Cis- and trans-acting gene regulation is associated with osteoarthritis

Abstract

Osteoarthritis (OA) is a complex disease of the skeleton and is associated with aging. Both environmental and genetic factors contribute to its pathogenesis. We set out to identify novel genes associated with OA, concentrating on regulatory polymorphisms allowing for differential expression. Our strategy to identify differentially expressed genes included an initial transcriptome analysis of the peripheral blood mononuclear cells of six patients with OA and six age-matched healthy controls. These were screened for allelic expression imbalances and potentially regulatory single-nucleotide polymorphisms (SNPs) in the 5' regions of the genes. To establish disease association, disparate promoter SNP distributions correlating with the differential expression were tested on larger cohorts. Our approach yielded 26 candidate genes differentially expressed between patients and controls. Whereas BLP2 and CIAS1 seem to be trans-regulated, as the absence of allelic expression imbalances suggests, the presence of allelic imbalances confirms cis-regulatory mechanisms for RHOB and TXNDC3. Interestingly, on/off-switching suggests additional trans-regulation for TXNDC3. Moreover, we demonstrate for RHOB and TXNDC3 statistically significant associations between 5' SNPs and the disease that hint at regulatory functions. Investigating the respective genes functionally will not only shed light on the disease association but will also add to the understanding of the pathogenic processes involved in OA and may point out novel therapeutic approaches.

Figure  1

Combination of two software tools, which reduces the number of differentially expressed candidate genes and lends more weight to genes identified via two different methods. For each cell type, lists of differentially expressed genes were compiled using DMT and Genes@work (G@W), before intersections were formed. Black numbers on the gray intersection indicate the results.

Figure  2

Workflow for the identification of cis-regulated and disease-associated genes among the 26 candidates. Our screening focuses on allelic expression imbalances, disease-associated promoter polymorphisms, and expression in articular chondrocytes.

Figure  3

Correlation between the presence of promoter SNPs and expression signals, which hints at disease-associated regulatory polymorphisms. Expression signals from CD14⁺ monocytes (RHOB, CIRBP, DKFZP434O1427, FIZZ3/RETN, and VNN1) and CD19⁺ B cells (BLK) were averaged for the patients and were compared to the pooled data from the controls in the left panels, detailing the differential expression. The right panels compare the frequencies of the minor alleles of promoter SNPs unevenly distributed between patients and controls. Whereas BLK is expressed exclusively in B cells and FIZZ3/RETN and VNN1 in monocytes, RHOB, CIRBP, and DKFZP434O1427 are expressed in all four cell types. The P values of .012, .009, and .002 summarize their differential expression and result from a Mann-Whitney test performed on the transcriptional values from all cell types.

Figure  4

Gene expression in chondrocytes supporting a role for candidate genes in the pathogenesis of OA. RNA isolated from the cartilage of patients with OA undergoing joint replacement surgery was transcribed into cDNA and was assayed for the presence of specific transcripts of BLK, CIRBP, DKFZP434O1427, FIZZ3/RETN, VNN1, and TXNDC3. The comparison to β-actin indicates the use of unequal amounts of template cDNA for the three patients. Size markers are indicated on the left of each panel, and the sizes of the specific PCR products are given on the right. MW = molecular weight.