Genome-wide DNA methylation profiles in the raphe nuclei of patients with autism spectrum disorder

doi:10.21203/rs.3.rs-4880533/v1

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Genome-wide DNA methylation profiles in the raphe nuclei of patients with autism spectrum disorder

https://doi.org/10.21203/rs.3.rs-4880533/v1

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Background:

Autism spectrum disorder (ASD) has a strong genetic basis, yet its genetic complexities remain elusive. Current research highlights environmental factors and epigenetic processes, such as DNA methylation, as crucial in ASD development. This study explored epigenetic modifications using postmortem brain samples from ASD subjects and controls.

Methods:

We comprehensively analyzed genome-wide DNA methylation profiles in the dorsal raphe brain region using the Infinium HumanMethylation450 BeadChip (Illumina). In addition, quantitative PCR was used to investigate mRNA expression levels of genes that demonstrated differential methylation in ASD.

Results:

We identified differentially methylated regions (DMRs) and individual-specific DMRs (IS-DMRs) between ASD and control subjects. These DMRs and IS-DMRs were located across various genomic regions, including promoters, gene bodies, 3ʹ UTRs, and intergenic regions. Notably, we found hypermethylation in genes related to olfaction (e.g., OR2C3), which is regulated by serotonin. Hypomethylated genes in IS-DMRs were linked to ASD and developmental disorders. Additionally, we observed that the hypomethylation of promoter-associated CpG islands in RABGGTB, a gene related to autophagy and synaptic function, corresponded with its increased expression.

Conclusions:

Our findings reveal extensive DNA methylation changes in critical genomic regions, shedding light on potential mechanisms underlying ASD. The identification of RABGGTB as a novel candidate gene, not listed in the SFARI database, underscores its significance and warrants further research to explore its role in ASD diagnosis. This study enhances our understanding of the epigenetic landscape in ASD, emphasizing the interplay between genetic and environmental factors in its pathophysiology.

DNA methylation

autism spectrum disorder

dorsal raphe nuclei

human brain

serotonergic system

CpG repeat sites

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by severe and sustained impairment of social interaction, deviance in communication, and patterns of behavior and interest that are restricted or stereotyped. The worldwide prevalence of ASD has been estimated at approximately 0.1% [1]. Although twin studies have provided evidence for a strong genetic component for the condition, and many candidate genes have been reported, the genetic mechanisms underlying ASD remain unclear. It has been reported that genetic heritability is lower than previously estimated and that environmental factors have a greater impact on the development of ASD [2]. Several reports have suggested that exposure to environmental factors during the earliest days of fetal life, such as chemicals, stress, and viral infections, may contribute to ASD [3–7].

The epigenetic processes that modulate gene expression, such as DNA methylation and histone modification, are considered to be at the interface between genetic and environmental factors. Parental imprinting of a chromosome (such as Angelman syndrome and Prader–Willi syndrome) and X chromosome inactivation (such as Fragile X syndrome and Rett syndrome) are two well-characterized epigenetic processes [8–10].These syndromes share neurological and behavioral symptoms with ASD.

Several investigators have reported differentially methylated regions (DMRs) in ASD-related genes in individuals with ASD, such as RORA in lymphoblasts [11] and UBE3A, MECP2, OXTR, and SHANK3 in the temporal cortex, frontal cortex, cerebral cortex, and cerebellum, respectively [12–16]. However, one study did not find any differentially methylated CpG sites in MECP2, UBE3A, BCL2, RORA, or OXTR in the occipital or cerebellar cortex in individuals with ASD [17]. Moreover, using a bump-hunting approach, PRRT1, C11orF21, ZFp57, and SDHAP3 were identified as differentially methylated genes in the temporal cortex and cerebellum of individuals diagnosed with ASD [18]. In addition, within-twin and between-group analyses using whole-blood samples identified numerous DMRs associated with ASD, as well as significant correlations between DNA methylation and quantitatively measured autistic trait scores [19]. Collectively, these studies provide both indirect and direct support for the role of DNA methylation in ASD, although their results remain inconsistent.

