Sex-dependent association of DNA methylation in the coding region of the corticotropin-releasing hormone gene and schizophrenia

Schizophrenia (SCZ) is a common mental disease causing severe chronic disability. Epigenetic changes in HPA axis-related genes are considered to play an important role in SCZ pathogenesis. Unfortunately, the methylation status of the corticotropin-releasing hormone ( CRH ) gene, which is the central driving force in the HPA axis, has not been reported in SCZ patients. Here, we used the sodium bisulfite and MethylTarget methods to detect the DNA methylation status of the CRH gene coding region in peripheral blood samples from 70 schizophrenic patients and 68 healthy controls. The results showed that the methylation level of the CRH gene CDS was significantly increased in SCZ patients, especially in the male subgroup. In conclusion, this study showed that differences in CRH gene CDS methylation were detectable in the peripheral blood of SCZ patients and that epigenetic abnormalities in the CRH gene were closely related to SCZ, revealing that epigenetic processes may mediate the pathophysiology of SCZ. Further research should address the underlying mechanism whereby CRH gene methylation regulates the occurrence and development of SCZ. effective promoter methylation gene expression CpG methylation in the coding region of MCT3 its Testicular germ cell-specific CD demethylation of the TACT1 /actl7b and PDHA2 genes can inhibit their expression 2003; Pinheiro al. 2012). The methylation of + 10 and + 88 CpG sites in the CDS of the MAT1A gene can reduce promoter activity by 60% (Tomasi et al. 2012). These results indicate that CpG-site or CpG-island methylation in the CDS region is also very important for gene expression regulation. Unfortunately, there have been no studies on the methylation of the CRH CDS in SCZ. this methylation difference is not clear, it suggests that epigenetic abnormalities in the CRH gene are related to the pathophysiology of SCZ. This is the first study to show an association between SCZ and CpG-island methylation in the CRH gene coding region in peripheral blood. These results improve our understanding of the role of the HPA stress response system in the pathological development of mental disorders (especially SCZ) and fill in a gap in research on the methylation of the CRH gene coding region in SCZ. Our results suggest that epigenetic processes may mediate psychopathology by influencing HPA axis activity. The effect of CRH methylation differences on gene expression and function needs further study, which may help to clarify the pathophysiology of SCZ.


