Fig 2. The T/T haplotype at rs1990760 and rs3747517 leading to histidine at position 843 and threonine at position 946 encodes a highly active MDA-5 variant.

HEK 293 cells were transfected with plasmids coding for the indicated MDA-5 variants (50 ng/well) defined by the four possible amino acid combinations at the amino acid positions 843 and 946 coded by the SNP alleles T/C at rs3747517 and T/C at rs1990760 respectively. R843/T946 the most common allele combination (WT) in the European population is highlighted in grey. An empty vector and in B) two MDA-5 variants, I923V and E627stop, known to be functionally inactive, were used as negative controls. 24 hours after transfection cells were stimulated using poly I:C (125 ng/ml) complexed with lipofectamine for cytoplasmic delivery. A) 24 h after stimulation, production of IP-10 and IL-28B was measured in the supernatants by ELISA. Results are depicted as means + SEM of n = 9 (IP-10) and n =3 (IL-28B). B) Prior to stimulation and 6 h after stimulation RNA was isolated and the expression levels of the indicated genes were analysed by quantitative rt-PCR. Target expression was normalized to the NPT gene and is depicted as fold induction of the WT (R843/T946). Data are depicted as means + SEM of at least n = 5. p-values were determined using Student’s t-test and are depicted as *p<0.05, **p<0.01 and ***p<0.001.