A novel long non-coding RNA, lnc-RNU12, influences the T-cell cycle via c-JUN and CCNL2 in rheumatoid arthritis.

OBJECTIVES: Long non-coding RNAs (lncRNAs) play important roles in RA pathogenesis. However, specific lncRNAs that regulate gene expression in RA pathogenesis are poorly known. This study was undertaken to characterize a novel lncRNA (lnc-RNU12) that has a lower-than-normal expression level in RA patients. METHODS: We performed initial genome-wide lncRNA microarray screening in peripheral blood mononuclear cells from 28 RA cases and 18 controls. Multiple methods were used to validate the detected associations between lncRNAs and RA. Furthermore, we identified the source and characteristics of the highlighted lncRNAs, detected the target genes, and determined the functional effect on immune cells through lncRNA knock-down in Jurkat T cell lines. RESULTS: lnc-RNU12 was downregulated in peripheral blood mononuclear cells and T cell subtypes of RA patients and was genetically associated with RA risk. lnc-RNU12 mediates the effect of microbiome alterations on RA risk. Activation of T cells caused low expression of lnc-RNU12. Knock-down of lnc-RNU12 in Jurkat T cells caused cell cycle S-phase arrest and altered the expression of protein-coding genes related to the cell cycle and apoptosis (e.g. c-JUN, CCNL2, CDK6, MYC, RNF40, PKM, VPS35, DNAJB6 and FLCN). Finally, c-JUN and CCNL2 were identified as target genes of lnc-RNU12 at the mRNA and protein expression levels. RNA-binding protein immunoprecipitation assays verified the interaction between lnc-RNU12 and the two proteins (c-Jun and cyclin L2) in Jurkat cells. CONCLUSIONS: Our study suggested that lnc-RNU12 was involved in the pathogenesis of RA by influencing the T cell cycle by targeting c-JUN and CCNL2.

Protein array test detected three osteoporosis related plasma inflammatory cytokines in Chinese postmenopausal women.

Inflammatory cytokines were involved in pathological conditions of osteoporosis (OP). However, the specific OP-associated inflammatory cytokines are still awaiting to be detected by using a systemic method. Herein, we adopted an extreme sampling scheme and examined inflammatory cytokines between subjects with low and high bone mineral density (BMD) through protein microarray. First, 8 candidate cytokines including B lymphocyte chemoattractant (BLC), osteopontin (OPN) and insulin-like growth factor-binding protein 4 (IGFBP4) were identified in the discovery extreme sampling subgroup. Then, the different expressions for BLC, OPN and IGFBP4 were validated and replicated in two independent extreme sampling subgroups. Further functional experiments showed that the cytokine BLC was involved in bone metabolism by inhibiting bone formation and promoting bone resorption. Together, this study further revealed that inflammatory cytokines were closely related with OP, and that they highlighted critical roles of BLC in the pathogenesis of OP.

Multi-omics integrative analysis identified SNP-methylation-mRNA: Interaction in peripheral blood mononuclear cells.

Genetic variants have potential influence on DNA methylation and thereby regulate mRNA expression. This study aimed to comprehensively reveal the relationships among SNP, methylation and mRNA, and identify methylation-mediated regulation patterns in human peripheral blood mononuclear cells (PBMCs). Based on in-house multi-omics datasets from 43 Chinese Han female subjects, genome-wide association trios were constructed by simultaneously testing the following three association pairs: SNP-methylation, methylation-mRNA and SNP-mRNA. Causal inference test (CIT) was used to identify methylation-mediated genetic effects on mRNA. A total of 64,184 significant cis-methylation quantitative trait loci (meQTLs) were identified (FDR < 0.05). Among the 745 constructed trios, 464 trios formed SNP-methylation-mRNA regulation chains (CIT). Network analysis (Cytoscape 3.3.0) constructed multiple complex regulation networks among SNP, methylation and mRNA (eg a total of 43 SNPs simultaneously connected to cg22517527 and further to PRMT2, DIP2A and YBEY). The regulation chains were supported by the evidence from 4DGenome database, relevant to immune or inflammatory related diseases/traits, and overlapped with previous eQTLs from dbGaP and GTEx. The results provide new insights into the regulation patterns among SNP, DNA methylation and mRNA expression, especially for the methylation-mediated effects, and also increase our understanding of functional mechanisms underlying the established associations.

