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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 Paulo2 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 cysticfibrosis 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 insweat 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

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Figure 3. Orthogonal partial least squares discriminant analysis (o-

PLS-DA) with a clear separation between control and patients with

cystic fibrosis.