A growing number of studies indicate the involvement of the serotonergic system of the brain in the mechanisms underlying ASD. Serotonin signaling facilitates several neural processes, including neurogenesis, cell migration and survival, synaptogenesis, and synaptic plasticity. Prior studies have consistently found elevated serotonin levels in whole blood cells and platelets of patients with autism [20–24]. Short-term dietary depletion of tryptophan (the precursor of serotonin) has been shown to exacerbate repetitive behavior and elevate anxiety and feelings of unhappiness in adults with autism [25]. Moreover, the membrane expression of the serotonin transporter, which is expressed in serotonergic neurons and regulates neurotransmission through the reuptake of serotonin, and its serotonin transport capacity is suggested to be decreased in the brains of patients with ASD [26, 27]. The dorsal raphe nucleus (DRN) contains serotonergic neurons that project widely throughout the forebrain and peripheral regions [28]. Environmental factors, such as peripheral immune activation and stress hormones, can result in the alteration of neuronal activities in this brain region [29, 30].

Here, we investigated the genome-wide DNA methylation profiles in the dorsal raphe (DR) region from postmortem brain tissues of individuals with ASD and individuals not diagnosed with ASD (controls). Given the recognized importance of methylation non-promoter regions in addition to promoter regions [31–33], we investigated the methylation status using the Infinium HumanMethylation450 BeadChip (Illumina), which can measure the DNA methylation levels of CpG dinucleotides in the promoter regions as well as in the intragenic (corresponding gene bodies), intergenic, and 3ʹ-untranslated regions (UTR) [34, 35].

Postmortem brain tissues

The Autism Tissue Program (Princeton, New Jersey; http://www.autismtissueprogram.org), the National Institute of Child Health and Development Brain and Tissue Bank for Developmental Disorders (Baltimore, Maryland; http://medschool.umaryland.edu/btbank/), and the Harvard Brain Tissue Resource Center (Belmont, Maryland; http://www.brainbank.mclean.org/) provided frozen postmortem brain tissues from the DR regions (from five individuals with ASD and seven control subjects). The demographic characteristics of the samples are described in Table S1. The Mann–Whitney U test was used to evaluate the differences in age and postmortem interval (PMI) between the ASD and control groups. Fisher’s exact test was used to evaluate the differences in sex and ethnicity between the ASD and control groups. Significance was set at P < 0.05. All statistical analyses were performed using statistical analysis software (SPSS version 12.0 J, SPSS, Inc.).

Genome-wide DNA methylation array (GWMA)

Genomic DNA was extracted from the brain tissues from male subjects (Table S1, C1-C7 and A1-A5) using an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). The DNA samples were bisulfite converted using the EZ DNA methylation kit (Zymo Research, Orange, CA). Genome-wide DNA methylation was assessed using the Infinium HumanMethylation450 BeadChip kit [34] (Illumina, San Diego, CA). The Illumina GenomeStudio software (Illumina) was used to extract signal intensities for each probe and perform initial quality-control checks. To ensure stringent data quality, probes with a detection P-value > 0.05 or a blank β-value in any sample were removed across all individuals. Because SNPs may affect methylation in nearby CpG dinucleotides [36–38], we excluded probes associated with SNPs (Okamura et al. Genomics Data: http://www.sciencedirect.com/science/article/pii/S2213596015300933).

Methylation array data analysis

The relative methylation level of each CpG site was calculated as the ratio of the normalized signal from the methylated probe to the sum of the normalized signals of the methylated and nonmethylated probes. This gave an average DNA methylation value, which was termed the average β-value for each CpG site and ranged from 0 (nonmethylated) to 1 (completely methylated). Differentially methylated CpG sites were determined by calculating the differences in the average β-values between the groups. For group analysis, a filtering criterion (Δβ = average β-value of ASD – average β-value of control) > 0.1 or < − 0.1 was applied, and P < 0.05 in the Mann–Whitney U test was used to assign significant differentially methylated sites. We did not use the Bonferroni method to adjust the statistical significance to the number of tests performed [39]. Individual abnormalities in methylated CpG sites were also analyzed. Individual abnormalities (outliers) were determined as values that exceeded a 30% difference from control values and those for which the standard deviation of the control was smaller than 0.05. Categorical variables were analyzed using Fisher’s exact test. All statistical analyses were performed using SPSS version 12.0 J (SPSS, Inc.). The data discussed in this publication have been deposited in the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) of NCBI and are accessible through the GEO Series accession number GSE242427.

