Abstract
Background
Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy.
Objective
To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment.
Methods
Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI 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, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations.
Results
Marked atopy and autoimmunity were associated with increased TH2 and TH17 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 cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological 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 UDP-GlcNAc, 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, Immune deficiency, Hyper-IgE, neurocognitive impairment, phosphoglucomutase 3, glycosylation, Allergy, Autoimmunity
INTRODUCTION
Mendelian atopic diseases provide opportunities to discover new pathophysiologic mechanisms contributing to allergy. Often, such diseases are identified because of features besides atopy, such as immunodeficiency (Hyper IgE syndrome, DOCK8 deficiency, Omenn’s syndrome, Wiskott-Aldrich syndrome, ADA-SCID), autoimmunity (IPEX syndrome), and non-immune abnormalities (developmental delay and abnormal skin in prolidase deficiency). When they co-segregate with atopy, these associated features help characterize the disease as a familial disorder, and demonstrate the full phenotypic spectrum caused by the underlying molecular lesion(s).
The prototype of such monogenic disorders is the autosomal dominant Hyper IgE syndrome (HIES), which was initially described because of recurrent staphylococcal infections 1, but was later found to be associated with elevated IgE 2. The identification of autosomal dominant STAT3 mutations in HIES established a role for this transcription factor in marked IgE elevation 3, 4, and more recently in protection from mast cell degranulation 5. By contrast, autosomal recessive DOCK8 mutations lead to viral skin infections, mucocutaneous candidiasis, and severe atopic disease including eczema, asthma, food allergies, and anaphylaxis 6–8. Such patients have increased TH2 cells (IL-4, IL-13), pointing to a role for DOCK8 in T cell regulation of allergic responses 9. Although STAT3 and DOCK8 mutations account for many cases of marked IgE elevation, the majority of patients with increased serum IgE and atopic disease in addition to syndromic features still have no identified genetic cause. These include an unusual kindred previously described at our center, which had recurrent infections, cutaneous vasculitis, motor and neurocognitive impairment, and other non-immune abnormalities 10.
Diseases that impact multiple organ systems, such as the one in the kindred mentioned above, include Congenital Disorders of Glycosylation (CDG). Typical features of CDG are extremely broad, but can include motor and neurologic deficits, hematologic abnormalities, dysmorphism, and other malformations. Abnormal immune function has been observed, including hypogammaglobulinemia with decreased B cell numbers in ALG12-CDG (also called CDG-Ig) due to mutations in ALG1211, leukocyte adhesion deficiency type 2 or SLC35C1-CDG due to mutations in SLC35C1 (also called CDG-IIc) 12, glucosidase I deficiency MOGS-CDG or CDG-IIb13. The widespread clinical manifestations are thought to be due to the ubiquity of glycosylation and its central roles in an array of normal cellular functions. During glycosylation, sugar chains are added to either proteins or lipids, using basic sugar building blocks such as UDP-N- acetyl-glucosamine (UDP-GlcNAc). After being generated through the hexosamine biosynthetic pathway or through the salvage pathway, UDP-GlcNAc is used to make N- glycans, O-glycans, proteoglycans, and glycosylphosphatidylinositol (GPI)-anchored proteins within the cell. These glycosylated proteins are found in various cellular compartments, on the cell surface, or in the plasma and extracellular matrix. Additionally, UDP-GlcNAc is also used for O-GlcNAc addition in the cytosol or nucleus, where it participates in cell signaling14.
Here we report the discovery of a genetic defect in glycosylation precursor synthesis causing a novel disease in eight patients from two families. The patients have severe atopy with marked serum IgE elevations, recurrent bacterial and viral infections, and motor and neurocognitive impairment most likely associated with hypomyelination. Their mutations, which affect an enzyme crucial in the generation of UDP-GlcNAc, point to a previously unappreciated role for glycosylation in the regulation of atopic disease, as well as associated comorbidities. Our findings suggest that altered glycosylation may be important in the pathophysiology of allergic diseases in the general population.
