Enhancer variants on chromosome 2p14 regulating SPRED2 and ACTR2 act as a signal amplifier to protect against rheumatoid arthritis.

Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA.

High-throughput functional dissection of noncoding SNPs with biased allelic enhancer activity for insulin resistance-relevant phenotypes.

Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer-indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.

Transcriptome-wide association study identifies multiple genes and pathways associated with thyroid function.

Thyroid dysfunction is a common endocrine disease measured by thyroid-stimulating hormone (TSH) level. Although >70 genetic loci associated with TSH have been reported through genome-wide association studies (GWASs), the variants can only explain a small fraction of the thyroid function heritability. To identify novel candidate genes for thyroid function, we conducted the first large-scale transcriptome-wide association study (TWAS) for thyroid function using GWAS-summary data for TSH levels in up to 119 715 individuals combined with precomputed gene expression weights of six panels from four tissue types. The candidate genes identified by TWAS were further validated by TWAS replication and gene expression profiles. We identified 74 conditionally independent genes significantly associated with thyroid function, such as PDE8B (P = 1.67 × 10-282), PDE10A (P = 7.61 × 10-119), NR3C2 (P = 1.50 × 10-92) and CAPZB (P = 3.13 × 10-79). After TWAS replication using UKBB datasets, 26 genes were replicated for significant associations with thyroid-relevant diseases/traits. Among them, 16 genes were causal for their associations to thyroid-relevant diseases/traits and further validated in differential expression analyses, including two novel genes (MFSD6 and RBM47) that did not implicate in previous GWASs. Enrichment analyses detected several pathways associated with thyroid function, such as the cAMP signaling pathway (P = 7.27 × 10-4), hemostasis (P = 3.74 × 10-4), and platelet activation, signaling and aggregation (P = 9.98 × 10-4). Our study identified multiple candidate genes and pathways associated with thyroid function, providing novel clues for revealing the genetic mechanisms of thyroid function and disease.

ExsgRNA: reduce off-target efficiency by on-target mismatched sgRNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has been widely used to facilitate efficient genome editing. Current popular sgRNA design tools only consider the sgRNA perfectly matched to the target site and provide the results without any on-target mismatch. We suppose taking on-target gRNA-DNA mismatches into consideration might provide better sgRNA with similar binding activity and reduced off-target sites. Here, we trained a seq2seq-attention model with feedback-loop architecture, to automatically generate sgRNAs with on-target mismatches. Dual-luciferase reporter experiment showed that multiple sgRNAs with three mismatches could achieve the 80% of the relative activity of the perfect matched sgRNA. Meanwhile, it could reduce the number of off-target sites using sgRNAs with on-target mismatches. Finally, we provided a freely accessible web server sgRNA design tool named ExsgRNA. Users could submit their target sequence to this server and get optimal sgRNAs with less off-targets and similar on-target activity compared with the perfect-matched sgRNA.

Assessment of bidirectional relationships between brain imaging-derived phenotypes and stroke: a Mendelian randomization study.

BACKGROUND: Stroke is a major cause of mortality and long-term disability worldwide. Whether the associations between brain imaging-derived phenotypes (IDPs) and stroke are causal is uncertain. METHODS: We performed two-sample bidirectional Mendelian randomization (MR) analyses to explore the causal associations between IDPs and stroke. Summary data of 587 brain IDPs (up to 33,224 individuals) from the UK Biobank and five stroke types (sample size range from 301,663 to 446,696, case number range from 5,386 to 40,585) from the MEGASTROKE consortium were used. RESULTS: Forward MR indicated 14 IDPs belong to projection fibers or association fibers were associated with stroke. For example, higher genetically determined mean diffusivity (MD) in the right external capsule was causally associated with an increased risk of small vessel stroke (IVW OR = 2.76, 95% CI 2.07 to 3.68, P = 5.87 × 10-12). Reverse MR indicated that genetically determined higher risk of any ischemic stroke was associated with increased isotropic or free water volume fraction (ISOVF) in body of corpus callosum (IVW ß = 0.23, 95% CI 0.14 to 0.33, P = 3.22 × 10-7). This IDP is a commissural fiber and it is not included in the IDPs identified by forward MR. CONCLUSIONS: We identified 14 IDPs with statistically significant evidence of causal effects on stroke or stroke subtypes. We also identified potential causal effects of stroke on one IDP of commissural fiber. These findings might guide further work toward identifying preventative strategies at the brain imaging levels.