Detection of lncRNA-mRNA interaction modules by integrating eQTL with weighted gene co-expression network analysis.

One major function of lncRNA is to regulate the expression of mRNA, but the patterns of their interactions were largely unknown. We attempted to construct lncRNA-mRNA interaction modules at a genome-wide scale. We performed a genome-wide lncRNA-mRNA eQTL analysis in peripheral blood mononuclear cells of 43 individuals, followed by weighted gene co-expression network analysis and functional enrichment analysis which sought to detect functional modules. There were 4627 significant cis lnc-eQTL pairs (P < 1.4 × 10-6) and 1,587,128 significant trans lnc-eQTL pairs (P < 3.46 × 10-9). We detected 11 eQTL modules for the lnc-eQTL networks. Among them, five modules showed significant enrichments in GO terms, and three modules showed significant enrichments in specific KEGG pathways (e.g., Toll-like receptor, PI3K-Akt, NF-kappa B, and TNF signaling pathways). lncRNA-protein interaction analysis showed that some well-known functional lncRNAs (HOTAIR, CCDC26, RHPN1-AS1, WT1-AS, and TCL6) in the eQTL module interacted with genes in focal adhesion and PI3K-Akt signaling pathway. We identified biologically functional lncRNA-mRNA interaction modules by integrating eQTL and weighted gene co-expression network analysis. Integrative analysis of lncRNA and mRNA data by applying eQTL analysis and weighted gene co-expression network analysis methods could be helpful for functional annotation of lncRNAs.

Rheumatoid arthritis-associated DNA methylation sites in peripheral blood mononuclear cells.

OBJECTIVES: To identify novel DNA methylation sites significant for rheumatoid arthritis (RA) and comprehensively understand their underlying pathological mechanism. METHODS: We performed (1) genome-wide DNA methylation and mRNA expression profiling in peripheral blood mononuclear cells from RA patients and health controls; (2) correlation analysis and causal inference tests for DNA methylation and mRNA expression data; (3) differential methylation genes regulatory network construction; (4) validation tests of 10 differential methylation positions (DMPs) of interest and corresponding gene expressions; (5) correlation between PARP9 methylation and its mRNA expression level in Jurkat cells and T cells from patients with RA; (6) testing the pathological functions of PARP9 in Jurkat cells. RESULTS: A total of 1046 DNA methylation positions were associated with RA. The identified DMPs have regulatory effects on mRNA expressions. Causal inference tests identified six DNA methylation-mRNA-RA regulatory chains (eg, cg00959259-PARP9-RA). The identified DMPs and genes formed an interferon-inducible gene interaction network (eg, MX1, IFI44L, DTX3L and PARP9). Key DMPs and corresponding genes were validated their differences in additional samples. Methylation of PARP9 was correlated with mRNA level in Jurkat cells and T lymphocytes isolated from patients with RA. The PARP9 gene exerted significant effects on Jurkat cells (eg, cell cycle, cell proliferation, cell activation and expression of inflammatory factor IL-2). CONCLUSIONS: This multistage study identified an interferon-inducible gene interaction network associated with RA and highlighted the importance of PARP9 gene in RA pathogenesis. The results enhanced our understanding of the important role of DNA methylation in pathology of RA.

Integrative Analysis Confirmed the Association between Osteoprotegerin and Osteoporosis.

