A Higher Dysregulation Burden of Brain DNA Methylation in Female Patients Implicated in the Sex Bias of Schizophrenia

Sex differences are pervasive in schizophrenia (SCZ), but the extent and magnitude of DNA methylation (DNAm) changes underlying these differences remain uncharacterized. In this study, sex-stratified differential DNAm analysis was performed in postmortem brain samples from 117 SCZ and 137 controls, partitioned into discovery and replication datasets. Three differentially methylated positions (DMPs) were identified (adj.p < 0.05) in females and 29 DMPs in males without overlap between them. Over 81 % of these sex-stratified DMPs were directionally consistent between sexes but with different effect sizes. Down-sampling analysis revealed more DMPs in females than in males when the sample sizes matched. Females had higher DNAm levels in healthy individuals and larger magnitude of DNAm changes in patients than males. Despite similar proportions of female-related DMPs (fDMPs, 8%) being under genetic control compared with males (10%), significant enrichment of DMP-related SNPs in signals of genome-wide association studies was identified only in fDMPs. One DMP in each sex connected the SNPs and gene expression of CALHM1 in females and CCDC149 in males. PPI subnetworks revealed that both female- and male-related differential DNAm interacted with synapse-related dysregulation. Immune-related pathways were unique for females and neuron-related pathways were associated with males. This study reveals remarkable quantitative differences in DNAm-related sexual dimorphism in SCZ and that females have a higher dysregulation burden of SCZ-associated DNAm than males.


Introduction
Sex differences in susceptibility to schizophrenia (SCZ) are involved in almost all features of the disorder, from prevalence, age of onset, clinical phenotype, to treatment response [1][2][3]. Males are 1.42 times more likely than females to develop SCZ and have an earlier onset [4,5]. Female SCZ patients are more likely to have affective symptoms, whereas males are more likely to have language disruption, positive symptoms, and a severe course of illness [6,7]. Sex differences are also noted in responses to antipsychotic medications. Some studies suggested that rst-episode female patients showed better treatment responses than males, and male patients often required higher doses of drugs [8,9]. Together this information suggests that the pathophysiology of SCZ is likely different between sexes.
Understanding the molecular mechanisms of sex differences contributing to SCZ could improve the precision of diagnoses and treatment for individual patients.
Higher male prevalence of SCZ led to the development of the female protective model [10,11] which posits that a greater minimum liability or higher threshold is required for females to develop SCZ as compared with males. Evidence from family studies [11,12] and the study of mutations [10] supports the hypothesis that female SCZ patients carry greater genetic liability than males. Although SCZ is thought to be highly heritable, environmental factors, gene-environment interactions [13], and epigenetics are also important contributors. However, no study has been completed on epigenetics or DNA methylation (DNAm) pertaining to the female protective model in SCZ.
The hypothesis of this study is that the dysregulation burden of DNAm differs by sex, and contributes to the sex bias of SCZ. The dysregulation burden of DNAm is measured by the number of differentially methylated positions (DMPs) and the magnitude of DNAm changes between healthy individuals and patients. Sex-strati ed DNAm analysis can directly compare the dysregulation burden in females and males. Only a few studies to date have assessed SCZ-related differential DNAm in different sexes [24,25]. Montano et al. [24] conducted a male-only differential DNAm analysis on blood and identi ed 23 replicated male-related DMPs. About 30% of these DMPs could be missed in a regular sex-combined analysis. Mill et al. [25] partitioned frontal cortex samples by sex and found that the DNAm changes in SCZ males and females were weakly correlated (r 2 = 0.13, p = 8.1e-26), and inferred that the SCZassociated DNAm changes were common to both sexes. These studies provided intriguing ndings into the sex-differential DNAm in SCZ. However, the hypothesis of sex-dependent dysregulation burden of DNAm in SCZ has not be formally tested yet.
