Genotype-specific molecular and functional trajectories in NGLY1 deficiency: a comparative multi-omics study of patient-derived iPSC neurons

NGLY1 deficiency is an ultra-rare congenital disorder of deglycosylation with prominent neurodevelopmental impairment and marked allelic heterogeneity. Prior human neuronal models have characterized NGLY1 loss largely within single genetic backgrounds, leaving open whether distinct patient genotypes converge on a shared molecular state or follow divergent routes. Because clinical presentations overlap across genotypes, resolving this question requires a direct, within-system comparison of multiple genotypes rather than characterization of any single line.

We generated iPSC-derived glutamatergic neurons from three patients carrying distinct biallelic NGLY1 genotypes (R401X/R401X, Q208X/G310G, L318P/R390P) and two healthy controls, and profiled them head-to-head using matched transcriptomics and proteomics at day 28, RNA to protein concordance and differential exon usage analysis, pathway and phenotype network integration, and longitudinal multielectrode array (MEA) recordings during early network maturation.

Across both molecular layers, samples segregated strongly by genotype rather than forming a unified disease signature. Differential expression revealed pronounced between-genotype heterogeneity with only a small shared molecular core. RNA to protein concordance was moderate and genotype-dependent, and differential exon usage was enriched among discordant genes in a mutation-dependent manner, implicating splicing-level regulation in the transcript to protein divergence. Integrated networks linked genotype-specific concordant changes to synaptic signaling, calcium handling, metabolism, and extracellular matrix remodeling, and connected them to overlapping clinical phenotypes through distinct molecular routes. MEA recordings showed delayed maturation and genotype-dependent shifts in network excitability, with functional clustering that mirrored the molecular separation.

Distinct NGLY1 genotypes produce divergent molecular and functional trajectories that converge on impaired network maturation through non-identical routes. These findings reframe NGLY1 deficiency as a set of genotype-specific molecular states rather than a single phenotype, with direct implications for mechanistic subgrouping and the design of preclinical models and validation studies.