Germline C1GALT1C1 mutation causes a multisystem chaperonopathy

Abstract

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Keywords: C1GALT1C1; COSMC-CDG; Cosmc; O-glycosylation; Tn-antigen.

Fig. 1.

(A) Schematic of O-glycan biosynthesis. Cosmc is an ER-localized molecular chaperone required for T-synthase function. T-synthase is critical for the generation of normal elongated O-glycans at serine, threonine, and tyrosine (S/T/Y) residues in glycoproteins. In the absence of functional Cosmc, T-synthase remains inactive and is degraded, resulting in truncated O-glycans. (B) Visualization of the transmembrane topology of full-length Cosmc protein. The red-filled alanine residue is mutated in patients M1, M2, F1, and F2. The orange-filled residues were affected by somatic mutations in previously reported patients with Tn syndrome. The light blue residue was affected in a recent single case publication of a patient carrying a de novo variant (c.266C>T; p.Thr89Ile) in C1GALT1C1 (11). Protein visualization modified from Protter output (12).

Fig. 2.

Pedigree and photographs of reported patients. (A) Frontal facial photograph of patient F1 at the age of 38. (B and C) Frontal facial photograph of patient M1 at the age of 15 d and 4 y, respectively. (D and E) Frontal facial and whole-body photographs of patient M2 at the age of 20. Shared facial features are retrognathia, prominent nasolabial folds and a tall forehead, patient M2 shows clear disproportionate short stature. (F) Sanger sequencing data on patients M1, M2, F1, and F2, and the tested unaffected family members. The unaffected brother (III-3) and unaffected maternal half-brother (III-5) of patient F1 do not carry the variant. The C1GALT1C1 c.59C>A variant is marked by a red box. Both male patients carry the variant hemizygously, the mother is heterozygous. (G) Pedigree. Black-filled symbols indicate affected C1GALT1C1 c.59C>A hemizygous male patients, gray-filled symbols indicate affected heterozygous female patients. Individual IV-2 died on postnatal day three from kidney failure. Since no detailed clinical information was available, this individual has not been marked as affected by COSMC-CDG. Genotype information is indicated to the left of the respective symbol. Individuals without genotype information were unavailable for genetic testing.

Fig. 3.

Serum creatinine (A), urinary albumin/creatinine ratio (B) and complement activity (C) for patient M1 over 2 y starting at the initial presentation of AKI. Complement activity was measured in two laboratories with different CH50 assays. Laboratory 1 reported CH50 in percent activity, laboratory 2 in U/mL. After September 2020, samples were no longer regularly analyzed by laboratory 1. Peritoneal dialysis was performed during the gray-shaded time period. Initial incomplete suppression of complement activity was due to an increased urinary Eculizumab loss and difficulties in dosage finding. In November 2020, biweekly Eculizumab treatment was switched to four-weekly Ravulizumab infusions. AP50: alternative complement pathway activity; CH50: classical complement pathway activity; Ecu: Eculizumab; Ravu: Ravulizumab.

Fig. 4.

A20D-Cosmc expression and its chaperone function on T-synthase activity. (A) Whole-cell lysates from cultured lymphoblastoid cells were analyzed by SDS-PAGE western blot, and probed for endogenous Cosmc, T-synthase, and BiP (loading control). (B) Similar to A, whole-cell lysates were assayed for T-synthase activity (n = 9, three independent experiments of n = 3). (C) As in B, hexosaminidase activity was measured (n = 12, three independent experiments of n = 4). (D) Wild-type C1GALT1C1 (+C) partially rescues T-synthase activity in the patients’ cells and control cell line (CosmcKO). Lysates were analyzed for T-synthase activity (n = 12, three independent experiments of n = 4). (E) Expression of A20D-Cosmc. Whole-cell lysates from transiently transfected cells as indicated on the bottom were analyzed by SDS-PAGE-WB, and probed as indicated (n = 3, a representative example). (F) In vitro expression of transiently transfected A20D-Cosmc-HPC4 can generate functional T-synthase. T-synthase activity from whole-cell lysates was measured (n = 9, three independent experiments of n = 3). Additionally, the same preparation of the lysates was analyzed by SDS-PAGE-WB and probed for the expression of A20D-Cosmc, B-actin, WT-Cosmc and T-synthase as indicated (n = 3, representative example). (G) Detectable level of endogenous A20D-Cosmc expression in M2 fibroblasts (M2-fib). Whole-cell lysates as shown, analyzed using SDS-PAGE-WB, and probed for endogenous Cosmc and T-synthase as indicated. Arrow (Left) shows A20D-Cosmc. Representative examples of n = 2. (H) Whole-cell lysates prepared as in G were analyzed for T-synthase activity (n = 6, two independent experiments of n = 3). HPC4, tagged Cosmc. CosmcKO: Cosmc-knockout SimpleCells, Jurkat: Jurkat T lymphocyte-derived cancer cell line without endogenous Cosmc expression, HEK: HEK-293 human embryonic kidney cell line with high endogenous Cosmc expression. Lymphoblastoid cells: Healthy control (HC) and male patients (M1 and M2). Fibroblasts: M2-fib. Error bars represent ±SD. *nonspecific bands.

