The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media

Abstract

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Keywords: FUT2; RNA-sequencing; microbiota; otitis media; p.Trp154*; rs601338.

Figure 1

Study flowchart. The flowchart shows the number of saliva and microbial samples included for genotyping, RNA-seq and microbiota analyses.

Figure 2

Volcano plot of differentially expressed genes based on carriage of the FUT2 c.461G>A variant in patients with OM. In variant carriers, KMT2D/MLL2, MUC16, NBPF20 and FN1 were downregulated (FDR-adjusted p < 0.05, log2 fold change < -2) and MTAP was upregulated (FDR-adjusted p < 0.05, log2 fold change > 2).

Figure 3

Network and pathway enrichment analysis of differentially expressed genes. (A) A single PPI network was constructed using the FUT2, RASIP1 and the DE genes as input. (B) KEGG and (C) PANTHER GO-slim:BP pathway enrichment analysis results, showing the top 10 pathways with the smallest p-values. MUC16 and FUT2 are not connected to this network, suggesting a different mechanism for the interaction of these two genes in relation to OM.

Figure 4

Gene array expression data for select genes post-inoculation with non-typeable Haemophilus influenzae (NTHi). Mouse middle ear expression of select genes across different time points, shown as fold change in middle ears inoculated with NTHi as compared to placebo. Fut2, Muc16 and Mtap reached peak change in expression at 24 hours post-inoculation while Muc16 demonstrated sustained downregulation. On the other hand, Rasip1 and Fn1 reached peak upregulation at 3 hours post-inoculation. In this experiment, time point 0h represents uninfected middle ear. *p < 0.05; see Table 3 for gene expression values by time point and gene.

Figure 5

Single-cell RNA-seq expression data from mouse middle ear 6 hours after inoculation with NTHi. Expression of Fut2, Fn1, Muc16, Kmt2d and Rasip1 in wildtype mouse middle ear, 6 hours post-infection. Cell types of the various PCA clusters were identified by the expression of unique marker genes. Darkness of color (red for each gene) indicates level of UMI expression by each cell, according to the associated log₂ scale. Epithelial cell clusters were identified based on the expression of Krt18 and/or Krt19. Basal epithelial cells also express Krt14, while ciliated epithelial cells express Hydin. Stromal cells are identified by Col1a2. Vascular endothelial cells express Egfl7 and Flt4, lymphatic endothelial cells Egfl7 and Flt1. Melanocytes express Mlana and pericytes Rgs5. Monocytes express Csf1r, lymphocytes Ptprcap, polymorphonuclear cells Il1f9 and Stfa2l1, and red blood cells Hba-a1.

Figure 6

Relative abundance of individual taxa in middle ears (ME) and nasopharynges (NP) of carriers and non-carriers of the FUT2 c.461G>A variant. (A) Cumulative relative abundance profiles in the ME of wildtype (n=8) and carriers (n=26) of FUT2 c.461G>A. (B) Cumulative relative abundance profiles in the NP of wildtype (n=14) and carriers (n=51) of FUT2 c.461G>A. Plots showing p-values for relative abundance of individual bacterial taxa in the (C) ME and (D) NP of wildtype versus variant carriers after adjusting for batch. Blue lines indicate taxa that were increased in wildtype, red lines for carriers. Dashed lines indicate significance thresholds where the red line is unadjusted-p=0.1 (non-significant) and green lines indicate unadjusted-p=0.05 and unadjusted-p=0.01. (C) In the ME, Haemophilus (1) and Moraxella (2) were nominally associated with wildtype, whereas Propionibacterium (3) and Anoxybacillus (4) were nominally associated with variant carriage. (D) In the NP, Candidate Division TM7 (1) was significantly associated with wildtype (FDR-adj-p=0.009). Additionally, Selenomonas (2) and Actinobacillus (3) were nominally associated with wildtype whereas Propionibacterium (4) was nominally associated with variant carriage.

Figure 7

Relative abundance of individual taxa in the middle ears (ME) and nasopharynges (NP) of carriers and non-carriers of the RASIP1 c.1801C>T variant. (A) Cumulative relative abundance profiles in the ME of wildtype (n=8) and carriers (n=26) of RASIP1 c.1801C>T. (B) Cumulative relative abundance profiles in the NP of wildtype (n=15) and carriers (n=50) of RASIP1 c.1801C>T. Plots showing p-values for relative abundance of individual bacterial taxa in the (C) ME and (D) NP of wildtype versus carriers after adjusting for batch. Blue lines indicate taxa that were increased in wildtype, red lines for carriers. Dashed lines indicate significance thresholds where the red line is unadjusted-p=0.1 (non-significant) and green lines indicate unadjusted-p=0.05 and unadjusted-p=0.01. (C) In the ME, Gammaproteobacteria (1) was nominally associated with wildtype, whereas Propionibacterium (2) was nominally associated with variant carriage. (D) In the NP, Candidate Division SR1 (1) and Candidate Division TM7 (2) were significantly associated with wildtype, and Chloroplast (5) with variant carriage (FDR-adj-p=0.05). Additionally, Actinobacillus (3) was nominally associated with wildtype, whereas Propionibacterium (4), Staphylococcus (6) and Escherichia-Shigella (7) were nominally associated with variant carriage.