Background Type VII collagen (col7) is the main component of the anchoring fibrils that connects the basement membrane to the underlying interstitial matrix and has mainly been investigated for its role in blistering skin diseases. It has been investigated for its role in dystrophic epidermolysis bullosa, a severe skin disease. Furthermore, increased levels of type VII collagen in skin has been reported for patients with systemic sclerosis (SSc).
Objectives The objective was to develop and characterize a blood-based marker assessing col7 degradation in patients with SSc.
Methods We identified a specific fragment of col7 in serum from COPD patients, which was not found in controls, using mass spectrometry. A monoclonal antibody was raised against the first ten amino acids of the neo-epitope (KLH-CGG-GPPGPPGRLV) and employed in a competitive ELISA (C7M). The C7M assay was validated technically and was subsequently evaluated in 2 cohorts including SSc patients. The first cohort (SSc#1; n=35) consisted of early (<2 years of SSc symptoms; n=16) and late (>10 years of disease with stable skin for at least 6 months, n=19) diffuse SSc patients, while the second cohort (SSc#2; n=119) consisted of limited (n=78) and diffuse (n=41) SSc patients. Serum C7M levels were likewise measured in healthy subjects and compared to the levels of SSc patients using the Kruskal-Wallis test with Dunn's multiple comparisons test comparing healthy individuals with the two SSc cohorts.
Results A technically robust competitive ELISA (C7M), which was highly specific for a col7 fragment was developed. The assay showed acceptable inter- (13%) and intra-assay (9%) variation, linearity (102% dilution recovery), analyte stability (102% recovery after 4 freeze/thaw cycles), and interference.
The C7M marker was evaluated by comparing serum levels in healthy donors with patients with SSc (Figure). Serum C7M levels were not associated with age, gender, BMI, or disease duration. The geometric mean serum C7M level in healthy donors was 4.6 ng/mL (95% CI 3.7–5.6 ng/mL). The geometric mean serum C7M levels were significantly elevated in both cohorts of patients with SSc (SSc#1, 13.6 ng/mL [95% CI 11.1–16.5], p<0.0001; SSc#2, 9.2 ng/mL [95% CI 8.3–10.2], p<0.0001). Furthermore, a significant difference were observed between the two cohorts (P=0.05).
Conclusions The C7M ELISA enabled quantification of type VII collagen degradation in serum. Elevated serum C7M levels indicated that the remodeling of type VII collagen was significantly increased in patients with SSc, suggesting a pathological role.
Acknowledgements We thank Biogen Idec's SSc department for their contribution.
Disclosure of Interest None declared
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