LDBlockShow: a fast and convenient tool for visualizing linkage disequilibrium and haplotype blocks based on variant call format files.

The triangular correlation heatmap aiming to visualize the linkage disequilibrium (LD) pattern and haplotype block structure of SNPs is ubiquitous component of population-based genetic studies. However, current tools suffered from the problem of time and memory consuming. Here, we developed LDBlockShow, an open source software, for visualizing LD and haplotype blocks from variant call format files. It is time and memory saving. In a test dataset with 100 SNPs from 60 000 subjects, it was at least 10.60 times faster and used only 0.03-13.33% of physical memory as compared with other tools. In addition, it could generate figures that simultaneously display additional statistical context (e.g. association P-values) and genomic region annotations. It can also compress the SVG files with a large number of SNPs and support subgroup analysis. This fast and convenient tool will facilitate the visualization of LD and haplotype blocks for geneticists.

Surgical methods influence on the risk of anastomotic fistula after pancreaticoduodenectomy: a systematic review and network meta-analysis.

BACKGROUND: Pancreaticoduodenectomy is the first choice surgical intervention for the radical treatment of pancreatic tumors. However, an anastomotic fistula is a common complication after pancreaticoduodenectomy with a high mortality rate. With the development of minimally invasive surgery, open pancreaticoduodenectomy (OPD), laparoscopic pancreaticoduodenectomy (LPD), and robotic pancreaticoduodenectomy (RPD) are gaining interest. But the impact of these surgical methods on the risk of anastomosis has not been confirmed. Therefore, we aimed to integrate relevant clinical studies and explore the effects of these three surgical methods on the occurrence of anastomotic fistula after pancreaticoduodenectomy. METHODS: A systematic literature search was conducted for studies reporting the RPD, LPD, and OPD. Network meta-analysis of postoperative anastomotic fistula (Pancreatic fistula, biliary leakage, gastrointestinal fistula) was performed. RESULTS: Sixty-five studies including 10,026 patients were included in the network meta-analysis. The rank of risk probability of pancreatic fistula for RPD (0.00) was better than LPD (0.37) and OPD (0.62). Thus, the analysis suggests the rank of risk of the postoperative pancreatic fistula for RPD, LPD, and OPD. The rank of risk probability for biliary leakage was similar for RPD (0.15) and LPD (0.15), and both were better than OPD (0.68). CONCLUSIONS: This network meta-analysis provided ranking for three different types of pancreaticoduodenectomy. The RPD and LPD can effectively improve the quality of surgery and are safe as well as feasible for OPD.

Causal Associations Between Blood Lipids and COVID-19 Risk: A Two-Sample Mendelian Randomization Study.

[Figure: see text].

An Osteoporosis Risk SNP at 1p36.12 Acts as an Allele-Specific Enhancer to Modulate LINC00339 Expression via Long-Range Loop Formation.

Genome-wide association studies (GWASs) have reproducibly associated variants within intergenic regions of 1p36.12 locus with osteoporosis, but the functional roles underlying these noncoding variants are unknown. Through an integrative functional genomic and epigenomic analyses, we prioritized rs6426749 as a potential causal SNP for osteoporosis at 1p36.12. Dual-luciferase assay and CRISPR/Cas9 experiments demonstrate that rs6426749 acts as a distal allele-specific enhancer regulating expression of a lncRNA (LINC00339) (∼360 kb) via long-range chromatin loop formation and that this loop is mediated by CTCF occupied near rs6426749 and LINC00339 promoter region. Specifically, rs6426749-G allele can bind transcription factor TFAP2A, which efficiently elevates the enhancer activity and increases LINC00339 expression. Downregulation of LINC00339 significantly increases the expression of CDC42 in osteoblast cells, which is a pivotal regulator involved in bone metabolism. Our study provides mechanistic insight into how a noncoding SNP affects osteoporosis by long-range interaction, a finding that could indicate promising therapeutic targets for osteoporosis.

Transcriptome-wide association study identifies multiple genes associated with childhood body mass index.