Objective This study aimed to verify the association between osteoprotegerin gene (OPG) and its variants with osteoporosis (OP) by performing integrative analysis.Methods We used the KGG software to perform gene-based association analysis, which integrated all publicly available single-nucleotide polymorphism (SNP)-based P values and obtained an overall P value for the OPG. The significant SNPs were screened for expression quantitative trait loci (eQTLs). Meta-analysis was used to combine the associations between the variants of OPG and bone mineral density (BMD) reported in the literatures. Then we performed dual-luciferase reporter gene systems for the functional verification of the variants of OPG in vitro.Results In the gene-based association analysis, the over all P value of OPG was 6.24×10 -13for BMD at femoral neck (FN) and 7.37×10 -17 for BMD at lumbar spine (LS), indicating the importance of OPG for OP. The publicly available eQTL database identified 5 eQTLs which exert cis-regulation effects on OPG at FN and LS. Literature searching found that rs2073617 (known as T950C) was the hot spot SNP. There were 13 relevant studies on rs2073617 besides the GEFOS-2 study identified from the PubMed. Significant differences among TT, TC and CC genotypes at FN (P= 0.047) and LS (P= 0.025) were shown by meta-analysis, demonstrating the associations between T950C polymorphism and BMD. Luciferase gene expression was significantly higher at the presence of allele C than allele T in the 293T cells (t=-9.47, P<0.01). Conclusion The integrative analysis further confirmed the importance of OPG in OP and the correlation of T950C polymorphism with BMD of OP. The strategy can be used as a reference for functional interpretation of other disease-related genes.

Relative abundance of mature myostatin rather than total myostatin is negatively associated with bone mineral density in Chinese.

Myostatin is mainly secreted by skeletal muscle and negatively regulates skeletal muscle growth. However, the roles of myostatin on bone metabolism are still largely unknown. Here, we recruited two large populations containing 6308 elderly Chinese and conducted comprehensive statistical analyses to evaluate the associations among lean body mass (LBM), plasma myostatin, and bone mineral density (BMD). Our data revealed that total myostatin in plasma was mainly determined by LBM. The relative abundance of mature myostatin (mature/total) was significantly lower in high versus low BMD subjects. Moreover, the relative abundance of mature myostatin was positively correlated with bone resorption marker. Finally, we carried out in vitro experiments and found that myostatin has inhibitory effects on the proliferation and differentiation of human osteoprogenitor cells. Taken together, our results have demonstrated that the relative abundance of mature myostatin in plasma is negatively associated with BMD, and the underlying functional mechanism for the association is most likely through inhibiting osteoblastogenesis and promoting osteoclastogenesis.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3'-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA-mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA-mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA-mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA-mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P < 0.05) and ~70% were negative. The correlation network was complex and highlighted key miRNAs/genes in PBMC. Some miRNAs, such as hsa-miR-29a, hsa-miR-148a, regulate a cluster of target genes. Some genes, e.g., TNRC6A, are regulated by multiple miRNAs. The identified genes tend to be enriched in molecular functions of DNA and RNA binding, and biological processes such as protein transport, regulation of translation and chromatin modification. The results provided a global view of the miRNA-mRNA expression correlation profile in human PBMCs, which would facilitate in-depth investigation of biological functions of key miRNAs/mRNAs and better understanding of the pathogenesis underlying PBMC-related diseases.

The distribution and functional relevance analysis of runs of homozygosity (ROHs) in Chinese Han female population.

Extended homozygosity is a genomic region in which the copies inherited from parents are identical, and has obvious inter-individual differences in length and frequency. Runs of homozygosity (ROHs), regarded as a type of structure variations, may have potential capacity in regulating gene transcription. To learn more about the genome-wide distribution of ROH regions in humans and understand the potential roles, this study applied ROH-based approach to quantify and characterize ROHs in 41 Chinese Han female subjects, and test potential associations between ROHs and mRNA expressions by eQTL analysis to ascertain whether ROHs are relevant to gene transcription in peripheral blood mononuclear cells (PBMCs). 10,884 ROH regions were identified in human genome. The average cumulative length of ROH regions was 217,250 ± 20,241 kb. The number of core segments in each chromosome generally matched the total length of corresponding chromosome, i.e., the longer the chromosome, the more the core segments. Genes located in the core regions of ROH were significantly enriched in multiple basic metabolism pathways. A total of 226 cis-eQTLs and 178 trans-eQTLs were identified. The cis-effect size was mainly concentrated at ± 0.5; and the trans-effect size was mainly concentrated at -1.5 and 1.0. Genes with eQTL effects were significantly enriched in functions related to protein binding, cytosol, nucleoplasm, nuclear membrane, protein binding and citrate metabolic process. This study described comprehensive distributions and characteristics of ROH in Han female Chinese, and recognized the significant role of ROH associated with gene transcription in human PBMC.

