By analyzing the interaction between DNAm and ASPH on case-control status, we identified DMRs in genes encoding LINGO3, BLCAP;NNAT, NANOS2 and SLC2A14. In the absence of ASPH, PT and HC have the same methylation level; while ASPH exposure corresponded to an increase of methylation in HC and a decrease of methylation in PT. Consistently, we found a weak global correlation in methylation profiles associated with ASPH between groups.
The increase of methylation at these DMRs in HC may be part of an adaptive epigenetic response to ASPH, which could be absent in PT, who indeed have lower DNAm at these DMRs, in the context of ASPH. ASPH may induce different responses in the epigenome, and some of these paths may increase the risk for psychiatric disorders. Since previous studies have found alterations with LINGO3 (42–45), BLCAP;NNAT (46–49) and SLC2A14 (50, 51) genes or associated functions in SZ and BD, it might be possible that an altered response to ASPH at the DNAm and/or gene expression levels may disrupt brain development and function in PT. DNAm increases in HC might represent epigenetic modifications influenced by environmental exposure of ASPH, which may be associated with increased susceptibility to somatic diseases later in life, but not necessarily the transition to severe mental disorders. It may also represent protective-adaptive alterations that may increase resilience in healthy individuals later in life.
Due to its availability and ease of collection, this EWAS used blood as a surrogate tissue rather than disease specific tissue; however, methylation levels in blood and brain-tissue have a concordance (52). To further address this issue, we performed secondary analyses on the probes associated with LINGO3, BLCAP;NNAT, NANOS2 and SLC2A14 to investigate the relationship between DNAm in blood and at the same genomic loci in four brain regions (prefrontal cortex, entorhinal cortex, superior temporal gyrus and cerebellum) (38) and found significant correlations. However, because the response to ASPH is systemic and given the stability of DNAm profiles, our findings have relevance to psychiatric disorder even in the absence of a proven brain-blood correlation. In other words, changes in DNAm detected in blood may reveal an epigenetic mechanism of response to ASPH which is relevant for the development of psychiatric disorders.
Nonetheless, we detected significant interactions between ASPH and DNAm at the level of DMR mapping to loci containing genes with relevant brain functions. Particularly, LINGO3, a paralog to LINGO1, is expressed in neurons and a highly restricted population of Olig2-expressing oligodendroglia cells. A functional overlap between LINGO3 and LINGO1 has been suggested (53). The more studied, LINGO1, is a potent negative regulator of neuron and oligodendrocyte survival, axon regeneration, neurite extension, oligodendrocyte differentiation, axonal myelination and functional recovery; these processes are highly involved in numerous brain functions (54). Reduced oligodendrocyte densities have been reported in both SZ and BD (42–45). In SZ, LINGO1 has been found to be upregulated in the dorsolateral prefrontal cortex and hippocampus (55) and associated with the dysregulation in apoptosis of neurons (56), which might explain previously observed reductions in mean total neuron number in the putamen and caudate nucleus of individuals with SZ (57). So, if the DNAm changes that we detected in blood are mirrored by DNAm changes in the brain, as our results suggest, we could speculate that ASPH may lead to a decrease of methylation in individuals who later in life developed SZ or BD. Such DNAm decrease may be associated with an increased LINGO3 expression, consistent with the findings of increased LINGO1 expression and reduced oligodendrocyte densities in SZ and BD.
For the overlapping BLCAP;NNAT genes, the BLCAP gene encodes a protein that reduces cell growth by stimulating apoptosis, and the imprinted gene NNAT encodes a protein associated with brain development, assembly, maturation and maintenance of the central nervous system (CNS) (58). A recent EWAS study found that the DNAm of BLCAP;NNAT were strongly associated with left-handedness (59), a trait with low heritability and where epigenetic mechanisms have been proposed as an underlying etiological mechanism. Numerous scientific studies have demonstrated a higher occurrence of left-handedness in an array of psychiatric disorders, supporting the view that there is a genetic link between handedness and brain lateralization (46–49). Consistent with these studies, we report here a higher frequency of non-right handers among PT who experienced ASPH compared to ASPH in the HC.
