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. Author manuscript; available in PMC: 2024 Jan 5.
Published before final editing as: J Med Genet. 2022 Jul 5:jmedgenet-2021-108177. doi: 10.1136/jmedgenet-2021-108177

Clinical, biochemical and genetic characteristics of MOGS-CDG, a rare congenital disorder of glycosylation

Shino Shimada 1,2,#,✉, Bobby G Ng 3,#, Amy L White 4,#, Kim K Nickander 4, Coleman Turgeon 4, Kristen L Liedtke 4, Christina T Lam 5, Esperanza Font-Montgomery 6, Charles M Lourenço 7,8, Miao He 9, Dawn S Peck 4, Luis A Umaña 10, Crescenda L Uhles 11, Devon Haynes 12,*, Patricia G Wheeler 12, Michael J Bamshad 13,14, Deborah A Nickerson 13, Tom Cushing 15, Ryan Gates 16, Natalia Gomez-Ospina 16, Heather M Byers 16; University of Washington Center for Mendelian Genomics**, Fernanda B Scalco 17, Noelia N Martinez 18, Rani Sachdev 18,19, Lacey Smith 20, Annapurna Poduri 20, Stephen Malone 21, Rebekah Harris 22, Ingrid E Scheffer 22,23,24, Sergio D Rosenzweig 25, David R Adams 1,2, William A Gahl 1,2, May CV Malicdan 1,2,26, Kimiyo M Raymond 4,26, Hudson H Freeze 3,26, Lynne A Wolfe 1,2,26,✉
PMCID: PMC9813274  NIHMSID: NIHMS1817980  PMID: 35790351

Abstract

Purpose

To summarize the clinical, molecular and biochemical phenotype of MOGS-CDG, a congenital disorder of glycosylation that presents with variable clinical manifestations, and to analyze which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS.

Methods

Phenotypic characterization was performed through an international and multicenter collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG.

Results

Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurologic, immunologic, and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis, was not consistently abnormal.

Conclusion

The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via MALDI-TOF can be used as a reliable biochemical test for screening and confirmation of disease.

Keywords: Congenital disorders of glycosylation (CDG), MOGS, MOGS-CDG, Urine oligosaccharide, MALDI-TOF MS

INTRODUCTION

Congenital disorders of glycosylation (CDG) are an increasingly recognized group of disorders due to inborn errors of glycosylation. Over 150 types of CDG due to impaired lipid or defective protein glycosylation have been reported. Disorders affecting the N-glycosylation pathway are the most common and can be subdivided into defects that impair lipid-linked oligosaccharide biosynthesis and transfer of this tri-branched glycan to proteins (CDG type I), or those altering processing of protein-bound glycans (CDG type II)1. The first obligate N-glycan processing step occurs in the endoplasmic reticulum (ER) and involves the removal the first of three consecutive glucose (Glc) residues located on one branch, catalyzed by α-glucosidase 1, also known as mannosyl-oligosaccharide glucosidase (MOGS)2 3, and subsequently followed by several steps of glycan processing4

Bi-allelic pathogenic variants in MOGS underlie MOGS-CDG, previously known as CDG-IIb (OMIM;606056), presenting with systemic manifestations and variable severity5–11. Recently, there has been an increasing utilization of next generation sequencing for clinical application, but there is also a corresponding increase in the number of variants of unknown significance (VUS) that could not readily be subjected for functional validation, posing a challenge to clinicians in determining a precise diagnosis. Thus, it is important to define molecular and biochemical tools that can solidify a diagnosis. While the majority of CDGs can be identified through carbohydrate deficient transferrin (CDT) testing in blood, some CDGs, including MOGS-CDG, will not be reliably detected using this methodology5 9 11 12.

Here, we report twelve individuals of MOGS-CDG, doubling the total number of known cases and further delineating the clinical heterogeneity of this multisystem disorder. The wide range of observed symptoms, lack of consistent dysmorphic features, and variable severity of presentation pose a challenge for the specific diagnosis of MOGS-CDG. As a part of diagnostic evaluation, we compared the diagnostic specificity of serum and urine biochemical tests. Overall, we highlight the importance of combining biochemical and molecular testing to provide diagnoses for individuals with MOGS-CDG.

MATERIAL AND METHODS

Subjects and clinical information

Twelve probands from eleven unrelated families with MOGS variants were enrolled from seven clinical centers. Probands P1, P2 and P3 were enrolled in the NIH Undiagnosed Diseases Program 76-HG-0238, “Diagnosis and Treatment of Patients with Inborn Errors of Metabolism or Other Genetic Disorders (http://clinicaltrials.gov, NCT00369421)” (P1 and P2) and NIH protocol 14-HG-0071, “Clinical and basic investigations into known and suspected congenital disorders of glycosylation (http://clinicaltrials.gov, NCT02089789)” (P1, P2 and P3), approved by the National Human Genome Research Institute Institutional Review Board (IRB). P4 was enrolled in Seattle Children’s hospital in IRB protocol STUDY00001831 in 2019. P12 was enrolled in the Epilepsy Genetics Research Program at Austin Health, University of Melbourne. Written informed consent was provided by each proband’s parents.

Molecular analysis of MOGS

Exome/genome and targeted gene panel sequencing were performed at different institutions. All variants were confirmed by Sanger sequencing of DNA from all probands and available family members.

Biochemical Testing

Urine oligosaccharides analysis

Urine oligosaccharides were prepared by permethylation13 and analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A volume of urine equivalent to 0.09 mg creatinine was prepared as described13 for the samples analyzed at Emory (P1, P2 and P3). For the urine samples analyzed at Mayo (P1, P3, P4, P5, P8, P10, P11 and P12), an equivalent volume of urine was applied to Waters Oasis HLB Extraction Cartridges preactivated using 1 mL methanol followed by 1 mL water. The urine specimens were applied to the columns and collected along with a 1 mL water wash. The collected urine with water was applied to preactivated carbograph columns as described by Xia et al13. The eluates were lyophilized overnight, permethylated, then either extracted into chloroform (Emory analysis) as described13 or directly using Waters Oasis HLB Extraction Cartridges (analysis from Mayo). The eluates were evaporated under speed-vac (Emory analysis) or lyophilized overnight (Mayo analysis), then resuspended in 50 µL of 50:50 methanol:water. MALDI plates were prepared as described by Xia et al. and analyzed using a SCIEX 4800 MALDI TOF/TOF (Emory analysis) or SCIEX 5800 MALDI TOF/TOF (Mayo analysis) operated in MS reflector mode across the range of m/z 500 to 3500 with a laser pulse rate of 400Hz, collecting 1000 shots per spectrum.

