Biomarker discovery in systemic sclerosis: state of the art

Figures & data

Table 1 Principal tools and techniques used in biomarker discovery

Technologies	Tools	Investigations
Genomic	Genome-wide methods  Microarrays: whole-genome expression array Serial analysis of gene expression (SAGE)  Expressed sequence tags (ESTs) analysis  Gene expression profiling based on alternative RNA splicing  Tangerine expression profiling Individual sequences  Real-time (RT)-polymerase chain reaction (PCR)  Competitive RT-PCR  RNase protection assay  T-cell receptor expression analysis Analysis of single-cell gene expression RNA amplification Monitoring in vivo gene expression by magnetic resonance imaging (MRI) or positron emission tomography (PET)	Gene expression in healthy and diseased tissues, identification of single nucleotide polymorphisms (SNPs), correlation genotype-phenotype
Epigenomic	DNA methylation profiling  Methylation at cytosine based sequences (CpG)  Quantitative methyl-SNP analysis  Histone endonuclease-linked detection of methylation sites of DNA (HELMET) Histone modification profiling acetylation, methylation, phosphorylation, SUMOylation Noncoding (nc)RNA profiling: long ncRNA and micro (mi)RNA - PCR and nanostring	Changes in the regulation of gene activity and expression independent from the DNA sequence
Proteomic	Two-dimensional (2D) gel electrophoresis (GE) and difference gel electrophoresis (DiGE) Isotope-coded affinity tag (ICAT) Mass spectrometry (MS)  Matrix-assisted laser desorption/ionization (MALDi)  Time-of-flight MALDI (MALDI-TOF)  Quantitative tandem MS imaging MS (iMS)  Liquid chromatography-MS  Surface-enhanced laser desorption/ionization (SELDI) and SELDI-TOF	Identification of proteins in fluids and tissue, detecting structural and functional variations of proteins (post-translational modifications, turnover, transport, and excretion)
Metabolomic	MS-based kits Urinary profiling by capillary electrophoresis Lipomic technologies	Technology that complements proteomics and transcriptomics, focusing on metabolites

Note: Copyright © 2010, Humana Press, a part of Springer Science+Business Media, LLC. Adapted with permission from Jain KK. Technology for Discovery of Biomarkers. In: The Handbook of Biomarkers. Vol 1. Totowa, NJ: Humana Press; 2010:23–72.²³¹

Table 2 miRNAs investigated in SSc, their potential function, and molecular targets

miRNA	Regulation	Tissue	Function	Target	Reference
miR-21	Up	Skin FBs, murine dermal FBs	Profibrotic	SMAD7	52
miR-29	Down	FBs, murine dermal FBs	Antifibrotic	COL1A1, COL1A2, COL3A1, FBNI	52,56
miR-31	Up	Skin, FBs	Unclear	COL1A1, COL1A2	52
miR-l29-5p	Down	FBs	Unclear	COL1A1, COL1A2	232
miR-145	Down	Skin, FBs	Unclear	SMAD3	52
miR-146	Up	Skin, FBs	Unclear	SMAD4	52
miR-503	Up	Skin, FBs	Unclear	SMAD7	52
Circulating miRNAs
miR-7	Up in dcSSc, down in lcSSc	FBs, skin, serum	Antifibrotic	COL1A1, COL1A2	233,234
miR let-7a	Down	FBs, serum	Unclear	COL1A1, COL1A2	235
miR-29a	Down	Hair shaft, serum	Antifibrotic	COL1A1, COL1A2,	236
miR-92-a	Up	FBs, serum	Unclear	MMP-1	237
miR-142	Up	Serum	Unclear	Integrin av	238
miR-150	Down	FBs, serum	Profibrotic	Integrin β3	53
miR-l96a	Down	FBs, serum, and hair shafts	Antifibrotic	COL1A1, COL1A2	239,240

Note: Human cells in origin, unless otherwise specified.

Abbreviations: miRNA, microRNA; SSc, systemic sclerosis; FB, fibroblast; SMAD, intracellular proteins that transduce extracellular signals from transforming growth factor-β ligands; COL1, type 1 collagen; dcSSc, diffuse systemic sclerosis; lcSSc, limited cutaneous systemic sclerosis; MMP-1, matrix metalloproteinase 1.

Table 3 Specific nuclear antigen and nonantinuclear autoantibodies and organ involvement in SSc

	SSc organ involvement
	Lung	Skin	DU	Joint/muscle	GI, renal, cardiac
Specific nuclear antigen antibodies
Histones (AHA)#	+ (ILD)
U1-RNP**	+ (PH)			+ (myositis)
U3-RNP*	+ (ILD, PH)	+ (limited)		+ (myositis, joint)
U11-RNP**	+ (severe ILD)
UI2-RNP**	+ (severe ILD)
RNP¶	+ (PH)			+ (myositis, joint)
SSA (R060)**	+ (ILD)
R052 (TRIM21)**	+ (ILD)			+/− (myositis)
Ku*				+ (myositis)	+ (dysphagia)
Scl-70**	+ (ILD)	+ (diffuse)	+		+ (cardiac)
ACA/CENP (A-E)	+ (PH, ILD)	+ (limited)	+	+ (myositis)	+ (PBC)
Th/To*	+ (ILD, PH)	+ (limited)
RNA-pol-I*		+ (diffuse)			+ (GAVE, renal crisis)
RNA-pol-II*		+ (diffuse)			+ (renal crisis)
RNA-pol-III*	+ (ILD)	+ (diffuse)			+ (GAVE, renal crisis)
Anti-RuvBLI/2		+ (diffuse)		+ (myositis, joint)
Anti-tRNA synthetases PL-7**	+ (ILD)			+/− (myositis)
PM-Scl**	+ (ILD)		+	+ (myositis)
Non-ANA
AECA	+ (ILD)		+
EIF-2	+ (ILD)
AFA		+ (diffuse)	+
Anti-M3R					+ (GAVE)
AMA M2					+ (PBC)

