Variant in human POFUT1 reduces enzymatic activity and likely causes a recessive microcephaly, global developmental delay with cardiac and vascular features

Abstract

Protein O-fucosyltransferase-1 (POFUT1) adds O-fucose monosaccharides to epidermal growth factor-like (EGF) repeats found on approximately 100 mammalian proteins, including Notch receptors. Haploinsufficiency of POFUT1 has been linked to adult-onset Dowling Degos Disease (DDD) with hyperpigmentation defects. Homozygous deletion of mouse Pofut1 results in embryonic lethality with severe Notch-like phenotypes including defects in somitogenesis, cardiogenesis, vasculogenesis and neurogenesis, but the extent to which POFUT1 is required for normal human development is not yet understood. Here we report a patient with a congenital syndrome consisting of severe global developmental delay, microcephaly, heart defects, failure to thrive and liver disease with a previously unreported homozygous NM_015352.1: c.485C>T variant (p.Ser162Leu) in POFUT1 detected by exome sequencing. Both parents are heterozygotes and neither manifests any signs of DDD. No other detected variant explained the phenotype. This variant eliminated a conserved N-glycosylation sequon at Asn160 in POFUT1 and profoundly decreased POFUT1 activity in patient fibroblasts compared to control fibroblasts. Purified p.Ser162Leu mutant protein also showed much lower POFUT1 activity with a lower affinity for EGF acceptor substrate than wild type POFUT1. Eliminating the N-glycan sequon by replacing Asn160 with Gln had little effect on POFUT1 activity, suggesting that loss of the glycan is not responsible for the defect. Furthermore, the p.Ser162Leu mutant showed weaker ability to rescue Notch activity in cell-based assays. These results suggest that this N-glycan of POFUT1 is not required for its proper enzymatic function, and that the p.Ser162Leu mutation of POFUT1 likely causes global developmental delay, microcephaly with vascular and cardiac defects.

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs

“…The pSecTag2c-based N-terminally 6xHis-tagged human POFUT1 lacking C-terminal RDEF sequence expression plasmid was described previously [ 69 ]. The pCMV6-based Myc-DDK-tagged mouse protein O -fructosyltransferase 2 (POFUT2) expression plasmid was purchased from Origene.…”

Multiple roles for O‐glycans in Notch signalling

“…A recently identified heterozygous mutation in POFUT1 , associated with DDD4 is also accompanied by Hidradenitis Suppurativa (HS) which is marked by recurrent painful nodules and abscesses [82]. A homozygous recessive mutation in POFUT1 ablates a N-glycan site and is correlated with more severe developmental defects [83]. However, the loss of the N-glycan is not the basis of the reduced activity of POFUT1.…”

Effects of Notch glycosylation on health and diseases

“…This process is considered to occur in the ER (Luo & Haltiwanger, 2005). Notably, it was recently reported that the p.Ser162Leu mutation of POFUT1 in humans likely causes global developmental delays, such as microcephaly with cardiovascular defects (Takeuchi, Wong, et al, 2018). Additionally, the loss of Pofut1 in mice reportedly causes embryonic lethality due to a global loss of Notch signaling (Shi & Stanley, 2003).…”