In P1 and P2, the fragmentation pattern of urine free oligosaccharide was analyzed by MALDI-TOF/TOF13. For sugar composition analysis, urine free oligosaccharides were purified and labeled with 2-aminobenzamide (2-AB) at the reducing end and hydrolyzed by 2M trifluoroacetic acid (TFA)5, The 2-AB labeled monosaccharides were separated by HPLC using the Shimadzu HPLC CBM-20A system, coupled with an online fluorescence detector RF-10Axl (Shimadzu Scientific Instruments, Columbia, MD)5. For sugar composition analysis, urine oligosaccharides were purified and hydrolyzed by 2M TFA. The resultant monosaccharides were filtered using a spin-X column and subjected to high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Dionex ICS-3000 system (Dionex Corporation, Sunnyvale, CA)5 14. In P6 and P9, specifically, urine oligosaccharide was analyzed by HPLC (P6) and by LC-MS/MS with ESI-MS (electrospray mass spectrometry) detection (P9).

RESULTS

Clinical cohort and molecular diagnosis

Twelve individuals (7 males and 5 females with ages 16 months to 20 years) from eleven families are described herein (Supplementary table S1). All affected individuals had bi-allelic pathogenic variants in MOGS (NM_0063012.3), including missense (17), nonsense (6), or frameshift (3) (Table 1), with no obvious hotspot (Figure 1A). Most missense variants are seen in the C-terminal domain (Supplementary figure S1). Homozygous variants in three consanguineous families, one with nonsense variants and two with missense variants (Table 1 and Supplementary table S1) were identified. All variants found in this study were either ultra-rare (with allele frequency (AF) of up to 0.001%), rare (with AF< 0.01%), or absent in gnomAD. These variants were predicted to be pathogenic by several in silico prediction programs and all had combined annotation-dependent depletion (CADD) Phred scores above 20 (Table 1).

Table 1.

Molecular data of MOGS-CDG

1, 2 3 4 5 6 7 8 9 10 11 12
A1 cDNAa c.65C>A c.1787G>A c.2098C>T c.370C>T c.882delT c.1766C>T c.1461G>C c.1619G>A c.2090T>C c.1212_1239 dup c.224A>C
Protein p.Ala22Glu p.Arg596Gln p.Arg700* p.Gln124* p.Glu295Asn fs*10 p.Ser589Leu p.Glu487Asp p.Arg540His p.Leu697Ser p.Asp414Leu fs*17 p.His75Pro
gnomADb 0.0015% 0.0032% 0.0008% 0.0018% 0.0044% NA 0.0008% NA 0.0004% 0.0004% NA
Pathogenicity C:15, S:0.02, P:0.001 C:25, S:0.29, P:1.0 C:35, S:NA, P:NA C:39, S:NA, P:NA C:25, S:NA, P:NA C:28, S:0.01, P:1.0 C:24, S:0, P:1.0 C:29 S:0.01 P:1.0 C:24 S:0.01 P:0.6 C:NA, S:NA, P:NA C:23, S:0.27, P:1.0
A2 cDNAa c.329G>A c.1801C>T c.2098C>T c.1461G>C c.1681C>T c.1766C>T c.1461G>C c.2126T>C c.2090T>C c.1348T>G c.1286–1287del
Protein p.Arg110His p.Arg601* p.Arg700* p.Glu487Asp p.Arg561Cys p.Ser589Leu p.Glu487Asp p.Leu709Pro p.Leu697Ser p.Trp450Gly p.Phe429Ser fs*23
gnomADb NA 0.0008% 0.0008% 0.0008% 0.0016% NA 0.0008% NA 0.0004% NA 0.0081%
Pathogenicity C:32, S:0, P:1.0 C:36, S:NA, P:NA C:35, S:NA, P:NA C:24, S:0, P:1.0 C:31, S:0.01, P:1.0 C:28, S:0.01, P:1.0 C:24, S:0, P:1.0 C:25, S:0, P:0.6 C:24 S:0.01 P:0.6 C:28 S:0, P:1 C:NA, S:NA, P:NA
A3 cDNAa c.370C>T
Protein p.Gln124*
gnomADb 0.0018%
Pathogenicity C:39, S:NA, P:NA
Method of sequencing WES WES WES Target array WES N/A WES WES WES WES WES WES
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Abbreviations: A, Allele; NA, data not available; WES, whole exome sequencing; CADD, Combined Annotation Dependent Depletion (https://cadd.gs.washington.edu/); SIFT, Sorting Intolerant from Tolerant (https://sift.bii.a-star.edu.sg/); Polyphen2 (http://genetics.bwh.harvard.edu/pph2/); gnomAD, Genome Aggregate consortium (https://gnomad.broadinstitute.org/);

a,

based on NM_006302.2

b,

last accessed March 2021.

Figure 1.

Figure 1

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A. Schematic presentation of MOGS and MOGS protein structure. MOGS consists of 4 exons, and the MOGS protein shows different functional domains. Previously reported variants are shown below the structure, and variants identified in this study are above the structure. Newly reported variants are in red.

B. Dysmorphic features of probands with MOGS variants. Proband 1 (P1) at age 20 (a), proband 2 (P2) at age 15 (b and c), proband 3 (P3) at age 11 (d), proband 8 (P8) at age 4 (e and f) and proband 11 (P11) at 1.5 month of age (g). Both P1 and P2 had broad nasal tip and retrognathia (a and b). P2 had 2nd and 3rd toe syndactyly with overlapping toes (c). P3, P8 and P11 had broad nasal tip, smooth philtrum, downturned corner of mouth and retrognathia (d, e, f, g).

