Fig. 3

A common genetic structural variant is significantly associated with 22q13.33 DNA methylation and ASD. A Insertion location (orange) relative to the 22q13.33 hypomethylated block (blue), and the novel transcript, NHIP (red) in the UCSC genome browser. The 22q13.33 co-methylated block was 117,974 bp in length (blue). NHIP TSS was located 7881 bp downstream from the start of the 22q13.33 hypomethylated block. The insertion (not in the reference genome) is 15,013 bp upstream from the start of the 22q13.33 hypomethylated block. B The association matrix shows ANOVA p values for the comparison of the insertion genotype (homozygous for insertion versus not) with smoothed methylation levels within each of 12 DMRs located in the 22q13.33 hypomethylated block from the discovery group (ASD n = 41, TD n = 37). C Association was tested between insertion genotype (Y, homozygous for insertion; N, not) and 22q13.33 co-methylated block methylation levels (discovery group, ASD n = 41, TD n = 37). ASD showed significantly lower DNA methylation levels compared to TD placenta samples within the entire 22q13.33 co-methylated block (Mann-Whitney-Wilcoxon, p value = 0.008, ASD n = 41, TD n = 37, effect size = −0.645). Samples homozygous for the insertion had significantly lower methylation than those not having insertion on one or both alleles (Mann-Whitney-Wilcoxon, p value = 0.004, Y n = 29, N n = 49, effect size = − 0.644). When broken down by diagnosis, samples with insertion had significantly lower methylation specifically in ASD samples (Mann-Whitney-Wilcoxon, p value = 0.005, Y n = 20, N n = 21, effect size = −0.878), not TD samples (Mann-Whitney-Wilcoxon, p value = 0.847, Y n = 9, N n = 28, effect size = −0.173). D Periconceptional prenatal vitamin use was a significant modifier of 22q13.33 block methylation in placenta (discovery group, ASD n = 41, TD n = 37). Lower percent methylation at the 22q13.33 co-methylated block was significantly associated with not taking prenatal vitamins during the first month of pregnancy (Mann-Whitney-Wilcoxon, p value = 0.001), which was in the same direction as ASD risk. E UCSC genome browser map shows the insertion location (orange vertical line) relative to two adjacent CTCF sites (green arrows) and NHIP. Both undifferentiated and differentiated LUHMES cells have both CTCF sites, consistent with them being homozygous for the reference sequence. Additional brain tracks show the variability of the upstream CTCF site between human samples. ChromHMM tracks were derived from fetal brain, multiple brain regions, ovary, and placenta. Red, active promoter; yellow, active enhancer; green; active transcriptional elongation; purple, bivalent poised chromatin. F Working model to explain ASD risk associated with SV homozygosity. Illustrations created with BioRender.com