FUT2 Variants Confer Susceptibility to Familial Otitis Media

Abstract

Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154^∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202^∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10^-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154^∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10^-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154^∗, and p.Arg202^∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.

Keywords: 16S rRNA sequencing; A antigen; FUT2; TDT; fucosyltransferase; microbiome; middle ear infection; otitis media; transient expression; transmission disequilibrium test.

Figure 1

Indigenous Filipino (IPOM) Subpedigree and Microbiome Results for Middle Ear Samples from the IPOM and Colorado Cohorts (A) Indigenous Filipino subpedigree with the FUT2 c.604C>T (p.Arg202^∗; rs1800028) variant co-segregating with otitis media status, as follows: IPOM112 (87 y.o.), cholesteatoma (red); IPOM8 (24 y.o.) and IPOM76 (9 y.o.), chronic otitis media (yellow); IPOM141 (5 y.o.), effusive otitis media (orange); IPOM15 (12 y.o.), IPOM26 (50 y.o.), IPOM90 (47 y.o.), IPOM105 (36 y.o.), and IPOM156 (17 y.o.), healed otitis media with healed perforations, sclerosis, and/or previously documented otitis media (gray); IPOM43 (25 y.o.), IPOM80 (32 y.o.), and IPOM162 (40 y.o.), normal (clear with black border); unknown phenotype, clear with gray border. dup, A2ML1 duplication; wt, wild-type. (B) Average family-level relative abundances for individuals with otitis media from the indigenous Filipino community (IPOM) and Colorado (CU) defined by genotype for FUT2 stop variants. Nominally significant differences were identified in the Colorado cohort according to genotype by permutation-based multiple analysis of variance (PERMANOVA) test (p < 10⁻⁷) using Bray-Curtis dissimilarities. (C) Manhattan plot showing log₁₀-transformed p values per bacterial family, ascertained by Kruskal-Wallis tests of wild-type versus rs601338 (p.Trp154^∗) carriers within the Colorado cohort. Families with p < 0.1 are labeled on the plot and named to the right of the plot.

Figure 2

Additional Microbiome Results in the IPOM and CU Cohorts (A) Inter-individual variability in middle ear samples by cohort and FUT2 variant. (B) Boxplots comparing biodiversity indices by cohort and FUT2 variant: richness measures the number of OTUs per sample (estimated by Chao1), evenness measures the uniformity of OTU distributions (estimated by Shannon H/H_max), while complexity (Shannon diversity) combines both richness and evenness. Error bars indicate range of values; boxes show the first and third quartiles.

Figure 3

Fold Expression of Fut2 across Different Time Points after Inoculation of Non-typeable Haemophilus influenzae into the Mouse Middle Ear Affymetrix probes 1434862_at (filled circles) and 1450246_at (open circles), targeting Fut2 transcripts, revealed enhanced expression that peaked at 24 hr post-infection. The 0 hr data point represents uninfected middle ears. Vertical bars represent the range of fold values observed for each probe on all arrays.

Figure 4

Flow Cytometry Profiles of GFP and Cell Surface A Blood Group Epitope Levels by COS-7 Cells Transfected with Human GFP-FUT2 Variant Constructs (A) COS-7 cells were transiently transfected for 72 hr with GFP-FUT2^WT (green-filled traces) or GFP-FUT2 variant constructs (red traces). GFP-positive (green traces) and non-transfected (gray traces) cells were used as negative controls. GFP-FUT2^WT, GFP-FUT2^Ala104Val, and GFP-FUT2^Arg138Cys transfected cells show the same GFP expression profiles while GFP-FUT2^Trp154∗ and GFP-FUT2^Arg202∗ transfected cells show a decrease of GFP levels as compared to GFP-FUT2^WT cells, meaning that those variants are less expressed or degraded quicker in COS-7 cells. (B) Quantification of the flow cytometry experiment shown in (A). ^∗∗∗p ≤ 0.001; n.s.: non-significant. Error bars indicate ± SEM. (C) Western blot analyses of COS-7 cells transfected with GFP-FUT2 variants. GFP and GAPDH blots are shown. (D) COS-7 cells were transiently transfected for 72 hr with GFP, GFP-FUT2^WT (green), or GFP-FUT2 variant constructs (red). Cell surface A blood group epitope levels on GFP-positive cells were analyzed for each condition. 42.5% of GFP-FUT2^WT cells have A antigen as compared to GFP-transfected cells (21.2%). The accumulation of A antigen at the surface of cells transfected with GFP-FUT2^Ala104Val, GFP-FUT2^Arg138Cys, GFP-FUT2^Trp154∗, or GFP-FUT2^Arg202∗ variant constructs was significantly reduced (28.3%, 24%, 11.1%, and 13.2%, respectively).