Here we describe a new COG4 patient of Indian ethnicity who presented with a very severe clinical phenotype compared to the previously reported COG4 patient [8
]. Although the clinical presentations were similar in several respects, our patient died around 2 years of age. While clinical phenotypes can vary between COG patients, the biochemical characteristics tend to be consistent. Thus far, defects have been identified in 6 of the 8 COG subunits. In each instance, patient serum glycoproteins were deficient in both sialylation and galactosylation and fibroblasts were defective in BFA-induced retrograde transport of resident Golgi proteins.
Consistent with the BFA defect, our patient’s fibroblasts exhibited a dramatic delay in retrograde movement of the Golgi marker Giantin back into the ER following BFA treatment. Analysis of all 8 subunits showed that only COG4 was significantly reduced in protein expression. This observation is unique when compared to other COG deficiencies in which loss of one subunit resulted in destabilizing multiple subunits. Why COG4 deficiency does not destabilize the complex is still unclear, although it is possible the position of the mutations lack direct interaction with the other subunits.
Sequencing genomic DNA revealed two novel mutations in the COG4 gene, the first c.697 G/T (p.E233X) and the second c.2318 T/G (p.L773R), that were absent from available public databases. Further analysis of transcript abundance showed the p.E233X was likely targeted for NMD.
It has been established that COG deficiencies can result in both N- and O-glycosylation abnormalities. Since Miura et al thoroughly characterized the N-glycan defect, we sought to understand more about the O-glycan defect in this patient.
To determine O-glycan structures in patient and control fibroblasts, we used a freely permeable GAP acceptor that acts as a primer for O-glycosylation and mimics O-glycan structures synthesized by the cells. Core1 β1,3-Galactosyl transferase adds a galactose to a Ser/Thr linked GalNAc also known as Tn antigen leading to Core1 O-GalNAc glycan (Galβ1,3GalNAc) or T antigen. Both T and Tn antigen can then be further modified by sialic acid to form mono or di-sialylated structures. Core1 O-GalNAc glycans can act as a substrate for two competing enzymes, Core2 β1,6-GlcNAc transferase and α2,6-sialyl transferase that catalyze two mutually exclusive reactions. Core2 β1,6-GlcNAc transferase, adds GlcNAc to the Core1 GalNAc thus branching it into a Core2 structure which can be extended by the addition of β1,4- galactose to GlcNAc. In addition one or more N-Acetyllactosamine repeats (Galβ1,4GlcNAcβ1,3) can be added and terminally capped by α2,3 sialic acid. The addition of sialic acid by α2,6-sialyl transferase on Core1 generates sialylated Core1 structure which is a substrate for α2,3 sialic acid on galactose but not for Core2 GlcNAc transferase [17
HPLC analysis of purified, metabolically labeled, GAP molecules from patient fibroblasts revealed an increase in Galβ1,3GalNAc as well as Core1 monosialylated structures, with a decrease in extended Core2 based glycans. One explanation for this is that Core2 β1,6-GlcNAc transferase and α2,6- sialyltransferase can compete for the same Core1 structure in the medial Golgi and it is possible the accumulation of Core1 in patient fibroblasts was due to a preference for α 2,6-sialyl transferase over Core2 β1,6- GlcNAc. But since there was no difference in disialylated Core1, this possibility was ruled out.
The precise mechanism for Core1 accumulation in the patient cells is still unclear. However, the COG complex is known to regulate the retrograde transport of key glycosyltransferase enzymes and sugar nucleotide transporters in the Golgi. COG4 deficiency in the patient fibroblasts might affect cellular localization as well as residence time of Core2 β1,6-GlcNAc transferase with protein cargo which could lead to decreased Core2 and more Core1 glycans.
Lentiviral complementation of patient fibroblasts with the wild type human COG4
gene failed to restore the normal BFA response. It was previously shown by Reynders et al
that overexpression of wild type COG4 protein in apparently normal fibroblasts invoked an abnormal BFA response suggesting a dominant negative effect [8
]. It is not apparent why COG4 has this effect since overexpression of several other individual subunits is capable of correcting the defective cellular phenotype [7
Here we present data on the first reported COG patient of Indian origin who had a novel set of mutations in the COG4 gene. Consistent with other COG deficiencies, this patient had deficiencies in sialylation and galactosylation of serum N-glycans, abnormal O-Glycans and a delay in BFA-induced retrograde transport. Identification of this patient underscores the geographic breath of this disorder. We highly recommend CDG testing in patients with similar clinical presentation.