C. Clinical summary for 12 probands with bi-allelic MOGS variants. Note all probands showed developmental delay, dysmorphic features, hypogammaglobulinemia. The denominator reflects the availability of clinical information. AST, aspartate aminotransferase.

Clinical features

We reviewed clinical records for 12 individuals with bi-allelic MOGS variants. All individuals showed developmental delay, hypotonia, dysmorphic features and hypogammaglobulinemia.

Dysmorphic features

Most individuals with MOGS variants had hypotonic facial muscles with broad nasal tip, retrognathia, and finger or toe deformities (e.g., clenched overlapping fingers, 5th finger clinodactyly or brachydactyly, camptodactyly, overlapping toes) (Figure 1B and Supplementary table S1). Pierre-Robin sequence (micrognathia, cleft palate and glossoptosis, and airway blockade) was identified in P4, necessitating respiratory support in the neonatal period.

Neurological and ophthalmological findings

Most probands (8 of 12 individuals; 68%) had focal seizures, tonic-clonic seizures or epileptic spasms seizures, with onset between the neonatal period (day 2) to early infancy with ages (6 months) (Supplementary table S1). Abnormal electroencephalogram (EEG) patterns included a burst-suppression pattern (P3 and P12), hypsarrhythmia (P6), and brief rhythmic discharges compatible with subclinical seizures at two months of age (P4). Sural nerve biopsy in P1 showed mild axonal degeneration with regeneration. P1, P2 and P3 had intermittently elevated CK levels. Ten probands were examined for ophthalmologic features, and most (10 of 11 cases; 91%) had abnormalities including optic nerve atrophy, cortical visual impairment (CVI), refractive errors, nystagmus, astigmatism, esotropia or hypertropia (Supplementary table S1)7. Sensorineural hearing impairment was seen in seven probands (Supplementary table S1)7. P3 and P9 had bradycardia, possibly reflecting brainstem dysfunction. P3 suffered severe episodes of asystole.

Brain imaging

Brain magnetic resonance imaging (MRI) was available in twelve probands from two weeks to 20 years of age. Seven (P1, P2, P3, P4, P6, P9 and P11) had non-specific brain MRI findings, including thin corpus callosum, cortical atrophy with enlarged ventricles, diminished white matter and delayed myelination (Table 2 and Supplementary figure S2)7. Magnetic resonance spectroscopy (MRS) in four individuals revealed decreased NAA (N-acetylaspartate) at different ages and in different areas (pons, cerebellar, subcortical) (Table 2 and Supplementary figure S2A). P4 had striking rapidly progressive atrophy (brainstem and cortex) and neuronal migration abnormality (periventricular heterotopia) detected at age six months (Supplementary figure S2B). Normal brain images were confirmed in three probands. Diffusion-weighted imaging (DWI) in P12 showed restricted diffusion involving the thalamus (ventral thalami), midbrain (red nucleus) and cerebellum (superior cerebellar peduncle and dentate nucleus) when the proband presented with seizures at 2 weeks of age (Supplementary figure S2C).

Table 2.

Neuroradiological features of MOGS-CDG.

Proband 1 2 3 4 5 6 7 8 9 10 11 12
Age of examination 15 years 10 months 10 years 11 months 11 years 1 month 6 months 30 months NA NA NA 9 months 5 months 1.5 months 2 weeks
 MRI
 Cerebral atrophy		 + + + + - + - - + - + -
Enlarged ventricles + + + + - + - - + - + -
Abnormal corpus callosum + + + - - + - - + - - -
White matter atrophy + + + + - + - - + - NA -
Delayed myelination + + NA + - + - - + Borderline + -
High signal intensity lesion of white matter + + + - - + - - - - - -
 MRS
 NAA		 Decreased (G, P, C) Decreased Decreased (G, P) NA NA Normal NA NA NA NA NA Slightly Decreased
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Abbreviations: MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NA, data not available; G, gray matter; P, pons; C, cerebellar region.

Skeletal and dental findings

Most probands with skeletal system evaluation had abnormalities (67%; 6 of 9 cases) (Figure 2 A–E and Supplementary table S1). Although data were limited, osteopenia was the most common finding. Additionally, P1 showed advanced bone age and partial pseudoepiphysial formation at 10 years. In P2 and P3, bone age was within the normal range with diffuse osteopenia (Figure 2A). P1, P2 and P3 had long bones with widened epiphysis and narrow metaphysis (Figure 2B and C and D). P4 showed delayed ossification of the femoral head at 19 months of age, suggesting delayed bone development (Figure 2C and D). P1, the only proband who acquired the ability to walk with support, had a history of recurrent bone fractures7. He received replacement therapy for Vitamin D deficiency and calcium supplementation for secondary hyperparathyroidism with elevated osteocalcin (60.9 ng/ml; reference, 9–40 ng/ml); insulin-like growth hormone-1 (IGF-1) was within the normal range7. Urine NTX-telopeptides, which are released during bone resorption, were also normal. Bone histopathological findings showed mineralization defect, osteopenia, high bone turnover, and prominent hyperosteoidosis. P4 had a congenital bell-shaped thorax (Figure 2B) and was suspected to have an osteoclast defect such as osteopetrosis because of his dense skull base and occipital bones, abnormal widening of the diaphysis with a permeative pattern (Figure 2C) and delayed dental eruptions. Other findings included pars interarticularis defects (P1) that imply stress fracture of the bones of the lower spine, an elongated spine (P4, P11) and rhizomelic shortening of the limbs in P11 (Figure 2B, 2C and 2E).

Figure 2. Radiographs of skeletal findings in individuals with MOGS-CDG.

Figure 2.

