Mutations in MAGT1 lead to a glycosylation disorderwith a variable phenotypeEline Blommaerta, Romain Péannea, Natalia A. Cherepanovab, Daisy Rymenc, Frederik Staelsd, Jaak Jaekene,Valérie Racea, Liesbeth Keldermansa, Erika Souchea, Anniek Corveleyna, Rebecca Sparkesf, Kaustuv Bhattacharyag,Christine Devalckh, Rik Schrijversd, François Foulquieri, Reid Gilmoreb, and Gert Matthijsa,1

aLaboratory for Molecular Diagnosis, Center for Human Genetics, KU Leuven, 3000 Leuven, Belgium; bDepartment of Biochemistry and MolecularPharmacology, University of Massachusetts Medical School, Worcester, MA 01655; cDivision of Metabolic Diseases, University Children’s Hospital, 8032Zürich, Switzerland; dKU Leuven Department of Microbiology, Immunology and Transplantation, Allergy and Clinical Immunology Research Group, KULeuven, 3000 Leuven, Belgium; eDepartment of Pediatrics, Center for Metabolic Diseases, KU Leuven, 3000 Leuven, Belgium; fAlberta Children’s Hospital,Calgary, AB T3B 6A8, Canada; gGenetic Metabolic Disorders Service, Children’s Hospital Westmead Clinical School, University of Sydney, NSW 2145Westmead, Australia; hDepartment of Hemato-Oncology, Hôpital Universitaire Des Enfants Reine Fabiola, Université Libre de Bruxelles, 1020 Brussels,Belgium; and iUnité de Glycobiologie Structurale et Fonctionnelle, University Lille, CNRS, UMR 8576, F-59000 Lille, France

Edited by Randy Schekman, University of California, Berkeley, CA, and approved March 7, 2019 (received for review October 18, 2018)

Congenital disorders of glycosylation (CDG) are a group of raremetabolic diseases, due to impaired protein and lipid glycosylation.We identified two patients with defective serum transferrin glycosyl-ation and mutations in the MAGT1 gene. These patients presentwith a phenotype that is mainly characterized by intellectual anddevelopmental disability. MAGT1 has been described to be a subunitof the oligosaccharyltransferase (OST) complex and more specificallyof the STT3B complex. However, it was also claimed that MAGT1 is amagnesium (Mg2+) transporter. So far, patients with mutations inMAGT1 were linked to a primary immunodeficiency, characterizedby chronic EBV infections attributed to a Mg2+ homeostasis defect(XMEN). We compared the clinical and cellular phenotype of our twopatients to that of an XMEN patient that we recently identified. Allthree patients have an N-glycosylation defect, as was shown by thestudy of different substrates, such as GLUT1 and SHBG, demonstratingthat the posttranslational glycosylation carried out by the STT3B com-plex is dysfunctional in all three patients. Moreover, MAGT1 deficiencyis associated with an enhanced expression of TUSC3, the homologprotein of MAGT1, pointing toward a compensatory mechanism.Hence, we delineate MAGT1-CDG as a disorder associated with twodifferent clinical phenotypes caused by defects in glycosylation.

congenital disorders of glycosylation | CDG | XMEN |oligosaccharyltransferase complex

Congenital disorders of glycosylation (CDG) are a rapidlygrowing group of genetic diseases caused by defects in glycan

synthesis, processing, and/or attachment. Glycosylation is animportant co- and posttranslational modification of proteins andlipids, mediating their function, stability, and dynamics (1, 2). Inthe N-glycosylation of proteins, the lipid-linked oligosaccharide(LLO) is first built in the endoplasmic reticulum (ER) and sub-sequently transferred en bloc by the oligosaccharyltransferase (OST)complex from a lipidic dolichol carrier to an N-X-S/T residue of anascent protein. Next, remodeling of the glycan structure continuesin the Golgi apparatus (3). Patients with CDG show an extremelyvariable phenotype, ranging from intellectual disability (ID) to severemultiorgan failure and death (1).Indispensable in this meticulously orchestrated glycosylation

machinery is the transfer of glycans by the OST, a multisubunitprotein complex consisting of a catalytic subunit (STT3A or STT3B),six shared subunits, and complex specific accessory subunits (4). Thetwo complexes have distinct roles: STT3A is associated with theprotein translocation channel and acts in a cotranslational fashion,while sites that are missed by STT3A can be posttranslationally gly-cosylated by STT3B (5). This interplay ensures the full N-glycosylationof proteins in mammalian cells. Both have accessory proteinsthat are specific for each of the catalytic subunits: DC2 and KCP2are indispensable for STT3A function (6), while STT3B requireseither MAGT1 or TUSC3 (7, 8). These two mutually exclusive

