Transition of allele-specific DNA hydroxymethylation at regulatory loci is associated with phenotypic variation in monozygotic twins discordant for psychiatric disorders.

BACKGROUND: Major psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BPD) are complex genetic mental illnesses. Their non-Mendelian features, such as those observed in monozygotic twins discordant for SCZ or BPD, are likely complicated by environmental modifiers of genetic effects. 5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark in gene regulation, and whether it is linked to genetic variants that contribute to non-Mendelian features remains largely unexplored. METHODS: We combined the 5hmC-selective chemical labeling method (5hmC-seq) and whole-genome sequencing (WGS) analysis of peripheral blood DNA obtained from monozygotic (MZ) twins discordant for SCZ or BPD to identify allelic imbalances in hydroxymethylome maps, and examined association of allele-specific hydroxymethylation (AShM) transition with disease susceptibility based on Bayes factors (BF) derived from the Bayesian generalized additive linear mixed model. We then performed multi-omics integrative analysis to determine the molecular pathogenic basis of those AShM sites. We finally employed luciferase reporter, CRISPR/Cas9 technology, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), PCR, FM4-64 imaging analysis, and RNA sequencing to validate the function of interested AShM sites in the human neuroblastoma SK-N-SH cells and human embryonic kidney 293T (HEK293T) cells. RESULTS: We identified thousands of genetic variants associated with AShM imbalances that exhibited phenotypic variation-associated AShM changes at regulatory loci. These AShM marks showed plausible associations with SCZ or BPD based on their effects on interactions among transcription factors (TFs), DNA methylation levels, or other epigenomic marks and thus contributed to dysregulated gene expression, which ultimately increased disease susceptibility. We then validated that competitive binding of POU3F2 on the alternative allele at the AShM site rs4558409 (G/T) in PLLP-enhanced PLLP expression, while the hydroxymethylated alternative allele, which alleviated the POU3F2 binding activity at the rs4558409 site, might be associated with the downregulated PLLP expression observed in BPD or SCZ. Moreover, disruption of rs4558409 promoted neural development and vesicle trafficking. CONCLUSION: Our study provides a powerful strategy for prioritizing regulatory risk variants and contributes to our understanding of the interplay between genetic and epigenetic factors in mediating SCZ or BPD susceptibility.

Allele-specific DNA methylation maps in monozygotic twins discordant for psychiatric disorders reveal that disease-associated switching at the EIPR1 regulatory loci modulates neural function.

The non-Mendelian features of phenotypic variations within monozygotic twins are likely complicated by environmental modifiers of genetic effects that have yet to be elucidated. Here, we performed methylome and genome analyses of blood DNA from psychiatric disorder-discordant monozygotic twins to study how allele-specific methylation (ASM) mediates phenotypic variations. We identified that thousands of genetic variants with ASM imbalances exhibit phenotypic variation-associated switching at regulatory loci. These ASMs have plausible causal associations with psychiatric disorders through effects on interactions between transcription factors, DNA methylations, and other epigenomic markers and then contribute to dysregulated gene expression, which eventually increases disease susceptibility. Moreover, we also experimentally validated the model that the rs4854158 alternative C allele at an ASM switching regulatory locus of EIPR1 encoding endosome-associated recycling protein-interacting protein 1, is associated with demethylation and higher RNA expression and shows lower TF binding affinities in unaffected controls. An epigenetic ASM switching induces C allele hypermethylation and then recruits repressive Polycomb repressive complex 2 (PRC2), reinforces trimethylation of lysine 27 on histone 3 and inhibits its transcriptional activity, thus leading to downregulation of EIPR1 in schizophrenia. Moreover, disruption of rs4854158 induces gain of EIPR1 function and promotes neural development and vesicle trafficking. Our study provides a powerful framework for identifying regulatory risk variants and contributes to our understanding of the interplay between genetic and epigenetic variants in mediating psychiatric disorder susceptibility.

Accurate identification of genes associated with brain disorders by integrating heterogeneous genomic data into a Bayesian framework.

BACKGROUND: Genome-wide association studies (GWAS) have revealed many brain disorder-associated SNPs residing in the noncoding genome, rendering it a challenge to decipher the underlying pathogenic mechanisms. METHODS: Here, we present an unsupervised Bayesian framework to identify disease-associated genes by integrating risk SNPs with long-range chromatin interactions (iGOAT), including SNP-SNP interactions extracted from ∼500,000 patients and controls from the UK Biobank, and enhancer-promoter interactions derived from multiple brain cell types at different developmental stages. FINDINGS: The application of iGOAT to three psychiatric disorders and three neurodegenerative/neurological diseases predicted sets of high-risk (HRGs) and low-risk (LRGs) genes for each disorder. The HRGs were enriched in drug targets, and exhibited higher expression during prenatal brain developmental stages than postnatal stages, indicating their potential to affect brain development at an early stage. The HRGs associated with Alzheimer's disease were found to share genetic architecture with schizophrenia, bipolar disorder and major depressive disorder according to gene co-expression module analysis and rare variants analysis. Comparisons of this method to the eQTL-based method, the TWAS-based method, and the gene-level GWAS method indicated that the genes identified by our method are more enriched in known brain disorder-related genes, and exhibited higher precision. Finally, the method predicted 205 risk genes not previously reported to be associated with any brain disorder, of which one top-risk gene, MLH1, was experimentally validated as being schizophrenia-associated. INTERPRETATION: iGOAT can successfully leverage epigenomic data, phenotype-genotype associations, and protein-protein interactions to advance our understanding of brain disorders, thereby facilitating the development of new therapeutic approaches. FUNDING: The work was funded by the National Key Research and Development Program of China (2024YFF1204902), the Natural Science Foundation of China (82371482), Guangzhou Science and Technology Research Plan (2023A03J0659) and Natural Science Foundation of Guangdong (2024A1515011363).

