Final Report Summary - CD40RA (Identification of functional common and rare variants in the CD40 signaling pathway in Rheumatoid Arthritis)
Rheumatoid Arthritis is a common chronic autoimmune disease that affects approximately 1% of the adult population worldwide. Although its causes are largely unknown, it is clear that there is a considerable heritable component. In 2007, three genome-wide association studies (GWAS) as well as candidate gene approaches successfully increased the number of RA gene loci from two to five. More recently, a meta-analysis of these GWAS has been completed. The most significant finding outside of the known RA risk loci was of a common polymorphism near the CD40 gene (odds ratio = 0.87 p = 5.1 x 10-9). While this represents a true disease gene, several questions remain to be answered to fully comprehend the role of CD40 mutations in RA pathogenesis. While this study provides direct evidence that CD40 is critical in RA pathogenesis, at least three crucial questions remain: 1) what is the exact common causal mutation? 2) Are there rare mutations that contribute to disease not accounted for in the genome-wide studies as they are designed to identify common mutations? 3) What is the biological relevance of both types of CD40 mutations?
The proposed research aimed to address these three critical questions by applying next generation genetic technologies (e.g. high-throughput Solexa sequencing) in large RA case-control collections available in the Plenge laboratory at Harvard Medical School (Outgoing Host Institute), together with functional immunology available in the Huizinga laboratory at Leiden University Medical Center (Return Host Institute). To achieve these goals, genetic and immunological resources have been combined to identify, validate and functionally characterize both common and rare functional mutations involved in the establishment of RA. This comprehensive approach has generated insight into a pathway relevant to disease development.
• a description of the work performed since the beginning of the project.
Since the beginning of the project, we have successfully resequenced and fine-mapped the CD40 locus to determine the common causal mutations (Objective 1). Given the rapid change in sequencing technology, we were able to extend our ability to not only sequence the coding exons of CD40 but created a study design that encompassed 25 candidate genes from regions associated with RA from previous genome-wide association studies (Objective 2) to identify low-frequency or rare variants that contribute to disease.
Given that we have identified the likely common causal mutations in the CD40 gene, we have further investigated its functional consequence on the mRNA and protein level (Objective3).
• a description of the main results achieved so far
Through this approach we fine-mapped the association at the CD40 locus, identifying a few potential common causal SNPs. We have also performed deep-requencing of coding exons of this gene in a large number of cases and controls and have not identified rare variants in this gene that would contribute to RA disease development. Given the rapid changes in sequencing technology though, we did extend the approach to encompass an additional 24 candidate genes from GWAS studies. Through this approach we identified for the first time two genes (IL2RA and IL2RB) that harbour an excess of rare variants in RA patients versus controls. We also identified novel associations in a low-frequency non-coding variant in the CD2 genes that contributes to risk of RA.
We have also observed that the CD40 mutation results in higher CD40 levels at the mRNA and protein level. We used retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe direct correlation between the amount fo CD40 protein and phosphorylaion of RelA (p65), a subunit of NF-KB transcription factor. We developed a high-throughput NF-Kb luciferase reporter assay in BL2 cells and conducted a screen of 1,982 chemical compounds and FDA-approved drugs. 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-kb signaling were identified implying a likely development of novel therapies.
• the expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
Given that we have identified the likely common causal mutations in the CD40 gene, we will further investigate its functional consequences on the mRNA and protein level. We have observed that this mutation likely results in higher CD40 levels at the mRNA and protein level and have investigated what the downstream effects are. We have performed high-throughput small-molecule drug screens and identified two novel compounds that may be of relevance for novel therapies.
The proposed research aimed to address these three critical questions by applying next generation genetic technologies (e.g. high-throughput Solexa sequencing) in large RA case-control collections available in the Plenge laboratory at Harvard Medical School (Outgoing Host Institute), together with functional immunology available in the Huizinga laboratory at Leiden University Medical Center (Return Host Institute). To achieve these goals, genetic and immunological resources have been combined to identify, validate and functionally characterize both common and rare functional mutations involved in the establishment of RA. This comprehensive approach has generated insight into a pathway relevant to disease development.
• a description of the work performed since the beginning of the project.
Since the beginning of the project, we have successfully resequenced and fine-mapped the CD40 locus to determine the common causal mutations (Objective 1). Given the rapid change in sequencing technology, we were able to extend our ability to not only sequence the coding exons of CD40 but created a study design that encompassed 25 candidate genes from regions associated with RA from previous genome-wide association studies (Objective 2) to identify low-frequency or rare variants that contribute to disease.
Given that we have identified the likely common causal mutations in the CD40 gene, we have further investigated its functional consequence on the mRNA and protein level (Objective3).
• a description of the main results achieved so far
Through this approach we fine-mapped the association at the CD40 locus, identifying a few potential common causal SNPs. We have also performed deep-requencing of coding exons of this gene in a large number of cases and controls and have not identified rare variants in this gene that would contribute to RA disease development. Given the rapid changes in sequencing technology though, we did extend the approach to encompass an additional 24 candidate genes from GWAS studies. Through this approach we identified for the first time two genes (IL2RA and IL2RB) that harbour an excess of rare variants in RA patients versus controls. We also identified novel associations in a low-frequency non-coding variant in the CD2 genes that contributes to risk of RA.
We have also observed that the CD40 mutation results in higher CD40 levels at the mRNA and protein level. We used retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe direct correlation between the amount fo CD40 protein and phosphorylaion of RelA (p65), a subunit of NF-KB transcription factor. We developed a high-throughput NF-Kb luciferase reporter assay in BL2 cells and conducted a screen of 1,982 chemical compounds and FDA-approved drugs. 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-kb signaling were identified implying a likely development of novel therapies.
• the expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
Given that we have identified the likely common causal mutations in the CD40 gene, we will further investigate its functional consequences on the mRNA and protein level. We have observed that this mutation likely results in higher CD40 levels at the mRNA and protein level and have investigated what the downstream effects are. We have performed high-throughput small-molecule drug screens and identified two novel compounds that may be of relevance for novel therapies.