METHODS
Subjects
Patients and their families provided informed consent on NIH IRB-approved research protocols designed to study atopy (NCT01164241), hyper-IgE syndromes (NCT00006150), general host defense defects (NCT00001355), and/or lymphocyte homeostasis disorders (NCT00246857). Comprehensive histories, review of all available outside records, serial clinical evaluations, and therapeutic interventions were all performed at the Clinical Center of the National Institutes of Health (NIH). Clinical immunologic laboratory tests were performed by the Department of Laboratory Medicine at NIH, Bethesda, MD. Glycan profile quantitation and analysis in blood and urine were performed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy by Emory Genetics Laboratory, Decatur, GA.
Detailed procedures and additional information on genetic analysis, PCR and DNA sequencing, immunoblot analysis, structural analysis, enzyme activity assay, sugar phosphate and nucleotide sugar analysis, flow cytometric analysis, and MRI are provided in the Methods section in this article’s Online Repository.
RESULTS
Clinical phenotype of individuals with hypomorphic PGM3 mutations
Much of the clinical phenotype of this syndrome was first reported in Family I as an autosomal recessive immunodeficiency-vasculitis-myoclonus syndrome 10. A second family comprising three male children of two consanguineous couples from Egypt was more recently identified with many phenotypic similarities (Figure 1). Both families were initially referred for evaluation because of atopic dermatitis, recurrent skin and pulmonary infections, and high serum IgE; DOCK8 and STAT3 sequences were found to be wild type.
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.
All affected individuals had atopic dermatitis that was frequently severe, as well as atopic diatheses including asthma, food, drug, and environmental allergies, and elevated serum IgE. Two individuals from Family I (I.1 and I.3) required repeated inpatient wet wrap therapy with topical corticosteroids and emollients; both individuals displayed a marked response to therapy, consistent with that seen in other patients with severe atopic dermatitis (Figure 2A). Those in Family I also developed severe blistering skin disease as toddlers, on the spectrum of Stevens-Johnson syndrome. Because they occurred without identifiable exogenous provocation, we refer to them as erythema multiforme major (Table I). Skin infections were prominent, with recurrent staphylococcal soft tissue infections (SSTI) in all patients, molluscum contagiosum in patient I.3, and flat warts in patient II.1 (Table I). Mild defects in T cell function were also suggested by persistent low-level EBV viremia despite detectable EBV IgG in the older living patients in Family I. This was associated with the development of EBV+ Nodular Sclerosing Hodgkin’s Lymphomas (NSHL) in identical twin patients I.3 and I.4, which were successfully treated with Adriamycin / Bleomycin / Vinblastine / Dacarbazine (ABVD) chemotherapy (Table I).
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).
Table I.
Clinical characteristics of individuals with hypomorphic PGM3 mutations.
| Pt. | Age/Sex | Allergic | Dermatologic | Infectious | Neurologic | Connective Tissue | Pulmonary | Renal | Hematologic | |
|---|---|---|---|---|---|---|---|---|---|---|
| Family I | I.1 | 35yo M | allergic rhinitis | atopic dermatitis, vasculitis, erythema multiforme major | pneumonia, otitis, SSTI | low IQ, dysarthria, ataxia, myoclonus, sensorineural hearing loss, CNS myelination defect, abnormal EEG, prolonged VEP | scoliosis, degenerative disc disease, dilated aortic root | bronchiectasis | MPGN on hemodialysis | polycythemia |