The DNAm data of postmortem brains from 117 SCZ and 137 controls were collected and analyzed. The sex-dependent dysregulation burden of DNAm in SCZ, and their related protein-protein interaction (PPI) subnetworks were assessed. The underlying regulatory networks in each sex were investigated and compared by integrating the genetic variants from methylation quantitative trait loci (meQTLs) and gene expression from correlated CpG-gene expression pairs (GCPs). Together these provided important insight into how DNAm may contribute to the sex-biased risk of SCZ.

Data information
The DNAm data of DLPFC and frontal cortex were obtained from Gene Expression Omnibus (GEO) [26] and ArrayExpress [27]. Raw microarray DNAm data of 117 SCZ and 137 controls were obtained from  [29]. These DNAm pro les were generated using the Illumina Human Methylation450 arrays. The GSE74193 data was used as the discovery dataset. We removed two batches from discovery dataset, including 66 subjects, putting case and control samples in separate batches, leaving DLPFC data of 82 SCZ patients (41 females, 41 males) and 96 sex-matched healthy controls (24 females, 72 males) for the further analyses. The other two datasets were used as the replication datasets, containing 35 SCZ (9 females, 26 males) and 41 controls (11 females, 30 males). Each study was preprocessed separately and analyzed according to the work ow below.

Sex-strati ed differential methylation analyses
The changes in DNAm (quanti ed as ) between SCZ cases and controls in females and males were examined by champ.DMP function. Multiple testing corrections using Benjamini-Hochberg adjusted pvalue (adj.p) as false discovery rate (FDR) were done in two separate groups of CpGs: all tested CpGs and subsets of sex chromosomes CpGs only. Signi cant DMPs were considered those with the adj.p < 0.05.
Next, a more inclusive p-value threshold of 1e-04 was considered primarily for enrichment analyses which need input of more genes/loci. For replication, sex-strati ed meta-analysis using the linear mixed-effect model were adopted to combine studies (GSE61431 and GSE61380) for identi cation of DMPs. The multiple testing correction in the replication data considered only the signi cant DMPs from the discovery data that can be tested in the replicate. The "replicable" were de ned as adj.p < 0.05 in the replication data and with the same direction of effect as the discovery results.

Sex-by-schizophrenia interaction analysis
To examine whether the effect of SCZ differs between females and males, we systematically tested the interaction effect between sex and diagnosis. For each CpGs, we applied the interaction term "sex*diagnosis" using the Limma package in R.

Δ β
Comparing the direction and magnitude of DNAm changes between females and males with SCZ The rank-rank hypergeometric overlap (RRHO) test [41] was used to evaluate the overall consistency of differential DNAm in females and males with SCZ. The CpGs were ranked by the -log10 of DMP p-value multiplied by the direction of effect size. A one-sided p-value of the overlapped DMPs from two datasets was calculated by the hypergeometric distribution. Furthermore, Spearman's correlation and Student's ttest were performed to compare the magnitude of DNAm changes between sexes for three classes of CpGs, including the RRHO detected concordant CpGs, X chromosome (chrX) CpGs, and all tested CpGs.
DMPs relate to single nucleotide polymorphisms by meQTLs and genes by GCPs The meQTLs from Jaffe et al.
[28] and Ng et al. [42] were used to search for female-and male-related SNP-DMP pairs (fSDPs and mSDPs). We only used the reproducible meQTLs that were signi cant in both two datasets, which contained 434,312 meQTLs (253,471 SNPs and 45,049 CpGs, with FDR < 0.05). We searched for our sex-strati ed DMPs of p < 1e-04 in these reproducible meQTLs for fSDPs and mSDPs. The partitioned linkage disequilibrium score regression (pLDSR) was applied to measure the enrichment of genome-wide association studies (GWAS) risk variants in SNPs of the fSDPs and mSDPs (within a 200kb window around the CpG site) [43]. LD scores were calculated for each SNPs in the SDPs using an LD window of 1cM in 1000 Genomes European Phase 3. The latest GWAS summary statistic (PGC3) was downloaded from the PGC websites (https://www.med.unc.edu/pgc/downloads).
