Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

Abstract

Background: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy.

Objective: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment.

Methods: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations.

Results: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation.

Conclusions: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.

Keywords: Atopy; allergy; autoimmunity; glycosylation; hyper-IgE; immune deficiency; neurocognitive impairment; phosphoglucomutase 3.

Figure 1. Pedigrees of the two families in the study

Solid symbols denote affected status and a slash through a symbol represents a deceased person.

Figure 2. Clinical findings in patients

A, Severe atopic dermatitis in Patient I.1, before and after wet wrap therapy with topical corticosteroids and emollients. Note scoliosis. B, Brain MRI, axial T2-FLAIR images of Patient I.2 showing stable hyperintensity in the white matter semiovale (blue arrows) at ages 22 (left), 31 (middle) and 35 (right) years, suggestive of stable dysmyelination. These findings were typical across our cohort. C, Visual evoked potentials in Patient II.1 demonstrate a prolonged p100 in both eyes (OS 200 ms and OD 197 ms) at check size 16, blue arrows, consistent with demyelinating optic neuropathy; x-axis 30ms per box, y-axis 10µV per box; normal p100 peak latency indicated by grayed areas (84.1–119.5 ms).

Figure 3. Genetic analysis of PGM3 deficiency

A. Compound heterozygous PGM3 mutations in Family I, and homozygous missense PGM3 mutation in Family II B. PGM3 domain structure. The mutations detected in Family I (blue) and Family II (yellow) are indicated by arrowheads. C, Multiple sequence alignment of PGM3 from different species, showing conservation of residues that are mutated in the patients. D, Structural model of PGM3. Left panel, three-dimensional structural model of PGM3 protein, based on the structure of the closest available homolog from Candida albicans. Relative locations of the patient missense mutations are denoted as circles (D325 in yellow, E529 in blue). Right panel, positions of both mutations relative to the active site and bound substrate. Hydrogen bonds between the focal amino acid and any adjacent residues are shown as yellow dashes. The charge of the side chain is denoted, if applicable. E, PGM3 protein levels in cell lysates from cycling T cells (left) and EBV cell lines (right), prepared from patients of both families, parents of Family II, and healthy control subjects. β-actin levels were measured as a loading control.

Figure 4. Impaired functions in PGM3-deficient patients

A, PGM3 enzymatic activities. The rate of GlcNAc1-P formation from GlcNAc6-P was measured in fibroblast lysates from patients (n=3) or normal healthy controls (n=2). B, Sugar phosphate analysis, quantitated on samples from A. C. Nucleotide sugar phosphate analysis, after overnight treatment of fibroblasts (three patients with two normal controls) with (+) or without (-) 10 mM GlcNAc. Exogenous GlcNAc supplementation increased UDP-GlcNAc and UDP-GalNAc levels. D. Elevated T_H2 and T_H17 cytokines in patients with PGM3 mutations. Percentage of CD3⁺ CD4⁺ CD45RO⁺ cells producing IL-4, IL-5, IL-17 or IFN-γ in response to PMA and ionomycin (PMA/I) in PBMCs from normals and patients. Horizontal bars represent mean percentage for each of the groups. Experiments were performed 2–4 times for each patient.