paralogues share 66% amino acid sequence identity and are orthologsto the yeast OST subunits Ost6 and Ost3 (9). Both proteins possess athioredoxin fold in the luminal domain, which has been de-scribed to be necessary for the glycosylation of STT3B substratesthat are bracketed by disulfides. Remarkably, MAGT1 is also re-quired for full STT3B glycosylation in an oxidoreductase-independentmanner (7, 9). MAGT1 and TUSC3 are physically associated withSTT3B, as was shown by native coimmunoprecipitation (7).Intriguingly, MAGT1 has been swayed back and forth over the

past years between the role as a subunit of the ER-localizedOST, or as a magnesium (Mg2+) transporter, located at the plasmamembrane. Indeed, MAGT1 was described to be required for Mg2+

uptake by vertebrate cells (10, 11). Mutations in this gene havebeen described to cause XMEN (X-linked immunodeficiency withmagnesium defect, Epstein–Barr virus infection and neoplasia)(12). T lymphocytes of these patients displayed altered kinetics ofMg2+ influx, although the cellular levels of Mg2+ remained normal.The link between MAGT1 and glycosylation has not been assessedin these patients.Here we describe three patients with pathogenic mutations in

MAGT1. We demonstrate that MAGT1 deficiency causes a gly-cosylation defect and that the consequences of the MAGT1 muta-tions can be very broad. We studied two patients with a differentclinical phenotype that is mostly characterized by intellectual and

Significance

MAGT1 is a controversial protein that has been described as anendoplasmic reticulum (ER) localized subunit of the oligosaccharyl-transferase (OST) complex involved in the posttranslational transferof glycans onto proteins, but also as a magnesium (Mg2+) trans-porter at the plasma membrane. So far, mutations in MAGT1 havebeen associated with Mg2+ defects causing an immunodeficiency.We demonstrate that MAGT1-deficient patients have a defect inglycosylation, and, in addition, we describe a different phenotypefor the disorder. These results confirm the presumed role ofMAGT1as a subunit of the OST.

Author contributions: E.B., R.P., N.A.C., and R.G. designed research; E.B., F.S., and L.K.performed research; E.B., D.R., F.S., J.J., V.R., E.S., A.C., R. Sparkes, K.B., C.D., R. Schrijvers,and R.G. analyzed data; A.C. supervised diagnostic investigations; G.M. supervised re-search; and E.B., R.P., N.A.C., F.F., R.G., and G.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Pathogenic variant data related to this paper have been deposited inClinVar (accession nos. SCV000898467.1, SCV000898468.1, and SCV000898469.1).1To whom correspondence should be addressed. Email: gert.matthijs@kuleuven.be.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1817815116/-/DCSupplemental.

Published online April 29, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1817815116 PNAS | May 14, 2019 | vol. 116 | no. 20 | 9865–9870

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developmental disability. The third reported patient in this studyhas a MAGT1 mutation and the clinical phenotype of XMEN.

ResultsClinical Data and Mutation Analysis.We identified three male patientswith mutations in the X-linked gene MAGT1. Patient 1 (P1) andpatient 2 (P2) were referred for metabolic screening because ofdevelopmental disability. This work-up revealed an abnormal(“type 1”) serum capillary zone electrophoresis and serum trans-ferrin isoelectric focusing (sTf IEF) in both patients. Patient 3 (P3)was diagnosed with a primary immunodeficiency disorder (PID).The clinical data of the patients are summarized in Table 1.P1 and P2 received a molecular diagnosis after the use of our

in-house CDG gene panel. Both boys were diagnosed with ahemizygous mutation inMAGT1 (NM_032121.5). P1 has a c.1068A >C mutation encoding a p.Lys356Asn. The mutation is not present ingnomAD nor in any other population database such as dbSNP, 1000Genomes, and the ESP database. The nucleotide change affects a verywell-conserved amino acid (AA) (SI Appendix, Fig. S1B). In addition,different prediction programs estimate that this variant is damaging.Furthermore, skewing analysis revealed that the mothers’ Xchromosome containing the mutation is almost fully skewed(Table 1). All these genetic data imply the causal nature of themissense mutation in P1.P2 has a de novo hemizygous loss-of-function mutation in

MAGT1: c.991C > T, p.Arg331*.A molecular diagnosis was reached for P3, after the use of a tar-

geted panel covering immune genes. A hemizygous nonsense muta-tion c.938T >G, pLeu313* was found inMAGT1, which classifies P3as an XMEN patient. In addition, P3 NK cells have reduced NKG2Dsteady state levels (SI Appendix, Fig. S2), which has previously beenlinked to the pathophysiology of XMEN disorder (13). Both loss-of-function mutations are not present in the aforementioned populationdatabases. Interestingly, P3 also has a sTf IEF type 1 pattern.