| I.2 | 32yo F | food allergy, allergic rhinitis, drug allergy | atopic dermatitis, vasculitis, erythema multiforme major | EBV viremia, sinopulmonary | low IQ, dysarthria, ataxia, myoclonus, sensorineural hearing loss, CNS myelination defect, seizure, abnl EEG | esophageal stricture | bronchiectasis s/p bilateral lung transplantation | lithiasis | ||
| I.3 | 30yo M | asthma, food allergy | atopic dermatitis, vasculitis, erythema multiforme major | EBV viremia, pneumonia, molluscum, otitis, SSTI | low IQ, dysarthria, ataxia, myoclonus, reduced vibratory sensation, sensorineural hearing loss | scoliosis, esophageal diverticulis | bronchiectasis | Hodgkin’s lymphoma (EBV+) | ||
| I.4 | 30yo M | food allergy, drug allergy | atopic dermatitis, vasculitis, erythema multiforme major | EBV viremia, otitis, SSTI | low IQ, dysarthria, ataxia, myoclonus, sensorineural hearing loss | scoliosis | bronchiectasis | MPGN | Hodgkin’s lymphoma (EBV+) | |
| I.5 | deceased (27yo F) | asthma, allergic rhinitis | atopic dermatitis, eczema herpeticum, erythema multiforme major | HSV esophagitis, otitis, sinopulmonary | ataxia, myoclonus | esophageal stricture | bronchiectasis, chronic failure | |||
| Family II | II.1 | 12yo M | food allergy, allergic rhinitis, drug allergy | atopic dermatitis | SSTI, fungal pneumonia, otitis, sinopulmonary | low IQ, developmental delay, ataxia, CNS myelination defect, meningoencephalopathy, febrile seizure, prolonged VEP | neutropenia (+anti-neutrophil Ab) | |||
| II.2 | 10yo M | allergic rhinitis | atopic dermatitis | flat warts, SSTI, otitis | low IQ, developmental delay, ataxia, CNS myelination defect, autism, febrile seizure | microcephaly, scoliosis | unilateral agenesis | hemolytic anemia, hepatosplenomegaly | ||
| II.3 | 1yo M | food allergy, FPIES | atopic dermatitis | RSV, SSTI, otitis | developmental delay, hypotonia | neutropenia |
FPIES, food protein-induced enterocolitis syndrome; EBV, Epstein-Barr virus; SSTI, recurrent staphylococcal skin and soft tissue infections; sinopulmonary, recurrent sinopulmonary infections; otitis, chronic otitis externa and/or recurrent otitis media; HSV, herpes simplex virus; RSV, respiratory syncytial virus; VEP, visual evoked potentials; MPGN, membranoproliferative glomerulonephritis.
Patients had recurrent pneumonias (n = 6), recurrent acute otitis media (n = 3), chronic otitis externa (n = 6), and chronic sinopulmonary disease (n = 6) (Table I). Most pneumonias were presumed bacterial in origin, although patient II.1 developed a fungal pneumonia after receiving high dose inhaled corticosteroids (Table I). The recurrent infections occurred in spite of elevated serum immunoglobulins and protective antibody titers (Table II). Anti-microbial prophylaxis proved moderately beneficial, but immunoglobulin replacement trials failed to reduce these infections in a clinically significant way. Moderate to severe bronchiectasis developed among older patients, leading to chronic respiratory failure, death of patient I.5 at age 27 years, and bilateral lung transplantation in patient I.3 at age 28 years.
Table II.
Significant immune laboratory findings of individuals with hypomorphic PGM3 mutations.
| Immunoglobulins | CBC | CD3 | CD20 | NK | EBV | RF | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient | IgE IU/mL (0–90) |
IgG mg/dL (759–1,550) |
IgA mg/dL (58–359) |
IgM mg/dL (35–239) |
WBC 103 cells/ µL |
ANC 103cells/ µL |
ALC 103cells/ µL |
AEC 103cells/ µL |
CD4 cells/µL (359–1,565) |
CD8 cells/µL (178–853) |
CD4/CD 8 ratio |
Total cells/µL (61–321) |
CD27+ Memory cells/µL (12–68) |
cells/µL (126–729) |
103 copies/ mL |
(<15 IU/mL) |
|
| Family I | I.1 | 11,200 | 1610–1,770 | 284–547 | 41–74 | 3.1–5.3 | 2.1–3.3 | 0.6–0.9 | 0.1–0.5 | 128 | 129 | 1 | 253 | 7 | 113 | 5.0 | POS |
| I.2 | 5,836–27,200 | 1,510–1,890 | 749–1,660 | 125–176 | 4.8–9.2 | 2.9–7.1 | 0.6–1.1 | 0.2–0.5 | 195–487 | 70–137 | 2.8–3.6 | 31–161 | 7 | 202–270 | 30.0 | POS | |