EM-amplicon sequencing (EMAS)

Genomic DNA (50 ng) was fragmented using an S220 focused ultrasonicator (Covaris). The sheared DNA was then treated with the NEBNext® Enzymatic Methyl-Seq Conversion Module (New England Biolabs, E7120) to enzymatically convert the unmethylated cytosine to uracil while leaving the 5-methylcytosine unaltered. PCR amplification was performed in a total volume of 20 µL, which contained 10 ng of enzymatically converted DNA, 20 pmol of each primer, dNTPs (2.5 mM each), 1.5 U of ExTaq HS (Takara), and the supplied buffer. The sequences of the PCR primers used are 5′-GTATTTATGTTTTAAAATGGTATTTA-3′ and 5′-AAAAATATTACCCCTAACTACAAAAAAA-3′, which were designed to amplify a 214 bp fragment corresponding to chr1:76,253,358–76,253,571 (hg19). The PCR products were purified using AMPure XP beads (Beckman Coulter). The purified PCR products (1 ng) were ligated with sequencing adaptors using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, E7645), which were amplified by six cycles of PCR and purified using Sample Purification Beads. The final products were sequenced using the MiSeq Reagent Nano Kit v2 (Illumina, MS-103-1001) on a MiSeq platform (Illumina). Fastq files were trimmed using trim_galore (v. 0.6.6) (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and aligned using Bismark (v. 0.22.3) (https://www.bioinformatics.babraham.ac.uk/projects/bismark/). The resultant bam files were subjected to methylKit [40] to determine the methylation ratio at cg08702915 (chr1: 76,253,688 (hg19)).

Quantitative polymerase chain reaction (qPCR)

mRNA was extracted from the brain tissues from both male and female subjects (Table S1, C1-C11 and A1-A6) using an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). First-strand complementary DNA (cDNA) was synthesized from the extracted RNA samples using the SuperScript III First-Strand Synthesis System (Invitrogen). cDNA was used for qRT-PCR, which was performed using the SYBR Green Master Mix (Bio-Rad). The relative quantification of the OR2C3, COLEC11, DCAKD, RABGGTB, and ZBTB40 expression levels was performed using the ΔΔC_T method, with the expression of the ACTB gene used as the internal control. Primer sequences are available upon request. The Mann–Whitney U test was used to evaluate the gene expression levels. P values of < 0.05 were considered statistically significant (SPSS, version 28.0 J, SPSS, Inc.).

Characteristics of the subjects and brain samples

We used only male subjects for methylation analysis because the DNA methylation levels at several autosomal loci in the brain are sex-dependent [41, 42]. The demographic characteristics of subjects (five patients with ASD and seven control subjects) are described in Tables S1. There were no significant differences in age, ethnicity, or PMI between the ASD and control groups (Table S2).

Brain genome-wide methylation analysis—average of the controls vs. average of the subjects with ASD

We used a DNA methylation microarray that assays the DNA methylation status of over 450 000 CpG sites, including promoters, as well as the corresponding gene bodies and 3ʹ-untranslated regions, in addition to intergenic regions derived from GWAS studies [34, 35]. This microarray platform produces DNA methylation data with an accuracy similar to that of other approaches, such as methylated DNA immunoprecipitation sequencing, methylated DNA capture by affinity purification, and reduced-representation bisulfite sequencing [43].

We identified 51 differentially methylated regions (DMRs) between the ASD and control groups. The DMRs were distributed across almost all human chromosomes (Fig. 1A). As shown in Fig. 1B, DMRs were identified in the promoter (25 CpG sites), body (10 CpG sites), and intergenic (16 CpG sites) regions, and none of them were identified in 3ʹ UTR regions. In individuals with ASD, 35% (18 sites) of the DMRs were hypermethylated and 65% (33 sites) were hypomethylated (Fig. 1C). Further analysis by regions, 28% of the DMRs in the promoter region (7), 40% in the gene body region (4), and 44% in intergenic region (7) corresponded to sequences with increased methylation, whereas 72% of the DMRs in the promoter region (18), 60% in the gene body region (6), and 56% in the intergenic regions (9) represented sequences with increased hypomethylation (Fig. 1D). There were no significant differences in the ratios of hypermethylation to hypomethylation between these three regions (promoter vs. body, P = 0.689; promoter vs. intergenic, P = 0.332; body vs. intergenic, P = 1.000; Fisher’s exact test).

