A novel nonsense and inactivating variant of ST3GAL3 in two infant siblings suffering severe epilepsy and expressing circulating CA19.9.

Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.

Total loss of GM3 synthase activity by a normally processed enzyme in a novel variant and in all ST3GAL5 variants reported to cause a distinct congenital disorder of glycosylation.

ST3GAL5-CDG is a rare syndrome which is caused by variant GM3 synthases, the enzyme involved in the biosynthesis of a-b-c-series gangliosides. Here we report a novel homozygous ST3GAL5 variant, p.Gly342Ser, in a patient suffering from failure to thrive, severe hearing, visual, motor, and cognitive impairment, and respiratory chain dysfunction. A GM3 synthase assay towards the natural acceptor substrate lactosylceramide was performed upon transfection in HEK-293T cells of expression plasmids carrying wild type and mutated ST3GAL5 cDNAs. The assay revealed a complete loss of enzyme activity. Identical results were obtained with the other four ST3GAL5 variants which have been reported to be pathogenic. HEK-293T clones permanently expressing HaloTag-ST3GAL5 carrying each of the five variants were assessed by quantitative PCR, flow cytometry, western blotting and confocal microscopy. The results indicated that transcription, translation, stability and intracellular localization of the tagged protein were identical to those of the wild type construct. Compared with the very mild phenotype of st3gal5 KO mouse models, the results suggest that unknown mechanisms, in addition to the lack of a-b-c-series gangliosides, contribute to the syndrome. Direct enzyme assay upon transfection in model cells appears to be an effective tool for characterizing variants of glycosyltransferases involved in glycosphingolipid biosynthesis.

Diseases of ganglioside biosynthesis: An expanding group of congenital disorders of glycosylation.

Among the numerous congenital disorders of glycosylation concerning glycoproteins, only a single mutation in ganglioside biosynthesis had been reported until a few years ago: one in the ST3GAL5 gene, encoding GM3 synthase. More recently, additional mutations in the same gene were reported, together with several distinct mutations in the B4GALNT1 gene, encoding GM2/GD2/GA2 synthase. Patients suffering from ST3GAL5 deficiency present a devastating syndrome characterized by early onset and dramatic neurological and cognitive impairment, sometimes associated with dyspigmentation and an increased blood lactate concentration. On the other hand, B4GALNT1 mutations give rise to a form of complicated hereditary spastic paraplegia (HSP), previously referred to as HSP26. It is characterized by the late onset of lower limb weakness and mild to moderate intellectual impairment, which is usually not progressive. In addition to the most typical signs, some patients present ocular and endocrine signs, pes cavus, and psychiatric illness. Since the nineties, mice lacking genes for single glycosyltransferases involved in ganglioside biosynthesis, including ST3GAL5 and B4GALNT1, were created and studied. The resulting phenotypes were frequently mild or very mild, so double knock-out animals were created to effectively study the function of gangliosides. The main clinical and biochemical features of patients suffering from GM3 synthase or GM2/GD2/GA2 synthase deficiency, compared with the phenotypes described in mice that are null for single or multiple glycosyltransferase genes, provide suggestions to improve the recognition of novel mutations and potentially related disorders.