| I.3 | 4,672–35,400 | 422–1,750 | 515–1,140 | 121–411 | 1.7–5.5 | 0.9–4.4 | 0.2–0.8 | 0.0–1.1 | 93–151 | 11–19 | 8–11 | 100–123** | 2–3** | 15–62 | 2.2 | POS | |
| I.4 | 23,594–35,445 | 673–1,470 | 509–877 | 103–130 | 2.7–4.7 | 1.4–3.5 | 0.3–0.7 | 0.0–0.9 | 150–248 | 22–43 | 5.8–6.8 | 37–140** | 4–10** | 30–131 | 28.2 | POS | |
| I.5 | ND | ND | ND | ND | 11.4–19.3 | 11–12.0 | 0.6–11.4 | 0–3.6 | ND | ND | ND | ND | ND | ND | ND | ND | |
| Family II | II.1 | 1,244–30,766 | 1,080–1,690 | 334–497 | 86–147 | 1.3–5.4 | 0.1–3.3* | 0.7–1.3 | 0–0.4 | 368–692 § | 111–247§ | 2.8–5.1§ | 16–108§ | 1§ | 150–260§ | NEG | NEG |
| II.2 | 5,396–11,405 | 1,660–2,180 | 407–677 | 42–144 | 1.0–3.5 | 0.4–1.7 | 0.4–2.0 | 0–0.2 | 403–878 | 189–295 | 2.1–3.0 | 4–112 | 1 | 160–503 | naive | POS | |
| II.3 | 207 | 554 | 84 | 38 | 1.9–5.5 | 0.7–1.0 | 0.4–2.3 | 0–1.7 | 324 (1,300–4,300) † | 263 (500–2,000) † | 1.23 | 711 (773–1,990) † | 11 (56–172) † | 664 (160–920) † | ND | ND * | |
All values within normal range were obtained while receiving G-CSF;
flow data obtained during acute meningoencephalitis were excluded;
received rituximab historically, prior to complex immunophenotyping; rheumatoid factor (RF) is reported as positive if ever detected in peripheral blood;
normal pediatric values for 1 year old 19, 20
Autoimmune and idiopathic immune-mediated disease was also prevalent. Cutaneous leukocytoclastic vasculitis was present in all surviving patients in Family I, as previously reported. Membranoproliferative glomerulonephritis developed in two of these patients (Table I); one of whom progressed to end-stage renal disease (ESRD) at age 30 years and remains on hemodialysis (HD) after failing deceased-donor renal transplantation (DDRT). Severe autoimmune/idiopathic neutropenia requiring G-CSF (patient II.1), and hemolytic anemia (patient II.2) with osmotic fragility also occurred.
Finally, neurologic impairment was evident from early in life in all patients. Features included developmental delay (n = 7), low IQ (n = 7), ataxia (n = 7), dysarthria (n = 5), sensorineural hearing loss (n = 4), myoclonus (n = 5), and seizures (n = 1). Facial dysmorphism was appreciated in several patients. Baseline electroencephalography (EEG) showed moderate background slowing, poor organization, and a slowed posterior dominant rhythm in two patients. In one patient, voluntary movement induced sharp waves and slowing in the contralateral motor cortex seen on scalp EEG, which were consistent with cortical intention myoclonus15. The neurologic impairment appeared to be associated with diminished myelination, as T2-FLAIR MRI showed stable diffuse hyperintensity on images within the central white matter (n = 4 of 6 patients scanned) (Figure 2B); these findings were stable over time. Furthermore, visual evoked potentials (VEPs) showed prolonged p100 latencies in both eyes (OS 200.2 ms and OD 197.1 ms) at check size 16 (normal 84 -120 ms) (n = 2) (Figure 2B). Somatosensory evoked potentials (SSEPs) also showed delayed central conduction in the same individuals, after stimulation of bilateral upper extremities as well as “giant” SSEPs (peak-to-peak amplitudes of the N20-P22 waveforms approximately 18.7 µV left and 15 µV right). Of note, patient II.1 developed Streptococcus pneumoniae meningoencephalitis; perivenular white matter lesions were seen on MRI following this episode.
Clinical laboratory findings
Patients had significant leukopenia, lymphopenia, and neutropenia, especially in Family II (Table II). Further immunophenotyping showed the lymphopenia to be predominantly due to low CD8+ T cells and CD27+ memory B cells (Table II). Patients had significant elevations in serum IgE, IgG, and IgA (Table II), and protective antibodies to protein and polysaccharide antigens. Of the autoantibodies screened, only rheumatoid factor (RF) was consistently present, occurring in five of seven patients tested (Table II).