The GCPs in DLPFC samples from Wang et al. [44] were used to de ne female-and male-related DMPgene pairs (fDGPs and mDGPs). Furthermore, we identi ed DGP genes that were reported to be differentially expressed in SCZ based on the PsychENCODE results [45].

DMP-related protein-protein interaction subnetworks
The functional epigenetic modules (FEM) [46] were used to identify female-and male-related PPI subnetworks (fPNs and mPNs) by the champ.EpiMod function. FEM is a functional supervised algorithm to identify PPI subnetworks that contain differentially methylated genes focusing on the promoter regions. The PPI data was derived from the Pathway Commons resource [47] described by West et al.
[48]. The PPI subnetworks in uenced by the differential methylation in females and males were extracted separately. The DNAm levels of genes were assigned according to the average DNAm of CpGs in the promoter regions.
Functional enrichment of PPI subnetwork-related genes was performed using R package clusterPro ler [49]. The minimum number of genes annotated by the ontology term was set to 10, and the maximum was 500. We used adj.p < 0.05 as the signi cance threshold. The direction of a PPI subnetwork was calculated by an area under the receiver operating curve (AUROC), summarizing its enriched differential DNAm genes. Genes were ranked from the most signi cant in the negative direction to the most signi cant in the positive direction [signed -log(p-values)] to calculate the AUROC. An AUROC less than 0.5 indicates the pathway is down-regulated as it is enriched in genes hypomethylated in SCZ in that sex.
An AUROC larger than 0.5 represents the up-regulated pathways in which enriched genes were hypermethylated in that sex.

Results
Female SCZ patients carried more differentially methylated CpGs than males Analyses of sex-strati ed differential DNAm identi ed three female-related DMPs (fDMPs) and 29 malerelated DMPs (mDMPs) (adj.p < 0.05) ( Fig. 1A and 1B, Supplementary Table S1). All the fDMPs and mDMPs were located on autosomes. There was no overlap between fDMPs and mDMPs. More than 81% (all three fDMPs and 23 of 29 mDMPs) of these sex-strati ed DMPs had directionally consistent changes across sex though they were only signi cant in one sex. Nearly 13% of the detected sex-strati ed DMPs (4 of 29 mDMPs and no fDMPs) were missed in a regular sex-combined analysis (adj.p < 0.05). The DMPs that were missed in the sex-combined analysis were those DMPs with opposite directions of DNAm changes between sexes in the sex-strati ed analyses. The fDMPs have larger changes in the female subgroup than in the male subgroup and vice versa. In other words, the quantitative differences of DNAm change amounts between females and males dominate the sex differences in DNAm.
Additionally, there were 74 CpGs that reached a relatively relaxed threshold of p < 1e-04 in female SCZ (Fig. 1A, Supplement Table S1), and 214 CpGs in males (Fig. 1A, Supplementary Table S1). Results were not driven by differences in sex chromosomes, as only 4% (3 of 74) of fDMPs and no mDMPs were found on the chrX and no DMPs on the Y chromosome (chrY). Only one DMP (cg09247020, nearest the C14orf34, females: p = 4.0e-05, males: p = 1.6e-05) was shared between sexes. Nearly 82% (61 of 74 fDMPs and 175 of 214 mDMPs) of those DMPs with relaxed threshold were consistent in direction between sexes but with varying degrees of DNAm changes.
Since the number of detected signi cant DMPs is related to the sample size, the male subgroup was down-sampled to match the female sample size to study whether the female subgroup could yield more signi cant DMPs than the male subgroup. With 4,000 times random subsampling, signi cantly more DMPs were detected in female patients than males (one-sided 95% con dence interval 0 to 1.66, p < 0.05, Supplementary Fig. S1).