Of note, NNAT is an imprinted gene (58), and alterations in the DNAm of imprinted genes (i.e. genes that carry parental allele-specific methylation profiles) have been documented in studies of in utero exposure to dietary micronutrients (60, 61), caloric restriction (62–64), protein restriction (65) and cigarette smoking (66). The ASPH related differences in the DNAm of NNAT gene reported here may be an example of a methylation alteration in an imprinted gene that can serve as a useful biosensor of ASPH exposure (67).
NANOS2 and Nanos genes are expressed in fetal and adult testis and ovary and the adult brain, particularly the hippocampus (68), and are known for their evolutionarily preserved role in germ cell pluripotency and survival (69). Nanos proteins are important in the development of the CNS (68, 70), but also their overexpression is associated with various human cancers (69, 71), consistent with the link between cancer and gene regulatory network acting during development. Specifically, alterations due to DNAm of Nanos genes have been associated with hepatocellular carcinoma (72), prostate cancer (73), thyroid carcinomas (74), adult (75) and childhood (76) asthma, type II diabetes (77), metabolic syndrome (78). Of interest, an animal study has detected increased NANOS2 expression in the hypothalamus following exposures to maternal deprivation, consistent with the possibility that other early life stressors, in addition to ASPH, may alter the epigenetic status of this gene, whose function as a zinc finger protein may contribute to regulate the translation of genes relevant for development (79).
SLC2A14 gene, a paralog of SLC2A3, is highly expressed in the brain and other tissues: cardiomyocytes, placenta, white blood cells, and others (80). In both the neocortex and in deeper cortical structures, SLC2A3 seems to be primarily localized to axonal and dendritic processes, suggesting an important role for SLC2A3 in ATP-dependent axonal transport processes and synaptic plasticity (81). Furthermore, SLC2A3 has been shown to strongly respond to hypoxic stress (82) and it has been shown that hypoxia-induced chromatin conformation changes influence SLC2A3 expression and functions in multiple cell types (83). Consistent with our findings, it has been suggested that alterations in SLC2A3 gene dosage interfere with neurodevelopment of individuals later diagnosed with a neuropsychiatric disorders, such as ADHD (84), SZ (50) and BD (51).
A strength of this study is the prospective birth registry information on ASPH that was available on a large sample of individuals with SZ or BD and HC. A limitation to the study is represented by our sample size, which reduces the power to detect changes at the differentially methylated position level. Stratifying by sex reduced the sample size considerably, so we interpret these findings and associations with disease severity and age of onset with caution. Another limitation might be the wide definition and lack of detailed reporting of ASPH in the MBRN, which might account for the high prevalence of ASPH (> 10%) in the sample. Inflammatory presentation is unclear in the MBRN. Since inflammation together with ASPH increases the risk for neural damage (85), milder insults may cause injury or alterations in those with a genetic liability for SZ development (86). Since ASPH is associated with neurodevelopmental deficits later in life, the design of the TOP study (i.e., exclusion based neurological diseases) might not fully capture the full range of ASPH exposure in our patient sample.
We identified four DMRs for LINGO3, BLCAP;NNAT, NANOS2 and SLC2A14 genes that significantly interacted with ASPH on case-control status, where methylation patterns differed with ASPH exposure between groups. These findings provide further evidence of alterations in the response to hypoxia and oxidative stress and may underscore the contribution of birth asphyxia as a source of the shared etiopathogenetic mechanisms leading to the abnormal brain development commonly observed in schizophrenia and bipolar disorder. Prevention strategies may benefit from understanding the possible divergent epigenetic responses to ASPH that give rise to a healthy brain development or to trajectories of risk for psychiatric disorders. We mainly assessed DNAm in adults but examining infants or young children might be of interest for future studies aimed at detecting epigenetic changes specific to early stages of development, which could represent biomarkers of risk.