Next, we focused on the DMRs identified in the genic regions (35 CpG sites), including the promoter and body (Table 1). We detected 11 significantly hypermethylated CpG sites in 10 genes, such as OR2C3 and SPIB, in subjects with ASD compared with the controls, as well as 24 hypomethylated CpG sites in 20 genes, such as PM20D1 and DCAKD, in subjects with ASD compared with the controls (Table 1). These genes represent novel candidate ASD genes. In the intergenic region, we found a notable hypermethylated DMR region on the X chromosome, located just 5'-upstream of HTR2C, which encodes the serotonin receptor 2C (Fig. S1). The serotonin receptor 2C is suggested to be involved in social behavior [44].

Brain genome-wide methylation analysis—subjects with ASD vs. average of the controls

Because ASD is a highly heterogeneous disorder, it is probable that many disorder-associated differentially methylated regions are individual-specific ones [45]. Therefore, we screened for individual-specific differential methylated regions (IS-DMRs) in each patient. We identified 56 IS-DMRs in the ASD group compared with the average of the controls using our criteria (Fig. 2A). The identified IS-DMRs were distributed across almost all human chromosomes (Fig. 2A). As shown in Fig. 2B, the IS-DMRs were detected in the promoter (11 CpG site), gene body (18 CpG site) and 3′ UTRs (4 CpG sites), 3′ UTRs (23 CpG sites) regions. In the ASD group, half of the IS-DMRs [50% (28 sites)] were hypomethylated, whereas the remaining half [50% (28 sites)] were hypermethylated (Fig. 2C). Further analysis by regions, 27%, 22%, and 91% of the DMRs were hypermethylated in promoter (3 sites), gene body (4 sites), and intergenic (21 sites) regions, respectively, whereas 73%, 78%, and 9% of the DMRs were hypomethylated in promoter (8 sites), body (14 sites), and intergenic (2 sites) regions (Fig. 2D). There were significant differences in the ratios of hypermethylation to hypomethylation between the promoter and intergenic regions (P < 0.001, Fisher’s exact test) and between the body and intergenic regions (P < 0.001, Fisher’s exact test), but not between the promoter and body regions (P = 1.000, Fisher’s exact test).

Next, we focused on the IS-DMRs located in genic regions (33 CpG sites). As shown in Table 2, we found significantly elevated levels of methylation in 7 CpG sites in 5 genes, and significantly decreased levels of methylation in 26 CpG sites in 26 genes in subjects with ASD compared with controls. Of these 26 genes, five (i.e., AUTS2, MEG3, HDC, TRPM1, and EFHC2) have been implicated in ASD and other developmental disorders [46–51] (Table 3). Moreover, four genes had significant IS-DMRs (AGER, GOLGA6L6, KLK5, and PLK5P), which have not been reported as ASD- or other developmental disorder-related genes but were hypomethylated by more than 50% in individuals with ASD (Table 4).

mRNA expression of differentially methylated genes in ASD

Our final analysis focused on the mRNA expression levels of genes that demonstrated differential methylation in ASD. The study involved 6 patients with ASD and 11 control subjects, and their demographic characteristics are outlined in Table S1. There were no significant differences between the two groups in terms of age, sex, ethnicity, or PMI (Tables S3). Among the top four DMRs (both hypermethylated and hypomethylated, as shown in Table 1), we considered the following genes: OR2C3, COLEC11, DCAKD, RABGGTB, and ZBTB40, which exhibited differential methylation in their promoter region. Our findings indicate a significant increase in the mRNA expression level of the RABGGTB gene in individuals with ASD compared to the control group (P = 0.027; Fig. 3), which supports a significant decrease in methylation of the gene. Furthermore, we confirmed a significant decrease in methylation at cg08702915 of RABGGTB; this DMR was identified by the genome-wide DNA methylation array (Table 1) through EM-amplicon sequencing (P = 0.0297; Table 5 and Fig. S2). However, OR2C3 was below the detection sensitivity threshold, and no significant differences in gene expressions were observed between the two groups for the COLEC11, DCAKD, and ZBTB40 genes (Fig. 3).

