Limits...
Cellular dissection of psoriasis for transcriptome analyses and the post-GWAS era.

Swindell WR, Stuart PE, Sarkar MK, Voorhees JJ, Elder JT, Johnston A, Gudjonsson JE - BMC Med Genomics (2014)

Bottom Line: Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%).Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, University of Michigan School of Medicine, Ann Arbor, MI 48109-2200, USA. wswindel@umich.edu.

ABSTRACT

Background: Genome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings.

Methods: We identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.

Results: Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4.

Conclusions: These findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.

Show MeSH

Related in: MedlinePlus

Identification of 91 genes co-expressed with TNFSRF9 in neutrophils. (A) Expression of TNFSRF9 across 10 cell types. Expression is normalized to that observed in normal human skin. Values in yellow denote TNFSRF9 detection frequency. (B) Procedure used to identify TNFSRF9-co-expressed genes in neutrophils. For genes expressed in both skin and neutrophils, we calculated the Spearman correlation coefficient between the expression pattern of each gene and that of TNFSRF9 (n = 426 neutrophil microarray samples). Genes were ranked based upon the estimated Spearman correlation coefficient (raised to the 7th power). To identify an appropriate cut-off for TNFSRF9-co-expressed genes, we located the “bend” in the curve of descending correlation coefficient estimates (i.e., the rank minimizing the length of the red line). (C) The gene most strongly associated with TNFSRF9 expression in neutrophils was VAV1. The scatterplot compares normalized expression values (log2 scale) between TNFSRF9 and VAV1 in neutrophils. (D) Expression of 91 genes co-expressed with TNFRSF9 in neutrophils (n = 426 microarray samples). Microarray samples were ordered with TNFRSF9 expression descending from left to right. Gold regions outline the middle 50% of normalized expression values among the 91 genes. Blue regions outline the middle 80% of normalized expression values among the 91 genes. Expression of each gene was normalized to its baseline (average) expression across all 426 samples, such that positive values correspond to above-average expression and negative values correspond to below-average expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4060870&req=5

Figure 7: Identification of 91 genes co-expressed with TNFSRF9 in neutrophils. (A) Expression of TNFSRF9 across 10 cell types. Expression is normalized to that observed in normal human skin. Values in yellow denote TNFSRF9 detection frequency. (B) Procedure used to identify TNFSRF9-co-expressed genes in neutrophils. For genes expressed in both skin and neutrophils, we calculated the Spearman correlation coefficient between the expression pattern of each gene and that of TNFSRF9 (n = 426 neutrophil microarray samples). Genes were ranked based upon the estimated Spearman correlation coefficient (raised to the 7th power). To identify an appropriate cut-off for TNFSRF9-co-expressed genes, we located the “bend” in the curve of descending correlation coefficient estimates (i.e., the rank minimizing the length of the red line). (C) The gene most strongly associated with TNFSRF9 expression in neutrophils was VAV1. The scatterplot compares normalized expression values (log2 scale) between TNFSRF9 and VAV1 in neutrophils. (D) Expression of 91 genes co-expressed with TNFRSF9 in neutrophils (n = 426 microarray samples). Microarray samples were ordered with TNFRSF9 expression descending from left to right. Gold regions outline the middle 50% of normalized expression values among the 91 genes. Blue regions outline the middle 80% of normalized expression values among the 91 genes. Expression of each gene was normalized to its baseline (average) expression across all 426 samples, such that positive values correspond to above-average expression and negative values correspond to below-average expression.

Mentions: Individuals carrying the A allele at rs11121129 (chr1, 8268095) have elevated psoriasis risk (OR = 1.13; P = 1.7 × 10−8), but mechanisms underlying this association are not known, since the locus is non-coding and does not affect protein structure [26]. Candidate genes near this locus include solute carrier family 45 member 1 (SLC45A1) and tumor necrosis factor receptor superfamily member 9 (TNFRSF9). Expression of SLC45A1 (distance from SNP: 116.3 kb) was rarely detectable in any of the 10 cell types we considered (data not shown). On the other hand, expression of TNFRSF9 (distance: 264.9 kb) was frequently detected in immune cells and was almost always detected in neutrophils (>99%), which was the cell type for which TNFRSF9 expression was quantitatively highest (Figure 7A). Consistent with this, we used RT-PCR and flow cytometry to show that TNFRSF9 mRNA and protein expression was highest in neutrophils, as compared to other cell types, including T-cells and monocytes (Additional file 15). Moreover, in agreement with microarray data (Figure 5), RT-PCR confirmed significant elevation of TNFRSF9 expression in psoriasis lesions as compared to uninvolved and normal skin (Additional file 15). We thus hypothesized that genetic variation at rs11121129 (or linked SNPs) altered the affinity of sequence-specific DNA-binding factors to influence TNFRSF9 expression in neutrophils.


