Evaluation of IFITM3 rs12252 Association With Severe Pediatric Influenza Infection

Abstract

Background: Interferon-induced transmembrane protein 3 (IFITM3) restricts endocytic fusion of influenza virus. IFITM3 rs12252_C, a putative alternate splice site, has been associated with influenza severity in adults. IFITM3 has not been evaluated in pediatric influenza.

Methods: The Pediatric Influenza (PICFLU) study enrolled children with suspected influenza infection across 38 pediatric intensive care units during November 2008 to April 2016. IFITM3 was sequenced in patients and parents were genotyped for specific variants for family-based association testing. rs12252 was genotyped in 54 African-American pediatric outpatients with influenza (FLU09), included in the population-based comparisons with 1000 genomes. Splice site analysis of rs12252_C was performed using PICFLU and FLU09 patient RNA.

Results: In PICFLU, 358 children had influenza infection. We identified 22 rs12252_C homozygotes in 185 white non-Hispanic children. rs12252_C was not associated with influenza infection in population or family-based analyses. We did not identify the Δ21 IFITM3 isoform in RNAseq data. The rs12252 genotype was not associated with IFITM3 expression levels, nor with critical illness severity. No novel rare IFITM3 functional variants were identified.

Conclusions: rs12252 was not associated with susceptibility to influenza-related critical illness in children or with critical illness severity. Our data also do not support it being a splice site.

Keywords: IFITM3; genetic susceptibility; influenza; interferon-inducible transmembrane protein; pediatric.

Figure 1.

Linkage disequilibrium (r²) in the white and influenza-positive subjects (n = 259) using a minor allele frequency of 0.01 as calculated by Haploview (www.broadinstitute.org) [18]. The following color scheme applies: white: r² = 0; shades of gray: r² = 0 to <1; black: r² = 1. The r² values are listed within each cell.

Figure 2.

A, IFITM3 isoforms identified in peripheral blood mononuclear cells (PBMCs) from the FLU09 cohort using RNAseq data. Each isoform is labeled with the percentage of normalized exon junction reads (counts per million) detected from only IFITM3 exon junction reads. Canonical full length IFITM3 comprised >96% of total IFITM3 exon junction reads. Coordinates for the exons are found below each exon with italicized coordinates indicating an alternative splice junction. Each splice junction is labeled with a number corresponding to the rs12252 genotype analysis below. B, Genotype analysis displays the percentage of exon junction reads (cpm) graphed by genotype of rs12252. Full-length IFITM3 (1) is graphed separately from the alternative splice junctions (2–4). The putative rs12252 splice isoform ENST00000526811 was not observed. C, IFITM3 expression levels measured by RNAseq (log₂ [FPKM]) in PBMCs of influenza-positive individuals from the FLU09 cohort plotted based on genotype of rs12252 (P = .25).