A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation

Abstract

The conserved oligomeric Golgi (COG) complex is involved in intracellular vesicular transport, and is composed of eight subunits distributed in two lobes, lobe A (COG1-4) and lobe B (COG5-8). We describe fourteen individuals with Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features. All affected subjects harbored heterozygous de novo variants in COG4, giving rise to the same recurrent amino acid substitution (p.Gly516Arg). Affected individuals' fibroblasts, whose COG4 mRNA and protein were not decreased, exhibited delayed anterograde vesicular trafficking from the ER to the Golgi and accelerated retrograde vesicular recycling from the Golgi to the ER. This altered steady-state equilibrium led to a decrease in Golgi volume, as well as morphologic abnormalities with collapse of the Golgi stacks. Despite these abnormalities of the Golgi apparatus, protein glycosylation in sera and fibroblasts from affected subjects was not notably altered, but decorin, a proteoglycan secreted into the extracellular matrix, showed altered Golgi-dependent glycosylation. In summary, we define a specific heterozygous COG4 substitution as the molecular basis of Saul-Wilson syndrome, a rare skeletal dysplasia distinct from biallelic COG4-CDG.

Figure 1

Clinical Features of Individuals with Saul-Wilson Syndrome (A) P2.1 as an infant, showing a progeroid appearance, sparse eyebrows, and thin vermillion of the upper lip. (B) P8.1 at 6 months of age, showing prominent forehead, veins, and eyes, thin vermilion of the upper lip, and micrognathia. (C) P6.1 at 7 months of age, showing a progeroid appearance, relative macrocephaly, prominent forehead and veins, sparse eyebrows, low-set ears, low hanging columella, thin vermillion of the upper lip, and use of boots and bar orthosis as therapy for clubfoot. (D) P10.1 at 7 months of age, showing prominent scalp veins and low-set ears. (E) P4.1 at 1 year of age, showing relative macrocephaly, prominent forehead, veins and eyes, sparse eyebrows, low hanging columella, and thin vermillion of the upper and lower lips. (F) P1.1 at 4 years of age, showing prominent forehead, veins, and eyes, and thin vermillion of the upper and lower lips. (G) P7.1 at 20 years of age, showing prominent forehead, sparse eyebrows, narrow nasal bridge, low hanging columella, and thin vermillion of the upper lip. (H) P2.1 at 29 years of age, showing prominent forehead, sparse eyebrows, narrow nasal bridge, low hanging columella, and thin vermillion of the upper and lower lips. (I) P2.1 at 29 years of age, showing telebrachydactyly.

Figure 2

COG Subunit Expression (A) mRNA expression of each of the COG-subunits in P1.1 (gray) compared to control (GM08398, black). No significant changes in expression were observed. Measurements were performed in quadruplicate. Error bars represent 95% confidence interval. (B) Infrared fluorescent western blot analysis (left) of COG4 in three control (GM08398, GM01582 and ATCC63792061) and three affected subject (P1.1, P4.1 and P5.1) fibroblast lines. Vinculin served as a loading control and in the calculation of normalized signal intensity. Quantitative analysis showing normalized signal intensity of COG4 in individual samples (middle). Mean protein level of COG4 in affected subjects versus controls; error bars represent SD.

Figure 3

Golgi Morphology and Volumetric Analysis (A) Compared to control fibroblasts (GM01582) that had normal Golgi morphology in 93.9% of the cells, P1.1, P4.1, and P5.1 only had normal morphology in 51.1%, 52.8%, and 54.9% of their cells, respectively. ^∗∗∗p < 0.0005 compared to the control. Affected subjects did not significantly differ from each other. (B) Representative images of a Golgi with normal morphology in the control fibroblasts and a Golgi with abnormal morphology in P1.1 fibroblasts. Images were taken using the 63× oil-based objective. Cis-Golgi is stained with GM130 (red), trans-Golgi with TGN46 (green), and DNA with Hoescht 33342 (blue). The yellow signal in the affected subject cells represents co-localization of GM130 and TGN46, suggesting that the cis- and trans-Golgi are collapsed. (C) The volumes of Giantin-labeled Golgi and DAPI-labeled nuclei were determined by Imaris software. A significant decrease in the ratio of Golgi-to-nucleus volume was seen in affected subject cell lines. Controls represent the average ratio for five cells from ATCC63792061 and GM8398 cell lines. Affected subjects represent the average ratio for 13 cells from P1.1, P4.1 and P5.1 cell lines. Error bars represent SD. ^∗∗p < 0.005.