Analysis of MAGT1. In humans, MAGT1 is localized on the longarm of the X chromosome, at position Xq21.1. For the func-tional analysis of the mutations we selected, as described in theMaterial and Methods, two transcripts, MAGT1-204 (RefSeqNM_032121.5) and MAGT1-205, that respectively encode a 367-and 335-AA protein (SI Appendix, Fig. S1A). In-silico predictionmodels (CBS Prediction Servers; www.cbs.dtu.dk/services/FeatureP/)suggest that the 335-AA isoform harbors four transmembrane (TM)domains, and the 367-AA isoform has one more. The sequence ofthe shorter transcript is identical to the MAGT1-204 form, as thetranslation initiation site is found further upstream. It is importantto note that efficiency of initiation is strongly dependent on the

consensus sequence surrounding the start site (14). The first AUG inMAGT1-204 has a very poor context for translation. It is more likelythat the second AUG will be used as translation initiation site, henceresulting in the exact same 335-AA product as the MAGT1-205transcript. In addition, there is also no evidence for MAGT1 doubletson Western blots. In summary, the 335 isoform is most likely theonly functional MAGT1 isoform (SI Appendix, Fig. S1C). Analysisof gene expression in the GTEx database (www.gtexportal.org)shows that MAGT1 is expressed in all studied tissues and organs.To look at the impact of the mutations, the relative expression levels

of MAGT1 in patient-derived fibroblasts were assessed (Fig. 1A). InP1, relativeMAGT1 transcript expression levels remained similar to theones observed in controls. However, P2 fibroblasts showed a 75% re-duction of MAGT1 transcript. That decrease was even more dramaticin P3, where only 6% residual MAGT1 transcript could be observed.An additional EBV-transformed lymphocyte cell line was available forP3, where about a 50% decrease of MAGT1 transcript could be seencompared with controls. This resource was unfortunately notavailable for P1 and P2. MAGT1 steady state levels were similarin P1 compared with control cell lines, hence suggesting that thep.Lys356Asn mutation does not affect MAGT1 stability (Fig. 1B).In P2 and P3, we observed an (almost) complete absence of theprotein, confirming the loss-of-function nature of these mutations.

Mutations Alter the Expression Levels of TUSC3. MAGT1 has beendescribed as a subunit of the OST STT3B complex (7). Therefore,we studied the protein steady state levels of the catalytic subunits ofthe OST (STT3A and STT3B) and TUSC3, the homolog protein ofMAGT1 (Fig. 2). These studies were performed in patient fibro-blasts, in EBV-transformed lymphocytes, and in CRISPR/Cas9knockout (KO) HEK293 cell lines. These cells were designed to notexpress either STT3A, STT3B, MAGT1, or TUSC3 (15).The expression levels of STT3B were variable in the different

patient cell lines, but no significant differences were observed.Similar results were obtained for STT3A, except for P3 lymphocytes,in which a twofold increase in expression levels was observed. Re-markably, the assessment of steady state levels showed a markedincrease of TUSC3 in all three patient fibroblast cells, which pointstoward a compensatory mechanism. Importantly, in both the controland patient lymphocytes there was no expression of TUSC3 (Fig. 2).To frame the observations in patient-derived cells, steady state

levels of the different subunits were assessed in KO HEK293cells. The observed TUSC3 up-regulation in patient fibroblasts wasalso confirmed in the MAGT1−/− cell line. In summary, these resultsshow that the mutations do not affect the stability of the catalyticsubunits of the OST complex and that there is a strong enhancementof TUSC3 steady state levels in patient fibroblasts.