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A. Radiographs of hands of proband 1 (P1) at age 10 (1) and 20 (2), proband 2 (P2) at age 10, proband 3 (P3) at age 11 and proband 11 (P11) at 1.5 months of age. Note osteopenia in P1, P2 and P3, partial pseudo-epiphysial formation P1 (1) and shortened metacarpals (P11).

B. Radiographs of upper extremities of P2 at age 10, P3 at age 11, P4 at 1 month of age (1 and 2) and P11 at 1.5 months of age, radiograph of thorax of P4 at 1 month (3). Note thin diaphysis and osteopenia in P2 and P3, bilateral ulnar deviation at the wrist in P4, and bell-shaped thorax.

C. Radiographs of lower extremities of P1 at age 15, P2 at age 10, P3 at age 11, P4 at 1 month of age and P11 at 1.5 months of age. Note thin diaphysis in P1, P2 and P3 especially in the fibulas (P2), bowing femur (P1), metaphyseal flaring (P2, P3 and P11) and widening of the diaphysis with a permeative pattern, consistent with expansion of the intramedullary spaces (P4).

D. Radiographs of pelvis in P3 at age 11, and P4 at age 1 month (1) and 19 months (2). Note protrusion acetabuli (P3) and deformity of femoral head and pelvis (P4).

E. Radiographs of lateral lumbar spine in P1 at age 11, P4 at 1 month of age and P11 at 1.5 months of age. Note the Pars interarticularis defects at the L5 in P1 and elongated spine in P2.

Cardiorespiratory, gastrointestinal, endocrine, reproductive and kidney phenotype

Fifty-eight percent (7 of 12 probands) showed cardiovascular structural abnormalities, including atrial septal defect (ASD), patent ductus arteriosus (PDA), patent foramen ovale (PFO), thickened left ventricle wall, and dilated aortic root (Supplementary table S1). Detailed respiratory and gastrointestinal system evaluations were available in 11 individuals. Hypoventilation with apnea was seen in 58% (7 of 12 probands), attributable to either central causes (central sleep apnea), subclinical seizures, aspiration, restrictive chest wall, or laryngomalacia. Hepatomegaly was noted in 45% (5 of 11 probands), while elevated liver transaminases were present in 64% (7 of 11 probands) (Supplementary table S1). P4 had gut dysmotility with a history of gastrointestinal bleeding at the ages 5, 8 and 17 months (PT; 16.8 sec, APTT; 52 sec) and P5 had a history of liver thrombosis with a negative coagulation evaluation. In addition, aminoaciduria (n=4), congenital hypothyroidism (n=1), hyperinsulinemia (n=2), premature adrenarche (n=3), hypoplastic genitalia (n=1) and hydronephrosis (n=3) were seen (Supplementary table S1).

Immunological features

All 9 probands in whom data were available showed variable degrees of hypogammaglobulinemia with low IgA and/or IgG, IgM and IgE. Immunoglobulin levels also fluctuated in these patients, and two probands (P3 and P9) had normal and abnormal levels at different timepoints. Recurrent infections (viral, in P3, P7; bacterial in P4) were identified, and poor wound healing was seen in one (P5) (Supplementary table S1). Of note, P1 had an episode of severe pneumococcal pneumonia with empyema (no previous vaccination), with residual bronchiectasis on chest CT scan7. P3 had recurrent viral infections, on occasion requiring ventilatory assistance; respiratory syncytial virus (RSV) was identified at ages of 7m, 16m and 5y4m. P11 had a Pneumocystis jiroveci pneumonia (PJP) with Serratia marcescens colonization.

The clinical features of the twelve probands including the previously reported siblings (P1 and P2)7 are detailed in Figure 1B, Figure 2, Table 2, Supplementary figure S2, Supplementary table S1, and summarized in Figure 1C.

Biochemical analysis

Various biochemical tests were performed to support the diagnosis of MOGS-CDG, including serum CDT (ESI-MS or MALDI-TOF), isoelectric focusing (IEF), analysis of serum/plasma total N-glycan (MALDI-TOF), and urine oligosaccharides analysis (MALDI-TOF MS, HPLC or LC-MS/MS). Overall, serum CDT in several probands was not consistently abnormal. Nine had normal CDT, whereas only 2 of 11 probands (P5 and P7) had abnormal CDT detected via ESI-MS or IEF (Table 3). Five showed abnormal total serum/plasma N-glycans (MALDI-TOF MS), including one glycan with a proposed structure of Glc3Man7GlcNAc2. Three had markedly abnormal glycans, and two had borderline total glycans (Table 3 and Supplementary figure S3).

Table 3.

Biochemical findings of MOGS-CDG.

Biochemical findings
Proband 1 2 3 4 5 6 7 8 9 10 11 12
CDT (serum) Normal Normal Normal Normal Abnormal Normal Abnormal Normal Normal Normal Normal Normal
Method ESI-MS and MALDI-TOF ESI-MS and MALDI-TOF ESI-MS and MALDI-TOF ESI-MS and MALDI-TOF ESI-MS IEF ESI-MS ESI-MS and MALDI-TOF ESI-MS ESI-MS and MALDI-TOF ESI-MS HPLC
Total N-glycan (Serum/ plasma) Normal Abnormal Normal Borderline Abnormal NA Abnormal Borderline NA Normal NA NA
Method MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF NA MALDI-TOF MALDI-TOF MALDI-TOF NA NA
IgG N-glycan (Plasma) Abnormal Abnormal NA NA NA NA NA NA NA NA NA NA
Method MALDI-TOF MALDI-TOF MALDI-TOF NA NA NA NA NA NA NA NA NA
Urine oligosaccharides Abnormal Abnormal Abnormal Abnormal Abnormal Abnormal NA Abnormal Abnormal Abnormal Abnormal Abnormal
Method TLC and MALDI-TOF TLC and MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF TLC and HPLC NA MALDI-TOF ESI-MS MALDI-TOF ESI-MS and MALDI-TOF MALDI-TOF
Fibroblast Abnormal Abnormal NA NA NA NA NA NA NA NA NA NA
Method MALDI-TOF MALDI-TOF
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Abbreviations: CDT, carbohydrate deficient transferrin; ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; NA, data not available; TLC, thin-layer chromatography.

