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. 2010 Sep 9;5(9):e12646.
doi: 10.1371/journal.pone.0012646.

Reduced expression of IFIH1 is protective for type 1 diabetes

Kate Downes  1 , Marcin Pekalski, Karen L Angus, Matthew Hardy, Sarah Nutland, Deborah J Smyth, Neil M Walker, Chris Wallace, John A Todd
Affiliations

Reduced expression of IFIH1 is protective for type 1 diabetes

Kate Downes et al. PLoS One. .

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, Ala(946), 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Allele specific expression of IFIH1 pre mRNA transcript for the common T1D haplotype in IFN-β stimulated PBMCs.
The mean allelic ratio is a measure of the ratio of the sequencing trace peak heights for the heterozygous SNP within genomic and cDNA from each individual (overall sign-rank p = 0.012).
Figure 2
Figure 2. Detection of aberrant splicing of IFIH1 mRNA for the two rare SNPs predicted to alter splicing.
Levels of IFIH1 pre mRNA and spliced RNA transcript were measured using qPCR assays designed to the corresponding exon to intron and exon to exon sequence. The delta Ct is calculated using the pre mRNA transcript Ct minus the mRNA transcript Ct. A larger delta Ct indicates more pre-mRNA and less mRNA transcript of IFIH1. Donor 1 and 2 are control individuals, donor 3 and 4 heterozygous for rs35337543 (IVS8+1) and donor 5 and 6 are heterozygous for rs35732034 (IVS14+1).
Figure 3
Figure 3. Reduced IFIH1 expression within monocytes, B and T cells in donors heterozygous for the rare premature stop codon SNP, rs35744605 (Glu627X).
Mean fluorescent intensity (MFI) of IFIH1 expression for each donor heterozygous for rs35744605 is normalised to the MFI of the paired control individual.
Figure 4
Figure 4. Levels of pre-mRNA and mRNA transcript of IFIH1 are not altered by the rare nsSNP, rs35667974, Ile923Val.
Delta Ct calculated using the IFIH1 qPCR minus the single copy gene β2 microglobulin (B2M) qPCR. Donor 17 is the control individual and donor 18 is heterozygous for Ile923Val. E8-E10: exon 8 to exon 10 qPCR assay, E14-E16: exon 14 to exon 16 qPCR assay and E8-I8: exon 8 to intron 8 qPCR assay.
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References

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