Table 1. Clinical and molecular summary of patients

Characteristics Patient 1 Patient 2 Patient 3

Age at evaluation (years) 13 11 17cDNA change c.1068A > C c.991C > T c.938T > GProtein change p.Lys356Asn p.Arg331* p.Leu313*Inheritance Maternal transmission De novo Maternal transmissionSkewed X inactivation Yes (98%) NA Yes (100%)Intellectual/developmental disability Yes Yes NoBehavior abnormalities Yes NT NoFacial dysmorphism Mild Mild NoHepatomegaly No Yes NoImmunological phenotype

CA EBV infection No No YesOther infections No No YesCD4:CD8 ratio NT NT NormalCD4 counts Normal NT Decreased

Serum transferrin IEF Type 1: 2-sialo form: 5.0%(normal range: 0–2.6)

Type 1 Type 1: 2-sialo form: 7.0%(normal range: 0–2.6)

CA EBV, chronic active Epstein Barr virus; IEF, isoelectric focusing; NA, not applicable; NT, not tested.

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STT3B-Dependent Substrates Are Hypoglycosylated in Patient Cells.The glycosylation of glycoproteins was studied directly in patient-derived fibroblasts to evaluate whether the MAGT1 mutationshave an effect on the glycosylation of STT3B or STT3A substrates.Patients’ and control fibroblasts were transfected with either sexhormone binding globulin (SHBG), preprocathepsin C (pCatC), orpreprosaposin (pSap) expression vectors for 24 h followed by pulse-chase labeling (Fig. 3A).Pulse-chase labeling of transfected fibroblasts and HEK293

cells showed a mild, but significant (P < 0.05), hypoglycosylationpattern of SHBG in all patient cell lines (Fig. 3B). SHBG is awell-studied and characterized reporter of STT3B glycosylationand has two C-terminal glycosylation sites (N380RS and N396GT)(Fig. 3A). C-terminal sites are often skipped by the STT3Acomplex and are required to be glycosylated by STT3B (16). In addi-tion, the reduction of the SHBG 2-glycan form by roughly 50% com-pared with controls and the 1.5-fold increase of the 1-glycan formconfirm that the glycosylation of SHBG is affected in patient fibroblasts(Fig. 3C).Next, we studied pCatC (Fig. 3A). This substrate has four N-

glycosylation sites, of which one is a MAGT1 substrate (N29CT)(5). Pulse-chase labeling of pCatC revealed an increase in theglycoform carrying three glycans in P1 and P3 cells (Fig. 3C), but nodifference in average total glycan count was observed (Fig. 3B). Thedefect was much less obvious in the P2 cells, possibly due to a very lowincorporation of 35S label in this cell line. Therefore, this patient wasnot included in the calculation of the different pCatC glycoforms.Third, the glycosylation of pSap was assessed. pSap has five N-

glycosylation sequons that are glycosylated exclusively by the STT3Acomplex (5) (Fig. 3A). We therefore used pSap as a negative control.Pulse-chase labeling showed no hypoglycosylation defects, except inthe STT3A−/− cell line (Fig. 3B). This confirms that our patients donot have a general glycosylation defect, but are only deficient in theSTT3B-dependent glycosylation.The patient fibroblasts show a tendency for an STT3B glyco-

sylation defect in both pCatC and SHBG, but the reduction inthe average glycan number was not as strong as in STT3B−/− orMAGT1−/−TUSC3−/− cells. This is in line with reports showingthat enhanced expression of TUSC3 mitigates the MAGT1 de-fect (15). Indeed, our results suggest that the marked increase inTUSC3 steady state levels observed in patient cell lines (Fig. 2)compensates for the MAGT1 defect in patients.

MAGT1 Mutations Are Pathogenic in the Absence of TUSC3. To assesswhether the patients’ variants are pathogenic, the wild-type(WT) MAGT1-205 cDNA sequence (SI Appendix, Fig. S1) wasmutagenized to harbor the corresponding point mutations. HEK293MAGT1−/−TUSC3−/− cells were cotransfected with SHBG and thedifferent MAGT1 expression vectors. The double-KO cell line waschosen to circumvent the issue of the increased expression ofTUSC3 in the MAGT1−/− cells. Pulse-chase labeling showed a re-duction of 70% in the average glycan number of the SHBG reporter

protein in MAGT1−/−TUSC3−/− cells compared with WT cells.Complemented with the WT construct, the average number ofglycans doubled (Fig. 4A). None of the MAGT1 cDNA se-quences carrying the patients’ mutations was able to improve theglycosylation of SHBG, thereby indicating that these mutationsare impairing the STT3B-mediated glycosylation. The sameresults were observed for complementation with theMAGT1-204WT and mutagenized cDNA (SI Appendix, Fig. S3).Next, the glycosylation status of two endogenous substrates,

