Multiple phenotypes in phosphoglucomutase 1 deficiency

Abstract

Background: Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest.

Methods: Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation.

Results: Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls.

Conclusions: Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.).

Figure 1. Glycoprotein Biosynthesis and Congenital Disorders of Glycosylation

For the N-glycosylation of proteins, a glycan precursor is assembled from monosaccharide units on an anchoring isoprenoid alcohol, dolichol, in the membrane of the endoplasmic reticulum. Each glycan precursor is then transferred en bloc to a specific asparagine residue in the nascent peptide chain of a protein being synthesized by a ribosome and entering the endoplasmic reticulum. Because transferrin (brown line) is abundant in blood, it is used as an indicator of the efficiency of the glycosylation system. Transferrin has two glycosylation sites. The protein is transported to the Golgi apparatus, and the glycans are modified in multiple steps. Each fully modified glycan is terminated with two negatively charged sialic acid residues. Thus, the transferrin molecule carries a total of four sialic acid residues. If a genetic defect impairs synthesis of the precursor glycan in the endoplasmic reticulum, one or both glycosylation sites of transferrin may remain unoccupied (white arrow). Such defects are classified as congenital disorders of glycosylation type I (CDG-I). In congenital disorders of glycosylation type II (CDG-II), the defect occurs in the modification of the already-transferred glycan. Both glycosylation residues may carry a glycan, but those glycans may be missing terminal sialic acid residues and end with galactose, mannose, or N-acetylglu-cosamine. Isoelectric focusing (IEF), which separates molecules according to differences in their isoelectric point, is used for investigating congenital disorders of glycosylation with the use of an agarose-gel–based system, followed by immunoprecipitation in gel with an antibody to transferrin and final staining of the precipitate with Coomassie blue. Under conditions of normal glycosylation, most transferrin molecules carry four sialic acid residues (tetrasialo-transferrin) and form a single major band on IEF. A few transferrin molecules carry five or six sialic acid residues that appear as minor bands on IEF (gray numbers; not shown in the molecular-structure diagrams). CDG-I defects are easily identified owing to the occurrence of transferrin isoforms with two or no sialic acid residues detected by means of IEF. No signal for monosialotransferrin (transferrin with one sialic acid residue) is seen in such disorders. In CDG-II, the defect can result in transferrin molecules with no, one, two, three, or four sialic acid residues. A patient with a CDG-II defect typically has bands of approximately equal intensity at all positions from 0 through 3 or a smooth gradient of intensities, whereas in most cases, tetrasialotransferrin shows a more intense signal owing to residual activity. In our patients, the signal of transferrin with two sialic acid residues (disialotransferrin) may be more intense than those for either one or three residues (as in the sample from Patient 1.1). The signal with two sialic acid residues presumably arises from a mixture of transferrin molecules that have one unoccupied glycosylation site as well as transferrin molecules that are occupied at both sites by glycans but have only two terminal sialic acid residues. The patients in this study had features of both CDG-I and CDG-II, as shown by IEF, suggesting a novel disorder.

Figure 2. Role of Phosphoglucomutase 1 in Sugar Metabolism

When blood glucose is low, glycogen degradation (yellow box) generates glucose-1-phosphate, which is converted by phosphoglucomutase 1 (PGM1) to glucose-6-phosphate for glucose release from the liver or subsequent catabolism in other tissues. In the reverse reaction, glucose-6-phosphate is converted to glucose-1-phosphate by means of PGM1 and is used as a substrate in the production of uridine diphosphate (UDP)–glucose, which can be used for the synthesis of glycogen or for protein glycosylation. Dietary galactose may be converted to UDP-galactose by galactose-1-phosphate uridyl-transferase (GALT) and used for protein glycosylation. UDP-galactose may also be converted into UDP-glucose by UDP-galactose epimerase (GALE). The GALT reaction is reversible; for clarity, only one direction of the coupled GALT reactions is shown. UGP denotes UDP-glucose pyrophosphorylase.

Figure 3. Effects of Dietary Galactose on Protein Glycosylation

The results of mass spectrometry of the intact transferrin protein and corresponding transferrin IEF profiles are shown before (Panel A) and after (Panel B) the intake of supplementary galactose. The glycan structures are shown in detail with the transferrin protein backbone (brown line). Patients with PGM1 deficiency have highly specific transferrin glycosylation profiles. Data from Patient 2 show several peaks indicating the presence of glycoforms completely lacking one or both N-glycans (white arrows), a finding that is also observed in CDG-I. There are also peaks indicating the presence of glycoforms with truncated glycans lacking galactose (ending with N-acetylglucosamine; yellow arrows). Several structures end this way, which is typically seen in CDG-II. Numbers at main peaks are deconvoluted mass (amu). Numbers next to the IEF results indicate individual bands corresponding to numbers of sialic acid residues (black numbers indicate structures shown along the respective mass spectrum, and gray numbers structures not shown). After the patient increased dietary intake of galactose, protein glycosylation was substantially improved (Panel B). Peaks indicating the presence of glycoforms with truncated glycans lacking galactose almost completely disappeared, and peaks indicating the presence of glycoforms with complete loss of one or both N-glycans were considerably reduced. For details on the method and quantification of isoforms by means of liquid chromatography–mass spectrometry, see the Supplementary Appendix.

Figure 4. Overview of Clinical Features of PGM1 Deficiency

Most patients had a bifid uvula (Panel A). Symptoms or findings related to PGM1 deficiency are listed according to frequency (Panel B). Hepatopathy was defined as elevated aminotransferase levels, steatosis, fibrosis, or a combination of these features. Myopathy was defined as a maximal creatine kinase level of more than 300 U per liter. Growth retardation was defined as a height at or below the 5th percentile. Hypoglycemia was defined as a fasting glucose level of less than 2.2 mmol per liter (40 mg per deciliter).