Genetic analyses
Because patients of both sexes in Family I were affected but their parents were not, we assumed an autosomal recessive model in searching for causal genetic variants. Whole exome sequencing produced about 80–110 million paired-end reads per exome and provided an average 80–100 fold sequencing coverage of >90% protein-coding regions. About 70,000 high confidence single nucleotide variants (SNVs) and small insertions/deletions (indels) were called from individually sequenced exomes. Variants were flagged if they met the following criteria: 1) they were nonsense, missense, splice site mutations/indels, or frameshift indels, which altered protein-coding sequence; 2) they were novel or rare when compared to several public variation databases including the dbSNP database and the NHLBI exome variant Server; and 3) they were shared by all 4 sequenced patients in the same family. Any gene that was identified to carry two or more such variants was further investigated by filtering against the genotype of those variants in the parents. After this step, PGM3 (Phosphoglucomutase 3, NM_001199917.1, OMIM 172100) emerged as the only possible candidate gene. We identified two novel compound heterozygous mutations in Family 1’s PGM3, which completely segregated with disease: a missense variant (c.1585G>C, p. E529Q) was inherited from the mother; a 5 bp deletion (c.1438_1442del , p. L480Sfs*10), causing a frameshift and premature stop was inherited from the father (Figure 3A). PGM3 genotyping was confirmed by Sanger sequencing.
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.
In Family II, WES was initially performed on patients II.1 and II.2. We used a similar strategy as in Family I for filtering, except that homozygous variants were favored because the parents were consanguineous. After filtering, only 15 rare/novel variants compatible with all the criteria remained. One of these was a missense mutation in PGM3 (c.975T>G, p.D325E, Figure 3A). Sanger sequencing of PGM3 in an affected cousin identified the same homozygous mutation as well as heterozygous carrier status in that patient’s unaffected parents.
Expression of PGM3 variants
PGM3 is a member of the hexose phosphate mutase family and catalyzes the reversible conversion of GlcNAc-6-phosphate (GlcNAc-6-P) to GlcNAc-1-P. This is a key step in the synthesis of UDP-GlcNAc, which is critical for multiple glycosylation pathways including N- and O-linked. The structure of PGM3 and the locations of the mutations in the gene are shown in Figure 3B. Both missense mutations in the patients result in the substitution of highly conserved amino acids (D325E and E529Q, Figure 3C). By analogy to the structure of its closest available homolog, yeast N-acetylglucosamine- phosphate mutase, both missense mutations are proximal to a central active site cavity that is surrounded by active serine, metal binding, sugar binding, and phosphate binding domains (domains 1 to 4, respectively) (Figure 3D). E529 is in the conserved substrate- binding loop of PGM3. Replacement of glutamine with glutamate removes a negatively charged moiety that normally interacts with positively charged K481; disruption of this interaction likely destabilizes a defined substrate-binding conformation within the loop. By contrast, D325 is located at the interface between the metal binding and sugar binding domains, where it is expected to neutralize its net negative charge by interacting with positively charged K328. Replacement of aspartic acid with glutamic acid increases the length of the side-chain, which likely forces rotation of the side-chain into the interdomain region; this would break several critical hydrogen bonds and perturb interdomain regions near the active site that are critical for catalysis. Thus, both missense mutations are expected to be hypomorphic.
To assess whether the mutations affected PGM3 protein levels, we performed immunoblotting using lysates from EBV-transformed B cell lines or cycling T cells derived from patients and controls. PGM3 protein levels were significantly decreased in patients from Family II but were not changed or only slightly decreased in Family I (Figure 3E). qRT-PCR showed normal PGM3 mRNA expression in cells derived from Family II (Figure E1), which further argued for a protein-destabilizing effect of the D325E variant. For Family I, the 5 bp deletion is predicted to result in a truncated protein (L480fs), but no such truncated protein band was detected (Figure 3E). This mutation is also predicted to cause nonsense-mediated decay of transcript, which likely contributes to the mild diminution of PGM3 protein expression (Figure 3E). The mild decrease in protein expression in Family I also suggests that the E529Q protein is expressed and has normal protein stability.
