v v SKIN BIOMARKERS FOR CYSTIC FIBROSIS SCREENING Esteves, C. Z. 1 ; Dias, L. A. 1 ; Dias, E. O. 1 ; de Oliveira, D. N. 1 ; Melo, C. F. O. R. 1 ; Delafiori, J. 1 ; Gomez, C. C. S. 2 ; Ribeiro, J. D. 2 ; Ribeiro, A. F. 2 ; Levy, C. E. 3 ; Catharino, R. R. 1 1 Innovare Biomarkers Laboratory, School of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo 2 Pediatric Department, University of Campinas, Campinas, São Paulo, Brazil 3 Clinical Pathology Department, University of Campinas, Campinas, São Paulo, Brazil Cystic fibrosis (CF) is a genetic disorder caused by mutations at the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which leads to impaired regulation of CFTR ion channel. As a result, the transportation of electrolytes and fluids through cell membranes become abnormal affecting overall physiological functioning and patients’ quality of life. The most common mutation at the CFTR gene is called F508del mutation. However, the great amount of known CFTR mutations, generates genetic and clinical symptoms heterogeneity, which might compromise a conclusive and fast diagnosis. The sweat test, a gold standard method, consists in the determination of chloride concentration in stimulated sweat production, an uncomfortable method for the patient with the limitation of inconclusive diagnosis in borderline chloride concentrations. DNA test is also limited due to the expensive and time-consuming process and is dependent of the variance of monitored genes. Due to the present limitations, the association of methods and the development of new techniques are required to reduce the borderline window and improve the accuracy of the diagnosis. The identification of CF biomarkers through metabolomics is a promising tool in clinical area and has been previously reported for other biological samples. This MS-based approach proposes a simplified method for the collection of skin surface molecules, without sweat stimulation and sample preparation. The sample collection method presents a simplified approach with good adsorption and stabilization of molecules, advantageous for sample preserving and transportation, being a non-invasive and comfortable method. This metabolomic strategy using HRMS elucidated molecules as biomarkers of CF dysfunctional metabolism, that may be useful to assist in a potential accurate CF diagnosis and monitoring the progression of the disease, which may improve patient’s quality of life. Nevertheless, further investigation of others CF mutations, the increase of subjects and inclusion of borderline patients are required. 1. Sosnay, P.R. et al. (2013). Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nature genetics 45(10), 1160- 1167. 2. Gibson, L.E., and Cooke, R.E. (1959). A test for concentration of electrolytes in sweat 504 in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23(3), 545-549. 3. Esteves, C. Z. et al. (2018). Skin Biomarkers for Cystic Fibrosis: a Potential Non- Invasive Approach for Patient Screening. Frontiers in Pediatrics 5, 290. 4. Xia, J. et al. (2015). MetaboAnalyst 3.0—making metabolomics more meaningful. Nucleic acids research 43(W1), W251-W257. INTRODUCTION METHODOLOGY Patient selection and sample collection: A retrospective study was performed for the collection of patients’ samples at Cystic Fibrosis Clinic of the Clinics Hospital at University of Campinas according to protocol approved by the Research Ethics Committee of the School of Medical Sciences – University of Campinas/Brazil (Protocol Number: 1.100.978). The subjects were composed of healthy individuals for control (CT) group as the same age and gender of cystic fibrosis (CF) patients with sweat test chloride concentration > 60 mEq/L and presence of homozygosis for F508del mutation. Samples preparation, mass spectrometry and statistical analysis: RESULTS The sample collection was performed with 16 CF patients. The ages of CF patients and CT group (16 subjects) ranged from 5 to 19 years old (mean: 12 years; median: 13 years). The average concentration of chloride in sweat test for CF patients was 111 mEq/L (minimum: 76 mEq/L; maximum: 166 mEq/L). The comparison of spectral data (Fig. 2) using o-PLS-DA plot (Fig. 3) showed complete separation of CT and CF groups, which allowed the selection of 7 biomarkers for CF, all with VIP score above 2. The biomarkers observed for CF group are related to the dysfunctional metabolism of patients with F508del CFTR mutation, highlighting the importance of the metabolomics as complementary approach for the diagnosis. This group of biomarkers translate not only the sweat gland ionic imbalance but also the pathophysiology and progression of the disease. Figure 2. Representative mass spectra comparing the skin imprints of control individuals and CF patients. Negative ion mode, m/z 200-700. REFERENCES DISCUSSION AND CONCLUSIONS ACKNOWLEDGEMENTS 17g Figure 3. Orthogonal partial least squares discriminant analysis (o- PLS-DA) with a clear separation between control and patients with cystic fibrosis.