LncRNA RP5-998N21.4 promotes immune defense through upregulation of IFIT2 and IFIT3 in schizophrenia.

Schizophrenia is a complex polygenic disease that is affected by genetic, developmental, and environmental factors. Accumulating evidence indicates that environmental factors such as maternal infection and excessive prenatal neuroinflammation may contribute to the onset of schizophrenia by affecting epigenetic modification. We recently identified a schizophrenia-associated upregulated long noncoding RNA (lncRNA) RP5-998N21.4 by transcriptomic analysis of monozygotic twins discordant for schizophrenia. Importantly, we found that genes coexpressed with RP5-998N21.4 were enriched in immune defense-related biological processes in twin subjects and in RP5-998N21.4-overexpressing (OE) SK-N-SH cell lines. We then identified two genes encoding an interferon-induced protein with tetratricopeptide repeat (IFIT) 2 and 3, which play an important role in immune defense, as potential targets of RP5-998N21.4 by integrative analysis of RP5-998N21.4OE-induced differentially expressed genes (DEGs) in SK-N-SH cells and RP5-998N21.4-coexpressed schizophrenia-associated DEGs from twin subjects. We further demonstrated that RP5-998N21.4 positively regulates the transcription of IFIT2 and IFIT3 by binding to their promoter regions and affecting their histone modifications. In addition, as a general nuclear coactivator, RMB14 (encoding RNA binding motif protein 14) was identified to facilitate the regulatory role of RP5-998N21.4 in IFIT2 and IFIT3 transcription. Finally, we observed that RP5-998N21.4OE can enhance IFIT2- and IFIT3-mediated immune defense responses through activation of signal transducer and activator of transcription 1 (STAT1) signaling pathway in U251 astrocytoma cells under treatment with the viral mimetic polyinosinic: polycytidylic acid (poly I:C). Taken together, our findings suggest that lncRNA RP5-998N21.4 is a critical regulator of immune defense, providing etiological and therapeutic implications for schizophrenia.

Loss of schizophrenia-related miR-501-3p in mice impairs sociability and memory by enhancing mGluR5-mediated glutamatergic transmission.

Schizophrenia is a polygenetic disease, the heterogeneity of which is likely complicated by epigenetic modifications yet to be elucidated. Here, we performed transcriptomic analysis of peripheral blood RNA from monozygotic twins discordant for schizophrenia and identified a schizophrenia-associated down-regulated microRNA, miR-501-3p. We showed that the loss of miR-501-3p in germline knockout (KO) male mice resulted in dendritic structure defects, glutamatergic transmission enhancement, and sociability, memory, and sensorimotor gating disruptions, which were attenuated when miR-501 expression was conditionally restored in the nervous system. Combining the results of proteomic analyses with the known genes linked to schizophrenia revealed that metabotropic glutamate receptor 5 (mGluR5) was one of the miR-501-3p targets and was elevated in vivo upon loss of miR-501. Treatment with the mGluR5 negative allosteric modulator 3-2((-methyl-4-thiazolyl) ethynyl) pyridine or the N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid ameliorated the deficits observed in Mir501-KO mice. The epigenetic and pathophysiological mechanism that links miR-501-3p to the modulation of glutamatergic transmission provides etiological implications for schizophrenia.

Epigenetic Age Acceleration Was Delayed in Schizophrenia.

Schizophrenia is a serious neuropsychiatric disorder with abnormal age-related neurodevelopmental (or neurodegenerative) trajectories. Although an accelerated aging hypothesis of schizophrenia has been proposed, the quantitative study of the disruption of the physiological trajectory caused by schizophrenia is inconclusive. In this study, we employed 3 "epigenetic clock" methods to quantify the epigenetic age of a large sample size of whole blood (1069 samples from patients with schizophrenia vs 1264 samples from unaffected controls) and brain tissues (500 samples from patients with schizophrenia vs 711 samples from unaffected controls). We observed significant positive correlations between epigenetic age and chronological age in both blood and brain tissues from unaffected controls and patients with schizophrenia, as estimated by 3 methods. Furthermore, we observed that epigenetic age acceleration was significantly delayed in schizophrenia from the whole blood samples (aged 20-90 years) and brain frontal cortex tissues (aged 20-39 years). Intriguingly, the genes regulated by the epigenetic clock also contained schizophrenia-associated genes, displaying differential expression and methylation in patients with schizophrenia and involving in the regulation of cell activation and development. These findings were further supported by the dysregulated leukocyte composition in patients with schizophrenia. Our study presents quantitative evidence for a neurodevelopmental model of schizophrenia from the perspective of a skewed "epigenetic clock." Moreover, landmark changes in an easily accessible biological sample, blood, reveal the value of these epigenetic clock genes as peripheral biomarkers for schizophrenia.

