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.