Cellular dissection of psoriasis for transcriptome analyses and the post-GWAS era.

Swindell WR, Stuart PE, Sarkar MK, Voorhees JJ, Elder JT, Johnston A, Gudjonsson JE - BMC Med Genomics (2014)

Identification of 91 genes co-expressed with TNFSRF9 in neutrophils. (A) Expression of TNFSRF9 across 10 cell types. Expression is normalized to that observed in normal human skin. Values in yellow denote TNFSRF9 detection frequency. (B) Procedure used to identify TNFSRF9-co-expressed genes in neutrophils. For genes expressed in both skin and neutrophils, we calculated the Spearman correlation coefficient between the expression pattern of each gene and that of TNFSRF9 (n = 426 neutrophil microarray samples). Genes were ranked based upon the estimated Spearman correlation coefficient (raised to the 7th power). To identify an appropriate cut-off for TNFSRF9-co-expressed genes, we located the “bend” in the curve of descending correlation coefficient estimates (i.e., the rank minimizing the length of the red line). (C) The gene most strongly associated with TNFSRF9 expression in neutrophils was VAV1. The scatterplot compares normalized expression values (log2 scale) between TNFSRF9 and VAV1 in neutrophils. (D) Expression of 91 genes co-expressed with TNFRSF9 in neutrophils (n = 426 microarray samples). Microarray samples were ordered with TNFRSF9 expression descending from left to right. Gold regions outline the middle 50% of normalized expression values among the 91 genes. Blue regions outline the middle 80% of normalized expression values among the 91 genes. Expression of each gene was normalized to its baseline (average) expression across all 426 samples, such that positive values correspond to above-average expression and negative values correspond to below-average expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4060870&req=5

Figure 7: Identification of 91 genes co-expressed with TNFSRF9 in neutrophils. (A) Expression of TNFSRF9 across 10 cell types. Expression is normalized to that observed in normal human skin. Values in yellow denote TNFSRF9 detection frequency. (B) Procedure used to identify TNFSRF9-co-expressed genes in neutrophils. For genes expressed in both skin and neutrophils, we calculated the Spearman correlation coefficient between the expression pattern of each gene and that of TNFSRF9 (n = 426 neutrophil microarray samples). Genes were ranked based upon the estimated Spearman correlation coefficient (raised to the 7th power). To identify an appropriate cut-off for TNFSRF9-co-expressed genes, we located the “bend” in the curve of descending correlation coefficient estimates (i.e., the rank minimizing the length of the red line). (C) The gene most strongly associated with TNFSRF9 expression in neutrophils was VAV1. The scatterplot compares normalized expression values (log2 scale) between TNFSRF9 and VAV1 in neutrophils. (D) Expression of 91 genes co-expressed with TNFRSF9 in neutrophils (n = 426 microarray samples). Microarray samples were ordered with TNFRSF9 expression descending from left to right. Gold regions outline the middle 50% of normalized expression values among the 91 genes. Blue regions outline the middle 80% of normalized expression values among the 91 genes. Expression of each gene was normalized to its baseline (average) expression across all 426 samples, such that positive values correspond to above-average expression and negative values correspond to below-average expression.
Mentions: Individuals carrying the A allele at rs11121129 (chr1, 8268095) have elevated psoriasis risk (OR = 1.13; P = 1.7 × 10−8), but mechanisms underlying this association are not known, since the locus is non-coding and does not affect protein structure [26]. Candidate genes near this locus include solute carrier family 45 member 1 (SLC45A1) and tumor necrosis factor receptor superfamily member 9 (TNFRSF9). Expression of SLC45A1 (distance from SNP: 116.3 kb) was rarely detectable in any of the 10 cell types we considered (data not shown). On the other hand, expression of TNFRSF9 (distance: 264.9 kb) was frequently detected in immune cells and was almost always detected in neutrophils (>99%), which was the cell type for which TNFRSF9 expression was quantitatively highest (Figure 7A). Consistent with this, we used RT-PCR and flow cytometry to show that TNFRSF9 mRNA and protein expression was highest in neutrophils, as compared to other cell types, including T-cells and monocytes (Additional file 15). Moreover, in agreement with microarray data (Figure 5), RT-PCR confirmed significant elevation of TNFRSF9 expression in psoriasis lesions as compared to uninvolved and normal skin (Additional file 15). We thus hypothesized that genetic variation at rs11121129 (or linked SNPs) altered the affinity of sequence-specific DNA-binding factors to influence TNFRSF9 expression in neutrophils.

Bottom Line: Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%).Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, University of Michigan School of Medicine, Ann Arbor, MI 48109-2200, USA. wswindel@umich.edu.

ABSTRACT

Background: Genome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings.

Methods: We identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.

Results: Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4.

Conclusions: These findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.

Show MeSH
Related in: MedlinePlus