Urine oligosaccharide profiles were abnormal in all probands who had urine analysis (P7 not tested). MALDI-TOF analysis revealed a characteristic diagnostic profile for MOGS-CDG. A species corresponding to the peak at m/z 885 was consistently elevated, with the molecular weight and fragmentation pattern of an oligosaccharide with four hexoses (Hex4) (Figure 3B) and consistent with the Hex4 described previously5 11. In addition to the peak at m/z 885, there was an accompanying peak at m/z 1742 (Figure 3A) in the MOGS-CDG profile from MALDI-TOF (Fig 3A), which is proposed to be Hex7GlcNAc. The 885 m/z peak was also seen in Pompe disease, which is due to the deficiency of the enzyme acid-α-glucosidase leading to (Glc4) free oligosaccharides, but Pompe disease urine profiles are different from MOGS-CDG in that, many larger glucose polymers are also elevated (Fig 3A). Sugar composition analysis (P1 and P2) using reducing end fluorescence confirmed the reducing sugar was mannose rather than glucose (Figure 3C). In total, sugar composition analysis showed markedly increased glucose and mannose with a glucose/mannose ratio of 3:1 (Glc3Man) (Figure 3D).

Figure 3. Urine oligosaccharide profile.

Figure 3.

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A. MALDI-TOF MS glycan profile in urine. Glycan profiles of urine oligosaccharide from control, P3 and P4. MALDI-TOF MS profile demonstrates a consistently larger than normal peak at 885 m/z (P3 and P4).

B. MALDI-TOF/TOF analysis in urine oligosaccharide. Representative profile of urine oligosaccharides from MOG-CDG proband (P1). Fragmentation pattern of the compound was done by using MALDI TOF/TOF to fragment 885 m/z. The m/z of Y1, B2, Y2, B1 and Y3 fragments of Hexose 4 are: 259.1, 445.2, 463.2, 649.2, 667.2. Mannose is shown as green and glucose as blue.

C. Urine oligosaccharide fluorescent labeling and HPLC. The fluorescence chromatograph of 2-aminobenzimide (2-AB) labeled reducing end monosaccharide. Standards are glucose (light blue), mannose (pink), and polyglucose (green). Urine oligosaccharides from a proband diagnosed with Pompe disease are in brown, and MOGS-CDG (P1) is in blue. Note the retention time of reducing end mono-saccharides from polyglucose, and urinary oligosaccharides of a known Pompe proband are consistent with that of the glucose standard, while the retention time of the reducing end mono-saccharide from our MOGS-CDG probands is the same as the mannose standard. Fluorescence (330 nm excitation; 420 nm emission) was used to detect the 2-AB derivatives. 2-AB, 2-aminobenzamide; TFA, trifluoroacetic acid.

D. HPAEC-PAD component analysis of urine monosaccharides. Analysis shows chromatograms from normal control and proband with MOGS-CDG using Dionex ICS-3000 with a Carbpac PA-10 column. The eluent gradient was set to 2.5–125 mM sodium acetate over 100 minutes in 100 mM sodium hydroxide. The levels of both glucose and mannose are increased in the patient’s urine post TFA hydrolysis. The ratio of fluorescence intensity between Peak 4 (glucose) and Peak 5 (mannose) is close to 3.0. Small amounts of galactosamine (GalNH2), glucosamine (GlcNH2), and galactose (Gal), are seen as components of normal urinary oligosaccharides.

In P6, abnormal urine oligosaccharide analysis by TLC showed a prominent band in Hex4, and TLC pattern was different from that seen in Pompe disease, which usually includes another Glc polymer such as Hex7. The Hex4 peak was estimated by HPLC to be 254.73 mmol per mol creatinine (reference, 1.4–9.7 mmol per mol creatinine). In P9, the abnormal elevation of a tetrasaccharide distinct from glucose tetrasaccharide (Glc4) was identified by LC-MS/MS with ESI-MS detection. The overall biochemical findings are summarized in Table 3.

DISCUSSION

Detailed clinical investigations into our MOGS-CDG proband cohort revealed clinical heterogeneity that included a diverse range of systemic manifestations. Skeletal features included short stature and osteopenia. Other types of CDG have been associated with short stature, including disorders of O-glycosylation defects15 16 involving several glycosylation pathways17 and disorders of N-glycosylation18. In addition, most MOGS-CDG probands have a high arched palate or cleft palate with retrognathia. Micrognathia, as a part of a Pierre Robin sequence in P4, might be the most severe phenotype, and can result from defects in different pathways, including those involving cranial neural crest cells (CNCCs), cell metabolism, regulation of bone remodeling, bone and cartilage development, and/or neuromuscular function.

Osteopenia is a common clinical finding in CDGs19, including PMM2-CDG, ALG12-CDG, TMEM165-CDG and ATP6V0A2-CDG19–22. Markedly reduced bone density was a characteristic finding, especially in older probands, indicating bone fragility and the risk for bone fractures7 12. Osteopenia could result from defective control of remodeling, involving imbalance of bone forming osteoblasts and bone resorbing osteoclasts despite increased osteoblastic activity, as suggested from the histopathology findings in P1 and the characteristic findings of defective osteoclasts in P4. In mice, N-glycosylation is required for the optimal function of a variety of transporters, receptors and carbohydrate-binding molecules23 24; the non-collagen SIBLING (small integrin-binding ligand, N-linked glycoprotein family), for example, is believed to play key biological roles in the mineralization of bone and dentin and suspected to be mutated in CDG19 25. Although the mechanism of skeletal abnormalities in our probands requires further study, we note that angiopoietin-like protein 2 (ANGPTL2), a glycoprotein regarded as a pro-inflammatory cytokine, has been shown to mitigate osteoclast production and bone loss through regulation of precursor proliferation and inflammation26, suggesting that pro-inflammatory cytokines might contribute to CDG associated bone abnormalities.