Introduction
Schizophrenia (SCZ) is a common severe, chronic, disabling mental illness with affective disorder, cognitive disorder and volitional behavior disorder symptoms (Zhu et al. 2019) affecting approximately 1% of the worldwide population (McGrath et al. 2008). SCZ is characterized by a slow onset, prolonged disease course and poor prognosis, which not only cause great harm to an individual's life and health but also imposes heavy economic and psychological burdens on families. It imposes one of the heaviest disease burdens with the highest costs worldwide (Rössler et al. 2005). SCZ is a complex disease that is affected by both genetic and environmental factors (Nishioka et al. 2012;Misiak et al. 2018), but its pathogenesis is still unclear.
In fact, mental disorders are closely associated with stress responses in the brain (Watkeys et al. 2018;Palma-Gudiel et al. 2015). In normal organisms, appropriate responses to stress are the basis of survival and adaptation (de Kloet et al. 2005), while inappropriate and long-term stress responses can lead to the occurrence and symptoms of mental disorders, including SCZ and bipolar disorder (Sinclair et al. 2012). The hypothalamic-pituitary-adrenal (HPA) axis is an important stress response system that maintains homeostasis through the responses to environmental stressors (Russo et al. 2012). Our previous studies have shown that DNA methylation of the NR3C1 and NR3C2 genes in the HPA axis leads to a risk of mental illness, including SCZ and aggressive behavior (Liu et al. 2020a and2020b;Qing et al. 2020;Qing et al. 2021). As the central driving force in the HPA axis (Zhou and Fang, 2018), the CRH gene has attracted our attention.
CRH plays a key role in the regulation of neuroendocrine, autonomic and behavioral adaptation to stress (de Kloet et al. 2005;Ishiwata et al. 2020). The CRH gene consists of two exons and one intron, and the coding region is located in the second exon ( Figure 1) (Cramer et al. 2019). CRH is mainly expressed by the parvocellular neurons of the hypothalamic paraventricular nucleus (PVN), which drives the activation of the HPA axis, thus regulating the stress response (Zhou & Fang, 2018;Ishiwata et al. 2020). Elevated CRH levels in the central nervous system are associated with stress-related physiological and behavioral disorders (Linthorst et al.1997). It has been confirmed in a large number of studies that CRH levels are upregulated in patients with depression disorders and suicide victims, CRH concentrations return to normal after recovery from depression (Naughton et al. 2014). The CRH concentration is also upregulated in individuals with posttraumatic stress disorder (Sautter et al. 2003). CRH also plays an important role in anxiety (Dedic et al. 2018), addiction (Carboni et al. 2018) and sleep (Kimura et al. 2010). The most recent study on this topic showed that the concentration of CRH in the cerebrospinal fluid (CSF) of schizophrenic patients was lower than that in the control group, and a lower CRH concentration was associated with negative symptoms of the illness (Ishiwata et al. 2020). This evidence suggests that CRH expression may be related to the pathogenesis of SCZ and other mental diseases.
As a mechanism of gene expression mediated by environmental factors, methylation plays a particularly important role in mental diseases. CRH methylation studies support this view. Under conditions of prenatal stress (Xu et al. 2014;Mueller and Bale, 2008), chronic variable mild stress (CVMs) (Sterrenburg et al. 2011), social stress (Elliott et al. 2010), and a lack of maternal love (Chen et al. 2012), CRH methylation decreases and CRH expression increases, leading to corresponding mental disorders or symptoms. Two CRH-related CpG sites (cg19035496 and cg23409074) were found to be significantly demethylated in attempted suicide patients. In subsequent adolescent cohort studies, one of the above cg19035496 sites was shown to be hypermethylated in subjects with higher general psychiatric risk scores (Jokinen et al. 2018). The results suggest that CRH methylation may be involved in the pathogenesis of mental disorders.
Previous studies on CRH methylation have focused on the promoter region (Xu et al. 2014;Mueller and Bale, 2008;Sterenburg et al. 2011;Elliott et al. 2010;Chen et al. 2012;Jokinen et al. 2018). With ongoing, increasingly in-depth research, it has been found that some CpGs may be located in exons or coding regions, which can affect promoter activity by affecting transcriptional elongation, and the DNA methylation of CDs is more effective than promoter methylation in inhibiting gene expression (Zhu et al. 2005;Hisano et al. 2003). CpG methylation in the coding region of MCT3 inhibits its expression (Zhu et al. 2005). Testicular germ cell-specific CD demethylation of the TACT1/actl7b and PDHA2 genes can inhibit their expression (Hisano et al. 2003;Pinheiro et al. 2012). The methylation of + 10 and + 88 CpG sites in the CDS of the MAT1A gene can reduce promoter activity by 60% (Tomasi et al. 2012). These results indicate that CpG-site or CpG-island methylation in the CDS region is also very important for gene expression regulation. Unfortunately, there have been no studies on the methylation of the CRH CDS in SCZ.
The purposes of this study were to analyze the methylation level of CpG islands in the CDS of the CRH gene in the SCZ population and to explore the relationship between the methylation level of the CRH gene CDS and SCZ.

Study participants
Blood samples were collected from 70 schizophrenic patients and 68 healthy controls living in Yunnan Province, China. Their demographic characteristics are shown in Table 1. Kunming. They were recruited and interviewed by psychiatrists. The exclusion and inclusion criteria of the disease group and the control group were described previously (Qing et al. 2020).
The study was approved by the ethics committee of Kunming Medical University. All subjects signed informed consent forms and donated peripheral blood.

DNA sample preparation and bisulfite modification
Blood samples were collected from all schizophrenic patients and controls. Genomic DNA was isolated from whole blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genomic DNA integrity was evaluated by agarose gel electrophoresis. Genomic DNA was quantified with a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the absorbance ratios at 260/280 and 260/230 were determined to assure DNA purity. The samples were treated with sodium bisulfite and processed by using the EZ DNA Methylation Gold Kit (Zymo, CA, USA) according to the manufacturer's instructions. Cytosine (C) residues without genomic DNA methylation were transformed into uracil (U) residues.

DNA methylation analysis
EBI database boss cpgplot software was used to predict CpG islands (http://emboss.bioinformatics.nl/cgi-bin/emboss/cpgplot) (Gardiner-Garden and Frommer, 1987), and the results predicted a CpG island containing 17 CpG sites in the CDS of the CRH gene (NCBI reference sequence: NG_016127.1 (+6013 to + 6231), Figure 1). By designing methylation primers for amplification with primer design software, the following primers were obtained: DNA methylation levels were analyzed by using MethylTarget® (

Statistical analyses
FastQC software was used to control the quality of the sequencing data. The FASTX Toolkit (http://hannonlab.cshl.edu/fastx_ toolkit/index.html) was used for the FASTA formatting steps of the FASTQ data. The FASTA format reads were mapped to the target bisulfite genome (hg19) using BLAST (Camacho et al. 2009). Methylation analysis was conducted with Perl scripts.
T-tests ere used for statistical analysis, and differences at the P<0.05 level were considered statistically significant. All statistical analyses were performed using SPSS 22.0; all graphical analyses were performed using GraphPad Prism version 8.