Concerning the chrX-speci c DMPs, no CpG reached signi cance (adj.p < 0.05) in either sex. Three CpGs on the chrX reached the threshold of p < 1e-04 in females (Fig. 1C, Supplementary Table S1) but were not signi cant in male patients (all p > 0.05, Fig. 1C). One of those three CpGs (cg10153260, p = 2.6e-05) was located at the 3'UTR region of the EDA2R gene. EDA2R was found to be associated with cytokine signaling in the immune system [50]. Another CpGs (cg13161621, p = 3.3e-05) mapped to the gene body of the IQSEC2 gene. IQSEC2 is an X chromosome inactivation (XCI)-escaped gene in healthy female subjects [22,51] and has been reported to participate in synapse organization [52].
Regarding the mDMPs on the chrY, only two CpGs showed case-control differences at adj.p < 0.05 (Supplementary Table S1). The most prominent mDMP on the chrY (cg10213302, adj.p = 9.0e-03) was located at the TSS1500 of the ZFY gene encoding a zinc nger-containing protein, a putative transcription factor [53]. Another mDMP (cg08160949, adj.p = 9.0e-03) was located at the intergenic regions.
In the sex-by-SCZ interaction analysis, no CpG passed adj.p < 0.05. Twenty-ve CpGs were identi ed at p < 1e-04, including four CpGs mapped to the chrX (Supplementary Table S1). One of the interaction CpGs (cg11884933, p = 3.8e-05) overlapped with the detected FDR-signi cant mDMPs (adj.p = 2.9e-02). The CpG, cg11884933, was mapped to the gene body of the GNA12 gene. The GNA12 gene is a previously identi ed differentially expressed gene (DEG) in SCZ and found to be involved in regulation of TOR signaling [45]. All these interaction CpGs had DNAm changes in opposite directions in female and male SCZ (Fig. 1D, Supplementary Table S1).
To replicate those sex-strati ed DMPs (adj.p < 0.05), sex-strati ed analysis was performed in another independent dataset, combining two datasets by meta-analysis. About 67% (2 of 3) of fDMPs and 28% (8 of 29) of mDMPs were well-replicated with same direction as the discovery dataset (adj.p < 0.05).
Female SCZ patients carried a larger magnitude of DNAm changes than males An unbiased RRHO analysis was used as a threshold-free method to compare the DNAm changes in females and males and to de ne "concordant CpGs" for those DMPs shared by both sexes. A statistically signi cant overlap of DNAm signatures for up-and down-regulated CpGs were identi ed in females and males with SCZ, particularly a strong sharing for up-regulated DNAm (maximum hypergeometric p < 1.0e-5240, Fig. 2A). There were 2,135 RRHO-determined concordant CpGs (961 up-regulated and 1174 downregulated CpGs) shared between female and male patients at nominal p < 0.05. Over 83% (1,780 of 2,135) of these shared CpGs showed larger changes of DNAm in females than in males. Comparison also showed a larger magnitude of differential DNAm in females than in males (slope = 0.78, = 0.01, = 0.008, t-test p < 2.2e-16, slope < 1 indicates larger effect in females SCZ, Fig. 2B and   2C).
Higher baseline DNAm levels in healthy females than in males at the DMP loci Given the previous hypothesis that the baseline sex differences in DNAm may cause the sex bias of SCZ, the correlation between sex-strati ed differential DNAm in SCZ (fDMPs or mDMPs at p < 1e-04) and the sex differences in healthy individuals (sex-related DMPs at baseline (sDMPs)) for all the CpGs tested for DMPs was explored. The innately sDMPs were calculated using the control subjects from our discovery dataset. A total of 9,116 sDMPs were identi ed (adj.p < 0.05, Fig. 2D, Supplementary Table S2).