Association of Plasma Irisin with Bone Mineral Density in a Large Chinese Population Using an Extreme Sampling Design.

Irisin, a myokine produced by skeletal muscle in response to physical exercise, promotes trans-differentiation of white adipose tissue into brown adipose tissue. Recent evidences suggested that irisin also plays an important role in the control of bone metabolism. This study aimed to ascertain the relationship between plasma irisin and bone mineral density (BMD) in Chinese population by adoption of an extreme sampling method. Based on a large and screened Chinese elderly population (N = 6308), two subgroups with extremely high and low hip BMD were selected for discovery (N = 80, high vs. low BMD = 44:36) and validation (N = 60, high vs. low BMD = 30:30), respectively. Plasma irisin, P1NP, and ß-CTx were measured using commercially available ELISA kits. Other metabolic parameters (e.g., blood glucose, total cholesterol and triglycerides) were collected. Student's t test and Spearman correlation analyses were conducted in SPSS. Significant difference was discovered for plasma irisin between females and age-matched males (N = 80, male vs. female = 42:38, P = 0.002). The plasma irisin levels were significantly higher in high BMD subjects than in low BMD subjects, which was observed in both discovery (P = 0.012) and validation samples (P = 0.022). However, such observation was limited to males only. Further correlation analyses in males showed that plasma irisin was correlated with BMD (r = 0.362, P = 0.025) and triglyceride (r = - 0.354, P = 0.032). Plasma irisin levels were associated with hip BMD in Chinese elderly men. This study represented the first effort of investigating the relationship of plasma irisin and BMD in elderly population. The positive correlation between plasma irisin and BMD hints intrinsic communication between muscle and bone.

Functional relevance for multiple sclerosis-associated genetic variants.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of central nervous system. Many genetic variants associated with MS have been identified by genome-wide association studies, but functional mechanism underlying the associations is largely unclear. Utilizing the publically available datasets, we carried out gene relationships among implicated loci (GRAIL) analyses to search for MS-associated SNPs/genes. Expression quantitative trait loci (eQTLs) analyses were conducted to identify eQTL SNPs/target genes. Further, functional prediction for SNP, differential gene expression, and functional annotation clustering analyses for gene were conducted to explore their functional relevance to MS. Among the 284 identified MS-associated SNPs (P < 10(-4)), eQTL analysis showed that 45 SNPs act as cis-effect regulators on 19 MS-associated genes. Among the 19 eQTL target genes, 14 showed significantly differential expressions in MS-related cells. Among the 45 SNPs, 15 were predicted most likely located in transcription factor (TF) binding sites, and five predicted SNPs (rs3095329 of TUBB, rs9469220/rs2647046 of HLA-DQB1, rs11154801 of AHI1, and rs1062158 of NDFIP1) have corresponding target genes with significantly differential expressions in multiple cell groups, while rs7194 of HLA-DRA was predicted in the has-miR-6507-3p binding site. The functional evidence, taken together, highlighted the functional relevance of the six SNPs to MS. The present findings provide novel insights into the functional mechanisms underlying the MS-associated genetic variants, which improve our understanding of the genetic association for MS.