This study revealed the presence of significant differences in DNA methylation profiles in the DR region of postmortem brains between ASD and control subjects. We also discovered individual-specific alterations in DNA methylation in patients with ASD. Moreover, DMRs and IS-DMRs were detected not only in promoter regions, but also in gene bodies, intergenic regions, and 3ʹ UTRs.

Among the DMR and IS-DMRs detected here, we found that several of them were located in genes associated with the serotonergic system. One of the most notable changes in methylation levels was identified in OR2C3, which encodes the olfactory receptor 2C3. This gene exhibited hypermethylation at two promoter CpG islands. Olfactory receptors of olfactory receptor neurons (ORNs) interact with odorant molecules in the nose, to initiate a neuronal response that triggers the perception of a smell. The olfactory bulb is one of the most densely innervated targets of serotonin fibers from the raphe brain region [28]. In Drosophila, serotonin was shown to excite both projection and local neurons and to increase the presynaptic inhibition of ORNs. Moreover, it has been proposed to suppress weak ORN responses [52]. Intriguingly, olfactory impairment and an atypical sensory response have been identified in individuals with ASD [53], as well as in other individuals diagnosed with a developmental disorder [54, 55]. Furthermore, olfactory dysfunction has been linked to the clinical phenotype of ASD [56]. Therefore, hypermethylation of the promoter region of the olfactory receptor may be implicated as a cause of this olfactory impairment in ASD.

We also observed that the serotonin-related gene HTR2C was located in the flanking region of the DMR detected in the intergenic region. HTR2C, which is located on the X chromosome, encodes the serotonin receptor 2C. Notably, Htr2c knockout mice exhibit social behavior deficits [44]. The expression of Htr2c is epigenetically regulated by the binding of the methyl-CpG binding protein 1 (Mbd1) to its promote, in mice [57]. The loss of Mbd1 has been shown to lead to the overexpression of Htr2c [57]. Moreover, mutant mice lacking Mbd1 display several core deficits associated with ASD [57]. Research has demonstrated that the methyl-CpG binding protein 2 preferentially binds to intragenic and intergenic regions, with less affinity for the methylated promoters of active genes [33]. However, it is not well understood whether Mbd1 binds intergenic methylated regions as well. It will be necessary to investigate the possibility of Mbd1 binding to intergenic methylated regions.

Here, IS-DMRs were found to be located in genes linked to ASD and other developmental disorders, such as AUTS2, MEG3, HDC, TRPM1, and EFHC2 [47–51, 58]. The hypomethylated MEG3 promoter is a noteworthy example, based on a study that reported the significant upregulation of MEG3 in children with ASD compared with controls [59]. This differential expression led to its identification as a promising diagnostic biomarker for children with ASD [59].

Including the aforementioned IS-DMRs located in ASD and other developmental disorders-related genes, a multitude of DMRs and IS-DMRs are discovered within gene bodies. DNA methylation principally occurs at cytosine residues located in CpG dinucleotides [60]. Although CpG dinucleotides are statistically underrepresented in the genome, they are concentrated at CpG islands (CGIs), which frequently coincide with promoter or gene-regulatory regions. Previous research indicates that gene body hypermethylation is linked to diminished gene expression, particularly in highly expressed genes [31]. In the context of human genomes, it has been confirmed that methylated gene body regions correlate with elevated levels of gene transcription [61, 62]. Moreover, gene body methylation impacts processes such as histone modification [63], alternative splicing [64], and spurious transcription [65]. Concurrently, numerous studies have revealed the close relationship between abnormal gene body methylation and gene expression, growth, differentiation, and development.