pCatC and glucose transporter 1 (GLUT1), was studied in lympho-cytes as these cells do not express TUSC3 (Fig. 2). Quantitativeglycoproteomics data and pulse-labeling experiments indicatedthat the N45QT site in the GLUT1 glucose transporter is an STT3Bsubstrate (Fig. 4D). Metabolic pulse-chase labeling showed amarked hypoglycosylation pattern in P3 lymphocytes, with a de-crease in the abundance of the fully glycosylated form of pCatC andan increase in the abundance of the band form carrying three andtwo glycans. Interestingly, GLUT1 is completely hypoglycosylatedin P3 lymphocytes, with no visible protein carrying one glycan (Fig.4B). Both results were similar to the glycosylation pattern observed inHEK293 MAGT1−/−TUSC3−/− and STT3B−/− cell lines. Takentogether with the data obtained in fibroblasts, these results suggestthat STT3B-dependent glycosylation is impaired in the XMENpatient (P3).Moreover, pCatC is known to be first synthesized as a pro-

enzyme and then processed into the mature cathepsin C (CatC).As a clear hypoglycosylation defect was demonstrated for pCatCin P3 lymphocytes, the steady state levels of endogenous pCatCwere assessed to determine the effect of the glycosylation de-ficiency on the stable expression and the maturation of CatC. Avariable glycosylation defect was observed in the three investigatedpatients (Fig. 4C). In P1, a strong hypoglycosylation defect could beobserved, as the upper band (four glycans) is completely absent. InP2 and P3, an increase of the hypoglycosylated form (three glycans)is found (Fig. 4E). In P3 lymphocytes this defect becomes morepronounced, with barely any fully glycosylated pCatC. Also, theamount of pCatC is reduced in the patient cell lines. The steadystate levels of the mature CatC were also severely reduced in P1 andP2 (Fig. 4F). In HEK293 cells, used as control, a hypoglycosylatedband appeared for MAGT1−/−TUSC3−/− and STT3B−/− cell lines.

DiscussionWe report an additional type of CDG caused by mutations in theX-linked gene MAGT1. Since numerous studies uncovered its

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Fig. 1. Expression levels of MAGT1. (A) Relative transcript levels of MAGT1and (B) protein immunoblotting for MAGT1 in fibroblasts and EBV-transformedlymphocytes. β-Tubulin was used as a loading control. Values below the cor-responding lane represent averaged normalized values (n = 3). Error bars rep-resent SE of mean (SEM). *P < 0.05.

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role as a subunit of the OST complex, MAGT1 has been a candi-date CDG gene (7, 15). It was proposed that MAGT1 or TUSC3,two mutually exclusive homologs, are specific accessory proteins ofthe STT3B subunit. They are crucial for the proper glycosylation ofSTT3B substrates due to their oxidoreductase activity (7, 8, 15). Onthe other hand, it was claimed that MAGT1 is a Mg2+ transporter,indispensable for Mg2+ uptake in vertebrate cells (10, 11). So far,mutations in MAGT1 have been associated with XMEN, an im-munodeficiency characterized by chronic active EBV infection (12),

but no glycosylation assays were performed in these patients. It wasproposed that the defect in Mg2+ transport leads to a deficient ac-tivation of T lymphocytes and natural killer (NK) cells (13).Here we describe two MAGT1-CDG patients with a pheno-

type that is mainly characterized by intellectual and develop-mental disability. In addition, we report a third patient with theclinical phenotype of XMEN. We showed that in P1 the missensemutation does not affect MAGT1 steady state levels. In sharpcontrast is the complete absence of MAGT1 in P2 and P3, both

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Fig. 4. Glycosylation assessment of different substrates. (A) Assessment of SHBG glycosylation. MAGT1−/− TUSC3−/− cells were cotransfected with SHBG andthe indicated MAGT1 transcripts. (B) Metabolic labeling of the endogenous pCatC and GLUT1 in HEK293 cells and EBV-transformed lymphocytes. (C) HEK293cells, primary fibroblasts, and EBV-transformed lymphocytes were analyzed for protein steady state levels of pCatC and CatC. β-Tubulin was used as a loadingcontrol. (D) Diagram showing the STT3B-dependent glycosylation site of GLUT1. The signal sequence is depicted in black. (E) Relative abundance of the 4- and3-glycans form for pCatC. (F) Relative steady state levels of pCatC and CatC in primary fibroblasts and EBV-transformed lymphoctyes. CatC was not quantifiable inHEK293 cells. Quantified values are shown below gel lanes and represent the average number of glycans for the respective reporter (n= 3). EH indicates endoglycosidaseH treatment. Error bars represent SEM. *P < 0.05.

