Reduced expression of IFIH1 is protective for type 1 diabetes.

Kate Downes,Sarah Nutland,Marcin Pekalski,Matthew Hardy,John A Todd,Karen L Angus,Chris Wallace,Deborah J Smyth,Neil M Walker,Matthias G Von Herrath

doi:10.1371/journal.pone.0012646

Abstract

IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation-associated protein 5), is one of a family of intracellular proteins known to recognise viral RNA and mediate the innate immune response. IFIH1 is causal in type 1 diabetes based on the protective associations of four rare variants, where the derived alleles are predicted to reduce gene expression or function. Originally, however, T1D protection was mapped to the common IFIH1 nsSNP, rs1990760 or Thr946Ala. This common amino acid substitution does not cause a loss of function and evidence suggests the protective allele, Ala946, may mark a haplotype with reduced expression of IFIH1 in line with the protection conferred by the four rare loss of function alleles. We have performed allele specific expression analysis that supports this hypothesis: the T1D protective haplotype correlates with reduced IFIH1 transcription in interferon-β stimulated peripheral blood mononuclear cells (overall p = 0.012). In addition, we have used multiflow cytometry analysis and quantitative PCR assays to prove reduced expression of IFIH1 in individuals heterozygous for three of the T1D-associated rare alleles: a premature stop codon, rs35744605 (Glu627X) and predicted splice variants, rs35337543 (IVS8+1) and rs35732034 (IVS14+1). We also show that the nsSNP, Ile923V, does not alter pre-mRNA levels of IFIH1. These results confirm and extend the new autoimmune disease pathway of reduced IFIH1 expression and protein function protecting from T1D.

Highlights

Susceptibility to T1D was originally mapped to a common SNP, rs1990760 in the IFIH1, FAP, GCA and KCNH7 region of chromosome 2q24 [1]
Owing to concerns over biases caused by RNA secondary structure [10], cDNA was synthesised using PBMC purified premRNA, poly A selected by priming with oligo-dT and using total RNA fragmented and primed using random hexamers
IFIH1, known as MDA5, is one of a family of intracellular proteins known to recognise viral RNA and mediate an immune response [15]

Summary

Introduction

Susceptibility to T1D was originally mapped to a common SNP, rs1990760 in the IFIH1, FAP, GCA and KCNH7 region of chromosome 2q24 [1]. Further SNP typing revealed several variants across the region in high linkage disequilibrium (LD) (Figure S1) and statistically indistinguishable for T1D association. The nsSNP, rs1990760 or Thr946Ala, in exon 15 of IFIH1, was considered the most likely functional candidate due to the highly conserved amino acid substitution and the role of IFIH1 in innate immunity, including mediating type 1 interferon production, which has been consistently reported to be associated with type 1 diabetes [1,2,3]. Four rare or low frequency variants within IFIH1 were associated with T1D, indicating the gene is causal [4] (Figure S2 and Table S1). The common variant or haplotype is still associated with T1D after taking into account the rare variants, and vice versa (Table S1)

Methods

Results

Conclusion