Targeted 3.34-Million CpG Site Sequencing Reveals Preliminary Cord Blood Epigenetic Alterations of Autism Spectrum Disorder: A Pilot Study Highlighting the PCDHA1-PCDHA8 Cluster.

Rising reported Autism Spectrum Disorder (ASD) prevalence underscores the need to explore early perinatal biological markers. To conduct an exploratory pilot investigation of cord blood DNA methylation patterns as potential early epigenetic candidate loci associated with ASD in infants born to mothers with obesity and gestational diabetes. This prospective pilot study analyzed 14 mother-infant pairs comprising infants born to mothers with obesity and gestational diabetes later diagnosed with ASD (n = 2); healthy infants born to mothers with obesity and diabetes (IODM, n = 6); and healthy controls (IHM, n = 6). Targeted methyl-capture sequencing (covering 3.34 million CpG sites) identified differentially methylated region (DMR)-associated genes. Neurodevelopment was evaluated at 24-26 months using Bayley-III scales. ASD infants showed lower Bayley-III scores across all domains. In this small cohort (n = 2 ASD cases), we identified 249 DMR-associated genes common to both ASD vs. IHM and ASD vs. IODM comparisons, representing preliminary candidate loci. Enrichment analysis identified homophilic cell-cell adhesion as the most significant pathway (fold enrichment = 8.28; FDR = 6.31 × 10-6), driven by the PCDH1-PCDHA8 cluster, alongside morphogenesis in neuron differentiation (fold enrichment = 3.45; FDR = 2.08 × 10-2). Although prospective validation in larger independent cohorts is required, these pilot results suggest that cord blood PCDHA1-PCDHA8 cluster methylation alterations represent preliminary candidate loci for hypothesis generation and future biomarker evaluation, regardless of maternal metabolic status.