All individuals in our cohort had neurological findings and variable manifestations of MOGS-related encephalopathy and developmental impairment. Many individuals also had optic nerve atrophy, malformed optic nerves, CVI, and brainstem dysfunction, including central apnea and bradycardia with asystole due to brainstem dysfunction. CVI has also been associated with defective GPI-anchor synthesis (PIGA, PIGN, PIGT and PGAP1)27 and multiple other pathways (SRD5A3-CDG and DPM1-CDG). Seven individuals had a developmental and epileptic encephalopathy (DEE) characterized by frequent epileptiform activity with developmental regression or plateauing28. EEG demonstrated typical DEE findings such as suppression-burst (P3 and P12), hypsarrhythmia (P6) and frequent multifocal epileptiform discharges as previously described5 9–11. Seizure onset occured early, with seven probands having neonatal onset5 9–11 (Supplementary table S1). Neonatal-onset DEE has been reported in individuals with genetic disorders of voltage-gated ion channels and ligand-gated ion channels encoded by KCNQ2, SCN2A, KCNT1, GABRA1, GABRB2 or GABRB3, among a range of other molecular mechanisms29 30. In the neonatal period the activation/deactivation of voltage-gated ion channels is tightly regulated; as most channel proteins contain sialylated N-glycans, N-glycosylation abnormalities may disrupt the function and change the balance of voltage-gated ion channels31–33.

Neuroradiological findings (MRI/MRS) included periventricular heterotopia, delayed myelination, enlarged ventricles, brainstem atrophy and decreased NAA (cortical, white matter and brainstem). These findings suggest that the neuronal migration phenotype, which is also seen in GPI anchor disorders or dystroglycanopathies33, is not common in the MOGS-CDG cohort. Severe deficiency of MOGS could disrupt neuronal migration during early development of the fetal brain. Recently, diffuse gliosis of cerebral white matter and enlarged cytoplasmic organelles in neuronal cells were reported as the pathological findings of a MOGS-CDG case, suggesting that the neuronal inflammation could be due to metabolic abnormalities11. Symmetrical restricted diffusion in the dentato-rubro-thalamic tract was identified in association with neonatal seizure episodes in P12, which may suggest that this finding is related to metabolic abnormalities or epilepsy in MOGS-CDG.

From both literature review and our proband experience, liver problems are common in CDG. Chronic transaminitis, which occurs in most CDG types, usually abates by 5 years of age with occasional fluctuations during illness34. In our cohort, hepatomegaly was noted in 45% (5/11), and one proband had liver steatosis without transaminitis. Considering that hepatomegaly was observed in most of the cases reported, our results suggest that liver dysfunction is prominent in MOGS-CDG5–11 35.

In terms of the immune system, hypogammaglobulinemia due to decreased immunoglobulin half-life (as production and antibody function was mostly preserved7), is a common finding with variable severity. Some probands show only low IgA or fluctuating hypogammaglobulinemia (Supplementary table S1) although glycosylation changes of IgG may still present. While N-glycosylation defects in MOGS-CDG have been shown to reduce susceptibility to infection to particular viruses that undergo protein glycosylation (e.g., HIV or influenza), this concept might not apply to all N-glycosylated viruses7 36. On the other hand, reduced IgG level and altered IgG glycosylation can increase the risk of infection in general. From our investigation, only four (P3, P4, P7 and P12) individuals for which information was provided, had experienced recurrent respiratory infections that were likely viral. It is possible that the viral infections in these individuals were due to viruses that do not undergo protein glycosylation10–12. Review of literature shows that, on average, a child has four to eight episodes of respiratory infections per year37–39, while infants and young children have a few respiratory infections per year, especially if they have only limited contacts. If the frequency of viral respiratory tract infection is considered, P9 (at 23 months) and P10 (at 16 months) may have some resistance to viral infections, as reported for P1 and P27. Further studies using fibroblasts from additional probands identified in this cohort will be needed to confirm these findings. Interestingly, poor wound healing in P5 and recognition of a PJP infection in P11 among our cohort, suggests that other areas of the immune system beyond the B-cell compartment (i.e., innate or T-cell immunity) might also be affected in MOGS-CDG patients.

Our findings also highlight the importance of molecular analysis, which can expedite diagnosis in individuals suspected to have CDG, especially in combination with biochemical testing. The variants in MOGS in this study, including those that were previously reported, are largely found in the regions that encode catalytic domains, with no obvious hotspots. The six truncating variants (p.Gln124*, p.Arg601*, p.Arg700*, p.Glu295Asnfs*10, p.Asp414Leufs*17*, p.Phe429Serfs*23) are likely to be targeted for nonsense-mediated decay if not resulting to the removal of the catalytic domain (glycosyl hydrolase family 63) that hydrolyses the glycosidic bond, eliminating catalytic activity. All missense variants but two (p.Ala22Glu, p.His75Pro) were located in regions that encode a catalytic domain (Glycosyl hydrolase family 63) (Figure 1A). The prediction protein structure showed that there were no obvious hotspots, but most of variants were scattered in two catalytic domains, with more variants in the C-terminal domain (Supplementary figure S1). These variants in the C-terminal domain appear to be on the same plane, suggesting that these amino acid residues are important to the proper positioning of the enzyme for substrate recognition at the surface (Supplementary figure S1). In probands 1 and 2, variants p.Ala22Glu, along with another variant p.Arg110His, are both paternally inherited. It is interesting to note based on predicted structure and topology that p.Ala22Glu is not located in the catalytic domain and is in the cytoplasmic region; thus, it is likely that the reduced enzyme activity observed in probands 1 and 2 is mainly influenced by p.Arg110His (Supplementary figure S1). Regarding another variant p.His75Pro, which is close enough to the catalytic domain, we hypothesize that its proximity might influence catalytic activity.

