Background/Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease, believed to arise from environmental triggering events in genetically predisposed individuals. To date, more than 50 genes have been associated with SLE through genome-wide association studies. However, only about 15% of the heritability of SLE is explained by these findings.  In this study we aimed to identify novel rare genetic variants of functional importance for SLE.

Methods: One hundred and forty Swedish SLE patients, fulfilling the ACR classification criteria for SLE, and 12 healthy controls were included in the study. We sequenced the exons, promoter regions and putative regulatory regions of 219 genes selected on basis of their role in immune response, autoimmunity or known association with SLE or SLE-related disease complex in dogs. Selected gene regions were targeted using Roche NimbleGen capture array and the DNA samples were then divided into 10 pools according to the disease manifestations of the patients, with the 12 healthy controls in a separate pool. The pools were paired-end sequenced using Illumina HiSeq2000 reaching approximately 250x coverage per individual.

Results: We detected 4276 novel single-nucleotide polymorphisms (SNPs; not present in 1000genomes or dbSNP137) out of which 1258 SNPs were case-only variants. Seventeen genes showed ≥ 5 novel variants private to cases. Of these, three genes have previously been associated with human SLE and 14 are novel candidate genes. Six SNPs (allele frequencies 0.01-0.03 in patients) located in non-coding sequences with potential regulatory function were selected for further studies based on their characteristics in terms of conservation, DNAse hypersensitivity, ENCODE data on histone marks and ChIP-Seq peaks. The SNPs were validated by genotyping in all patients and in 96 additional healthy controls, to confirm their increased frequency in the patient cohort. Using electrophoretic mobility shift assay, binding of protein complexes was investigated for all six SNPs and they are currently evaluated for their effect on gene expression. The clinical disease manifestations of the patients harboring each of the six SNPs are being analyzed in order to identify any phenotype-genotype correlations.

Conclusion: This proof-of-principle study highlights the importance of analysis of non-coding putative regulatory DNA regions for the identification of rare variants associated with complex disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/high-throughput-sequencing-of-219-candidate-genes-for-identification-of-sle-associated-risk-variants/