9868 | www.pnas.org/cgi/doi/10.1073/pnas.1817815116 Blommaert et al.

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harboring stop mutations. Moreover, we demonstrated that theexpression levels of TUSC3 are elevated in the patients’ fibro-blasts. This observation is restricted to fibroblasts, as lymphocytesdo not express TUSC3. The molecular mechanism(s) by whichTUSC3 is stabilized in response to a defect in MAGT1 are notelucidated yet. It was proposed that, in HEK293 cells, TUSC3 maybe degraded because it does not compete efficiently with MAGT1for incorporation in the STT3B complex (15). We hypothesize thata similar mechanism may occur in patients’ fibroblasts, explainingthe increased levels of TUSC3.Most importantly, we show that the glycosylation of specific

STT3B substrates is altered in all three patient-derived cell lines.SHBG and pCatC were hypoglycosylated to a mild extent inpatients’ fibroblasts, thereby confirming the previously describedrole of MAGT1 as an important subunit of the STT3B complex (7).This was reinforced by the fact that the glycosylation of pSap, anSTT3A substrate, was not altered in MAGT1-deficient cells.Due to the enhanced expression of TUSC3 that can mitigate

the loss of the MAGT1 function, we confirmed the pathogenicityof the different mutations in MAGT1−/−TUSC3−/− cells. MAGT1cDNA constructs carrying the various mutations reported in thepatients were, contrarily to the WT sequence, not able to rescue thehypoglycosylation of the SHBG reporter. Moreover, the examina-tion of EBV-transformed lymphocytes showed a glycosylation de-fect of two STT3B substrates (GLUT1, pCatC) in patient-derivedcells. The lack of MAGT1 cannot be restored by TUSC3 in thesecells, and the glycosylation defect associated with mutations inMAGT1 becomes much more pronounced than in fibroblasts andcomparable to the STT3B−/− HEK293 cell line. As such, we wereable to confirm the role of MAGT1 as a subunit of the OST inpatient cells harboring mutations in MAGT1. Also, we demonstratethat EBV-transformed lymphocytes are the ideal patient-derivedcellular model to assess MAGT1 mutations. In addition, by assess-ing steady state levels of pCatC it could be observed that the muta-tions lead to different levels of hypoglycosylation.The missense mutation (p.Lys356Asn) in P1 does not affect

the steady state protein expression. However, Lys356 and Glu268form a salt bridge in MAGT1 that stabilizes a TM span, and wehypothesize that in P1 the AA substitution may destabilize thatTM span (17). It seems that in general, MAGT1 is preferablyincorporated over TUSC3 (15). Thus, the fact that MAGT1 isstill stably expressed (but not functioning) might prevent TUSC3to take over. On the other hand, P3 has a complete lack ofMAGT1. In tissues that express TUSC3, the glycosylation deficiencycan be bypassed by the latter. In other cells, such as lymphocytes,where TUSC3 is not expressed, the mutation becomes pathogenic.This may explain why XMEN patients in general only have an im-munodeficiency. This hypothesis was confirmed by the study ofthe steady state levels of CatC, where P1 showed a stronghypoglycosylation effect of the pCatC and, in addition, decreasedlevels of matured CatC even though TUSC3 is expressed in fi-broblasts. In P3 the defect was only partial in fibroblasts, but fullyobservable in the lymphocytes where the levels of the processedCatC were also lowered.It is important to note the phenotypical difference between

our MAGT1 patients and the previously reported ones. The twoMAGT1-CDG patients we report here are mainly characterizedby ID and developmental delay. On the other hand, patients withmutations in MAGT1 present with a PID that is characterized byCD4 lymphopenia and chronic active EBV infection (12). Neitherone of our MAGT1-CDG patients has a history of infections.Remarkably, in a family with five affected males, patients with anintronic mutation in MAGT1 and a mutation in ATRX were di-agnosed with ID and skin manifestations (18). The former wasattributed to ATRX, a protein that has been linked to ID onmultiple occasions, while the skin manifestations were linked toMAGT1. We wonder whether the ID could also be attributed tothe mutations in MAGT1, as is the case in our patients. It wasalso claimed that MAGT1 deficiency causes ID in a family with amissense mutation (19). This variant (c.1028C > T; pVal311Gly)was later marked as “highly questionable” and retracted, as it was