Looking at the location and severity of MOGS variants, genotype-phenotype correlation is not apparent, except in two circumstances. First, P4 with homozygous nonsense variants had the most severe phenotype, likely due to loss of function in the enzyme’s catalytic domain. As a complete loss of MOGS is reportedly embryonic lethal in mice40, we hypothesize that a complete deletion of all MOGS enzymatic activity in humans would also be lethal. In the case of P4 with the homozygous p.Arg700* variant, it is likely that the truncated protein still produces some residual catalytic activity. Second, attenuating factors, like mitotic recombination in P1 and P2, might have contributed to the seemingly milder clinical presentation41. Further studies will be needed to define genotype-phenotype and genotype-phenotype-biochemical correlations; future studies could include analysis of genetic variants in animal or cell models, in vitro analysis of enzyme activities, and determination of clinical severity using a validated rating scale in studies focusing on the natural history of MOGS-CDG.

Overall, biochemical analysis is important in establishing the diagnosis of MOGS-CDG. CDT testing in blood is often the initial step in a diagnostic evaluation for CDG. CDT can be performed on serum specimens via IEF or electrospray ionization mass spectrometry (ESI-MS), which is more sensitive and has advantages over IEF. While CDT analysis did detect MOGS-CDG in 2 of 11 individuals, it is not reliable for diagnosis as we confirmed (Table 3) and as previously suggested7. Therefore, CDT analysis alone can lead to a missed diagnosis of MOGS-CDG in clinical practice due to a normal finding. Total serum/plasma N-glycans via MALDI-TOF is better in terms of diagnosis (63%, 5 of 8 individuals; Table 3 and Supplementary figure S3), but it is still not consistently abnormal. Urine oligosaccharides measured by TLC, HPLC or MALDI-TOF were abnormal in all probands (100%, 12 of 12 individuals tested). MALDI-TOF MS-based urine oligosaccharide analysis has the highest sensitivity and specificity for detection of MOGS-CDG, however, whereas the other urine oligosaccharide test methods (TLC, HPLC) will yield nonspecific abnormal results. It is important to note that urine oligosaccharides via MALDI-TOF is the appropriate biochemical test to screen for or confirm a diagnosis of MOGS-CDG. Given the overlapping clinical phenotypes of CDG type I, II, or mixed type I and II, comprehensive biochemical testing for CDG should include urine oligosaccharides in addition to CDT.

In summary, we present clinical, molecular, and biochemical data that characterize the MOGS-CDG probands in this case series. Our findings expand the phenotype, describe the clinical heterogeneity in MOGS-CDG, and contribute to the understanding of the natural history of the disease, which will be important in exploring potential therapeutic options. Importantly, our results underscore the identification of a characteristic abnormal urine oligosaccharide profile via MALDI-TOF that could be used as a reliable biochemical screening test.

Supplementary Material

Supp1. Supplemental figure S1. Predicted structure of human MOGS.

The AlphaFold structure predictions of human MOGS were visualized with the PyMOL program to highlight the conserved Glycosyl hydrolase 63 family. The N-terminal domain is depicted in magenta, and the C-terminal domain is depicted in orange. The pink dots show the variants’ locations. The variant p.His75Pro is not located in the Glycosyl hydrolase family 63 domains, but it is close to it.

Supp2. Supplemental figure S2. Representative brain images in probands with MOGS-CDG.

A. Brain MRI and MRS in probands 1, 2 and 3. Proband 1 (P1) at age 20 (1–2). Proband 2 (P2) at age 15 (5–6). Proband 3 (P3) at age 11 (9–10). Sagittal T1 weighted images show thin corpus callosum (1, 5, 9). T2 axial weighted images show cortical atrophy, enlarged ventricles (2, 6, 10) and high signal intensity in decreased white matter. MRS shows mild decreased NAA in pons (4, 12) and cerebellar region (7). Axial MRI images show right posterior plagiocephaly (11).

B. Brain MRI findings in proband 4 (P4). Images obtained at the age of 1 month (1–3) and 6 months (4–6). Sagittal T1-weighted images show thin corpus callosum and progressive brainstem atrophy (1, arrow in 4). Progressive cortical atrophy and delayed myelination were seen. At the age of 6 months, myelination is seen only in the anterior and posterior limb of internal capsule (arrow in 5), which is equivalent to 3 months age pattern. Periventricular heterotopia was also seen (arrow in 6). Images shown are axial T2 FLAIR (3), T2-weighed (6) and T1-axial (2, 5).

C. Brain MRI findings in proband 12 (P12). Images obtained at the age of 2 weeks (1–2). Axial diffusion-weighted image (DWI) shows hyperintense signal involving the red nucleus (arrow in 1) with corresponding signal changes on apparent diffusion coefficient (ADC) (arrow in 2).

Supp3. Supplemental figure S3. MALDI-TOF total serum N-glycan profile.

MALDI-TOF profiles of PNGase released serum glycans (control, P5 and P7) yield an abnormal peak at 2600 m/z, with a proposed structure of Glc3Man7GlcNAc2. Subsequent CID (2 kV) TOT/TOF analysis for the precursor ion 2600 m/z is shown with the proposed fragments for Glc3Man7GlcNAc2 annotated. Blue square, N-acetylglucosamine; blue circle, glucose; green circle, mannose; yellow circle, galactose; pink diamond, sialic acid; red triangle, fructose; m/z ratio, mass to charge ratio.

Supp4
Supp5

Key messages.

What is already known on this topic –

In a clinical practice, serum carbohydrate deficient transferrin (CDT) is considered a common biochemical test in congenital disorders of glycosylation, which includes MOGS-CDG. Our results show that CDT analysis alone can lead to a missed diagnosis of MOGS-CDG, hence there is a need for an additional biochemical assay for diagnosis.

What this study adds –

Based on our study, we propose that urine MALDI-TOF MS-based urine oligosaccharide analysis is the most reliable biochemical assay for the diagnosis of MOGS-CDG. We also encourage that such results should be interpreted by experts in the field.

How this study might affect research, practice or policy –

With today’s increasing use of next-generation sequencing for clinical application, clinical geneticists and researchers are confronted with the interpretation of variants of unknown significance (VUS) in candidate genes for rare disorders, including MOGS. Biochemical confirmation, i.e., the use of urine oligosaccharide assay, is necessary in interpreting these VUS, and establishing the diagnosis of MOGS-CDG.