Figure 4

Decorin Immunoblotting in Control and Affected Individual Fibroblasts (A) Western blot analysis of secreted decorin. (B) Western blot analysis of intracellular decorin. The size ranges for the fully glycanated form of decorin and its core protein are indicated. A portion of decorin is extended with GAG chains (MW 100−130+kD), while a significant portion is not glycosylated and runs as a doublet at about 40kD. (C) Levels of glycanated decorin were quantified against total decorin levels. The decorin bands between 70–150kD and core decorin bands (∼40kD) in cell lysates were used to calculate the ratio of glycanated decorin to total decorin. Experiments were repeated in duplicate with similar results. Representative blots are shown. ^∗∗p < 0.005.

Figure 5

Brefeldin A Trafficking Assays (A) BFA-induced retrograde transport kinetics of control and affected subject cells. Between 80 and 100 cells were counted for each time point. The experiment was performed in duplicate. C1, ATCC63792061; C2, GM08398. (B) Golgi protein anterograde transport kinetics of control and affected subject cells was measured after treatment with BFA for 1 hr followed by washout (T = 0). Between 80 and 100 cells were counted for each time point. The experiment was performed in duplicate. C1, ATCC63792061; C2, GM08398; COG8, positive control. Error bars represent SD.

Figure 6

Morphogenesis and Function of the Inner Ear Are Abnormal in cog4-Deficient Zebrafish (A and B) Morphology of the inner ear in cog4 sibling (A) and cog4^−/− mutant (B) larvae. Red stars indicate the semicircular canals. (C–F) Phalloidin staining of the anterior macula in cog4 sibling (C) and cog4 mutant (D) larvae. Phalloidin staining of the second posterior neuromast in cog4 sibling (E) and cog4 mutant (F) larvae. (G) Number of hair bundles in the anterior macula of cog4 mutants (n = 15 larvae) and cog4 siblings (n = 14 larvae). (H) Number of hair bundles in the second posterior neuromast of cog4 mutants (n = 6 larvae) and cog4 siblings (n = 7 larvae). ^∗∗p < 0.01. All data obtained at 5 dpf, anterior to the left (A–F).

Figure 7

Zebrafish cog4-Deficient Mutants Have Morphological and Secretory Defects (A and B) Lateral views of (A) cog4 sibling and (B) biallelic cog4^−/− mutant larvae. Red dashed lines indicate the anterior (jaw extension) and posterior (caudal fin) ends of the wild-type larvae. Red double arrow in B marks the length difference between wild-type and mutant larvae. Blue arrowheads indicate the inner ear. (C and D) Dorsal views of cog4 sibling (C) and cog4^−/− mutant (D) larvae. Orange arrowheads indicate the eyes, and purple arrowheads the pectoral fins; green arrowhead indicates the forward extent of the jaw in (C). (E) Body length in homozygous wild-type cog4^+/+ (n = 7 larvae), heterozygous cog4^+/− (n = 37 larvae) and biallelic homozygous cog4^−/− mutant larvae (n = 17 larvae). Length was measured as in (A). (F) Inner ear size in cog4^+/+ (n = 14), cog4^+/− (n = 70), and cog4^−/− larvae (n = 21). Size was measured as distance along the antero-posterior axis of the inner ear. ^∗∗p < 0.01. (G and H) Alcian blue staining of the ceratohyal in cog4 sibling (G) and biallelic mutant (H) larvae. (I and J) col1a2 mRNA in situ hybridization in the pectoral fin of cog4 sibling (I) and mutant (J) larvae. (K and L) Immunolabeling of collagen type II in the ceratohyal of cog4 sibling (K) and mutant (L) larvae. All data obtained at 5 days postfertilization (dpf), anterior to the left (A–D, G–L).