Notes:

* Nucleolar pattern at Hep-2 immunofluorescence (IF);

** speckled pattern at IF;

# homogeneous pattern at IF;

¶ cytoplasmic pattern at IF. Antibodies with rare incidence (<5%) have been excluded. + indicates established association (several studies). +/− indicates insufficient body of evidence or controversial data. Blank spaces in the table indicate non reported. © Georg Thieme Verlag KG. Adapted with permission from Bonella F, Costabel U. Biomarkers in connective tissue disease-associated interstitial lung disease. Semin Respir Crit Care Med. 2014;35(2):181–200.²⁴¹

Abbreviations: ACA, anti centromere antibody; AECA, anti endothelial cells antibodies; AFA, antifibrillarin antibodies; AHA, anti histone antibodies; AMA, antimitochondrial antibodies; ANA, antinuclear autoantibodies; CENP, centromere associated protein; DU, digital ulcer; EIF-2, eukaryotic initiation factor 2; GAVE, gastric antral vascular ectasia; GI, gastrointestinal; ILD, interstitial lung disease; M3R, muscarinic receptor 3; PBC, primary biliary cyrrosis; PH, pulmonary hypertension; PM, polymyositis; RNA-pol, RNA polymerase; RNP, ribonucleoprotein; SSA, anti-Sjögren’s-syndrome-related antigen A; SSc, systemic sclerosis.

Table 4 Circulating biomarkers associated with organ involvement in SSc and their cellular source

Organ and cellular source	Protein	Evidence	Reference
Lung
Damaged alveolar epithelium	SP-A	+	129
	SP-D†	+++	93,108,127,130,145,148
	KL-6	+++	93,108,114-116,127,130,145,242,243
	CXCL9	+/−	152
	CXCL10	+/−	151,152,244,245
	CXCL12	+	153
Extracellular matrix (ECM)	MMP-7	++	137
	MMP-9	+	88
	MMP-12	++	89
	ADAM-12 (reduced)	+	−39
Activated macrophages	CCL8	++	144–146,148
	CCL2	+++	102,150–152,154,245
	IL-6	+++	102,152,154,246,247
	IL-10	+	102,152
	TNF-alpha	+	152,164
	CTGF	+	248
	YKL-40	+	133
Activated lymphocytes T
Subset Th2	IL-4	+/−	152
	IL-13	+	247
Subset Thl7	IL-17A	+/−	247
	IL-22	+	162
	IL-23	+	161^^
Activated lymphocytes B	sFLC*	+	168
Activated platelets	CXCL4	+++	46,155
Damaged endothelium	ET-1	++	171-173,175
	VCAM-1	+	174,175
	E-Selectin	+	175
	vEGF	+	175
Acute-Phase proteins	CRP^Table Footnote¶,Table Footnote§	+++	249
	PTX3	+	206
Skin
ECM	OPN	+	85
Epithelia (airway/intestine)	MMP 9 and I2	++	88,89
Adipocytes	KL-6	+/−	93
	APN (reduced)	+	95,96
Vascular involvement
Peripheral (DU)	PIGF	+	100
Pulmonary hypertension	EPC (reduced)	+	100
	IL-6	+	101
	BNP	+++	197-200,250,251
	ET-1	++	173,175
	GDF-15	++	202,203
	PTX3	++	205,206
	FGF (reduced)	+	206

Notes: Otherwise indicated, serum levels of the biomarkers are increased. +/− indicates controversial (non confirmatory studies); + indicates one single center study; ++ indicates more single center studies; +++ indicates large cohort studies;

† GENISOS cohort study;

* free light chains of immunoglobulins;

¶ CSRG (Canadian Scleroderma Research Group);

§ confirmed strong association with restrictive functional impairment but not with ILD. The evidence of clinical utility is scored in the table. © Georg Thieme Verlag KG. Adapted with permission from Bonella F, Costabel U. Biomarkers in connective tissue disease-associated interstitial lung disease. Semin Respir Crit Care Med. 2014;35(2):181–200.²⁴¹

Abbreviations: ADAM, A disintegrin and metalloproteinase; APN, adiponectin; BNP, brain natriuretic peptide; CCL, chemokine (C-C motif) ligand; CRP, C reactive protein; CTGF, connective tissue growth factor; CXCL, chemokine (C-X-C motif) ligand; DU, digital ulcers; ET-1, endothelin 1; EPC, circulating endothelial progenitor cells; FGF, fibroblast growth factor; GDF-15, growth differentiation factor 15; IL, interleukin; ILD, interstitial lung disease; KL-6, Krebs von den Lungen-6; MMP, matrix metalloproteinase; OPN, osteopontin; sFLC, free light chains of immunoglobulins; PIGF, placental growth factor; PTX3, pentraxin 3; SP, surfactant protein; SSc, systemic sclerosis; TNF-alpha, tumor necrosis factor alpha; YKL-40, chitinase-3-like protein 1; VCAM-1, vascular cell adhesion protein 1; VEGF, vascular endothelial growth factor.

Jain KK. Technology for Discovery of Biomarkers. In: The Handbook of Biomarkers. Vol 1. Totowa, NJ: Humana Press; 2010:23–72.

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Bonella F, Costabel U. Biomarkers in connective tissue disease-associated interstitial lung disease. Semin Respir Crit Care Med. 2014;35(2):181–200.

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