Identification of novel rheumatoid arthritis-associated MiRNA-204-5p from plasma exosomes.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by infiltration of immune cells in the synovium. However, the crosstalk of immune cells and synovial fibroblasts is still largely unknown. Here, global miRNA screening in plasma exosomes was carried out with a custom microarray (RA patients vs. healthy controls = 9:9). A total of 14 exosomal miRNAs were abnormally expressed in the RA patients. Then, downregulated expression of exosomal miR-204-5p was confirmed in both the replication (RA patients vs. healthy controls = 30:30) and validation groups (RA patients vs. healthy controls = 56:60). Similar to the findings obtained in humans, a decreased abundance of exosomal miR-204-5p was observed in mice with collagen-induced arthritis (CIA). Furthermore, Spearman correlation analysis indicated that plasma exosomal miR-204-5p expression was inversely correlated with disease parameters of RA patients, such as rheumatoid factor, erythrocyte sedimentation rate, and C-reactive protein. In vitro, our data showed that human T lymphocytes released exosomes containing large amounts of miR-204-5p, which can be transferred into synovial fibroblasts, inhibiting cell proliferation. Overexpression of miR-204-5p in synovial fibroblasts suppressed synovial fibroblast activation by targeting genes related to cell proliferation and invasion. In vivo assays found that administration of lentiviruses expressing miR-204-5p markedly alleviated the disease progression of the mice with CIA. Collectively, this study identified a novel RA-associated plasma exosomal miRNA-204-5p that mediates the communication between immune cells and synovial fibroblasts and can be used as a potential biomarker for RA diagnosis and treatment.

Integrative analysis identifies potential causal methylation-mRNA regulation chains for rheumatoid arthritis.

Genome-wide association studies have identified many genetic loci for rheumatoid arthritis (RA). However, causal factors underlying these loci were largely unknown. The aim of this study was to identify potential causal methylation-mRNA regulation chains for RA. We identified differentially expressed mRNAs and methylations and conducted summary statistic data-based Mendelian randomization (SMR) analysis to detect potential causal mRNAs and methylations for RA. Then causal inference test (CIT) was performed to determine if the methylation-mRNA pairs formed causal chains. We identified 11,170 mRNAs and 24,065 methylations that were nominally associated with RA. Among them, 197 mRNAs and 104 methylations passed the SMR test. According to physical positions, we defined 16 cis methylation-mRNA pairs and inferred 5 chains containing 4 methylations and 4 genes (BACH2, MBP, MX1 and SYNGR1) to be methylation→mRNA→RA causal chains. The effect of SYNGR1 expression in peripheral blood mononuclear cells on RA risk was found to be consistent in both the in-house and public data. The identified methylations located in CpG Islands that overlap promoters in the 5' region of the genes. The promoter regions showed long-range interactions with other enhancers and promoters, suggesting a regulatory potential of these methylations. Therefore, the present study provided a new integrative analysis strategy and highlighted potential causal methylation-mRNA chains for RA. Taking the evidences together, SYNGR1 promoter methylations most probably affect mRNA expressions and then affect RA risk.

Integrative analysis identifies the association between CASZ1 methylation and ischemic stroke.

OBJECTIVE: To highlight potential epigenetic risk factors for blood pressure (BP) and ischemic stroke (IS) in loci identified by genome-wide association studies (GWASs). METHODS: We detected DNA methylation for BP (317,756 individuals from UK Biobank) and IS (521,612 individuals from MEGASTROKE) in Europeans by using the summary data-based mendelian randomization (SMR) method. We selected the most relevant gene to validate the association in 1,207 patients with hypertensive IS and 1,269 controls from the Chinese populations. RESULTS: We first identified 173 CpG sites in 90 genes, 337 CpG sites in 142 genes, and 9 CpG sites in 7 genes that were significantly associated with systolic, diastolic BP, and IS, respectively. The methylation level of cg12760995 in CASZ1 was associated with systolic (P SMR = 1.74 × 10-12), diastolic BP (P SMR = 2.48 × 10-10), and IS (odds ratio [OR] = 0.92 [95% confidence interval [CI]: 0.91-0.94]; P SMR = 2.28 × 10-8) in Europeans. The methylation levels of 17 sites in the promoter of CASZ1 were measured in the Chinese individuals, and 10 of them were significantly associated with IS. The higher methylation level of CASZ1 was associated with a lower risk of IS (adjusted OR = 0.97 [95% CI: 0.96-0.99]). CASZ1 seemed to be hypomethylated in hypertensive cases, and the level was negatively correlated with BP. Systolic and diastolic BP mediated approximately 61.2% (p = 3.49 × 10-6) and 45.0% (p = 0.0029) of the association between CASZ1 methylation and IS, respectively. CONCLUSIONS: This study identified DNA methylations that were associated with BP and IS. CASZ1 was hypomethylated in Chinese patients with hypertensive IS.

