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Gene Screening Tool Allows Researchers to Zero in on Variants Influencing Lupus Risk

NEW YORK — Researchers have developed a new screening tool to home in on genetic variants that contribute to disease risk, which they applied to study the genetic factors influencing lupus.

Systemic lupus erythematosus (SLE) affects about 300,000 people in the US, the majority of whom are women. Previous studies have implicated both genetic and environmental contributors to the condition. While nearly 100 genetic loci have been associated with lupus, it is also closely tied to previous infection with Epstein-Barr virus.

Researchers from Cincinnati Children's Hospital applied a gene screening tool they developed to further investigate genetic loci associated with SLE. Through their massively parallel reporter assay (MPRA) approach, they narrowed in on key disease-linked genetic variants and began to tease out disease-associated biological mechanisms, as they reported Friday in Nature Communications.

"This study not only provides several critical new discoveries about lupus, it also provides a blueprint for dissecting the genetic mechanisms of many complex human diseases," co-senior author Leah Kottyan, interim director of the Center for Autoimmune Genomics and Etiology at Cincinnati Children, said in a statement.

With their MPRA tool, the researchers screened 91 loci associated with SLE through a previous genome-wide association study to gauge whether they lead to transcriptional dysregulation in cell lines.

In particular, the researchers first conducted linkage disequilibrium expansion analyses in different ancestral groups for the 91 risk loci to capture as many disease-linked variants as possible. In all, they identified 3,073 genetic variants, to which they added a further 20 variants highlighted in a previous study. For each of these, they generated a pair of 170-base-pair DNA oligonucleotides with random 20mers for barcoding and added the eGFR gene under a minimal promoter. In all, they generated 12,478 oligos, which they transfected into an Epstein-Barr virus-transformed B cell line.

In the cell line, the researchers identified 482 variants with enhancer activity and 51 with genotype-dependent enhancer activity at 27 loci. Most, 31 of the variants, decreased enhancer activity, as compared to the non-risk allele.

The researchers further found that certain transcription factors had altered binding at these SLE risk loci. For instance, the rs3101018 variant, for which the non-risk allele exhibits 1.7-fold stronger enhancer activity, the researchers uncovered 15 proteins that better bound the non-risk allele and two that preferred the risk allele. The ATF7, CREB1, and CREM proteins all bound the non-risk allele more tightly than the risk allele, suggesting that weaker binding to the risk allele could change how other proteins are recruited to the locus and affect the expression of the associated C4A gene.

They similarly found changed protein binding at the rs2069235 variant.

The researchers additionally transfected their SLE MRPA library into the Jurkat cell line of T cells, another key cell type in lupus. In this cell line, they uncovered 92 SLE risk variants that were allelic enhancer variants, a quarter of which were also found in the B cell line.

According to the researchers, this approach and findings could help in the development of targeted therapies for lupus. "In terms of new treatment, we are using what we learned from this study to identify medications that would affect many lupus risk SNPs. Because each SNP increases risk by a modest amount, targeting many at the same time will likely have a higher value for patients," Kottyan said.

They further added that their approach could be applied to study other diseases. They are in particular using it to study atopic dermatitis, eosinophilic esophagitis, and multiple sclerosis.