Hypomorphic PGM3 mutations encode proteins with reduced enzymatic activity
To assess the effects of PGM3 mutations, we developed a novel enzymatic assay and tested fibroblasts from three patients by determining the rate of GlcNAc1-P formation from GlcNAc6-P. All patients had substantially decreased PGM3 activity compared to control cells (Figure 4A). As this is an easily reversible reaction, we measured the intracellular amounts of GlcNAc1-P and GlcNAc6-P. Patient fibroblasts had no change in GlcNAc1-P but a significant increase in the amount of GlcNAc6-P (Figure 4B). Conversion of GlcNAc6-P to GlcNAc1-P is a key step in the synthesis of UDP-GlcNAc; thus, we also measured the amount of UDP-GlcNAc and other nucleotide sugars in cell extracts. PGM3-deficient cells showed significant reduction of UDP-GlcNAc and somewhat reduced level of UDP-GalNAc, which is derived from UDP-GlcNAc, but other nucleotide sugars remained normal (Figure 4C). Importantly, GlcNAc supplements in the medium restored depleted UDP-GlcNAc and UDP-GalNAc compared to controls (Figure 4C).
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 TH2 and TH17 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.
Abnormal glycosylation in patients with hypomorphic PGM3 mutations
O-linked glycosylation in 5 patients (2 from Family I, and all affected individuals in Family II) was studied by mass spectrometric analysis of single samples. All individuals showed increased T antigen (Galβ1,3GalNAc), a precursor of sialylated T antigen (Sia2,3 Galβ1,3GalNAc). (Table III). The high T-antigen/Sialyl T-antigen ratio suggests hypo-sialylation, and is consistent with decreased UDP-GlcNAc, a precursor to CMP- Sialic acid. Serum transferrin glycosylation was normal, but analysis of total N-linked glycans showed under-galactosylation of N-linked oligosaccharides in 3 individuals.
Table III.
MALDI-TOF quantification of glycan profiles in patients with hypomorphic PGM3 mutations.
| Patient | O-linked | ||
|---|---|---|---|
| Normal |  |
T antigen Sialyl-T antigen Ratio |
0.22–1.14 mmol/L 11.7–31.4 mmol/L 0–0.06 |
| I.2 |  |
T antigen Sialyl-T antigen Ratio |
3.27 mmol/L 10.9 mmol/L 0.25 |
| I.3 |  |
T antigen Sialyl-T antigen Ratio |
3.82 mmol/L 28.2 mmol/L 0.12 |
| II.1 |  |
T antigen Sialyl-T antigen Ratio |
2.24 mmol/L 14.5 mmol/L 0.13 |
| II.2 |  |
T antigen Sialyl-T antigen Ratio |
5.38 mmol/L 22.8 mmol/L 0.2 |
| II.3 |  |
T antigen Sialyl-T antigen Ratio |
1.54 mmol/L 22.5 mmol/L 0.06 |
MALDI-TOF, Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy; PGM3, phosphoglucomutase 3; T antigen, Hex1HexNAcitol; Sialyl-T antigen Neu5AcHex1HexNAcitol.
Immune abnormalities in the patients
We next assessed various T cell functions including cytokine production and proliferation. Ex vivo stimulation of CD4+ T cells from patients led to a marked increase in TH2 cytokine production compared to normal controls, as indicated by elevated levels of IL-4, IL-5 (Figure 4D and Figure E2), and IL-13 (data not shown). IL-17 production was increased in both families, especially in Family II. By contrast, IFN-γ expression in patients was not significantly different from controls (Figure 4D). While the patients had fewer T cells than controls, measurement of CFSE dilution in SEB-stimulated total PBMCs led to similar or increased proliferation of gated CD4+ and CD8+ cells in patients as compared to normal controls. Similar results were observed upon stimulation of PBMCs by the lectin PHA-P (Figure E3).
DISCUSSION
Whole exome sequencing has proven useful for the unbiased identification of mutations in genes whose functions are not limited to the immune system. We have now discovered a new syndrome of severe atopy, recurrent infections, autoimmunity, vasculitis, renal failure, and lymphoma, associated with motor and neurocognitive impairment. These patients have novel recessive mutations in phosphoglucomutase 3 (PGM3) and show hypo-sialylation of O-linked serum glycans, consistent with impaired PGM3 function. In conjunction with the nine patients identified by Barbouche et al (see accompanying article), a total of 17 patients from six families have now been identified, making a compelling argument for the pathogenicity of these mutations.
