Fig 1: Validation of spectrometry data for C7 and LUM with secondary methods. (A) Spectral counts of top changing proteins (Combined Cohort, FDR < 0.1, |log2FC| > 0.5) in FSHD vs. Healthy across cohorts. *p < 0.05, **p < 0.01, ***p < 0.001 by Wald‐test. (B) Western blot verification of C7 and LUM levels. Tetraspanin CD9 was used as an EV marker. Stain‐free SDS‐PAGE gel was used to visualize total protein levels. (C) Quantification of Western blot data. C7 band intensity was normalized to total protein intensity. **p < 0.01 by Wilcoxon test. (D) ELISA verification of EV LUM levels. *p < 0.05 by Wilcoxon test. (E) Proteinase K (PK) treatment of plasma EV isolated from an FSHD1 patient (20 μg/mL PK for 1 h). Protease inhibitor PMSF (20 μM) is used to timely quench PK. 1% Triton X‐100 is used to lyse open EVs. ApoB is used as a corona layer control. (F) Transmission Electron Microscopy (TEM) of plasma EVs isolated from an FSHD1 patient. Immunogold staining detected positive LUM signals (gold arrows) on EV surface (Mean AuNP/EV: 1.40 ± 0.96; percent positive EVs: 54% ± 14%). The graph on the right shows EV size distribution and concentration measured by Nanoparticle Tracking Analysis (NTA). Uncropped Western blots can be found in Figure S8.
Fig 2: Correlation of LUM with clinical outcome measures. Pearson R correlations of FSHD‐COM score, Self‐selected Gait Speed and Timed Up and Go with LUM levels in Cohort 1. Pearson R correlations with FSHD‐COM score are adjusted for sex. Confidence intervals (95%) for each correlation are indicated by blue shade.
Fig 3: Upregulated proteins in FSHD1 patients significantly correlate with clinical outcome measures of patients in Cohort 1. (A) Composite z‐scores of a panel of four upregulated proteins (log2FC > 0.5, FDR < 0.1 in Combined Cohort) were calculated and evaluated in FSHD1 patients and healthy controls of Cohort 1. All participants of Cohort 1 were ranked based on composite z‐scores for these select proteins. (B) Boxplots of composite z‐scores from (A). Points represent individual samples; boxes show interquartile range with median; red lines indicate means. p calculated using the Wilcoxon rank‐sum test. (C) Pearson R correlation of disease severity (as evaluated by FSHD‐COM score) with composite z‐score (FDR < 0.1 in Combined Cohort) in Cohort 1. (D) Composite z‐scores of a panel of eight upregulated proteins (log2FC > 0.5, p < 0.05 in Cohort 2) are calculated and evaluated in FSHD1 patients and healthy controls of Cohort 1. All participants of Cohort 1 are ranked based on composite z‐scores for these select proteins. (E) Boxplots of composite z‐scores from (D). Points represent individual samples; boxes show interquartile range with median; red lines indicate means. p calculated using the Wilcoxon rank‐sum test. (F) Pearson R correlation of disease severity (as evaluated by FSHD‐COM score) with composite z‐score (p < 0.05 in Cohort 2) Cohort 1. All Pearson R correlations with FSHD‐COM scores are adjusted for sex (Partial Pearson). Confidence intervals (95%) for each correlation are indicated by blue shade. (G) An elastic‐net–regularized logistic regression model trained on 75% of Cohort 2 identified LUM and C7 upregulation and CNDP1 downregulation as key predictors of FSHD1. The resulting model showed strong discrimination in the Cohort 2 hold‐out set and generalized well to the independent Cohort 1 validation set. Violin plots show model scores (linear predictor) for each dataset with Cohort 1 points colored by FSHD‐COM severity. Higher model scores correspond to higher predicted probability of FSHD1. AUC: Area Under Curve. Sensitivity (sens.) and specificity (spec.) scores indicate model's ability to predict healthy controls and FSHD1 patients, respectively. Scale: 0–1 where 1.0 is perfect. (H) Permutation testing of the elastic‐net model in Cohort 1. Histogram shows AUC values from 1000 label permutations; dashed line indicates the observed AUC. The permutation p‐value reflects the proportion of permuted models with AUC ≥ observed. (I) ROC curves for Lumican (LUM) alone and the composite biomarker panel (LUM, C7, C9, FBLN1). The composite panel shows higher AUC, indicating improved discrimination. The dashed diagonal represents random classification.
Supplier Page from Abcam for Human Lumican ELISA Kit