Integrative analysis highlighted susceptibility genes for rheumatoid arthritis.

Genetic factors underlying susceptibility to rheumatoid arthritis (RA) are largely unknown. The aim of this study was to identify potential genes for RA. We conducted summary statistic data-based Mendelian randomization (SMR) analysis to detect potential causal genes for RA. Further, we performed additional bioinformatics analysis to show the potential relevance of the identified genes to RA. We identified 140 genes that showed causal association with RA. Among these genes, 24 have not been reported to be associated with RA (e.g., IFNAR2, FLOT1, ITPR3, PPP2R3C and SLC35B2). The unreported genes were highly connected with some well-known RA-related genes (e.g., HLA-DQB1, CD226, PTPN22, CD40, IFNGR2, BLK, TRAF1, SYNGR1 and CCR6) that were also found to be causally associated with RA. The identified genes were involved in the significant enriched RA-related biological pathways. We found integrative evidence in support of IFNAR2 as a potential causal gene of RA in SMR, differential expression, weighted gene co-expression network, protein-protein interaction and functional enrichment analyses. The present study highlights a list of potential causal genes for RA. The findings provide new insights into the mechanism underlying known genome-wide associated RA susceptibility loci.

Epigenetically regulated co-expression network of genes significant for rheumatoid arthritis.

Aim: To identify epigenetically regulated network of genes in peripheral blood mononuclear cells significant for rheumatoid arthritis (RA). Methods: Differentially expressed genes (DEGs) and their associated differentially expressed miRNAs and differentially methylated positions (DMPs) were identified. Causal inference test (CIT) identified the causal regulation chains. The analyses, for example, weighted gene co-expression network (WGCNA), protein-protein interaction and functional enrichment, evaluated interaction patterns among the DEGs and the associated epigenetic factors. Results: A total of 181 DEGs were identified. The DEGs were significantly regulated by DMPs and/or differentially expressed miRNAs. Causal inference test analyses identified 18 causal chains of DMP-DEG-RA and 16 intermediate DEGs enriched in 'protein kinase inhibitor activity'. BTN2A1 was co-expressed with other 9 intermediate genes and 11 known RA-associated genes and played a pivotal role in the co-expression network. Conclusion: Epigenetically regulated network of genes in peripheral blood mononuclear cells (PBMC) contributed to RA. The causal DMPs and key intermediate genes may serve as potential biomarkers for RA.

Integrative Analysis Identified IRF6 and NDST1 as Potential Causal Genes for Ischemic Stroke.

