyotrophic lateral sclerosis (ALS) |
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The TNF gene is shared in 7 pathways, it is a multifunctional pro-inflammatory cytokine and represents an index of fibroblast activity in the regulation of a broad spectrum of biological processes, including cell proliferation, differentiation, apoptosis, metabolic lipids and coagulation within the bone marrow [53, 54].
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NFATC1 and LCP2 genes are present in 4 of the selected pathways. The function of the protein encoded by the NFATC1 gene is the regulation of multiple cytokines and other regulatory molecules, including some interleukins (IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, GM CSF), interferon (IFN)-\(\nu ,\) tumor necrosis factor (TNF)-\(\alpha ,\) CD40 ligand (CD154), and CD95 ligand (FasL). The NFATC1 protein is mainly studied in T cells (calcineurin-dependent 1) and is involved in the immune responses of lymphocytes B, NK cells, macrophages, mast cells, and eosinophils [20, 54, 57]. The LCP2 protein acts as a T cell substrate (TCR), therefore it plays a role in the transduction of the intracellular signal mediated by TCR [58].
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EGF, HGF, TGFB3, LAT, CASP9, and COMP genes are present in 3 of the 11 pathways, they encode for proteins involved in the growth, proliferation and differentiation of many cell types. In particular, EGF is a potent mitogen factor [59], while HGF binds to the hepatocyte growth factor receptor to regulate morphogenesis, growth and cellular motility and is secreted by mesenchymal cells. The HGF therefore acts as a multifunctional cytokine on predominantly epithelial cells and plays a role in angiogenesis, tumorigenesis, and tissue regeneration [60, 61]. TGFB3 protein is a ligand of the various TGF-beta receptors and leads to the recruitment and activation of SMAD family transcription factors and regulates a myriad of mainly immunosuppressive responses [18, 62‐64]. The LAT protein forms, along with several adaptive proteins, a complex that creates a multimolecular signaling network located at the TCR engagement site. The role played by LAT protein underlines the complex modulation performed by fibroblasts of uncommon metaMM subsets in regulating immune response [65]. The CASP9 gene encodes for a protein whose function is comparable to a tumor suppressor and its functional polymorphisms may be responsible for alterations in proliferation [66]. The protein encoded by the gene COMP family belonging to the thrombospondin family carries out direct action on the mechanical integrity of the extracellular matrix, intervening in the interface between mineralized and non-mineralized regions [67, 68]. This protein is involved in the interaction between fibroblasts and osteoblasts, and is related to the significant changes caused by the nature of mechanical stress by the mineralized fibrocartilage to the bone [69].
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GAB2, CRK, APC2, BMP4, SPI1, PDPK1 and FCGR3B genes are only present in 2 of the 11 pathways selected, specifically GAB2 and CRK, are adapters for the transmission of various signals, GAB2 protein responds to receptors stimuli cytokines, growth factors, and antigen receptors [70] while CRK protein binds several tyrosine-phosphorylated proteins [71]. The APC2 gene encodes a protein that promotes the assembly of a multiproteic complex responsible for the control of beta-catenin cytoplasmic levels and is crucial in interaction with cytoskeletal proteins [72]. The BMP4 gene belongs to the superfamily of TGF-beta proteins [73] and the SPI1 gene encodes for a transcription factor that activates gene expression in the myeloid line and the development of B-lymphoid cells [74]. The PDPK1 gene encodes for a serine-threonine kinase, crucial to regulating cell migration. PDPK1 is a signal transducer for PI3K and activates multiple downstream effectors, it represents a focal point in the coordination of signals from the extracellular environment to the cytoskeleton PLC\(\nu\) [75]. Finally, the FCGR3B gene encodes for a low affinity receptor for the Fc region of gamma immunoglobulins (IgG) and is capable of capturing immune complexes [76].
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Gene residues individually present in the 11 pathways, such as the CD3E gene and the CD28 gene may be involved in some cellular processes mainly related to an immune response [75]. The immune response is integrated with the CSF2RA [76] and SLIT2 genes [77]. A second set of genes promotes cell proliferation control; indeed, the ID2 gene belongs to transcriptional factor regulators and negatively regulates cell differentiation [78]. E2F5 genes and PTPN11 are important in cell cycle control and tumor suppressor genes in oncogenic transformation [79, 80]. The last group consists of FZD2 [81], ACTN1 [82], CFL1 [83] and RHOD [84, 85], these genes are involved in the organization of the cytoskeleton. Finally, MAPK gene encoding MAP3K5, responsible for the activation of several downstream effects, in particular transcription factors, which regulate different cellular responses [86]. The functional network created by the 30 genes in the 11 pathways selected and belonging to the uncommon subset of metaMM shows that fibroblasts have acquired additional properties from those belonging to the common metaMGUS subset that favor the “activated fibroblast” condition.