Establishment of a tear protein biomarker panel differentiating between Graves' disease with or without orbitopathy

Abstract

Background: Graves' orbitopathy (GO) is an autoimmune inflammatory ocular complication and one of the most frequent manifestations of Graves' disease (GD). Clinical judgment of GO is subjective sometimes leading to clinical and therapeutic challenges. Better tools to diagnose this severe complication are warranted.

Patients and methods: The aim of the present study was to evaluate tear levels of LYZ, LACRT and AZGP1 in GD patients with or without GO, as possible biomarkers for GO. Tear samples were collected from GD patients with moderate-to-severe GO (n = 21) and no clinical signs of GO (n = 21). Additionally, 18 GD patients with mild GO and 9 patients without GO were included in a further part of the study.

Results: Tear levels of LYZ (p < 0.001), LACRT (p = 0.004) and AZGP1 (p = 0.001) were significantly elevated in GD patients with moderate-to-severe GO compared to GD patients without GO. The discriminatory power of the three biomarkers, combined in a panel was confirmed by ROC plot analysis, with an AUC value of 0.93 (sensitivity of 95%; specificity of 80%). Since LYZ showed the best performance in discriminating between GD patients with (moderate-to-severe) and without GO (in combination with limited sample volume available), LYZ levels were also measured in tears from GD patients with mild GO and without GO. Significantly higher levels of LYZ were measured in GD patients with mild GO compared to those without GO (p = 0.003).

Conclusions: We have established a novel three-protein biomarker panel that is able to discriminate between GD patients with and without GO, which might aid in diagnostic evaluation of GO as well as an indicator for disease activity.

Fig 1. Tear levels of LYZ, LACRT and AZGP1 in moderate-to-severe GO patients vs GD patients without GO obtained with ELISA.

Box and-whisker plot showing median and interquartile range (IQR). (A) The median for LYZ (268.9 μg/mg vs 84.3 μg/mg), (B) LACRT (6.9 μg/mg vs 0.9 μg/mg) and (C) AZGP1 (42.5 μg/mg vs 22.1 μ/mg). Mann-Whitney U-tests were performed and p-values are indicated on each graph.

Fig 2. Diagnostic performance of the 3-protein biomarker panel in discriminating GD patients with moderate-to-severe GO from GD patients without GO.

ROC curve analysis showing LYZ, LACRT and AZGP1 combined in a panel, with an AUC of 0.93. The curve is created by plotting the true positive rate (sensitivity) against the false positive rate (1 –specificity).

Fig 3. Tear levels of LYZ in mild GO patients vs GD patients without GO obtained with ELISA.

Box and-whisker plot showing median and interquartile range (IQR). The median for LYZ (51.4 μg/mg vs. 13.9 μg/mg). Mann-Whitney U-test was performed and the p-value is indicated on the graph.