Background Autism spectrum disorder (ASD) involves complex genetics interacting with the perinatal environment, complicating the discovery of common genetic risk. The epigenetic layer of DNA methylation shows dynamic developmental changes and molecular memory of in utero experiences, particularly in placenta, a fetal tissue discarded at birth. However, current array-based methods to identify novel ASD risk genes lack coverage of the most structurally and epigenetically variable regions of the human genome. Results We use whole genome bisulfite sequencing in placenta samples from prospective ASD studies to discover a previously uncharacterized ASD risk gene, LOC105373085, renamed NHIP. Out of 134 differentially methylated regions associated with ASD in placental samples, a cluster at 22q13.33 corresponds to a 118-kb hypomethylated block that replicates in two additional cohorts. Within this locus, NHIP is functionally characterized as a nuclear peptide-encoding transcript with high expression in brain, and increased expression following neuronal differentiation or hypoxia, but decreased expression in ASD placenta and brain. NHIP overexpression increases cellular proliferation and alters expression of genes regulating synapses and neurogenesis, overlapping significantly with known ASD risk genes and NHIP-associated genes in ASD brain. A common structural variant disrupting the proximity of NHIP to a fetal brain enhancer is associated with NHIP expression and methylation levels and ASD risk, demonstrating a common genetic influence. Conclusions Together, these results identify and initially characterize a novel environmentally responsive ASD risk gene relevant to brain development in a hitherto under-characterized region of the human genome.

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Saliva is a widely used biological sample, especially in pediatric research, containing a heterogenous mixture of immune and epithelial cells. Associations of exposure or disease with saliva DNA methylation can be influenced by cell-type proportions. Here, we developed a saliva cell-type DNA methylation reference panel to estimate interindividual cell-type heterogeneity in whole saliva studies. Saliva was collected from 22 children (7-16 years) and sorted into immune and epithelial cells, using size exclusion filtration and magnetic bead sorting. DNA methylation was measured using the Illumina MethylationEPIC BeadChip. We assessed cell-type differences in DNA methylation profiles and tested for enriched biological pathways. Immune and epithelial cells differed at 164,793 (20.7%) DNA methylation sites (t-test p < 10 -8 ). Immune cell hypomethylated sites mapped to genes enriched for immune pathways (p < 3.2 x 10 -5 ). Epithelial cell hypomethylated sites were enriched for cornification (p = 5.2 x 10 -4 ), a key process for hard palette formation. Saliva immune and epithelial cells have distinct DNA methylation profiles which can drive whole saliva DNA methylation measures. A primary saliva DNA methylation reference panel, easily implemented with an R package, will allow estimates of cell proportions from whole saliva samples and improve epigenetic epidemiology studies by accounting for measurement heterogeneity by cell-type proportions.

Smoking impacts DNA methylation genome-wide in blood of newborns from maternal smoking during pregnancy and adults from personal smoking. We compared smoking-related DNA methylation in lung adenocarcinoma (61 never smokers, 91 current smokers, and 238 former smokers) quantified with the Illumina450k BeadArray in The Cancer Genome Atlas with published large consortium meta-analyses of newborn and adult blood. We assessed whether CpG sites related to smoking in blood from newborns and adults were enriched in the lung adenocarcinoma methylation signal. Testing CpGs differentially methylated by smoke exposure, we identified 296 in lung adenocarcinoma meeting a P < 10−4 cutoff, while previous meta-analyses identified 3,042 in newborn blood, and 8,898 in adult blood meeting the same P < 10−4 cutoff. Lung signals were highly enriched for those seen in newborn (24 overlapping CpGs, Penrichment = 1.2 × 10−18) and adult blood (66 overlapping CpGs, Penrichment = 1.2 × 10−48). The 105 genes annotated to CpGs differentially methylated in lung tumors, but not blood, were enriched for RNA processing ontologies. Some epigenetic alterations associated with cigarette smoke exposure are tissue specific, but others are common across tissues. These findings support the value of blood-based methylation biomarkers for assessing exposure effects in target tissues.

Background: Pregnancy measures of DNA methylation, an epigenetic mark, may be associated with autism spectrum disorder (ASD) development in children. Few ASD studies have considered prospective designs with DNA methylation measured in multiple tissues and tested overlap with ASD genetic risk loci.Objectives: To estimate associations between DNA methylation in maternal blood, cord blood, and placenta and later diagnosis of ASD, and to evaluate enrichment of ASD-associated DNA methylation for known ASD-associated genes.Methods: In the Early Autism Risk Longitudinal Investigation (EARLI), an ASD-enriched risk birth cohort, genome-scale maternal blood (early n = 140 and late n = 75 pregnancy), infant cord blood (n = 133), and placenta (maternal n = 106 and fetal n = 107 compartments) DNA methylation was assessed on the Illumina 450k HumanMethylation array and compared to ASD diagnosis at 36 months of age. Differences in site-specific and global methylation were tested with ASD, as well as enrichment of single site associations for ASD risk genes (n = 881) from the Simons Foundation Autism Research Initiative (SFARI) database.Results: No individual DNA methylation site was associated with ASD at genome-wide significance, however, individual DNA methylation sites nominally associated with ASD (P &lt; 0.05) in each tissue were highly enriched for SFARI genes (cord blood P = 7.9 × 10–29, maternal blood early pregnancy P = 6.1 × 10–27, maternal blood late pregnancy P = 2.8 × 10–16, maternal placenta P = 5.6 × 10–15, fetal placenta P = 1.3 × 10–20). DNA methylation sites nominally associated with ASD across all five tissues overlapped at 144 (29.5%) SFARI genes.Conclusion: DNA methylation sites nominally associated with later ASD diagnosis in multiple tissues were enriched for ASD risk genes. Our multi-tissue study demonstrates the utility of examining DNA methylation prior to ASD diagnosis.