Dynamic Changes in the Urinary Proteome of Normal Pregnant Mice Carrying Phenylketonuria-Affected Fetuses

Phenylketonuria (PKU) is an autosomal recessive disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene. Clinically, maternal PKU syndrome—in which affected mothers exhibit hyperphenylalaninemia—leads to irreversible fetal neurological injury during gestation. However, it remains unclear whether fetuses with intrinsic PKU, carried by metabolically normal mothers, trigger measurable molecular changes in maternal urine, and non-invasive prenatal monitoring methods for this scenario are lacking. In this study, Pah+/− female mice were mated with Pah+/− male mice and wild-type male mice, respectively. Urine samples were collected longitudinally from pregnant mice at 10 time points during gestation from Day 1 to Day 19. Data-independent acquisition (DIA) quantitative proteomics combined with GO/KEGG analysis, WGCNA, and a dual-strategy LASSO-random forest approach was used to screen candidate biomarkers. The results showed that group-specific proteomic signals were detectable as early as Day 1, with a small subset of differentially expressed proteins persisting across gestation and exhibiting temporal dynamics. Permutation tests indicated that the probability of random generation of the differential protein combination was low (0.04–0.06), suggesting that it was not randomly generated. The intersection of the two machine-learning screening strategies identified five proteins: GINM1, PRG4, LY6A, NTM, and YIPF3. GLO1 was further added as a candidate biomarker based on its significant differential expression and central role in the protein co-expression network, resulting in six candidate biomarkers in total. This study found that the maternal urine proteome can non-invasively reflect the systemic maternal response induced by fetal intrinsic PKU.