Galactose Supplementation in Patients With TMEM165-CDG Rescues the Glycosylation Defects

Abstract

Context: TMEM165 deficiency is a severe multisystem disease that manifests with metabolic, endocrine, and skeletal involvement. It leads to one type of congenital disorders of glycosylation (CDG), a rapidly growing group of inherited diseases in which the glycosylation process is altered. Patients have decreased galactosylation by serum glycan analysis. There are >100 CDGs, but only specific types are treatable.

Objective: Galactose has been shown to be beneficial in other CDG types with abnormal galactosylation. The aim of this study was to characterize the effects of galactose supplementation on Golgi glycosylation in TMEM165-depleted HEK293 cells, as well as in 2 patients with TMEM165-CDG and in their cultured skin fibroblast cells.

Design and setting: Glycosylation was assessed by mass spectrometry, western blot analysis, and transferrin isoelectrofocusing.

Patients and interventions: Both unrelated patients with TMEM165-CDG with the same deep intronic homozygous mutation (c.792+182G>A) were allocated to receive d-galactose in a daily dose of 1 g/kg.

Results: We analyzed N-linked glycans and glycolipids in knockout TMEM165 HEK293 cells, revealing severe hypogalactosylation and GalNAc transfer defects. Although these defects were completely corrected by the addition of Mn2+, we demonstrated that the observed N-glycosylation defect could also be overcome by galactose supplementation. We then demonstrated that oral galactose supplementation in patients with TMEM165-deficient CDG improved biochemical and clinical parameters, including a substantial increase in the negatively charged transferrin isoforms, and a decrease in hypogalactosylated total N-glycan structures, endocrine function, and coagulation parameters.

Conclusion: To our knowledge, this is the first description of abnormal glycosylation of lipids in the TMEM165 defect and the first report of successful dietary treatment in TMEM165 deficiency. We recommend the use of oral d-galactose therapy in TMEM165-CDG.

Figure 1.

Galactose specifically suppresses the observed LAMP2 glycosylation defect in TMEM165 KO cells. (A) Steady-state cellular level and gel mobility of LAMP2. Control and TMEM165 KO HEK293 cells were cultured in absence or presence of galactose (1 mM), MnCl₂ (100 µM), or MnSO₄ (100 µM) for 18 hours; cell lysates were prepared, subjected to SDS-PAGE, and western blot with the indicated antibodies. (B) Steady-state cellular level and gel mobility of LAMP2. Control and TMEM165 KO HEK293 cells were cultured in absence or presence of galactose (1 mM), glucosamine (5 mM), or N-acetylglucosamine (5 mM) for 36 hours. Cell lysates were prepared, subjected to SDS-PAGE, and western blot with the indicated antibodies.

Figure 2.

Galactose supplementation suppresses the observed galactosylation defect on N-glycans in TMEM165 KO cells. (A) MALDI-TOF-MS spectra of the permethylated N-glycans from control HEK293 cells. (B) MALDI-TOF-MS spectra of the permethylated N-glycans from control HEK293 cells treated with galactose (1 mM, 36 hours). (C) MALDI-TOF-MS spectra of the permethylated N-glycans from TMEM165 KO HEK293 cells. (D) MALDI-TOF-MS spectra of the permethylated N-glycans from TMEM165 KO HEK293 cells treated with Galactose (1 mM, 36 hours). The symbols representing sugar residues are as follows: closed square, N-acetylglucosamine; open circle, mannose; closed circle, galactose; open diamond, sialic acid; and closed triangle, fucose. Linkages between sugar residues have been removed for simplicity.

Figure 3.

Galactose supplementation only partially suppresses the observed glycosylation defect on glycolipids in TMEM165 KO cells. (A) MALDI-TOF-MS spectra of permethylated glycolipids from control HEK 293 cells. (B) MALDI-TOF-MS spectra of permethylated glycolipids from control HEK 293 cells treated with galactose (1 mM, 36 hours). (C) MALDI-TOF-MS spectra of permethylated glycolipids from TMEM165 KO HEK293 cells. (D) MALDI-TOF-MS spectra of permethylated glycolipids from TMEM165 KO HEK293 cells treated with galactose (1 mM, 36 hours). The symbols representing sugar residues are as follows: yellow closed square, N-acetylgalactosamine; blue circle, glucose; yellow circle, galactose; open diamond, sialic acid. Linkages between sugar residues have been removed for simplicity. All GSLs are present as d18:1/C16:0 and d18:1/C24:0 ceramide isomers.

Figure 4.

MnCl₂ supplementation totally suppresses the observed glycosylation defect on glycolipids in TMEM165 KO cells. (A) MALDI-TOF-MS spectra of permethylated glycolipids from control HEK293 cells. (B) MALDI-TOF-MS spectra of permethylated glycolipids from control HEK293 cells treated with MnCl₂ (100 µM, 36 hours). (C) MALDI-TOF-MS spectra of permethylated glycolipids from TMEM165 KO HEK293 cells. (D) MALDI-TOF-MS spectra of permethylated glycolipids from TMEM165 KO HEK293 cells treated with MnCl₂ (100 µM, 36 hours). The symbols representing sugar residues are as follows: yellow closed square, N-acetylgalactosamine; blue circle, glucose; yellow circle, galactose; open diamond, sialic acid. Linkages between sugar residues have been removed for simplicity. All GSLs are present as d18:1/C16:0 and d18:1/C24:0 ceramide isomers.

Figure 5.

Galactose therapy ameliorates the N-glycosylation in individuals with TMEM165deficiency. (A and C) MALDI-TOF-MS spectra of the permethylated N-glycans from sera of 2 patients with TMEM165 deficiency who carry the homozygous c.792+182G>A mutation. (B and D) MALDI-TOF-MS spectra of the permethylated N-glycans from sera after 18 weeks of galactose therapy. The symbols representing sugar residues are as follows: closed square, N-acetylglucosamine; open circle, mannose; closed circle, galactose; open diamond, sialic acid; and closed triangle, fucose. Linkages between sugar residues have been removed for simplicity.