ACKNOWLEDGMENTS

The authors would like to thank the proband families for their cooperation. We thank Dr. Michael Marble for clinical data collection (Department of Pediatrics, UNM Health Sciences Center and Division of Pediatric Genetics). Sequencing was, in part, provided by the University of Washington Center for Mendelian Genomics (UW-CMG).

FUNDING

This research was supported by the Intramural Research Program of the National Human Genome Research Institute and the Common Fund of the NIH Office of the Director. The Freeze Lab was supported by The Rocket Fund, and NIH R01DK99551. Dr. Shimada was partly supported by the JSPS Research fellowship for Japanese Biomedical and Behavior Research. Dr. Raymond, Dr. He and Dr. Freeze are partially supported by the NINDS/NCATS Frontiers in Congenital Disorders of Glycosylation Grant (1U54NS115198-01). Dr Scheffer is supported by the National Health and Medical Research Council of Australia. University of Washington Center for Mendelian Genomics (UW-CMG) was funded by NHGRI and NHLBI grants UM1 HG006493 and U24 HG008956.

** Consortium collaborators

Michael J. Bamshad, Deborah A. Nickerson, Peter Anderson, Tamara J. Bacus, Elizabeth E. Blue, Katherine Brower, Kati J. Buckingham, Jessica X. Chong, Colleen P. Davis, Chayna J. Davis, Christian D. Frazar, Katherine Gomeztagle-Burgess, William W. Gordon, Martha Horike-Pyne, Jameson R. Hurless, Gail P. Jarvik, Eric Johanson, J. Thomas Kolar, Colby T. Marvin, Sean McGee, Daniel J. McGoldrick, Betselote Mekonnen, Patrick M. Nielsen, Karynne Patterson, Aparna Radhakrishnan, Matthew A. Richardson, Gwendolin T. Roote, Erica L. Ryke, Kathryn M. Shively, Joshua D. Smith, Monica Tackett, Jeffrey M. Weiss, Marsha M. Wheeler, Qian Yi, and Xiaohong Zhang.

Footnotes

ETHNICS APPROVAL STATEMENT

This study involves human participants and was approved by the institutional review boards at National Human Genome Research Institute Institutional Review Board (IRB), Seattle Children’s hospital and Epilepsy Genetics Research Program at Austin Health, University of Melbourne. Written informed consent was provided by each proband’s parents.

COMPETING INTERESTS

The authors declare no competing interests.

DATA AVAILABILITY

All data in the paper are included in the Results Section, and in the Supplemental Data. Additional anonymized data will be shared upon request and after execution of Data Sharing Agreement that is mutually acceptable to both parties. All requests should be addressed to the corresponding authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp1. Supplemental figure S1. Predicted structure of human MOGS.

The AlphaFold structure predictions of human MOGS were visualized with the PyMOL program to highlight the conserved Glycosyl hydrolase 63 family. The N-terminal domain is depicted in magenta, and the C-terminal domain is depicted in orange. The pink dots show the variants’ locations. The variant p.His75Pro is not located in the Glycosyl hydrolase family 63 domains, but it is close to it.

NIHMS1817980-supplement-Supp1.pdf (218.3KB, pdf)
Supp2. Supplemental figure S2. Representative brain images in probands with MOGS-CDG.

A. Brain MRI and MRS in probands 1, 2 and 3. Proband 1 (P1) at age 20 (1–2). Proband 2 (P2) at age 15 (5–6). Proband 3 (P3) at age 11 (9–10). Sagittal T1 weighted images show thin corpus callosum (1, 5, 9). T2 axial weighted images show cortical atrophy, enlarged ventricles (2, 6, 10) and high signal intensity in decreased white matter. MRS shows mild decreased NAA in pons (4, 12) and cerebellar region (7). Axial MRI images show right posterior plagiocephaly (11).

B. Brain MRI findings in proband 4 (P4). Images obtained at the age of 1 month (1–3) and 6 months (4–6). Sagittal T1-weighted images show thin corpus callosum and progressive brainstem atrophy (1, arrow in 4). Progressive cortical atrophy and delayed myelination were seen. At the age of 6 months, myelination is seen only in the anterior and posterior limb of internal capsule (arrow in 5), which is equivalent to 3 months age pattern. Periventricular heterotopia was also seen (arrow in 6). Images shown are axial T2 FLAIR (3), T2-weighed (6) and T1-axial (2, 5).

C. Brain MRI findings in proband 12 (P12). Images obtained at the age of 2 weeks (1–2). Axial diffusion-weighted image (DWI) shows hyperintense signal involving the red nucleus (arrow in 1) with corresponding signal changes on apparent diffusion coefficient (ADC) (arrow in 2).

NIHMS1817980-supplement-Supp2.pdf (1.1MB, pdf)
Supp3. Supplemental figure S3. MALDI-TOF total serum N-glycan profile.

MALDI-TOF profiles of PNGase released serum glycans (control, P5 and P7) yield an abnormal peak at 2600 m/z, with a proposed structure of Glc3Man7GlcNAc2. Subsequent CID (2 kV) TOT/TOF analysis for the precursor ion 2600 m/z is shown with the proposed fragments for Glc3Man7GlcNAc2 annotated. Blue square, N-acetylglucosamine; blue circle, glucose; green circle, mannose; yellow circle, galactose; pink diamond, sialic acid; red triangle, fructose; m/z ratio, mass to charge ratio.

NIHMS1817980-supplement-Supp3.pdf (248.2KB, pdf)
Supp4
NIHMS1817980-supplement-Supp4.pdf (117KB, pdf)
Supp5
NIHMS1817980-supplement-Supp5.pdf (131.5KB, pdf)

Data Availability Statement

All data in the paper are included in the Results Section, and in the Supplemental Data. Additional anonymized data will be shared upon request and after execution of Data Sharing Agreement that is mutually acceptable to both parties. All requests should be addressed to the corresponding authors.

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