PGM3 is required for glycosylation through catalyzing the isomerization of N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate during the generation of UDP-GlcNAc. Consistent with this role, inactivating mutations in the Drosophila homolog nst cause severe defects in mesodermal development 16. Complete loss of Pgm3 is embryonically lethal in mice, while hypomorphic mutations in Pgm3 allow viability 17. Our patients had partial loss of PGM3 function, which allowed them to survive into adulthood, but they still had a distinct phenotype that recapitulated that seen in hypomorphic mice. Both humans and mice had lymphopenia with CD8+ and B cell defects, autoimmunity such as glomerulonephritis, and other hematologic abnormalities. Other phenotypes not originally examined such as atopic disease, high serum IgE, and neurological abnormalities, may well be present in the mouse.
Whereas PGM3 deficiency presented to us with elevated IgE, atopic dermatitis, bronchiectasis, and scoliosis, other clinical features helped to distinguish this disorder from the two best-known diseases with elevated IgE, STAT3 and DOCK8 deficiencies (Table IV). PGM3 deficiency is unassociated with cold abscesses, chronic mucocutaneous candidiasis, retained childhood dentition, and joint hypermobility, which are seen very commonly in patients with STAT3 mutations. Although PGM3-deficient patients occasionally have viral skin infections, they were not as prevalent as in DOCK8 deficiency. By contrast, primary neurocognitive deficits and probable hypomyelination, suggested by the evoked potential findings and consistent with the MRI scans, are seen in PGM3 deficiency but not in either STAT3 or DOCK8 deficiencies. The neurologic features are evocative of other glycosylation defects, such as DPAGT1-CDG (CDG Ij) and MOGS-CDG (CDG-IIb), which are both associated with primary hypomyelination. These overlaps in three distinct glycosylation disorders indicate an important role for glycosylation in normal axonal health 13.
Table IV.
Comparison of conditions with elevated serum IgE versus congenital disorders of glycosylation.
| PGM3 | DOCK8 | STAT3 | Severe atopic dermatitis |
CDGs | |
|---|---|---|---|---|---|
| IgE | +++ | ++ | +++ | +++ | NR |
| Immunoglobulins | high | Low (IgM)/normal/high | low/normal | normal | low/normal |
| T Lymphopenia | ++ | ++ | +/− | - | +/− |
| Atopy | +++ | +/+++ | ++ | +++ | - |
| Neurologic Deficits | +++ | +/- | - | − | +++ |
| EBV viremia | ++ | ++ | + | - | - |
| Cutaneous viral infections | ++ | +++ | + | + | - |
| Lymphoma | ++ | ++ | + | - | - |
| Vascular Abnormality | +/- | + | ++ | - | - |
| Bronchiectasis | ++ | ++ | +++ | - | - |
| Scoliosis | ++ | - | +++ | + | ++ |
| Retained Primary Teeth | - | - | +++ | +/− | - |
| Renal | ++ | - | + | - | +++ |
PGM3, phosphoglucomutase 3; DOCK8, dedicator of cytokinesis 8; STAT3, signal transducer and activator of transcription 3; atopic dermatitis, atopic dermatitis; CDGs, congenital disorders of glycosylation; NR, not reported; +, mild; ++, moderate; +++, severe; -, absent; +/−, reported but uncommon.
In our patients, we also observed abnormal effector cytokine production, which might contribute to their inflammatory and autoimmune symptoms. PGM3-deficient T cells produced excessive TH17 and TH2 cytokines while maintaining normal IFN-γ production (Figure 4D). While excessive TH2 cytokine production may explain the atopic disease and hypergammaglobulinemia, it is unclear whether it contributes to the vasculitis or autoimmune kidney disease. The excessive IL-17 production can be associated with demyelinating diseases, but the diffuse and stable hypomyelination observed in these patients argues against an inflammatory etiology. We have not yet been able to establish an immunologic mechanism to explain the link between glycosylation abnormalities and the immune dysregulation seen in these patients. Because glycosylation is known to be critical for a large number of immune-related proteins, these patients likely have additional abnormalities in numerous other pathways affecting leukocyte development and maturation. Such abnormalities will need to be examined in order to establish the precise mechanism by which these mutations and abnormal glycosylation lead to focal cellular and clinical defects in immune responses. Analysis of these pathways should illuminate how differential protein glycosylation contributes to the basic functioning of many primary immunologic and non-immunologic pathways, including those that lead to severe atopic phenotypes.
