Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection

Abstract

A typical clinical manifestation of growth hormone deficiency (GHD) is a short stature resulting from delayed growth, but GHD affects bone health, cardiovascular function and metabolic profile and therefore quality of life. Although early GH treatment during childhood has been shown to improve outcomes, no single biochemical parameter is currently available for the accurate diagnosis of GHD in children. There is hence a need for non-invasive biomarkers. In this study, the relative abundance of serum proteins from GHD children and healthy controls was measured by next-generation proteomics SWATH-MS technology. The data generated was analysed by machine-learning feature-selection algorithms in order to discover the minimum number of protein biomarkers that best discriminate between both groups. The analysis of serum proteins by a SWATH-MS approach yielded a useful method for discovering potential biomarkers of GHD in children. A total of 263 proteins were confidently detected and quantified in each sample. Pathway analysis indicated an effect on tissue/organ structure and morphogenesis. The top ten serum protein biomarker candidates were identified after applying feature-selection data analysis. The combination of three proteins - apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein - demonstrated the best classification performance for our data. In addition, the apolipoprotein group resulted in strong over-representation, thus highlighting these proteins as an additional promising biomarker panel.

Significance: Currently there is no single biochemical parameter available for the accurate diagnosis of growth hormone (GH) deficiency (GHD) in children. Simple GH measurements are not an option: because GH is released in a pulsatile action, its blood levels fluctuate throughout the day and remain nearly undetectable for most of that time. This makes measurements of GH in a single blood sample useless for assessing GH deficiency. Actually, the diagnosis of GHD includes a combination of direct and indirect non-accurate measurements, such as taking several body measurements, testing GH levels in multiple blood samples after provocative tests (GH peak <7.3ng/mL, using radioimmunoassay), and conducting magnetic resonance imaging (MRI), among others. Therefore, there is a need for simple, non-invasive, accurate and cost-effective biomarkers. Here we report a case-control study, where relative abundance of serum proteins were measured by next-generation proteomics SWATH-MS technology in 15 GHD children and 15healthy controls matched by age, sex, and not receiving any treatment. Data generated was analysed by machine learning feature selection algorithms. 263 proteins could be confidently detected and quantified on each sample. The top 10 serum protein biomarker candidates could be identified after applying a feature selection data analysis. The combination of three proteins, apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein, showed the best classification performance for our data. In addition, the fact that the pathway and GO analysis we performed pointed to the apolipoproteins as over-represented highlights this protein group as an additional promising biomarker panel for the diagnosis of GHD and for treatment evaluation.

Keywords: Biomarkers; Growth hormone deficiency; Quantitative proteomics; SWATH.