BACKGROUND: Childhood obesity is one of the most common and costly nutritional problems with high heritability. The genetic mechanism of childhood obesity remains unclear. Here, we conducted a transcriptome-wide association study (TWAS) to identify novel genes for childhood obesity. METHODS: By integrating the GWAS summary of childhood body mass index (BMI), we conducted TWAS analyses with pre-computed gene expression weights in 39 obesity priority tissues. The GWAS summary statistics of childhood BMI were derived from the early growth genetics consortium with 35,668 children from 20 studies. RESULTS: We identified 15 candidate genes for childhood BMI after Bonferroni corrections. The most significant gene, ADCY3, was identified in 13 tissues, including adipose, brain, and blood. Interestingly, eight genes were only identified in the specific tissue, such as FAIM2 in the brain (P = 2.04 × 10-7) and fat mass and obesity-associated gene (FTO) in the muscle (P = 1.93 × 10-8). Compared with the TWAS results of adult BMI, we found that one gene TUBA1B with predominant influence only on childhood BMI in the muscle (P = 1.12 × 10-7). We evaluated the candidate genes by querying public databases and identified 12 genes functionally related to obesity phenotypes, including nine differentially expressed genes during the differentiation of human preadipocyte cells. The remaining genes (FAM150B, KNOP1, and LMBR1L) were regarded as novel candidate genes for childhood BMI. CONCLUSIONS: Our study identified multiple candidate genes for childhood BMI, providing novel clues for understanding the genetic mechanism of childhood obesity.

Integrating regulatory features data for prediction of functional disease-associated SNPs.

Genome-wide association studies (GWASs) are an effective strategy to identify susceptibility loci for human complex diseases. However, missing heritability is still a big problem. Most GWASs single-nucleotide polymorphisms (SNPs) are located in noncoding regions, which has been considered to be the unexplored territory of the genome. Recently, data from the Encyclopedia of DNA Elements (ENCODE) and Roadmap Epigenomics projects have shown that many GWASs SNPs in the noncoding regions fall within regulatory elements. In this study, we developed a pipeline named functional disease-associated SNPs prediction (FDSP), to identify novel susceptibility loci for complex diseases based on the interpretation of the functional features for known disease-associated variants with machine learning. We applied our pipeline to predict novel susceptibility SNPs for type 2 diabetes (T2D) and hypertension. The predicted SNPs could explain heritability beyond that explained by GWAS-associated SNPs. Functional annotation by expression quantitative trait loci analyses showed that the target genes of the predicted SNPs were significantly enriched in T2D or hypertension-related pathways in multiple tissues. Our results suggest that combining GWASs and regulatory features data could identify additional functional susceptibility SNPs for complex diseases. We hope FDSP could help to identify novel susceptibility loci for complex diseases and solve the missing heritability problem.

Modeling circRNA expression pattern with integrated sequence and epigenetic features demonstrates the potential involvement of H3K79me2 in circRNA expression.

MOTIVATION: CircRNAs are an abundant class of non-coding RNAs with widespread, cell-/tissue-specific patterns. Previous work suggested that epigenetic features might be related to circRNA expression. However, the contribution of epigenetic changes to circRNA expression has not been investigated systematically. Here, we built a machine learning framework named CIRCScan, to predict circRNA expression in various cell lines based on the sequence and epigenetic features. RESULTS: The predicted accuracy of the expression status models was high with area under the curve of receiver operating characteristic (ROC) values of 0.89-0.92 and the false-positive rates of 0.17-0.25. Predicted expressed circRNAs were further validated by RNA-seq data. The performance of expression-level prediction models was also good with normalized root-mean-square errors of 0.28-0.30 and Pearson's correlation coefficient r over 0.4 in all cell lines, along with Spearman's correlation coefficient ρ of 0.33-0.46. Noteworthy, H3K79me2 was highly ranked in modeling both circRNA expression status and levels across different cells. Further analysis in additional nine cell lines demonstrated a significant enrichment of H3K79me2 in circRNA flanking intron regions, supporting the potential involvement of H3K79me2 in circRNA expression regulation. AVAILABILITY AND IMPLEMENTATION: The CIRCScan assembler is freely available online for academic use at https://github.com/johnlcd/CIRCScan. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phosphorus Co-Existing in Water: A New Mechanism to Boost Boron Removal by Calcined Oyster Shell Powder.