The fDMPs were signi cantly enriched in sDMPs (11 of  Genetic variants exhibit a sex-biased association with the sex-strati ed differential DNAm Given that DNAm has been hypothesized to mediate genetic risks, the GWAS signals might be related to the sex-strati ed DMPs (at p < 1e-04) by the previously identi ed brain meQTLs [28,42] as the SNP-DMP pairs (SDPs). SNPs were found to be associated with fDMPs as 39 female SDPs (fSDPs) involving six CpGs and 39 SNPs, and associated with mDMPs as 208 male SDPs (mSDPs) involving 22 CpGs and 208 SNPs (Supplementary Table S3). Thus, only a limited number of DMPs (8% of fDMPs; 10% of mDMPs) have evidence to be under genetic regulation in the brain.
The comparison of the SNPs in the fSDPs and mSDPs with the SCZ GWAS SNPs from PGC3 (at p < 5e-08) [54] showed no SDP SNPs directly overlapped with GWAS SNPs. Considering the SDP SNPs might be in linkage disequilibrium (LD) with SCZ SNPs, enrichment of GWAS signals were further estimated using pLDSR, taking LD into account. The fSDP SNPs were found to be signi cantly enriched in SCZ SNPs with a 2.29-fold enrichment (p = 0.03). In contrast, such enrichment was not detected in mSDP SNPs (1.23-fold enrichment, p = 0.60). This result suggested that DNAm changes mediated more SCZ genetic risks in females than in males.
Sex-strati ed differential DNAm in SCZ connected to changes of gene expression in patients DNAm has a primary function of regulating gene expression. The previously identi ed brain-related Gene-CpG pairs (GCPs) [44] were used to identify fDMP-and mDMP-related (at p < 1e-04) GCPs as DMP-gene pairs (DGPs). The negative DGPs refer to the DNAm levels negatively correlated with gene expression  Table S4).
The directions of DGPs were further compared with those of DEGs in SCZ according to the PsychENCODE results [45]. Of the ve fDGPs, only one positive fDGP, cg12864903-EFCAB5, was hypomethylated in female SCZ and had signi cantly down-regulated EFCAB5 expression in SCZ brains [45]. Another negative fDGP cg18018027-POU3F2 was also noted. Though POU3F2 was not a signi cant DEG in SCZ [45], our previous studies found POU3F2 as one of the critical hub regulators in a SCZ-related coexpression module [55,56]. Of the 14 mDGPs, two mDGPs with correlated genes were also signi cant SCZ DEGs. The hypermethylated mDMP cg11884933 was positively correlated with the increased expression of the GNA12 gene in SCZ. The PTMS gene was down-regulated in SCZ and negatively correlate with hypermethylation of cg04671742.

Sex-strati ed DMPs mediate genetic effects on gene expression
The DMPs were further used to link risk SNPs and gene expression. Only one fDMP (cg02167201) and one mDMP (cg24278948) were involved in both SDP (SNP-DMP) and DGP (DMP-gene expression).
The fDMP, cg02167201, was associated with four SNPs and negatively correlated with gene expression of the CALHM1 gene (Fig. 3A). The CALHM1 gene plays a critical role in calcium homeostasis and synaptic activity in cerebral neurons [57,58]. Previous GWAS studies prioritized this gene as a susceptibility gene for SCZ [59,60]. Of note, three (rs6580, rs942900 and rs7831) of these four SNPs were in LD (all r 2 > 0.2) with the reported susceptibility SNP (rs1163238) in SCZ [54].
One mDMP cg24278948 associated with eight SNPs was negatively correlated with the CCDC149 gene in males (Fig. 3B). Previous linkage studies indicated that this gene resided in a putative region of susceptibility for SCZ [61]. However, the eight associated SNPs were not in LD (all r 2 < 0.1) with the SCZ index SNPs.
Synapse-, neuron-, and immune-related pathway genes were enriched in the PPI subnetworks and functions related to sex-strati ed differential DNAm in SCZ To identify the PPI subnetworks that interact with differential DNAm, we focused on the genes with promoter CpGs methylated, where DNAm-gene relationship can be better de ned. Four female-related PPI subnetworks (fPNs) and six male-related PPI subnetworks (mPNs) were deduced (Supplementary Table  S5). These f(m)PNs were coded by numbers, like fPN1 and mPN1, etc.