The current study also proposed novel ASD candidate genes from an epigenetic perspective. Four notable IS-DMRs were identified in the AGER, GOLGA6L6, KLK5, and PLK5P genes. These IS-DMRs exhibited individual hypomethylation levels, exceeding 50%, in the ASD group. For example, KLK5, which encodes kallikrein-5, displayed the most significant change rate and exhibited hypomethylation at the promoter site of the gene. Intriguingly, previous evidence suggests a connection between kallikrein and the serotonergic system. In rats, the serotonin precursor induced substantial renal morphological changes and reduced kallikrein immunostaining [66]. Furthermore, the KLK5 mRNA is expressed in the raphe nuclei of the human brain (Allen Brain Atlas; http://human.brain-map.org/). Nevertheless, the role of KLK5 in the brain remains relatively unexplored. Further investigation into the relationship between KLK5 and the serotonergic system in the brain could shed light on the pathophysiological mechanisms underlying ASD.

Recently, it has been demonstrated that the intergenic DNA hypomethylation resulting from a dysfunction in the trans-regulatory pathways of the histone methyltransferase NSD1 and the DNA methyltransferase DNMT3A serves as a mechanistic link between two phenotypically overlapping human overgrowth syndromes, both of which also exhibit a developmental delay [67]. In the current study, although the ratios of the hypermethylation to the hypomethylation of DMRs were consistent across promoter, intergenic, and body regions, significant differences in these ratios were observed between promoter and intergenic regions, as well as between body and intergenic regions, within IS-DMRs. Notably, approximately 90% of IS-DMRs exhibited hypermethylation within intergenic regions. Interestingly, this outcome contrasts with observations from overgrowth syndromes [67]. Given the observed exceptional hypermethylation within intergenic regions, it will be crucial to investigate how this phenomenon influences gene expression and contributes to the pathophysiological mechanisms of ASD. Further exploration of this subject is warranted for a comprehensive understanding of this disorder.

Because approximately two-thirds of all CGIs in the genome are associated with promoters, the hypermethylation of promoter-associated CGIs is commonly believed to lead to gene-transcription silencing [68]. Speculatively, increased methylation at CGI-containing promoters might correspond to decreased gene expression. However, because of the relatively modest scale of many of these methylation changes and the intricate relationship between DNA methylation and gene expression, demonstrating the direct biological significance of these alterations at specific loci could prove challenging [69–71]. In this study, we confirmed that the increased expression of RABGGTB corresponds to the hypomethylation of the gene's promoter-associated CGIs. RABGGTB is a β subunit of GGTase II, one of the prenyltransferases responsible for protein prenylation [72]. Prenylation of proteins plays a vital role in their membrane binding and localization. Rab7, a member of the Ras GTPase superfamily, is involved in autophagy, a cellular process essential for maintaining cellular homeostasis by degrading and recycling damaged or redundant cellular components [73]. The function of Rab7 depends on its localization to the plasma membrane, and RABGGTB performs a critical role in modulating autophagy by mediating the prenylation of Rab7 [73]. The accumulation of abnormal proteins is a known hallmark feature of neurodegenerative diseases. Rab7 plays a crucial role in maintaining neuronal function through autophagy regulation, which has been shown to influence the progression of neurodegenerative diseases [74]. In neurodevelopmental disorders, mutations in genes involved in the mTOR-dependent autophagy process, such as TSC1/2, FMR1, and PTEN, have been reported, thus prompting the possibility of neurodevelopmental pathology [75]. Moreover, the loss of mTOR-dependent autophagy has been reported to be linked to autistic-like synaptic pruning deficits [76]. However, our findings revealed increased levels of RABGGTB in autism. Autism is thought to involve the impaired initiation of autophagy, particularly related to the mTOR pathways [75]. Because RABGGTB contributes to the later process, such as autophagosome-lysosome fusion, this stage may be a compensatory increase, but the underlying mechanism might be more complex. On the other hand, the prenylation of Rab3a induced by RABGGTB is essential for regulating vesicle fusion and neurotransmitter release at the nerve terminal [77, 78]. In addition, an increased gene expression level of RABGGT has been reported in the postmortem prefrontal cortex of individuals with schizophrenia compared to normal controls [79]. Moreover, decreased protein expression levels have been observed in the dorsolateral prefrontal cortex of individuals with schizophrenia compared to normal controls [80]. Accumulated evidence suggests that synaptic dysfunction contributes to the pathogenesis of ASD [81]. Therefore, considering prenylation’s role in regulating vesicle fusion and neurotransmitter release, our data provides a potential mechanism underlying the synaptopathology involved in ASD.

