Metabolic Signatures of Cystic Fibrosis Identified in Dried Blood Spots For Newborn Screening Without Carrier Identification

Cystic fibrosis (CF) is a complex multiorgan disorder that is among the most common fatal genetic diseases benefiting from therapeutic interventions early in life. Newborn screening (NBS) for presymptomatic detection of CF currently relies on a two-stage immunoreactive trypsinogen (IRT) and cystic fibrosis transmembrane conductance regulator (CFTR) mutation panel algorithm that is sensitive but not specific for identifying affected neonates with a low positive predictive value. For the first time, we report the discovery of a panel of CF-specific metabolites from a single 3.2 mm diameter dried blood spot (DBS) punch when using multisegment injection-capillary electrophoresis-mass spectrometry (MS) as a high-throughput platform for nontargeted metabolite profiling from volume-restricted/biobanked specimens with quality control. This retrospective case-control study design identified 32 metabolites, including a series of N-glycated amino acids, oxidized glutathione disulfide, and nicotinamide that were differentially expressed in normal birth weight CF neonates without meconium ileus (n = 36) as compared to gestational age/sex-matched screen-negative controls (n = 44) after a false discovery rate adjustment (q < 0.05). Also, 16 metabolites from DBS extracts allowed for discrimination of true CF cases from presumptive screen-positive carriers with one identified CFTR mutation and transient neonatal hypertrypsinogenemic neonates (n = 72), who were later confirmed as unaffected due to a low sweat chloride (<29 mM) test result. Importantly, six CF-specific biomarker candidates satisfying a Bonferroni adjustment (p < 7.25 × 10^–5) from three independent batches of DBS specimens included several amino acids depleted in circulation (Tyr, Ser, Thr, Pro, Gly) likely reflecting protein maldigestion/malabsorption. Additionally, CF neonates had lower ophthalmic acid as an indicator of oxidative stress due to impaired glutathione efflux from exocrine/epithelial tissue and elevation of an unknown trivalent peptide that was directly correlated with IRT (ρ = 0.332, p = 4.55 × 10^–4). Structural elucidation of unknown metabolites was performed by high-resolution MS/MS, whereas biomarker validation was realized when comparing a subset of metabolites from matching neonatal DBS specimens independently analyzed by direct infusion-MS/MS at an accredited NBS facility. This work sheds new light into the metabolic phenotype of CF early in life, which is required for better functional understanding of CFTR mutations of unknown clinical consequence and the development of more accurate yet cost-effective strategies for CF screening.