Two-dimensional differential in-gel electrophoresis proteomic approaches reveal urine candidate biomarkers in pediatric obstructive sleep apnea

Abstract

Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children.

Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA.

Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA.

Measurements and main results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver-operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%.

Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted.

Figure 1.

(A) Two-dimensional differential in-gel electrophoresis (2D-DIGE) gels of urinary proteins of a child with obstructive sleep apnea (OSA) and a matched child with primary snoring (PS) and fluorescence image overlay. (B) Candidate spot analysis showing spot location on gel, differential intensity analysis for two spots showing increased expression in OSA (spots 1 and 2), and two spots in which decreased expression occurred (spots 3 and 4).

Figure 2.

Representative Western blots for tenascin, tribbles homolog protein-2, and zinc finger protein-81 in urinary proteins from children with obstructive sleep apnea (OSA), those with primary snoring (PS), and healthy control subjects (CO).

Figure 3.

Receiver–operator curves for kallikrein-1, urocortin-3, orosomucoid, and uromodulin urine levels (after correction for corresponding urinary creatinine concentrations). Dashed lines indicate 95% confidence intervals.

Figure 4.

Box plots of urinary concentrations of orosomucoid-1 (after correction for corresponding urinary creatinine concentrations) in 30 control children (CO), 30 children with primary snoring (PS), and 60 children with obstructive sleep apnea (OSA). OSA versus PS or CO: P < 0.0001.

Figure 5.

(A) Receiver–operator curve for combinatorial analyses using one or more of the cutoff levels defined for kallikrein-1, urocortin-3, orosomucoid-1, and uromodulin urine levels (after correction for corresponding urinary creatinine concentrations). Use of two or more of these cutoffs yields a sensitivity of 100% and a specificity of 96.5%. Dashed lines indicate 95% confidence intervals. (B) Dot plot illustrating the discriminatory ability of three or more cutoff levels defined for kallikrein-1, urocortin-3, orosomucoid-1, and uromodulin urine levels (after correction for corresponding urinary creatinine concentrations). A sensitivity of 95% and a specificity of 100% were achieved.

Figure 5.

(A) Receiver–operator curve for combinatorial analyses using one or more of the cutoff levels defined for kallikrein-1, urocortin-3, orosomucoid-1, and uromodulin urine levels (after correction for corresponding urinary creatinine concentrations). Use of two or more of these cutoffs yields a sensitivity of 100% and a specificity of 96.5%. Dashed lines indicate 95% confidence intervals. (B) Dot plot illustrating the discriminatory ability of three or more cutoff levels defined for kallikrein-1, urocortin-3, orosomucoid-1, and uromodulin urine levels (after correction for corresponding urinary creatinine concentrations). A sensitivity of 95% and a specificity of 100% were achieved.