The removal of boron (B) from water by co-precipitation with hydroxyapatite (HAP) has been extensively studied due to its low cost, ease of use and high efficiency. However, there is no explicit mechanism to express how resolved B was trapped by HAP. Thus, in this work, the process of removing B from water was studied using a low-cost calcium (Ca) precipitation agent derived from used waste oyster shells. The results showed that the removal rate of B in the simulated wastewater by calcined oyster shell (COS) in the presence of phosphorus (P) is up to more than 90%, as opposed to virtually no removal without phosphate. For B removal, the treated water needs to be an alkaline solution with a high pH above 12, where B is removed as [CaB(OH)4]+ but is not molecular. Finally, the synergistic mechanism of co-precipitation between HAP and dissolved B, occlusion co-precipitation, was explained in detail. The proposed method discovered the relationship between Ca, P and B, and was aimed at removing B without secondary pollution through co-precipitation.

Sex-specific SNP-SNP interaction analyses within topologically associated domains reveals ANGPT1 as a novel tumor suppressor gene for lung cancer.

Genetic interaction has been recognized to be an important cause of the missing heritability. The topologically associating domain (TAD) is a self-interacting genomic region, and the DNA sequences within a TAD physically interact with each other more frequently. Sex differences influence cancer susceptibility at the genetic level. Here, we performed both regular and sex-specific genetic interaction analyses within TAD to identify susceptibility genes for lung cancer in 5204 lung cancer patients and 7389 controls. We found that one SNP pair, rs4262299-rs1654701, was associated with lung cancer in women after multiple testing corrections (combined P = 8.52 × 10-9 ). Single-SNP analyses did not detect significant association signals for these two SNPs. Both identified SNPs are located in the intron region of ANGPT1. We further found that 5% of nonsmall cell lung cancer patients have an alteration in ANGPT1, indicated the potential role of ANGPT1 in the neoplastic progression in lung cancer. The expression of ANGPT1 was significantly down-regulated in patients in lung squamous cell carcinoma and lung adenocarcinoma. We checked the interaction effect on the ANGPT1 expression and lung cancer and found that the minor allele "G" of rs1654701 increased ANGPT1 gene expression and decreased lung cancer risk with the increased dosage of "A" of rs4262299, which consistent with the tumor suppressor function of ANGPT1. Survival analyses found that the high expression of ANGPT1 was individually associated with a higher survival probability in lung cancer patients. In summary, our results suggest that ANGPT1 may be a novel tumor suppressor gene for lung cancer.

PopLDdecay: a fast and effective tool for linkage disequilibrium decay analysis based on variant call format files.

MOTIVATION: Linkage disequilibrium (LD) decay is of great interest in population genetic studies. However, no tool is available now to do LD decay analysis from variant call format (VCF) files directly. In addition, generation of pair-wise LD measurements for whole genome SNPs usually resulting in large storage wasting files. RESULTS: We developed PopLDdecay, an open source software, for LD decay analysis from VCF files. It is fast and is able to handle large number of variants from sequencing data. It is also storage saving by avoiding exporting pair-wise results of LD measurements. Subgroup analyses are also supported. AVAILABILITY AND IMPLEMENTATION: PopLDdecay is freely available at https://github.com/BGI-shenzhen/PopLDdecay.

Detecting epistasis within chromatin regulatory circuitry reveals CAND2 as a novel susceptibility gene for obesity.

BACKGROUND: Genome-wide association studies have identified many susceptibility loci for obesity. However, missing heritability problem is still challenging and ignorance of genetic interactions is believed to be an important cause. Current methods for detecting interactions usually do not consider regulatory elements in non-coding regions. Interaction analyses within chromatin regulatory circuitry may identify new susceptibility loci. METHODS: We developed a pipeline named interaction analyses within chromatin regulatory circuitry (IACRC), to identify genetic interactions impacting body mass index (BMI). Potential interacting SNP pairs were obtained based on Hi-C datasets, PreSTIGE (Predicting Specific Tissue Interactions of Genes and Enhancers) algorithm, and super enhancer regions. SNP × SNP analyses were next performed in three GWAS datasets, including 2286 unrelated Caucasians from Kansas City, 3062 healthy Caucasians from the Gene Environment Association Studies initiative, and 3164 Hispanic subjects from the Women's Health Initiative. RESULTS: A total of 16,643,227 SNP × SNP analyses were performed. Meta-analyses showed that two SNP pairs, rs6808450-rs9813534 (combined P = 2.39 × 10-9) and rs6808450-rs3773306 (combined P = 2.89 × 10-9) were associated with BMI after multiple testing corrections. Single-SNP analyses did not detect significant association signals for these three SNPs. In obesity relevant cells, rs6808450 is located in intergenic enhancers, while rs9813534 and rs3773306 are located in the region of strong transcription regions of CAND2 and RPL32, respectively. The expression of CAND2 was significantly downregulated after the differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cells (P = 0.0241). Functional validation in the International Mouse Phenotyping Consortium database showed that CAND2 was associated with increased lean body mass and decreased total body fat amount. CONCLUSIONS: Detecting epistasis within chromatin regulatory circuitry identified CAND2 as a novel obesity susceptibility gene. We hope IACRC could facilitate the interaction analyses for complex diseases and offer new insights into solving the missing heritability problem.