The mPN5 (centered around TJP1 gene, Fig. 4A) and fPN3 (centered around GRIA2 gene, Fig. 4B) were both enriched for synapse-related pathways, though the member genes of these two subnetworks were different. Given the pathways enriched genes contained both hyper-and hypo-methylated genes, the directions of pathways were determined by the receiver operating curve (AUROC) statistic of the DNAm changes of its enriched genes. The down-regulated pathway was de ned as enriched in hypomethylated genes in SCZ in that sex. The up-regulated pathway was de ned otherwise. Interestingly, the two subnetwork-related pathways had the synaptic and postsynaptic membrane-related functions affected in females and males in opposite directions, though de ned by DNAm of distinct genes (Fig. 4D, Supplementary Table S5). For instance, the synaptic and postsynaptic membrane pathways were enriched in genes hypomethylated in female SCZ patients, whereas the same pathways were enriched in hypermethylated genes in males.
The PPI subnetworks were further investigated by checking for the SCZ DEGs based on PsychENCODE results [45]. The hub genes in each f(m)PNs were always the most differentially methylated genes in the subnetworks, though the hub genes were not necessarily a signi cant DMP gene detected in each sex. The sex-strati ed DMP genes can be at any position of the network. Twenty-two percent of genes involved in fPNs and 28% in mPNs were SCZ DEGs (adj.p < 0.05) (Fig. 4A-C). Among each f(m)PNs, there was no overlap between detected DMP genes and involved SCZ DEGs, indicating changes in gene expression and DNAm in SCZ brains occurred at different components of the same biological networks.
Since DNAm is highly cell-type speci c, we assessed the cell composition differences between the female and male subgroups. The composition values of the estimated neuronal and non-neuronal cells did not differ in females and males comparing SCZ to controls (all p > 0.05).

Discussion
By analyzing differential DNAm in female and male SCZ patients separately, this study supported the hypothesis that female patients have a higher dysregulation burden of DNAm than males (Fig. 5), with three major ndings: 1) female SCZ patients carry signi cantly more differential DNAm and larger changes than male patients; 2) females had signi cantly higher DNAm levels in healthy individuals at the DMP loci and more baseline sex differences in fDMPs than males; and 3) genetic variants associated with SCZ risk contribute more to the differential DNAm in females than in males. Moreover, despite a limited effect of differential DNAm on gene expression detected in this study for both sexes, the DMPgene relationships represented by DGPs provided one possible mechanism of DNAm-related downstream regulation for SCZ risks. Nearly all of the detected sex-strati ed DMPs were mapped to autosomes, suggesting a major contribution of autosomal DNAm to the sex bias in SCZ. The differential DNAm in males and females participated SCZ risk through many different genes and pathways while sharing synapse-related pathways.
This study provided compelling evidence for the female protective model in SCZ where females have a higher dysregulation burden of DNAm than males. The magnitude of SCZ-associated DNAm changes was overall signi cantly larger in females than in males. Moreover, females had more DMPs than males when the sample sizes of each sex subgroup matched. Quantitative difference dominated the DNAm differences between sexes in SCZ, leading the females' DNAm liability away from the diagnostic threshold. These may explain why females have a lower prevalence of SCZ. Previous studies surveyed the sex difference in SCZ at the genetic levels instead of the DNAm levels. However, debate exists over whether the heritability of SCZ differs by sex [62-64], suggesting the sex differences in SCZ cannot be fully explained by genetic factors. As observed, sex differences at DNAm levels in SCZ were small but ubiquitous, and quanti cation of such differences can provide important insight into some biological functions that are perturbed in both sexes but may be more detectable in one sex.
