A rare regulatory variant in the MEF2D gene affects gene regulation and splicing and is associated with a SLE sub-phenotype in Swedish cohorts

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disorder with heterogeneous clinical presentation and complex etiology involving the interplay between genetic, epigenetic, environmental and hormonal factors. Many common SNPs identified by genome wide-association studies (GWAS) explain only a small part of the disease heritability suggesting the contribution from rare genetic variants, undetectable in GWAS, and complex epistatic interactions. Using targeted re-sequencing of coding and conserved regulatory regions within and around 215 candidate genes selected on the basis of their known role in autoimmunity and genes associated with canine immune-mediated diseases, we identified a rare regulatory variant rs200395694:G > T located in intron 4 of the MEF2D gene encoding the myocyte-specific enhancer factor 2D transcription factor and associated with SLE in Swedish cohorts (504 SLE patients and 839 healthy controls, p = 0.014, CI = 1.1-10). Fisher's exact test revealed an association between the genetic variant and a triad of disease manifestations including Raynaud, anti-U1-ribonucleoprotein (anti-RNP), and anti-Smith (anti-Sm) antibodies (p = 0.00037) among the patients. The DNA-binding activity of the allele was further studied by EMSA, reporter assays, and minigenes. The region has properties of an active cell-specific enhancer, differentially affected by the alleles of rs200395694:G > T. In addition, the risk allele exerts an inhibitory effect on the splicing of the alternative tissue-specific isoform, and thus may modify the target gene set regulated by this isoform. These findings emphasize the potential of dissecting traits of complex diseases and correlating them with rare risk alleles with strong biological effects.

Fig. 1

Flowchart of variant selection. Variant selection was based on a series of filters to remove variants without enough evidence for regulatory potential

Fig. 2

Functional annotation of the MEF2D region with SNP rs200395694. The variant is located in intron 4 of the MEF2D gene and overlaps with strong regulatory marks including DNase I hypersensitivity region (DNase Clusters), open regulatory region associated with active gene expression (ORegAnno), conserved transcription factor binding sites (TFBS Summary) and region conservation across 100 vertebrates (Cons 100 Verts) (http://genome.ucsc.edu/). The histone modification marks (H3K27ac, H3K4me1) associated with active enhancers mapped for blood cell populations according to Roadmap Epigenomics (http://egg2.wustl.edu/roadmap/web_portal/) indicate the presence of a cell type-specific enhancer. The known GWA signals located in the MEF2D gene region for migraine and blood cell traits are shown by green vertical lines (GWAS Catalog) [–33]

Fig. 3

Binding and regulatory potential of rs200395694 alleles. a EMSA results with nuclear extract from Jurkat cells. b Luciferase reporter assay performed in Jurkat. Bars represent mean values ± SD. RLU relative light units. Statistical analysis was done using an unpaired t-test

Fig. 4

Analysis of alternative splicing with minigenes. a Minigenes with alternative alleles were cloned into pcDNA3.1 vector between the CMV promoter and the polyadenylation site. Neomycin gene was used for transfection normalization. b–e Levels of alternative isoforms transcribed from minigenes transfected into THP-1 cells (b, c) and C2C12 cells (d, e) were measured by quantitative RT-PCR. THP-1 cells were stimulated with 100 ng/ml of LPS and 10 ng/ml of interferon gamma for 12 h. C2C12 cells were differentiated with 2% horse serum for 64 h. Bars represent mean values ± SEM