Limitation

The results of this study should be interpreted carefully due to several limitations. The main limitations of the present study include the exclusive use of male brain samples in the methylation analyses and the small sample size. The sample size in postmortem brain studies of autism, though dependent on the brain region, is generally not large [82]. This study is the first to conduct methylation analysis in the raphe region of individuals with autism, highlighting its unique value. Additionally, multiple comparison correction was not performed due to the relatively small sample size, which significantly reduces the statistical power of the analysis. Applying stringent correction methods, such as Bonferroni, could result in an overly conservative approach, potentially overlooking biologically meaningful differences (39). Instead, a more lenient significance threshold was applied to balance the risk of Type I errors with the need to detect relevant DNA methylation changes in this exploratory study. We will continue collecting more samples and conducting epigenomic and transcriptome analyses. Future studies will investigate the relationship between DNA methylation and gene expression alterations in the brain and blood, which could lead to the identification of candidate biomarkers.

Our findings reveal extensive DNA methylation changes in critical genomic regions, shedding light on potential mechanisms underlying ASD. Notably, we found that heightened expression of RABGGTB, a gene related to autophagy and synaptic function, aligns with the hypomethylation of its promoter-associated CGIs. RABGGTB is a novel candidate gene absent from the autism-related genes database (SFARI database), emphasizing the importance of further research into its involvement in autism and its potential as a diagnostic marker. Nevertheless, it is possible that the complex methylation systems identified in the present study, including the methylation of the promoter regions and the intragenic and intergenic regions, are involved in the pathogenesis of ASD. In future studies, it will be important to combine DNA methylation profiling with transcriptome analyses to evaluate the relationship between aberrant DNA methylation and RNA expression, including the association between DNA methylation status at alternative promoters and the expression levels of transcript variants.

ASD

Autism spectrum disorder

DMRs

differentially methylated regions

IS-DMRs

individual-specific DMRs

DRN

dorsal raphe nucleus

PMI

postmortem interval

GWMA

Genome-wide DNA methylation array

EMAS

EM-amplicon sequencing

qPCR

Quantitative polymerase chain reaction

ORNs

Olfactory receptors of olfactory receptor neurons

CGIs

CpG islands

FGD

Functional genomic distribution

CHR

chromosome

Acknowledgments and Funding

We thank Dr. Jane Pickett, Director of Brain Resources and Data, Autism Tissue Program, for facilitating brain tissue collection and Ms. Hiromi Kamura for technical assistance. Human tissue was obtained from the National Institute of Child Health and Development Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, Maryland. Tissue samples were provided by the Harvard Brain Tissue Resource Center. This study was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K. I. (19K08041 and 23K07019) and a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. M. (19H03581).

Contributions

Keiko Iwata: Study concept and design; acquisition of data; analysis and interpretation of data; writing of the manuscript for content. Kazuhiko Nakabayashi: Acquisition of data; analysis and interpretation of data; writing of the manuscript for content. Keisuke Ishiwata: Acquisition of data; analysis and interpretation of data. Kazuhiko Nakamura: Sample collection; writing of the manuscript for content. Yosuke Kameno: Acquisition of data; analysis and interpretation of data. Kenichiro Hata: Study concept and design; acquisition of data; analysis and interpretation of data; writing of the manuscript for content. Hideo Matsuzaki: Study concept and design; acquisition of data; analysis and interpretation of data; writing of the manuscript for content.

Data availability

DNA methylation data have been deposited in GEO (accession number GSE242427).

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Tables 1-5 are available in the Supplementary Files section.

No competing interests reported.

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Genome-wide DNA methylation profiles in the raphe nuclei of patients with autism spectrum disorder

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Version 1

Abstract

Figures

Introduction

Methods

Postmortem brain tissues

Genome-wide DNA methylation array (GWMA)

Methylation array data analysis

EM-amplicon sequencing (EMAS)

Quantitative polymerase chain reaction (qPCR)

Results

Characteristics of the subjects and brain samples

Brain genome-wide methylation analysis—average of the controls vs. average of the subjects with ASD

Brain genome-wide methylation analysis—subjects with ASD vs. average of the controls

mRNA expression of differentially methylated genes in ASD

Discussion

Limitation

Conclusions

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1