Multi-omic brain and behavioral correlates of cell-free fetal DNA methylation in macaque maternal obesity models (GC-FID dataset infant brains)

Maternal obesity during pregnancy is associated with neurodevelopmental disorder (NDD) risk. We utilized integrative multi-omics to examine maternal obesity effects on offspring neurodevelopment in rhesus macaques by comparison to lean controls and two interventions. Differentially methylated regions (DMRs) from longitudinal maternal blood-derived cell-free fetal DNA (cffDNA) significantly overlapped with DMRs from infant brain. The DMRs were enriched for neurodevelopmental functions, methylation-sensitive developmental transcription factor motifs, and human NDD DMRs identified from brain and placenta. Brain and cffDNA methylation levels from a large region overlapping mir-663 correlated with maternal obesity, metabolic and immune markers, and infant behavior. A DUX4 hippocampal co-methylation network correlated with maternal obesity, infant behavior, infant hippocampal lipidomic and metabolomic profiles, and maternal blood measurements of DUX4 cffDNA methylation, cytokines, and metabolites. Ultimately, maternal obesity altered infant brain and behavior, and these differences were detectable in pregnancy through integrative analyses of cffDNA methylation with immune and metabolic factors.

Methods
Samples were analyzed on a Perkin Elmer Clarus 500 GC-FID system (Perkin Elmer) equipped with a DB-FFAP polyethylene glycol fused capillary column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness; Agilent Technologies; 1223232). The injector and detector temperatures were 285°C and 300°C, respectively. The initial oven temperature was 80°C. It was held at 80°C for 2 min, increased by 10°C/min to 185°C, raised to 249°C at 6°C/min and lastly held at 240°C for 44 min. The total run time was 65 min. Helium was used as the carrier gas with a maintained flow rate of 1.3 mL/min. The injection volume was 1 µL per sample. The split ratio was 10:1. A custom-made mix of 29 FAME standards was used to identify each fatty acid based on retention time. Fatty acid concentrations were determined by comparing GC peak areas to the internal standard area. Cholesterol concentration was calculated based on a five-point standard curve of cholesterol and 5α-Cholestane as surrogate. The linear dynamic range of the calibration curve for cholesterol was 0.125 mg/mL to 2 mg/mL. All chromatograms were analyzed on Totalchrom Navigator Software Version 6.3.2 and converted to pdf formats.