Finally, we found that GlcNAc supplementation restored intracellular UDP- GlcNAc levels in PGM3-deficient cells. Thus, exogenous non-diabetogenic sugars might be used to bypass the metabolic defect and treat these patients. A similar therapeutic approach has been taken using oral mannose in patients with MPI-CDG (CDG-Ib), due to mutation in mannosephosphate isomerase 18. Identifying a role for glycosylation events in patients with atopic disease opens new avenues for pharmacological manipulation to treat common allergic diseases.
Supplementary Material
Clinical implications
Our findings define a new clinical entity of atopy, immune deficiency, autoimmunity with neurocognitive impairment in patients associated with diminished PGM3 function.
Capsule summary
This study reports a novel genetic defect leading to a syndrome that includes significant atopic disease and indicates an important role for glycosylated proteins in the pathogenesis of allergic disease.
ACKNOWLEDGEMENTS
We thank the following people for their help: For technical support, helpful discussion, and critically reading the manuscript, Guangping Sun and Qian Zhang; for clinical support, Nina Jones, Celeste Nelson, Michelle O’Brien, Lisa Boris, Dirk Darnell, Cathleen Frein, and Pamela Welch; for neurology evaluations, Irene Cortese, Andrea Gropman, Pascal Sati, Ariane Soldatos, and Brandon Loughridge; for genetic counseling, Joie Davis and Gillian Hooker; for clinical glycosylation studies, Miao He and William Gahl; and the patients and their families for participating in this study. This work was supported in part by Merck Research Laboratories and by the Intramural Research Program of the NIAID, NHGRI, and NINDS, all at the NIH. HHF and MI are supported by R01DK55615 and The Rocket Fund.
Declaration of sources of funding:
This study was supported by the Intramural Research Programs of the NIAID, NHGRI, and NINDS, all at the National Institutes of Health. HHF and MI were supported by R01DK55615 and The Rocket Fund.
Abbreviations used
- ABVD
Adriamycin / Bleomycin / Vinblastine / Dacarbazine
- ADA-SCID
Adenosine deaminase - severe combined immunodeficiency
- CDG
congenital disorder of glycosylation
- dbSNP
The Single Nucleotide Polymorphism Database
- DDRT
deceased-donor renal transplantation
- DOCK8
dedicator of cytokinesis 8
- EBV
Epstein-Barr virus
- EEG
electroencephalography
- ESRD
end-stage renal disease
- GlcNAc
N-acetyl-D-glucosamine
- GlcNAc-6-P
GlcNAc-6-phosphate
- GlcNAc-1-P
GlcNAc-1-phosphate
- GPI
glycosylphosphatidylinositol
- HD
hemodialysis
- HIES
Hyper-IgE syndrome
- IPEX
Immune Dysregulation Polyendocrinopathy Enteropathy X-linked
- MALDI-TOF MS
Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry
- NSHL
Nodular Sclerosing Hodgkin’s Lymphomas
- PBMC
Peripheral blood mononuclear cel
- PGM3
phosphoglucomutase 3
- PHA
phytohemagglutinin
- PMA
phorbol 13-myristate 13-acetate
- qRT-PCR
quantitative reverse transcription polymerase chain reaction
- SEB
Staphylococcal enterotoxin B
- SNV
Single Nucleotide Variant
- SSEP
Somatosensory evoked potentials
- SSTI
staphylococcal soft tissue infections
- STAT3
signal transducer and activator of transcription 3
- TH2
T helper type 2 cells
- TH17
T helper type 17 cells
- UDP-GalNAc
Uridine diphosphate N-acetylgalactosamine
- UDP-GlcNAc
Uridine diphosphate N-acetylglucosamine
- VEP
visual evoked potentials
- WES
whole-exome sequencing
Footnotes
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