Objective: To highlight potential functional variants and causal genes for ischemic stroke (IS) in genomic loci identified by genome-wide association studies (GWAS). Methods: We examined the association between m6A-SNPs and IS in large scale GWAS. Furthermore, eQTL analysis was performed to evaluate the effect of m6A-SNPs on gene expression. The top associations between m6A-SNPs and gene expressions were validated in 40 individuals from the Chinese Han population. Besides, we applied differential expression analysis and Mendelian randomization (MR) analysis to detect potential causal genes for IS. Results: We found 310 (7.39%) m6A-SNPs which were nominally associated with IS. The proportion of m6A-SNPs with P < 0.05 for IS was significantly higher than the non-m6A-SNPs (95%CI: [5.84%, 7.36%], P = 0.02). We found that the IS-associated m6A-SNP rs2013162 was associated with IRF6 expression (P = 6.30 × 10-23), meanwhile IRF6 was differentially expressed between IS cases and controls (P = 6.15 × 10-3) and showed a causal association with IS (P = 3.64 × 10-4). Similar results were found for m6A-SNP rs2273235 in the NDST1 gene which was associated with cardioembolic stroke (P = 8.47 × 10-3). The associations of rs2013162 and rs2273235 with the expression of IRF6 and NDST1 were validated in blood cells (P = 0.0247 and 0.0007), respectively. Conclusions: This study showed that m6A-SNPs may affect IS risk through altering gene expressions. The results suggested that m6A might play a role in IS etiology and gene expressions that affected by m6A may be causal factors for IS.

Examination of the associations between m6A-associated single-nucleotide polymorphisms and blood pressure.

N6-methyladenosine (m6A) has been shown to play critical roles in many biological processes and a variety of diseases. The aim of this study was to investigate the association between m6A-associated single-nucleotide polymorphisms (m6A-SNPs) and blood pressure (BP) in large-scale genome-wide association studies and to test whether m6A-SNPs are enriched among the SNPs that were associated with BP. Furthermore, gene expression analysis was performed to obtain additional evidence for the identified m6A-SNPs. We found 1236 m6A-SNPs that were nominally associated with BP, and 33 of them were significant genome wide. The proportion of m6A-SNPs with a P < 0.05 was significantly higher than that of non-m6A-SNPs. Using fgwas, we found that SNPs associated with diastolic BP (P < 5 × 10-8) were significantly enriched with m6A-SNPs (log 2 enrichment of 2.67, 95% confidence interval: [0.42, 3.68]). Approximately 10% of the BP-associated m6A SNPs were associated with coronary artery disease or stroke. Most of these m6A-SNPs were strongly associated with gene expression. We showed that rs56001051, rs9847953, rs197922, and rs740406 were associated with C1orf167 (P = 0.019), ZNF589 (P = 0.013), GOSR2 (P = 0.001), and DOT1L (P = 0.032) expression levels in peripheral blood mononuclear cells of 40 Chinese individuals, respectively. The present study identified many BP-associated m6A-SNPs and demonstrated their potential functionality. The results suggested that m6A might play important roles in BP regulation.

Integrative analysis revealed potential causal genetic and epigenetic factors for multiple sclerosis.

OBJECTIVE: Many genomic loci have been identified for multiple sclerosis (MS) by genome-wide association studies (GWAS). Discrimination of the most functionally relevant genes in these loci remains challenging. The aim of this study was to highlight potential causal genes for MS. METHODS: We detected potential causal DNA methylations and gene expressions for MS by integrating data from large scale GWAS and quantitative trait locus (QTL) studies using the summary data-based Mendelian randomization method. Potential functional SNPs in the identified genes were searched. RESULTS: We found 178 DNA methylation sites and mRNA expressions of 29 genes that were causally associated with MS. The identified genes enriched in 21 specific KEGG pathways and 80 GO terms (e.g., antigen processing and presentation, interferon gamma mediated signaling pathway). Among the identified non-MHC genes, METTL21B, METTL1 and TSFM were strongly connected. MS-associated SNPs in DDR1 were strongly associated with plasma MHC class I polypeptide-related sequence B (MICB) and Granzyme A levels. And plasma MICB and Granzyme A levels were causally associated with MS. Many SNPs in the causal genes showed QTL effects. The association between m6A-SNPs rs923829 and METTL21B expression level was validated in 40 unrelated Chinese Han individuals. CONCLUSIONS: This study identified many DNA methylations and genes as important risk factors for MS and provided novel evidence on the association between circulating MICB and Granzyme A and MS. We also showed that the interaction among DDR1, MICB and GZMA and interaction among METTL21B, METTL1 and TSFM may participate in the pathogenesis of MS.