AUTOIMMUNITY RASGRP1 · Functional analysis of RasGRP1 SNPs in autoimmune disease

Functional analysis of RasGRP1 SNPs in autoimmune disease.

Final summary report Project: Functional analysis of RasGRP1 SNPs in autoimmune disease. # 328666 2 years outgoing part: University of California San Francisco, CA, USA 1 year return part: University Medical Center Utrecht, the Netherlands. Investigator: Dr. Vercoulen (y.vercoulen@umcutrecht.nl) Scientific representative: Prof. Dr. Prakken (b.prakken@umcutrecht.nl) Project context and objectives: Development of autoimmunity is thought to involve recurrent breakdown in the mechanisms that select an appropriate lymphoid repertoire, leading to production of autoantibodies that can become pathologic and trigger immune responses to various organs that ultimately result in organ damage. Several types of immune cells and biological processes have been implicated in autoimmunity, including the aberrant stimulation of B cells by CD4+ T cells in germinal centers to produce autoantibodies. Dr Vercoulen is exploring mutations found in the autoimmune disease systemic lupus erythematosus (SLE). Modern technology has led to an increased understanding of the genetic basis of SLE through GWAS and fine-mapping approaches. Four GWAS of SLE in European populations identified more than 20 robust susceptibility genes or loci. Much less robust is our knowledge of dysregulated signaling pathways in T- or B-cells in SLE patients. Understanding of these dysregulated signaling pathways is critical for future development of specific therapies for SLE patients, as well as patients suffering from other autoimmune disorders characterized by the production of autoantibodies, such as Juvenile Idiopathic Arthritis and Juvenile Dermatomyositis. Single Nucleotide Polymorphisms (SNPs) near RasGRP1 are associated with susceptibility to autoimmune disease. Several RasGRP1 missense SNPs are currently known but have remained unstudied. In addition, splice variants of RasGRP1 have been documented for patients with SLE, and abnormal microRNA-driven downregulation of RasGRP1 expression has been postulated to play a role in lupus CD4+ T cells. The genetic basis underlying these splice variants or reduced RasGRP1 expression is not known, and it is unclear how this impacts CD4+ T cell function. Each person inherits ~12,000 missense (non-synonymous) SNPs (single-nucleotide polymorphisms) that cause amino acid substitutions, generating enormous genetic variation. It is not known how these relatively subtle SNPs may cause complex immune disease by impacting the network of immune cells. The Roose lab characterized a novel Rasgrp1Anaef ENU mutant mouse strain, encoding a point-mutated variant of Rasgrp1, and Dr. Vercoulen has established with the lab of John Kuriyan three regulatory mechanisms that keep RasGRP1 in an inhibited state in a dimerized form. Dr. Vercoulem is using these initial results and the Rasgrp1Anaef mouse model (Daley et al., eLife 2013) with autoimmune features as a platform for this research plan. These studies take a Rasgrp1-centric approach, but it is anticipated that results from the proposed work will function as paradigm for future studies on dysregulated signaling pathways in T cells of patients with autoimmune diseases Objectives: This project will lead to the understanding of how mutations in RasGRP1 are involved in development of autoimmune diseases. Dr Vercoulen investigated: 1) how SNPs in RasGRP1 lead to autoimmunity, 2) how a single mutated allele of RasGRP1 causes autoimmunity 3) whether RasGRP1 function and/or expression is aberrant in patients with autoimmune disease. Results In the lab of Dr. Jeroen Roose, Dr. Vercoulen has established a solid cell-line based system to test the effect of point-mutations on RasGRP1 function and downstream signaling. Using this system, she established that several SNP’s (single nucleotide polymorphisms) from public databases caused changes in RasGRP1 activity. Both mutations in the RasGRP1 protein, as well as point-mutations in enhancer regions that regulate RasGRP1 expression (in collaboration with Dr. Alexander Marson, UCSF) affected RasGRP1 expression and/or function. Dr. Vercoulen collaborated with structural biologists in the lab of Dr. John Kuriyan (UC Berkeley), and based on the effects by the protein point-mutations, established how RasGRP1 transforms from an inactive autoinhibited state (structure published in Iwig, Vercoulen et al., eLife 2013) to an active state. Furthermore, they established which cellular and structural mechanisms are responsible for this transition (manuscript submitted for publication). Furthermore, Dr Vercoulen, in collaboration with Dr. Limnander, Myers and Hartzell in the Roose lab, elucidated how a single allelic mutation can still result in decreased RasGRP1 function, in vitro and in vivo (manuscript in preparation). In the University Medical Center Utrecht, the Netherlands, Dr. Vercoulen continued her studies in the lab of Prof. Prakken and Dr. van Wijk. Here, she collected patient samples from patients with an autoimmune disease: Juvenile Idiopathic Arthritis. Dr. Vercoulen discovered that the patients with active disease display aberrant RasGRP1 expression levels, and that this can be regulated by the RasGRP1 enhancer regions (Manuscript in preparation). Notably, Dr. Vercoulen has initiated in another collaboration with Dr. Lindsey Criswell at UCSF, the screening for patients with SLE that have the SNP causing RasGRP1 hyperactivity (ongoing). Impact: The results of this project lead to a profound understanding of how RasGRP1 activity and expression are regulated, and that this is aberrant in patients with autoimmune disease. Therefore, in the future, this work will aid to provide targeted therapies (precision medicine) to these patients. Furthermore, the structure-function studies of RasGRP1 transition from inactive to active state can provide required information for future development of small-molecule inhibitors. Since aberrant RasGRP1 signaling and expression are also relevant in patients with T-ALL, and in murine models for colorectal cancer, these findings are not only relevant to inflammatory/autoimmune disease, but also to cancers.

Data: CORDIS, © European Union