The strong contribution of innately sex-differential DNAm to sex-speci c DMPs supported our previous hypothesis about the role of DNAm in the sex-biased risk of SCZ [15], and also extended the female protective model by highlighting their baseline functions. Two major hypotheses about the relationship between sexually dimorphic and disease risk genes are that risk genes are sex-differentially regulated, and/or they interact with sexually dimorphic pathways. This study offered support for both theories. On the one side, the baseline sex differences contributed more differential DNAm effects in female SCZ than in males. With differing baseline DNAm levels, the magnitude of the impact of the risk genes differed by sex, suggesting that baseline sex-differential DNAm were sex-speci cally contributing to the SCZ risk. On the other side, indeed, enrichments of sDMPs were slight, with ~ 15% of fDMPs showing baseline sex differences, while only 2% of mDMPs did. A previous study on sex differences in autism spectrum disorder (ASD) found that sex differentially expressed genes were enriched in ASD-related biological pathways but not ASD risk genes [65]. The female-and male-related differential DNAm were enriched in synapse-, immune-, and neuron-related pathways in this study, which were also previously identi ed as sexually dimorphic pathways [15,66]. Therefore, baseline sex differences of SCZ risk genes and sexually dimorphic biological processes play critical roles in the sex-biased risk of SCZ.
Nearly all of the detected sex-strati ed DMPs were located on autosomes. Previous studies demonstrated a prominent in uence of autosomal DNAm in innate sex differences [15,16,19,20]. Our previous study on healthy individuals also uncovered over 75% of autosomal sex-differential DNAm [15]. Consistently, the GTEx Consortium [66] and Hoffman et al.
[67] both identi ed a large fraction of sex-differential gene expression on autosomes in the prefrontal cortex, suggesting a genome-wide regulatory in uence of sex. Previous discovery of epigenetic sex differences indicated that sex chromosome genes could regulate autosomal methylation [68], but casual relationships will need further research. These ndings strongly implicated the importance of autosomal contribution to the sex-biased SCZ risks.
Although it seems intuitive that chrX CpGs would contribute to sex differences in SCZ risk, very few Xlinked DMPs were detected in sex-strati ed differential methylations and sex-by-disease interactions. This raises the possibility that the differential DNAm on the chrX were weak and swamped by strong signals from autosomes. When restricting the analysis to the chrX CpGs, only three X-linked CpGs passed the threshold of p < 1e-04 in females, while none were identi ed in males. Effect sizes of chrX CpGs had DNAm changes in female patients 1.94 times larger than in males. Females have two chrX, but epigenetic modi cations silence one to maintain the dosage of single-copy X-linked genes similar to that in males [22,51]. The limited contribution of chrX to the sex-biased SCZ risk likely involves the silencing of chrX. Out of three X-linked fDMPs genes, one was a previously identi ed XCI gene, and two were XCI-escaped genes [51]. Additionally, experimental and analytical procedures for autosomes applied to chrX may limit the power to identify chrX-related differential DNAm. Our ndings suggest that chrX has a minor but consistent contribution to the DNAm dysregulation burden for females.
Genetic variants were sex-speci cally associated with differential DNAm in SCZ. A recent study identi ed genetic correlation between sexes for SCZ was high (r g = 0.92), although it was signi cant different from this raises the question about how genetic variants contribute to the sex differences in SCZ. In this study, a similarly proportion of fDMPs (8%) was under genetic control as in males (10%), but signi cant enrichment of SCZ GWAS signals was only found in females. SCZ risk SNPs may regulate more DNAmrelated risks in females than in males. DNAm effects manifest through SDPs, demonstrating one possible mechanism by which a proportion of common genetic variants associated with altered DNAm indeed contribute to the sex-biased risk for SCZ.
