Functional Relevance for Associations between Genetic Variants and Systemic Lupus Erythematosus

<div><p>Systemic lupus erythematosus (SLE) is a serious prototype autoimmune disease characterized by chronic inflammation, auto-antibody production and multi-organ damage. Recent association studies have identified a long list of loci that were associated with SLE with relatively high statistical power. However, most of them only established the statistical associations of genetic markers and SLE at the DNA level without supporting evidence of functional relevance. Here, using publically available datasets, we performed integrative analyses (gene relationship across implicated loci analysis, differential gene expression analysis and functional annotation clustering analysis) and combined with expression quantitative trait loci (eQTLs) results to dissect functional mechanisms underlying the associations for SLE. We found that 14 SNPs, which were significantly associated with SLE in previous studies, have cis-regulation effects on four eQTL genes (HLA-DQA1, HLA-DQB1, HLA-DQB2, and IRF5) that were also differentially expressed in SLE-related cell groups. The functional evidence, taken together, suggested the functional mechanisms underlying the associations of 14 SNPs and SLE. The study may serve as an example of mining publically available datasets and results in validation of significant disease-association results. Utilization of public data resources for integrative analyses may provide novel insights into the molecular genetic mechanisms underlying human diseases.</p> </div

Expression quantitative trait locus (eQTL) analysis results between SNPs and the expressions of 11 genes from the 108 “identified” genes.

<p>Note: N/A:not available; “Score”: −log10 (P); LCLs: lymphoblastoid cell lines; transcript-QTL: transcript expression levels against SNPs; exon-QTL: quantified reads for known exons by RNA sequencing against SNPs; sQTL: splicing QTL; PP-eqtl: posterior probability-eqtl.</p><p>All the eqtls have cis-effects.</p><p>Database#: <a href="http://eqtl.uchicago.edu/cgi-bin/gbrowse/eqtl" target="_blank">http://eqtl.uchicago.edu/cgi-bin/gbrowse/eqtl</a>.</p><p>“Gene 1” and “Gene 2”are the nearby genes physically located at two sides of SNP.</p><p>“GRAIL P-value “and “Implicated gene” are the results from Gene Relationships Across Implicated Loci (GRAIL) analyses.</p

Differential expression analyses for the detected eQTL genes in multiple SLE related cells.

<p>Note: Sample size: SLE cases: controls; N/A: Not available;</p>*<p>:ratio of mean expression values of log2(sample/reference).</p><p>GSE NO: Gene Expression Omnibus Number, <a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">www.ncbi.nlm.nih.gov/geo/</a>.</p><p>Database#: The data downloaded from the database (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">www.ncbi.nlm.nih.gov/geo/</a>) according to GSE number.</p><p>We only listed the most significant expression results of probes if one gene has multiple detected probes.</p

Gene-Based Association Analysis Identified Novel Genes Associated with Bone Mineral Density

<div><p>Genetic factors contribute to the variation of bone mineral density (BMD), which is a major risk factor of osteoporosis. The aim of this study was to identify more “novel” genes for BMD. Based on the publicly available SNP-based <i>P</i> values, we performed an initial gene-based analysis in a total of 32,961 individuals. Furthermore, we performed differential expression, pathway and protein-protein interaction analyses to find supplementary evidence to support the significance of the identified genes. About 21,695 genes for femoral neck (FN)-BMD and 21,683 genes for lumbar spine (LS)-BMD were analyzed using gene-based association analysis. A total of 35 FN-BMD associated genes and 53 LS-BMD associated genes were identified (<i>P</i> < 2.3×10^-6) after Bonferroni correction. Among them, 64 genes have not been reported in previous SNP-based genome-wide association studies. Differential expression analysis further supported the significant associations of 14 genes with FN-BMD and 19 genes with LS-BMD. Especially, <i>WNT3</i> and <i>WNT9B</i> in the Wnt signaling pathway for FN-BMD were further supported by pathway analysis and protein-protein interaction analysis. The present study took the advantage of gene-based association method to perform a supplementary analysis of the GWAS dataset and found some BMD-associated genes. The evidence taken together supported the importance of Wnt signaling pathway genes in determining osteoporosis. Our findings provided more insights into the genetic basis of osteoporosis.</p></div

A PI3K inhibitor LY-294002 blocked the neuroprotection of CAR on cerebral I/R injury.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia and 5 µl LY-294002 (10 mM) was administered (i.c.v.) at 15 min before ischemia. (A) After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and p-Akt and t-Akt levels were determined by Western blot. The photographs show that LY-294002 treatment inhibited the activation of Akt by CAR. (B) After 75 min of ischemia and 24 h of reperfusion, cerebral infarct volume was determined by TTC staining. The representative TTC-stained coronal sections demonstrate that LY-294002 treatment abolished the protection of CAR on infarct volume. (C) Statistical analysis of cerebral infarct volume shows that PI3K inhibitor LY-294002 blocked the protective effects of CAR. I/R+CAR+LY: CAR and LY-294002-treated I/R group. Bars represent mean ± SEM of 6–9 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

The protection of CAR on infarct volume was in a dose-dependent manner.

<p>Mice were administered (i.p.) with CAR at doses of 5, 25, and 50 mg/kg at 2 h before ischemia. Cerebral infarct volume was determined by TTC staining after 75 min of ischemia and 24 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR post-treatment reduced infarct volume after cerebral I/R injury.

<p>CAR was administered (i.p. and i.c.v.) at different reperfusion times after 75 min of ischemia. TTC staining for cerebral infarct volume was performed after 24 h reperfusion. (A) Mice were administered CAR (50 mg/kg, i.p.) and saline at 0 h, 2 h, and 4 h after reperfusion. CAR had no protection on infarct volume when it was administered at 4 h after reperfusion. (B) Mice were administered with 10 µg CAR (i.c.v.) or saline at 2 h, 4 h, 6 h, and 7 h after reperfusion. CAR still had protective effect when mice treated with CAR after 6 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR treatment reduced cleaved caspase-3 level after cerebral I/R injury.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia. Cleaved caspase-3 level was determined by Western blot after 75 min of ischemia and 24 h of reperfusion. (A)The representative photographs show the levels of cleaved caspase-3 and beta-tubulin. Beta-tubulin was used as a loading control. (B)The quantitative analysis indicated that CAR treatment decreased ratio of cleaved caspase-3 to beta-tubulin. Bars represent mean ± SEM for samples from 4 brains in each group. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR treatment increased the activation of Akt after MCAO.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia. After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and protein levels were determined by Western blot. (A)The representative photographs show levels of p-Akt, t-Akt, and beta-tubulin. Beta-tubulin was used as a loading control. (B) Quantitative analysis of the ratio of p-Akt to t-Akt was performed. Bars represent mean ± SEM for samples from 4 brains in each group. *, <i>P</i><0.05 versus sham group; #, <i>P</i><0.05 I/R group versus I/R+CAR group.</p

CAR pre-treatment reduced infarct volume and improved neurological deficits after MCAO.

<p>Mice were administered (i.p.) with CAR or saline at 2 h before cerebral ischemia. TTC staining and neurological scores were examined after 75 min of ischemia and 24 h of reperfusion. (A)The molecular structure of CAR is shown. (B) The representative TTC-stained coronal sections in vehicle-treated mice and CAR-treated mice (50 mg/kg,i.p.) are shown. (C)Statistical analysis of cerebral infarct volume was performed in I/R group and I/R+CAR group; CAR reduced infarct volume after cerebral I/R injury. (D)Neurological scores were evaluated according to a 5-points scale system. CAR treatment significantly decreased neurological deficits. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p