Expression quantitative trait loci analysis identifies associations between genotype and gene expression in human intestine.

paper Cited Public

Authors: Kabakchiev, Boyko; Silverberg, Mark S
Year: 2013
Journal: Gastroenterology
PMID: 23474282
DOI: 10.1053/j.gastro.2013.03.001
PMCID: PMC3775712

BACKGROUND & AIMS: Genome-wide association studies have greatly increased our understanding of intestinal disease. However, little is known about how genetic variations result in phenotypic changes. Some polymorphisms have been shown to modulate quantifiable phenotypic traits; these are called quantitative trait loci. Quantitative trait loci that affect levels of gene expression are called expression quantitative trait loci (eQTL), which can provide insight into the biological relevance of data from genome-wide association studies. We performed a comprehensive eQTL scan of intestinal tissue. METHODS: Total RNA was extracted from ileal biopsy specimens and genomic DNA was obtained from whole-blood samples from the same cohort of individuals. Cis- and trans-eQTL analyses were performed using a custom software pipeline for samples from 173 subjects. The analyses determined the expression levels of 19,047 unique autosomal genes listed in the US National Center for Biotechnology Information database and more than 580,000 variants from the Single Nucleotide Polymorphism database. RESULTS: The presence of more than 15,000 cis- and trans-eQTL was detected with statistical significance. eQTL associated with the same expression trait were in high linkage disequilibrium. Comparative analysis with previous eQTL studies showed that 30% to 40% of genes identified as eQTL in monocytes, liver tissue, lymphoblastoid cell lines, T cells, and fibroblasts are also eQTL in ileal tissue. Conversely, most of the significant eQTL have not been previously identified and could be tissue specific. These are involved in many cell functions, including division and antigen processing and presentation. Our analysis confirmed that previously published cis-eQTL are single nucleotide polymorphisms associated with inflammatory bowel disease: rs2298428/UBE2L3, rs1050152/SLC22A4, and SLC22A5. We identified many new associations between inflammatory bowel disease susceptibility loci and gene expression. CONCLUSIONS: eQTL analysis of intestinal tissue supports findings that some eQTL remain stable across cell types, whereas others are specific to the sampled location. Our findings confirm and expand the number of known genotypes associated with expression and could help elucidate mechanisms of intestinal disease.

Figure 1

Twelve significant cis-eQTL. Box plot depiction of the 12 most significant, unique cis-eQTL using a 50-kb window. The x-axis of each plot corresponds to the 3 observed SNP genotypes in forward orientation, and the y-axis represents log2-normalized gene expression values. Listed P values are FDR corrected.

LLM interpretation

This figure consists of 12 individual box plots showing the relationship between specific SNP genotypes (x-axis) and $\log_2$-normalized gene expression values (y-axis) for 12 different genes. Each plot displays a trend where gene expression levels vary across the three observed genotypes, with FDR-corrected p-values ranging from $2.08 \times 10^{-24}$ to $9.22 \times 10^{-19}$. The visualizations demonstrate either a positive or negative correlation between the number of alternative alleles and the level of gene expression.

Figure 2

MAF distribution of eSNPs. Distribution of MAF among 13,907 significant eSNPs after cis-eQTL analysis with 50-kb windows and an α level of 5%.

LLM interpretation

This is a histogram showing the distribution of Minor Allele Frequency (MAF) for 13,907 significant eSNPs. The x-axis represents MAF (ranging from 0 to 0.5) and the y-axis represents the percentage of SNPs. The data shows a general upward trend, with a higher percentage of significant eSNPs occurring at higher MAF values.

Figure 3

Visualization of LD between SNPs surrounding the gene MMEL1. Visual representation of pairwise LD between markers about the MMEL1 gene. Color intensity and values in the triangular matrix indicate the corresponding r2 values. Bar plot heights represent the intensity of each –log10-transformed P value, and markers identified as an LD cluster by eQTLA are colored uniformly. White bars did not cluster with other markers. The horizontal red line corresponds to the Bonferroni cutoff for significance.

LLM interpretation

This figure consists of a bar plot aligned above a triangular linkage disequilibrium (LD) heatmap for SNPs surrounding the *MMEL1* gene. The bar plot displays $-\log_{10}(P)$ values, with a red horizontal line indicating the Bonferroni significance threshold and colored bars identifying eQTLA LD clusters. The heatmap uses a grayscale gradient and numerical $r^2$ values to represent the strength of pairwise LD between the 21 labeled markers.