present in population databases (20). According to our analysisbased on the American College of Medical Genetics and Genomics(ACMG) guidelines (21), we would also qualify this variant as likelybenign. An assay to study the glycosylation would have to be performedto conclude on its pathogenicity.The question arises whether MAGT1-CDG and XMEN rep-

resent two different phenotypes of a same clinical entity, orwhether these are different disorders? Nonetheless, we were ableto evidence glycosylation defects in cells derived from ourXMEN patient (P3). This suggests that XMEN is a glycosylationdisorder characterized mainly by an immunological phenotype.We favor the hypothesis describing that the defect in Mg2+ ho-meostasis occurs by an indirect mechanism involving the STT3B-dependent glycosylation of a protein involved in Mg2+ transport(22). We have attempted to assess Mg2+ fluxes, by following andadapting the protocol from Li et al. (12), but we have not beenable to overcome the nonspecific reaction of MagFluo4 to Mg2+

and Ca2+ in CTL T cells (SI Appendix, Fig. S4). A question wouldthen be why the defect in P1 and P2 shows no immunologicalinvolvement? We cannot exclude the possibility that the patientswill not present with an immunodeficiency after infection withEBV, as the onset of disease also varies in the XMEN patient.Likewise, the interplay of TUSC3 and MAGT1 is not well un-derstood, particularly in human tissues where TUSC3 and MAGT1are expressed at different levels. A better understanding of the in-corporation of these proteins into STT3B complexes in human tis-sues may be the key that will explain the different clinicalpresentation of the two ID patients compared with the XMENpatient. In addition, the previously reported STT3B-CDG patientpresented with a severe congenital and developmental disorder, butnot with immunological issues (23). Interestingly, this patient alsohad a mild type 1 sTf IEF pattern.In summary, we identified three disease-causing mutations in

MAGT1. Two of these mutations are responsible for a distinctphenotype, characterized by ID and developmental delay. All ofthese patients show anomalies in sTf IEF and the STT3B-dependent glycosylation, classifying these diseases caused bymutations in MAGT1 in the broad group of CDG. Further re-search still needs to be conducted to confirm whether otherXMEN patients also present with aberrant glycosylation.

Materials and MethodsEthics Statement. Research on patients’ DNA and cells was approved by theEthical Committee of the University Hospital of Leuven (approval nos. S59377,S58358, and S58466). All human subjects in this study provided informed consent.

Cell Lines. CRISPR/Cas9-engineered HEK293 WT cells, depleted for eitherSTT3A, STT3B, MAGT1, or TUSC3 were previously described (15). Primary fi-broblasts from patients and controls were grown from a skin biopsy. EBV-transformed lymphocytes were derived from patient and control individuals.All cell lines were cultured in DMEM/F12 (Life Technologies) supplementedwith 10% FBS (Clone III, HyClones) at 37 °C under 5% CO2.

Capillary Zone Electrophoresis and Isoelectric Focusing of Serum Transferrin.Capillary zone electrophoresis and isoelectric focusing of serum transferrinwere performed as previously described (24).

Gene Panel. For P1 and P2, libraries were prepared from genomic DNA withthe Illumina TruSeq DNA sample preparation kit and enriched for 79glycosylation-related genes using a custom in-solution targeted assay(NimbleGen SeqCap EZ kit; Roche). For P3, the library was prepared with theKAPA High-Throughput Library Preparation Kit before enrichment for 290immune-related genes using another custom in-solution targeted assay(NimbleGen SeqCap EZ kit; Roche). The enriched libraries were paired endsequenced on MiSeq (150 bp) or HiSeq2500 (100 or 125 bp, Illumina). Theresulting reads were mapped to the reference genome hg19 using BWA, andvariants were detected with GATK HaplotypeCaller after duplicate removal,realignment around indels, and base quality score recalibration.

X-Inactivation Assay. Genomic DNA, derived from lymphocytes, was subjectedto the androgen receptor (MIM# 313700) assay to determine X-inactivationratios, as previously described in ref. 25.

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RNA Extraction and Real-Time Quantitative PCR. Total RNA was isolated fromthe different cell lines with RNeasyMini kit (Qiagen). DNase treatment (RocheDiagnostics) was performed. Subsequently, 2 μg of purified total RNA wassubjected to reverse transcription with the First-Strand cDNA synthesis kit(GE Healthcare).