Fig. 5.

Characterization of cell surface and total O-glycans from A20D-Cosmc patients’ cells. (A–C) Primary PBMCs (panel A) and cultured lymphoblastoid cells (panels B and C) from healthy control (HC), both female patients (F1 and F2), and both male patients (M2 and M1) as indicated, analyzed for their surface glycan expression as indicated (ReBaGs6 antibody [binds to truncated O-glycan, Tn-antigen], PNA lectin [binds to normal O-glycan, T-antigen], ConA lectin [N-glycans, binds to mannosylated glycans]) using flow cytometry. For the analysis of sugar structures, cells were first treated with mock or neuraminidase. FACS plots for A (n = 1)/FACS plots for B (n = 3), P values: P = 0.0404 (M1 vs. healthy), P = 0.0283 (M2 vs. healthy); and normal O-glycans expression measured by PNA, without Neuraminidase (n = 3), P values: P = 0.2094 (M1 vs. healthy), P = 0.0981 (M2 vs. healthy); with Neuraminidase (n = 3), P values: P = 0.2919 (M1 vs. healthy), P = 0.3962 (M2 vs. healthy). (C) For normal N-glycans expression (n = 2), without Neuraminidase, P values: P = 0.0983 (M1 vs. healthy), P = 0.2239 (M2 vs. healthy); with Neuraminidase, P values: P = 0.1402 (M1 vs. healthy), P = 0.4183 (M2 vs. healthy). (D–F) Glycan characterization on whole-cell extracts. Similar to Fig. 4C, whole-cell extracts were prepared and treated with neuraminidase (NeuA, removes sialic acid), NeuA and O-glycosidase (NeuA+O, removes non-sialylated T-antigen), PNGase-F (PNG, removes all N-glycans), or untreated (Mock). The preparations and enzyme mixture (E-mix) were resolved on an SDS-PAGE gel. B-actin serves as a loading control. Blot images are one of the representative images of three independent experiments. Asterisk (*) indicates nonspecific binding from HRP-streptavidin. (G) Characterization of IgA1. Serum from patients and pooled healthy control were used to purify total IgA and treated with mock (left lane under each respective sample heading) or Neuraminidase (right lane). SDS-PAGE-WB/LB were used to probe for total IgA, Gd-IgA1 (VVA lane), and IgA1 with normal O-glycans (PNA lane). All patients have high levels of Gd-IgA1, but no normally O-glycosylated IgA1 (bottom, PNA stain). Control normally O-glycosylated IgA1 is stained by PNA after removal of sialic acid by Neuraminidase (PNA, last lane). Representative example of three independent experiments. (H) Transient transfection of wild-type Cosmc on patient cells reduces expression of the immature Tn-antigen, partially restoring normal O-glycosylation. Flow cytometry analysis was performed similar to A, on cells treated with neuraminidase. FACS plot for H (n = 3), M1 rescue, Tn antigen expression, P value = 0.0258 (M1 vs. M1+Cosmc) and Normal O-glycans expression measured by PNA, P value = 0.5053 (M1 vs. M1+Cosmc); similarly, for M2 rescue, Tn antigen expression, P value value = 0.0416 (M1 vs. M1+Cosmc) and Normal O-glycans expression measured by PNA, P value = 0.3051 (M1 vs. M1+Cosmc).