DNAm alterations in brains of female and male patients may impact the downstream expression of risk genes, further contributing to sex-biased SCZ risk. There were 7% of detected sex-strati ed DMPs in SCZ manifest as signi cant DNAm-gene pairs. Several f(m)DGP genes were also SCZ risk genes, such as POU3F2 and EFCAB5 in females and GNA12 and PTMS in males. The directions of DNAm changes for POU3F2-, EFCAB5-, and PTMS-correlated DMPs were consistent between sexes, but a prominent sexrelated quantitative bias of DNAm change exists and leads to the signi cant correlation only detected in one sex. For example, our previous studies found the POU3F2 gene was a risk gene for SCZ that could affect the expression of its co-expressed genes [55,56]. The negative DGP cg18018027-POU3F2 was prioritized in female SCZ brains. The aberrant expression of POU3F2 could lead to alterations in neuron number [55]. Together these may provide explanation for sex differences in neuron functions in SCZ [72,73]. Of note, the positive DGP cg11884933-GNA12 was unique for male SCZ brains, while the DNAm change for cg11884933 was in the opposite direction between sexes, although this mDMP was not signi cant in females. The GNA12 gene was a previously identi ed sex-differentially expressed gene in healthy brain cortex [66] and also an SCZ DEG [45]. This suggests that DNAm changes of cg11884933 may oppositely in uence the expression of GNA12 in females and males and then contribute to the SCZ risk. Thus, these de ned f(m)DGPs in SCZ could provide some functional explanations for how DNAm sex-speci cally regulates differential expression in SCZ brains.
The integration of SNP-DMP-gene expression also offered insights into sex-biased genomic regulation in SCZ. One DMP was found in each sex to be signi cantly correlated with several SNPs and expression of one unique gene, including CALHM1 in females and CCDC149 in males. Of note, three SNPs of these four SNP-cg02167201-CALHM1 clusters in females were in LD with one SCZ risk SNP [54]. By leveraging de ned SDPs overlapped with DGPs in each sex of SCZ patients, this highlights the role of sexdifferential DNAm in linking genetic variation to gene expression, further prioritizing the potential risk DMPs. Unlike genetic variants, DNAm and gene expression are both dynamic. Our recent study indicated that the concerted DNAm-gene expression relationship is highly tissue-and age-speci c [44]. More relationships among SNPs, DNAm, and gene expression may remain to be discovered in different cell types, particularly in the developing brains, which could deliver more functional explanations for sex differences in SCZ.
The differential DNAm-related PPI subnetworks in females and males mediate SCZ risk through several different pathways while sharing synapse-related pathways. The contribution of neuroimmune dysfunction to SCZ brains is well accepted [45,74,75], but this study showed that dysfunction of immune-related pathways was more extensive in female SCZ patients than in males. In contrast, this study found a male-biased dysregulation of neuron-related pathways. The neuron-related pathways were expected to be enriched for both sexes, but due to these pathways' enriched genes not being signi cant in female patients, perturbed in neuron-related function was more detectable in male patients. Sharing of synapse-related pathways between sexes was observed, though the speci c genes involved were different. Of note, the synaptic and postsynaptic membrane-related pathways had opposite directions of DNAm changes in females and males. Synaptic sexual dimorphism has been well characterized [76-78].
DNAm and transcriptome studies also noted the association between sex differences with synaptic functions [15,67,73]. Directional differences in DNAm changes of synaptic organization might contribute to the sex differences in SCZ brains functions. However, causal relationship of such opposite directions of DNAm changes in females and males needs follow-up studies in cellular or animal models.
Overall, this study provided solid support for the female protective model, where female SCZ patient brains had a higher dysregulation burden of DNAm than males. However, the data utilized here were from the human prefrontal cortex only. Sex differences in other brain regions and through other epigenetic mechanisms remain to be investigated. Investigation in sex-speci c epigenetics and its associated regulatory network and biological processes could help us to understand the biology of sex in SCZ.    Sex-speci c PPI subnetworks and biological processes. PPI subnetworks for (A) mPN5 (centered around TJP1gene), (B) fPN3 (centered around GRIA2 gene), (C) fPN1 (centered around PSMD14 gene) and fPN2 (centered around PSMB4gene). Every node represents a gene. The color of nodes represents differential methylation levels in corresponding promoters (yellow represents hypomethylation; blue means hypermethylation). The edges were built based on the protein-protein interaction in the Pathway