A functional SNP regulated by miR-196a-3p in the 3'UTR of FGF2 is associated with bone mineral density in the Chinese population.

Previous studies have identified FGF2 as a susceptibility gene for osteoporosis in Caucasians. Evaluating the genetic associations in different ethnicities is necessary. Moreover, elucidating the functional mechanism for the susceptibility loci is important to offer new targets for therapeutic studies. Here, we genotyped 10 SNPs in FGF2 and tested for associations with bone mineral density (BMD) in a discovery sample of 1,300 Chinese subjects. Nominally significant results were subjected to replication in another sample of 1,039 Chinese subjects. We identified one SNP rs1048201:C>T in FGF2 3'untranslated region significantly associated with spine BMD (combined cohorts, P = 1.53×10-3 ). Expression quantitative trait locus analyses revealed that rs1048201 also affected FGF2 gene expression (P = 7.03×10-4 ). Bioinformatics prediction suggested that rs1048201 T allele could disrupt miRNA binding. Luciferase assay validated that the C allele had a repressive effect on FGF2 gene expression. We found that hsa-miR-196a-3p affected expression on both mRNA and protein levels of FGF2. In conclusion, our study provided evidence that a functional SNP rs1048201 was associated with BMD, and SNP rs1048201:C>T variant may act by affecting binding of hsa-miR-196a-3p. The SNP-modified posttranscriptional gene regulation by miRNA could be a potentially pathogenetic mechanism of osteoporosis.

Regulatory element-based prediction identifies new susceptibility regulatory variants for osteoporosis.

Despite genome-wide association studies (GWASs) have identified many susceptibility genes for osteoporosis, it still leaves a large part of missing heritability to be discovered. Integrating regulatory information and GWASs could offer new insights into the biological link between the susceptibility SNPs and osteoporosis. We generated five machine learning classifiers with osteoporosis-associated variants and regulatory features data. We gained the optimal classifier and predicted genome-wide SNPs to discover susceptibility regulatory variants. We further utilized Genetic Factors for Osteoporosis Consortium (GEFOS) and three in-house GWASs samples to validate the associations for predicted positive SNPs. The random forest classifier performed best among all machine learning methods with the F1 score of 0.8871. Using the optimized model, we predicted 37,584 candidate SNPs for osteoporosis. According to the meta-analysis results, a list of regulatory variants was significantly associated with osteoporosis after multiple testing corrections and contributed to the expression of known osteoporosis-associated protein-coding genes. In summary, combining GWASs and regulatory elements through machine learning could provide additional information for understanding the mechanism of osteoporosis. The regulatory variants we predicted will provide novel targets for etiology research and treatment of osteoporosis.

Association analyses of FGFR2 gene polymorphisms with femoral neck bone mineral density in Chinese Han population.

Femoral neck (FN) bone mineral density (BMD) is the most important risk phenotype for osteoporosis and has been used as a reference standard for describing osteoporosis. Identification of genetic variations associated with FN BMD may provide potential targets for therapeutic studies. Given the important biological role of FGFR2 gene involved in bone, we tested the associations between FGFR2 polymorphisms and FN BMD in 1,300 Chinese Han subjects. Of the 28 total SNPs, 2 SNPs, namely rs11200014 and rs1078806, were significantly associated with FN BMD under dominant model (P = 0.0014 and 0.0012, respectively) after conservative Bonferroni correction. The two SNPs were in complete linkage disequilibrium. In addition, haplotype-based association tests identified two haplotypes significantly associated with FN BMD, including one haplotype in block 4 where the two SNPs located. However, different from previous studies in white older men, we did not detect any significant association in sex-stratified analyses. In summary, our findings suggest that the FGFR2 gene may play an important role in variation in FN BMD in Chinese Han population, independent of gender effects. Further studies performed in multiple and large samples are needed to elucidate the underlying molecular mechanism and pathophysiology of osteoporosis.