Interactions of the GABRG2 polymorphisms and childhood trauma on suicide attempt and related traits in depressed patients.

BACKGROUND: Previously, we reported that the longest variant of the GABA A receptor γ2 subunit (GABRG2) was associated with suicidal behavior. The present study therefore aimed to determine whether polymorphisms near the alternatively spliced exon of GABRG2 are associated with suicide attempt (SA) and its related traits, and how these variants might interact with reported childhood trauma (CT) in their association with suicidal behavior. METHODS: We examined 5 single nucleotide polymorphisms (SNPs) of GABRG2. Subjects were suicide Attempters (N = 94), non-suicide attempters (N = 168) with MDD or Bipolar depression, and healthy volunteers (N = 100). Data on demographics, depression severity and suicide attempts were collected. Participants also completed a set of instruments assessing CT, and lifetime aggression and impulsivity.. GABRG2 polymorphisms were genotyped using Sanger sequencing. RESULTS: Allele A of rs211034 was a protective factor for SA (OR = 0.50 (0.32, 0.80), p = 0.003), and had an interaction effect with emotional neglect (OR = 0.89 (0.82, 0.97), p = 0.006) on depression. One haploblock (consisting of rs211035 and rs211034) was identified within these SNPs, and subjects with haplotype GA (frequency = 7.3%), had lower rate of SA (OR=0.26(0.10, 0.67), p = 0.006). Cognitive impulsivity (OR=1.38)1.24,1.55), p < 0.001), non-planning impulsivity (OR = 1.18 (1.10,1.25), p < 0.001), anger (OR = 1.13 (1.07,1.19), p < 0.001), impulsivity total score (OR = 1.10(1.06,1.15), p < 0.001), hostility (OR = 1.10 (1.04, 1.15), p < 0.001), aggression total score (OR = 1.05 (1.03,1.07), p < 0.001) were associated with depression, meanwhile, hopelessness (OR = 2.18 (1.56, 3.04), p < 0.001) and impulsivity total score (OR = 1.05 (1.02,1.08), p < 0.001) were associated with the risk of SA, adjusted by age and gender. There was no mediation effect in the relationship among CT, gene polymorphisms and SA or depression through increased impulsivity or aggression. LIMITATIONS: The main limitation of this study is its modest sample size. More genetic variants as well as epigenetic markers should be examined in future studies. CONCLUSIONS: These findings add to evidence for the involvement of GABRG2 and impulsivity and hopelessness in SA independent from their association with depression. More research is needed on possible mediators of the relationship between GABA-related gene and SA.

[Clinicopathologic features and diagnosis of metastatic carcinoma to the spleen].

OBJECTIVE: To study the clinicopathologic features and diagnosis of metastatic carcinoma to the spleen (MCS). METHODS: Four patients (1 man and 3 women, mean age 43.5 years) with MCS were analyzed clinicopathologically. RESULTS: The four MCS patients accounted for 1.3% of 308 patients having spleen biopsy from 1959 to 1999. Their chief presentations were pain and mass in the left upper quadrant of the abdomen. The mass was located in the upper pole of the spleen (1 patient), the lower pole of the spleen (2), or the lower pole and hilum of the spleen (1). Macroscopically, all of the lesions were nodular. Histologic type of these MCSs included acinar cell carcinoma of the pancreas (2 patients), transverse colon adenocarcinoma (1), and hepatic cell carcinoma (1). Clinically, 1 patient was diagnosed as having MCS and 3 were misdiagnosed. According to Chinese literature, the clinicopathologic features of MCS were as follows: (1) 66.7% of the patients with MCS were aged 30 approximately 60 years, with a mean of 51.2 years. (2) 76.3% of the patients presented with pain in the left upper quadrant of the abdomen and 63.2% with splenomegaly and splenic masses. (3) Macroscopically, nodular lesions accounted for 68.4%. (4) Microscopically, 84.2% of the lesions were adenocarcinomas and 70.3% originated from carcinomas of the colon, liver, ovary and pancreas. (5) B-mode ultrasonography and/or CT showed occupying lesions or masses in the spleen in 76.7%, and MCS in 11.8%. (6) Clinically, 73.7% of the patients were misdiagnosed. CONCLUSIONS: MCS is uncommon but its clinical misdiagnosis rate is high. Image examination is of value in clinical diagnosis. Cooperation of clinicians and pathologists may enhance the diagnostic level of MCS.