Systematic dissection of lupus non-coding risk variants in primary B cells

John Ray, Ph.D., Benaroya Research Institute at Virginia Mason Franciscan Health 

While the causes of systemic lupus erythematosus (SLE) are not well understood, research has shown that genetic factors play a key role. Only a small amount of the DNA in our genome, or genetic map, contains information needed to make proteins required for the structure, function, and regulation of the body’s cells. Most of the genome is non-coding, meaning that portion of DNA does not provide instructions to make proteins. Studies that scan the genomes of many people to find genetic variations, or genetic differences between people, that associate with a particular disease have identified over 150 areas of the genome linked to SLE. However, over 90% of these identified areas are in non-coding regions of the genome, which we know much less about, making it hard to understand the specific effects of each variant. To better understand the mechanisms associated with SLE risk and to enable the development of personalized treatments, Dr. Ray will identify genetic variants that lead to disease-causing immune cell features often seen in SLE. 

B cells are immune cells that play a key role in SLE. Dr. Ray will analyze thousands of SLE-associated genetic variants in B cells from East Asian and European people, both healthy and with SLE, using two innovative approaches called massively parallel reporter assays and allele-specific ATAC-seq. These two methods will pinpoint genetic variants that alter B cell function and gene regulation—the process cells use to control which genes are expressed– and may cause SLE. To follow up on the top ten variants that likely cause SLE, Dr. Ray will use genome editing (a tool used to alter DNA sequences) to introduce the variants into the DNA of B cells, assessing how SLE-linked genetic variants alter B cell function. 

What this study means for people with lupus 

Our ability to develop personalized treatments is hindered by our incomplete understanding of the causes of SLE. Understanding how genetic variants alter B cells could enhance our knowledge of genes and pathways that promote risk and severity in SLE and could transform our ability to develop personalized treatments. 

Systemic Lupus Erythematosus (SLE) is a debilitating autoinflammatory syndrome that affects over 3 million people worldwide. Therapeutics that treat, prevent, or cure SLE are lacking, and this is in part due to a lack of knowledge of how SLE is caused. Genome-wide association studies (GWAS) have identified >150 regions of the genome associated with SLE, with risk regions differing across ancestries, but the genetic variants that cause SLE are often in tight linkage disequilibrium (LD) with 10s to 100s of non-causal variants, and >90% of risk loci are in non-coding regions, making causal variants and therefore the mechanisms of disease risk hard to identify. We and others have found that SLE-associated variants enrich in candidate regulatory elements of B cells and other immune cells, suggesting the genetic risk for disease acts through perturbing regulatory region activity in B cells. Thus, we hypothesize that SLE-causal variants alter cis-regulatory element activity in B cells to affect gene regulation and B cell function, and that this effect is exacerbated in the context of disease. To determine how genetic variants affect cis-regulatory region activity, we have previously utilized methods that test variant allelic effects on regulatory region activity in massively parallel reporter assays (MPRAs). We have recently conducted these assays in GM12878 B cells studying variants associated with autoimmune diseases, finding that variants that affect regulatory region activity enrich ~8-fold for likely causal variants according to statistical fine mapping. Combining these data with other measurements of B cell regulatory regions such as B cell DNase I Hypersensitivity-sequencing (DHS), we find that causal variants are enriched ~30-fold, suggesting that MPRA combined with DHS makes a highly effective readout for identifying disease-causal variants. Furthermore, others have assayed allele-specific chromatin accessibility as a readout for variant effects on cis-regulatory activity finding that these variants also enrich for disease risk variants, suggesting that this method used in conjunction with MPRA could highlight global effects of SLE risk variants across B cell states. To determine SLE variants that alter cis-regulatory activity, we will test thousands of SLE-associated variants found in East Asian and European individuals using these two high-throughput genomic approaches in primary human B cells. Upon completion of this study, we will have identified genetic variants that likely cause SLE and, for the top 10 variants, we will have assessed their target genes and effects on B cell function. Genes and pathways that are identified as a result of these analyses could lead to information about why and how lupus is heterogeneous and could help determine drug targets to treat and prevent SLE.