Biallelic Mutations in FUT8 Cause a Congenital Disorder of Glycosylation with Defective Fucosylation

Abstract

Fucosyltransferase 8 (FUT8) encodes a Golgi-localized α1,6 fucosyltransferase that is essential for transferring the monosaccharide fucose into N-linked glycoproteins, a process known as "core fucosylation." Here we describe three unrelated individuals, who presented with intrauterine growth retardation, severe developmental and growth delays with shortened limbs, neurological impairments, and respiratory complications. Each underwent whole-exome sequencing and was found to carry pathogenic variants in FUT8. The first individual (consanguineous family) was homozygous for c.715C>T (p.Arg239^∗), while the second (non-consanguineous family) was compound heterozygous for c.1009C>G (p.Arg337Gly) and a splice site variant c.1259+5G>T. The third individual (consanguineous family) was homozygous for a c.943C>T (p.Arg315^∗). Splicing analysis confirmed the c.1259+5G>T resulted in expression of an abnormal FUT8 transcript lacking exon 9. Functional studies using primary fibroblasts from two affected individuals revealed a complete lack of FUT8 protein expression that ultimately resulted in substantial deficiencies in total core fucosylated N-glycans. Furthermore, serum samples from all three individuals showed a complete loss of core fucosylation. Here, we show that loss of function mutations in FUT8 cause a congenital disorder of glycosylation (FUT8-CDG) characterized by defective core fucosylation that phenotypically parallels some aspects of the Fut8^-/- knockout mouse. Importantly, identification of additional affected individuals can be easily achieved through analysis of core fucosylation of N-glycans.

Figure 1

Identification of FUT8 Pathogenic Variants in Three Unrelated Families (A) Schematic of the FUT8 dependent α1,6 fucosyltransferase enzymatic reaction creating a core fucose motif. (B) Pedigrees showing FUT8 pathogenic variants and segregation in families 0448, 0459, and 0464. (C) Schematic of Human FUT8 with the relative positions of each pathogenic variant on mRNA (GenBank: NM_178155.2) and protein level (Uniprot: Q9BYC5).

Figure 2

Effect of FUT8 Pathogenic Variants on Protein Expression and mRNA Splicing (A) Western blot analysis of FUT8 protein in fibroblast from three controls, CDG-0448 and CDG-0459 showing loss of FUT8 protein expression. Two different monoclonal antibodies with separate epitopes were used to detect human FUT8 protein. The first monoclonal anti-FUT8 recognizes an epitope within amino acids 31–230 and was used at a 1:500 dilution and (Santa Cruz Biotechnology, sc-271244), while the second has an epitope within amino acids 276–575 and was used at a 1:2,000 dilution (Proteintech 66118-1-Ig). (B) Splicing analysis was performed using primers spanning exon 8 to exon 10 of FUT8 mRNA and determined that the c.1259+5G>T, found in CDG-0459, produced an abnormal transcript lacking exon 9. Two independent RNA preparations are shown for CDG-0459.

Figure 3

Characterization of N-Glycans from Whole Serum using LC-MS LC-MS chromatograms for control, CDG-0448, CDG-0459, and CDG-0464 showing the loss of multiple core fucosylated N-glycans (green shade). Unlike the other two affected individuals, CDG-0464 also had an absence of non-fucosylated neutral glycans (orange shade), which is not due to the loss of FUT8.

Figure 4

Characterization of N-Glycans from Primary Fibroblasts using LC-MS and Flow Cytometry LC-MS chromatograms for Control, CDG-0448, and CDG-0459 showing the loss of multiple fucosylated N-glycans (green shade). The control used, GM-05381, is representative of three different control lines (GM-00038, GM-03348, GM-05381).

Figure 5

Lectin Analysis of Cell Surface Core Fucosylated N-Glycans (A) Flow cytometry was used to evaluate the cell surface binding of FITC-LcH (Lens culinaris agglutinin) in the two affected individuals and in a representative control culture and showed reduced lectin staining in both affected individuals. (B) Flow cytometry analysis of cell surface FITC-PSA (Pisum sativum agglutinin) in the two affected individuals and in a representative control culture and showed reduced lectin staining in both affected individuals. GM-03348 is representative of three different control lines (GM-00038, GM-03348, GM-05381).