Results
In this study, we selected predicted CpG islands in the coding region for analysis, including males, 21 females) and 70 schizophrenic patients (46 males, 24 females). The sequencing depth and quality of all samples met the standards required for subsequent analysis.

Overall analysis of CpG-island methylation
The analysis of the DNA methylation status of CpG islands in the CRH gene CDS showed that compared with the control group (0.0204±0.0062), methylation was significantly increased in the case group (0.0242±0.0105) (p=0.0092). When the subgroups were further divided according to sex, significance was maintained in the male subgroup (p=0.0105). Although a trend of hypermethylation remained in the female disease subgroup, it was not significant (p=0.4632) ( Figure 2 and Table S1).

Methylation analysis of CpG sites
A total of 17 CpG sites were detected in the CpG island ( Figure 1 and Table S2). The methylation values of all CpG sites were below 6% in both the disease group and the control group. Compared with the control group, the DNA methylation levels of 7 CpG sites in the disease group were significantly higher (CpG3, P=0.0469; CpG9, P=0.0045; CpG11, P=0.0123; CpG12, P=0.0170; CpG14, P=0.0328; CpG16, P=0.0278; CpG17, P=0.0027) ( Figure 3A and Table S2).
For individual CpG sites, we also grouped the results by sex and compared the DNA methylation levels of all CpG sites according to sex. Compared with the control group, the methylation levels of 6 CpG sites (CpG9, P=0.0083; CpG11, P=0.0163; CpG12, P=0.0233; CpG14, P=0.0154; CpG16, P=0.0081; CpG17, P=0.0031) in the male group were still significantly higher than those in the control group, while the CpG3 methylation level was not significantly different ( Figure 3B and Table S2). Surprisingly, compared with the female control group, only the CpG3 site presented a significant difference in the female disease group (p=0.0383), and no significant difference was found at the other sites ( Figure 3C and Table S2).
In addition, we compared and analyzed the methylation levels of all male (n=93) and female (n=45) samples. The results showed that there was no significant difference in the methylation levels of either the whole fragments or the single CpG sites ( Figure 3D).  Table   S1). After division into subgroups according to sex, the significance of this increase was only maintained in the male subgroup. Among the individual CpG sites, a total of 7 CpG sites (CpG3, CpG9, CpG11, CpG12, CpG14, CpG16, and CpG17) were found to be significantly associated with SCZ. When the samples were divided into male and female subgroups, the CpG3 site showed significant differences only in the female subgroup, while the other 6 CpG sites maintained significant differences only in the male subgroup ( Figure 3 and Table S2).