Figure 4

Locus size distribution after LD correction with eQTLA. Distribution of locus sizes for 4196 significant clusters after cis-eQTL analysis with 50-kb windows, an α level of 5%, and subsequent LD correction.

LLM interpretation

This is a histogram showing the distribution of locus sizes for 4,196 significant clusters after LD correction. The x-axis represents "Locus size" and the y-axis represents "Percent." The data is heavily right-skewed, with the highest percentage of loci occurring at the smallest size (approximately 35%) and a rapid decline as locus size increases.

Figure 5

eQTL comparison of human ileum with other tissues. Percentage overlap between significant cis expression traits detected in other tissues (monocytes, lymphoblastoid cell lines, liver, T cells, and fibroblasts) and human small intestinal tissue. Results are presented with varying windows sizes and α levels for the analysis in intestinal tissue.

LLM interpretation

This grouped bar chart shows the percentage overlap of significant cis expression traits between the human ileum and five other tissues (Monocytes, LCL, Liver, T cells, and Fibroblasts). The data is categorized by two window sizes (50 kb and 1 mb) and two $\alpha$ levels (5% and 10%). Across all conditions, LCL consistently shows the highest percentage of overlap, while the highest overall overlap percentages are observed in the 50 kb window at the 10% $\alpha$ level.

#	Section	Preview
0	Materials and Methods — Subject Cohort	Individuals who underwent ileal pouch–anal anastomosis following colectomy were recruited at Mount…
1	Materials and Methods — Messenger RNA Extraction and Quantification	Two tissue biopsy specimens were obtained from endoscopically and histologically normal prepouch…
2	Materials and Methods — Messenger RNA Extraction and Quantification	A total of 400 ng of RNA from samples that passed quality control were amplified with the Ambion WT…
3	Materials and Methods — Messenger RNA Extraction and Quantification	The empirical Bayes method described by Johnson et al29 was applied to the normalized data to…
4	Materials and Methods — Genotyping	Parallel to gene expression quantification, genomic DNA was extracted from whole-blood samples…
5	Materials and Methods — Bioinformatics	A custom standalone software application, eQTLA, was created to address the analytical issues…
6	Materials and Methods — Bioinformatics	eQTLA is capable of performing both cis- and trans-eQTL analysis given a set of raw genetic marker…
7	Materials and Methods — Bioinformatics	An optional feature in eQTLA can identify markers in close proximity that are highly correlated due…
8	Materials and Methods — eQTL and Statistical Analysis	Information on the genomic coordinates of genes based on human genome build GRCh37.p5 was downloaded…
9	Materials and Methods — eQTL and Statistical Analysis	Two conservative variations of trans-eQTL analysis were also performed. One approach involved the…
10	Materials and Methods — eQTL and Statistical Analysis	A secondary method for trans-based analysis built on the initial cis-eQTL results. Genotypes that…
11	Materials and Methods — Comparison With Other Tissues	Significant cis-eQTL results from this study were compared with other previously reported cis-eQTL…
12	Materials and Methods — eQTL and Inflammatory Bowel Disease	Significant cis-acting eSNPs in this study were examined for possible replication of a small number…
13	Results — Basic Measures of Data Quality	Following quality control, 19,908 unambiguous Affymetrix Human Gene 1.0 ST array probe sets remained…
14	Results — Basic Measures of Data Quality	Of the 173 subjects, 153 (88.4%) described themselves as white, 18 (10.4%) as Asian, and 2 (1.2%) as…
15	Results — eQTL Analysis	Cis-eQTL analysis based on these quality-trimmed data and focusing on SNPs located within 50-kb…
16	Results — eQTL Analysis	for DNA damage repair in human cell lines.36 Overall, Gene Ontology classification based on the…
17	Results — eQTL Analysis	Cis-eQTL associated with the same expression trait were in high LD (Figure 3). Significant eSNPs…
18	Results — eQTL Analysis	Trans-eQTL analysis resulted in far fewer significant associations. Using human interactome data,…
19	Results — Comparison With Other Tissues	Comparative analysis with other eQTL studies showed that a substantial fraction of eQTL in the ileum…

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