L-Fucose treatment of FUT8-CDG

Abstract

FUT8-CDG is a severe multisystem disorder caused by mutations in FUT8, encoding the α-1,6-fucosyltransferase. We report on dizygotic twins with FUT8-CDG presenting with dysmorphisms, failure to thrive, and respiratory abnormalities. Due to the severe phenotype, oral L-fucose supplementation was started. Glycosylation analysis using mass spectrometry indicated a limited response to fucose therapy while the clinical presentation stabilized. Further research is needed to assess the concept of substrate supplementation in FUT8-CDG.

Keywords: Congenital disorders of glycosylation; Fucose; Mass spectrometry; Therapy.

Fig. 1

Phenotype of twins with FUT8-CDG. Clinical presentation of patient A (A) and patient (B) pretreatment. Both patients show typical dysmorphisms with an arched palate, low- set ears, a broad nasal bridge, and a prominent epicanthal fold. Strabismus is present in both individuals. Note the muscular hypotonia necessitating support to keep an upright position. Over the course of L-fucose the treatment, the clinical presentation improved with improvement of muscle tone and discontinuation of ventilatory support (C).

Fig. 2

FUT8-CDG leads to a lack of core fucosylation in MALDI TOF-MS N-glycome profiling. A) 5 μg of human plasma protein was analyzed as previously described (Mealer et al., 2020). Permethylated N-glycome profiles from batch controls and two FUT8-CDGs are shown, with relative intensity (R.I) on the y-axis and mass-to-charge (m/z) ratio on the x-axis. Profiles with glycans greater than 3% relative abundance are illustrated. B) MS/MS fractionation confirms the absence of core fucosylation in FUT8-CDG samples. C) MALDI-MS profiling of FUT8-CDGs confirms a lack of core fucosylation with a preservation of antennary fucosylation in both patients. D) MALDI-MS profiling of FUT8-CDGs identifies a unique loss of bisected N-glycans in FUT8-CDG, the majority of which are also core fucosylated, and minimal change in high mannose or hybrid glycans.

Fig. 3

Lectin blotting confirms a loss of core fucosylation in FUT8-CDG. FUT8-CDG has a near total absence of fucose-specific lectin binding, which does not improve with fucose supplementation, consistent with MALDI-MS glycomics results. Human plasma (0.5 μL) was loaded in each lane after treatment with no without PNGase F at 37 °C for 1 h. Protein visualized using biotinylated lectins (LCA, AAL, Con A, SNA) followed by fluorescently labelled streptavidin, and total protein stain (Total) using the LICOR system. Non-specific signal from PNGase F at 35kD is marked with an asterisk (*).

Fig. 4

N-glycome profiling using MALDI-TOF MS is unaltered following fucose supplementation in FUT8-CDG. A) N-glycome profiling using MALDI-TOF MS did not improve following oral L-fucose supplementation. B) The abundance of fucosylated as well as bisected glycans showed fluctuations in relative abundance without improving substantially. Total abundance of core fucosylated glycans and core fucosylated bisected glycan structures remains severely reduced while fluctuations of antennary fucosylated glycans and non-fucosylated bisected glycans are present.

Fig. 5

Targeted glycosylation analysis using ESI-TOF MS of immunopurified glycoproteins identifies subtle improvement of glycosylation following fucose substitution. A) ESI-TOF MS identified truncated and non-fucosylated glycan species that decreased to or close to ranges seen in controls under fucose supplementation. Fucosylated glycan species showed fluctuations, remaining below reference ranges (Supplementary Fig. 2). B) Targeted analysis of fucosylation of an agalactosylated glycan on IgG Fc showed an initial increase while later falling to the ranges of initial values under higher doses of fucose and additional galactose. Fucosylation of serum transferrin at one of the two glycosylation site showed fluctuations in % fucosylation of a disialo-glycan without any clear effect of fucose supplementation.

Fig. 6

ESI-QTOF MS detects reduced dysglycosylation following fucose/galactose treatment in FUT8-CDG. A) In exemplary pretreatment samples taken from Patient A, ESI-QTOF MS both show severe lack of fucosylation as well as hypogalactosylation based on analysis of selected reporter glycans. B) Following supplementation of 1 g/day fucose, a reduction in hypofucosylated glycans was noted while hypogalactosylated glycans remained elevated. Additional supplementation of 10 g galactose/day in conjunction with an increase of fucose supplementation lead to a further shift towards regular glycosylation and specifically a reduction in hypogalactosylation.