DNA methylation is an important link between environmental factors and genetic
background, and differences in DNA methylation may lead to SCZ and other mental diseases (Popov et al. 2012;Gürel et al. 2020). Previous studies have focused mainly on the promoter regions of genes and less on the methylation of CDS regions. This study confirmed that the methylation level of the CRH gene coding region is associated with SCZ. This phenomenon has been observed not only in human and mouse studies (Zhu et al. 2005;Hisano et al. 2003;Pinheiro et al. 2012;Tomasi et al. 2012) but also in studies on fungi, viruses and cells. In fungi, the effect of coding region methylation on transcriptional extension is greater than the effect on transcription initiation (Barry et al. 1993;Rountree and Selker, 1997). The methylation of the CDS of the herpes simplex virus TK gene has been shown to inhibit TK expression (Graessmann et al. 1994).
At the cellular level, Hsieh (Hsieh, 1997) and Irvine (Irvine et al. 2002)  Although the mechanism by which coding region methylation affects gene expression is not completely clear, more attention should be paid to the important role of coding region methylation.
In the past, most of the obtained evidence has shown that the methylation level of CRH decreases under a lack of maternal love and prenatal stress, leading to the upregulation of CRH mRNA or protein expression, which drives the HPA axis and eventually leads to mental or stress disorders (Chen et al. 2012;Sterenburg et al. 2011;Xu et al. 2014;Mueller andBale, 2008；Elliott et al. 2010). However, we found that the methylation level of the SCZ group was higher than that of the control group, suggesting low CRH expression in the disease group. Similar results were found in a recent study in which the concentration of CRH in the CSF of schizophrenic patients was shown to be lower than that in the control group, and a lower CRH concentration was associated with negative symptoms of the disease (Ishiwata et al. 2020). Similarly, in an adolescent cohort study, the cg19035496 CRH gene was found to be hypermethylated in subjects with higher psychiatric risk scores (Jokinen et al. 2018). Previous studies have shown that the expression of CRH in SCZ patients is not consistent. The expression of CRH in disease versus control groups has been found to be downregulated or upregulated or to shown no difference (Ishiwata et al. 2020). The results regarding the methylation and expression of the CRH gene are not consistent among different mental disorders or stress disorders, suggesting that the regulatory network associated with CRH may be highly complex.
In this study, we found that the methylation level of the CRH gene coding region was associated with SCZ. It is worth noting that this association was male specific. Previous studies have reported that males showing sex-specific differences in SCZ are more likely to be diagnosed and develop cognitive impairment than females (Gershon, 2002;Mendrek and Mancini-Marïe, 2016). Sex dependence of CRH function has also been found in previous studies, although the results are inconsistent. After adrenalectomy (ADX), the potent androgen dihydrotestosterone can promote a rapid CRH mRNA response in male rats (Heck and Handa, 2019). Male mice are more susceptible than female mice to memory impairment caused by CRH in the medial septum (Kimberly et al. 2019). Negative feedback from PVN CRH mediated by the glucocorticoid receptor (GR) shows a significant sex difference, especially in female mice (Heck et al. 2020).
This indicates that the mechanism of CRH action in stress responses and mental disorders may be sex-dependent. We speculate that data on sex-dependent DNA methylation may contribute to the understanding of sex differences in CRH gene expression, HPA axis activation and the pathogenesis of SCZ.
Similar to most previous studies on methylation in humans, DNA extracted from peripheral blood was used for methylation analysis in the present study. In the study of SCZ, although most DNA methylation markers in peripheral blood cannot reliably predict DNA methylation status in the brain, data from peripheral tissue can reflect the methylation status in brain tissue to some extent (Walton et al. 2016). A previous study on CRH methylation supports this view. CRH methylation in peripheral blood is significantly correlated with methylation in four different brain regions, indicating that CRH methylation detected in peripheral blood can reflect the situation in the central nervous system and brain (Jokinen et al. 2018). To verify this hypothesis, future studies could directly compare methylation levels in brain tissue and peripheral blood after death.
Our previous studies on the methylation of the NR3C1 and NR3C2 genes in the HPA axis and SCZ patients have produced very significant results. The methylation status of the NR3C1 and NR3C2 genes is significantly associated with SCZ and is sex dependent to some extent (Liu et al. 2020a and2020b;Qing et al. 2020;Qing et al. 2021). Based on the results of this study, we strongly believe that HPA axis methylation can mediate environmental and genetic interactions and affect the pathogenesis of SCZ.
In conclusion, this study revealed differences in the methylation of the CRH gene coding region in the peripheral blood of schizophrenic patients. Although the functional significance of this methylation difference is not clear, it suggests that epigenetic abnormalities in the CRH gene are related to the pathophysiology of SCZ. This is the first study to show an association between SCZ and CpG-island methylation in the CRH gene coding region in peripheral blood. These results improve our understanding of the role of the HPA stress response system in the pathological development of mental disorders (especially SCZ) and fill in a gap in research on the methylation of the CRH gene coding region in SCZ. Our results suggest that epigenetic processes may mediate psychopathology by influencing HPA axis activity. The effect of CRH methylation differences on gene expression and function needs further study, which may help to clarify the pathophysiology of SCZ.

Ethics approval and consent to participate
The ethics committee of Kunming Medical University approved this study. Informed consent was obtained from all individual participants included in the study.

Consent for publication
Manuscript is approved by all authors for publication.

Availability of data and materials
Not Applicable.

Authors' contributions
Shengjie Nie and Lili Qing participated in the conception and design of this study. Sample collection and material preparation were performed by Peng Xiong and Hongyan Jiang, and data collection and analysis were performed by Lili Qing, Peng Xiong, Hongyan Jiang and Yumei Lu.
The first draft of the manuscript was written by Lili Qing. Shengjie Nie performed manuscript review. All authors read and approved the final manuscript.     Table S1 DNA methylation levels of CRH in schizophrenia patients and matched healthy controls.

Table S2
All CpG site methylation in patients with schizophrenia and matched healthy controls.