Effects of GWAS-Associated Genetic Variants on lncRNAs within IBD and T1D Candidate Loci

<div><p>Long non-coding RNAs are a new class of non-coding RNAs that are at the crosshairs in many human diseases such as cancers, cardiovascular disorders, inflammatory and autoimmune disease like Inflammatory Bowel Disease (IBD) and Type 1 Diabetes (T1D). Nearly 90% of the phenotype-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) lie outside of the protein coding regions, and map to the non-coding intervals. However, the relationship between phenotype-associated loci and the non-coding regions including the long non-coding RNAs (lncRNAs) is poorly understood. Here, we systemically identified all annotated IBD and T1D loci-associated lncRNAs, and mapped nominally significant GWAS/ImmunoChip SNPs for IBD and T1D within these lncRNAs. Additionally, we identified tissue-specific <i>cis</i>-eQTLs, and strong linkage disequilibrium (LD) signals associated with these SNPs. We explored sequence and structure based attributes of these lncRNAs, and also predicted the structural effects of mapped SNPs within them. We also identified lncRNAs in IBD and T1D that are under recent positive selection. Our analysis identified putative lncRNA secondary structure-disruptive SNPs within and in close proximity (+/−5 kb flanking regions) of IBD and T1D loci-associated candidate genes, suggesting that these RNA conformation-altering polymorphisms might be associated with diseased-phenotype. Disruption of lncRNA secondary structure due to presence of GWAS SNPs provides valuable information that could be potentially useful for future structure-function studies on lncRNAs.</p></div

Structure disruption of lncRNA <i>NONHSAG044354</i>-associated with <i>BACH2</i> (implicated in both IBD and T1D) by GWAS SNPs rs3757247 and rs597325.

<p>The structure-disruptive effects of SNPs rs3757247 (A) and rs597325 (B) located in lncRNA <i>NONHSAG044354</i> associated with candidate gene <i>BACH2</i>. (a) UCSC genome browser view showing the location of the predicted local region disrupted by the SNP. The color of predicted local region (green in this case) is based on the RNAsnp p-value (0.129 and 0.120 for rs3757247 and rs597325 respectively). (b) Minimum free energy structures (MFE) of the global wild-type and mutant sequences displaying the secondary structure and the local region affected by the SNP position, colored green (wild-type) and red (mutant) (c) Dot plot representing the base pair probabilities of wild type and mutant RNA sequences corresponding to the predicted local region by RNAsnp. The upper and lower triangle of the matrix represents the base pair probabilities of wild-type (green) and mutant (red), respectively.</p

<i>cis</i>-eQTLs and gene-SNP associations for rs3757247 and rs597325 with <i>BACH2</i> candidate gene.

<p>(A) The gene SNP association for rs3757247 and <i>BACH2</i> candidate gene in whole blood. (B, C, D) The gene SNP association for rs597325 and <i>BACH2</i> in colon transverse, brain cortex and cell line fibroblasts. The gene SNP associations are calculated using linear regression model using Genotype Tissue Expression Portal (GTEx) in the selected tissues with more than 80 samples using a <i>cis</i> window of +/−1 MB around the transcription start site (TSS) at significance level of 0.05. For each gene SNP association plot, p-values are displayed at the bottom.</p

Comparison of expression levels of <i>NONHSAG044354</i> with its host gene (<i>BACH2</i>) in 14 tissues.

<p>(A)Tissue-specific gene expression profile of <i>BACH2</i> candidate gene and <i>NONHSAG044354</i> associated lncRNA across 14 tissues based on Human Body Map (HBM) data. (B) Sense exonic lncRNA <i>NONHSAG044354</i> positively correlating (r_s = 0.85) with <i>BACH2</i> candidate gene. Protein coding mRNA expression is plotted on the x-axis and lncRNA expression is shown on the y-axis both on log10 FPKM for 14 HBM tissues.</p

Regional LD plot for SNP rs3757247 associated with IBD and T1D loci.

<p>The regional LD plot for SNP rs3757247 was calculated using HapMap3 (release 2) in SNAP tool. SNPs are represented as diamonds and the brightness of each SNP is proportional to the r² threshold value for that SNP. SNPs rs11755527 and rs1847472 had an r2 value of 0.949 (T1D p-value_meta 5.38e-08, Barrett et al 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105723#pone.0105723-Barrett1" target="_blank">[52]</a>) and 0.565 (IBD p-value_ichip 1.19e-04, IBD p-value_meta 1.10e-09, Jostins et al 2012 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105723#pone.0105723-Jostins1" target="_blank">[71]</a>) respectively (shown in red).</p

Structure-disruptive SNPs and their-associated lncRNAs.

<p>Total numbers of structure-disruptive SNPs are reported after ranking based on RegulomeDB score, <i>cis</i>-eQTLs and recent positive selection.</p

Mapped lncRNA intervals within IBD and T1D susceptibility loci genes.

<p>Categorization of the genomic association of the lncRNAs to the IBD and T1D loci-associated genes, as sense exonic (A), sense non-exonic (B), antisense (C), and intergenic (D).</p

Correlations of expression for antisense IBD and T1D loci-associated lncRNAs.

<p>Spearman correlations for (A) antisense IBD and (B) antisense T1D loci-associated lncRNAs expressed across all the HBM tissues. The FPKM threshold of >1 was used and the values were log10 transformed.</p

Expression of antisense IBD and T1D loci-associated lncRNAs.

<p>Expression profiles for (A) 49 antisense IBD loci-associated lncRNAs and (B) 37 antisense T1D loci-associated lncRNAs expressed across all the HBM tissues. The FPKM threshold of >1 was used and the values were log10 transformed.</p

Common structure-disruptive SNPs within IBD and T1D loci-associated lncRNAs.

<p>Four SNPs common between an IBD and T1D loci-associated antisense lncRNA were in close proximity of a protein coding gene <i>HORMAD2</i>, which has been implicated in both IBD and T1D and three other T1D candidate genes <i>MTMR3</i> (protein coding gene), <i>CTA-85E5.10</i> (processed transcript), <i>CNN2P1</i> (pseudogene). Two SNPs rs3757247 and rs597325 were located within lncRNA <i>NONHSAG044354</i> and a protein coding gene <i>BACH2</i> and a pseudogene <i>RP3-453I5.2</i>. SNP rs602662 was found to be present within antisense lncRNA <i>NONHSAG026183</i> and protein coding gene <i>FUT2</i>. Strand is displayed in brackets for lncRNAs and their-associated IBD/T1D candidate genes.</p