PCRs were performed forMAGT1 (NM_032121), as well as HPRT (NM_000194)and GAPDH (NM_002046), which were used as an endogenous control fornormalization. PCR primers were designed using the primer-BLAST softwarefrom NCBI and synthesized by Integrated DNA Technologies. PCR primersequences will be provided on request. PCRs were performed using the 2×LightCycler 480 SYBR Green I Master. Data were analyzed using the Light-Cycler 480 Software (both Roche Applied Science). The comparative thresholdcycle method described by Pfaffl was used to quantify the results (26).

Expression Vectors. The different MAGT1 expression vectors were synthesizedby e-Zyvec. Four protein-coding transcripts are described in Ensembl:MAGT1-201, -202, -203, and -204. Considering the lack of homology between MAGT1-203 and the yeast ortholog Ost3, we excluded this isoform.

Both MAGT1-201 and -204 encode the same 367-AA protein. For thosetwo transcripts, we decided to choose the transcript MAGT1-204, in accor-dance with RefSeq. MAGT1-205 only differs from MAGT1-204 by shorter 3′and 5′ UTRs. These two transcripts were used for the design of the WTMAGT1 expression vectors. Each one of the patient mutations were introducedin these vectors by site-directed mutagenesis. The expression vectors for SHBG(27), pCatC-HA (5), and pSAP-DDK-His (6) have all been described.

Protein Expression. HEK293 cells were seeded in 60-mm dishes 24 h beforetransfection. Plasmid transfection was performed using 6 μg plasmid andLipofectamine 2000 (Invitrogen) as transfection reagent in serum-free Opti-MEMmedium (Gibco). Primary fibroblasts were transfected similarly, but withLipofectamine LTX (Invitrogen). Cotransfections were performed with 4 μgof the MAGT1 vector and 4 μg of SHBG. All cells were assayed 24 h later.

Antibodies. Anti-MAGT1 (17430-1-AP) and anti-TUSC3 (16039-1-AP) poly-clonal antibodies were purchased from Proteintech, anti-BIP (3177S) fromBioké. Anti-STT3B and anti-STT3A antibodies were described previously

(4, 5). Goat polyclonal antisera specific for CatC (AF1071) and SHBG (VFJ01)were purchased from R&D Systems and mouse monoclonal GLUT1 (ab40084)and β-tubulin (ab101019) from Abcam. The following antibodies againstepitope tags were used: anti-HA (11867423001; Roche) and anti-DDK (F3165anti-FLAG M2; Sigma).

Immunoblotting. Tenmicrograms of proteins were analyzed by SDS/PAGE andimmunoblotted onto a nitrocellulose membrane (Thermo Fisher Scientific)with the indicated antibodies, as previously described (28). Signal detectionwas performed by autoradiography with ImageQuant LAS 4000, andquantification was performed with the Image Quant TL software (both GEHealthcare).

Pulse-Chase Radiolabeling and Immunoprecipitation. Glycoprotein substratesin HEK293 and fibroblast cells were pulse-chase–labeled with Tran35S label(Perkin–Elmer), as previously described (7). Dry gels were exposed to a phosphorscreen (Fujifilm), scanned in Typhoon FLA 9000, and quantified using ImageQuant TL software (GE Healthcare).

Statistics. Statistical analyses was performed in R. The Wilcoxon signed-ranktest was used to compare control versus patient samples. P < 0.05 was consideredsignificant.

ACKNOWLEDGMENTS. We thank Dr. D. Sinasac, Dr. N.Wright, Dr.M. Alessandro,and K. Klassen, RN, for their valuable contribution; L. Coorevits and J. Cremersfor technical assistance with flow cytometry; and I. Meyts for setup of thediagnostic gene panel for primary immune deficiencies. This research wassupported by Research Foundation Flanders (FWO): under the frame of E-Rare-3, the ERA-Net for Research on Rare Diseases (ERA-NET Cofund action N°64578)(to G.M.), a research stay grant (FWO V417818N) (to E.B.), a PegasusMarie Curiepostdoctoral fellow (FWO 1207416N, 2012–2018) (to R.P.), a senior clinical in-vestigator fellowship (to R. Schrijvers), and GLYCO4DIAG, an International As-sociated Laboratory grant from National Centre for Scientific Research (CNRS)and FWO (to F.F. and G.M.). The work was also supported by the NationalInstitute of General Medical Sciences of the National Institutes of Health underaward GM43768 (to R.G.), C1 KU Leuven fund (to R. Schrijvers), and by theJaeken-Theunissen CDG Fund.

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