Gains of Glycosylation Comprise an Unexpectedly Large

Gains of glycosylation comprise an unexpectedly largegroup of pathogenic mutationsGuillaume Vogt1, Ariane Chapgier1, Kun Yang1,2, Nadia Chuzhanova3,4, Jacqueline Feinberg1,Claire Fieschi1,5, Stephanie Boisson-Dupuis1, Alexandre Alcais1, Orchidee Filipe-Santos1, Jacinta Bustamante1,Ludovic de Beaucoudrey1, Ibrahim Al-Mohsen6, Sami Al-Hajjar6, Abdulaziz Al-Ghonaium6, Parisa Adimi7,Mehdi Mirsaeidi7, Soheila Khalilzadeh7, Sergio Rosenzweig8,17, Oscar de la Calle Martin9, Thomas R Bauer10,Jennifer M Puck11, Hans D Ochs12, Dieter Furthner13, Carolin Engelhorn14, Bernd Belohradsky14,Davood Mansouri7, Steven M Holland8, Robert D Schreiber15, Laurent Abel1, David N Cooper4,Claire Soudais1 & Jean-Laurent Casanova1,2,16

Mutations involving gains of glycosylation have been considered rare, and the pathogenic role of the new carbohydrate chainshas never been formally established. We identified three children with mendelian susceptibility to mycobacterial disease whowere homozygous with respect to a missense mutation in IFNGR2 creating a new N-glycosylation site in the IFNcR2 chain.The resulting additional carbohydrate moiety was both necessary and sufficient to abolish the cellular response to IFNc. Wethen searched the Human Gene Mutation Database for potential gain-of-N-glycosylation missense mutations; of 10,047 mutationsin 577 genes encoding proteins trafficked through the secretory pathway, we identified 142 candidate mutations (B1.4%) in77 genes (B13.3%). Six mutant proteins bore new N-linked carbohydrate moieties. Thus, an unexpectedly high proportion ofmutations that cause human genetic disease might lead to the creation of new N-glycosylation sites. Their pathogenic effectsmay be a direct consequence of the addition of N-linked carbohydrate.

Mendelian susceptibility to mycobacterial disease (MSMD; OMIM209950) is a rare syndrome that confers predisposition to illnesscaused by moderately virulent mycobacterial species, such as BacillusCalmette-Guerin (BCG) vaccines and nontuberculous environmentalmycobacteria, and by the more virulent Mycobacterium tuberculosis1.Other types of microorganism rarely cause severe clinical disease inindividuals with MSMD, with the exception of Salmonella, whichinfects o50% of these individuals. The demonstration that thiscondition was associated in some affected individuals with deficiencyof interferon g receptor ligand-binding chain (IFNgR1) provided thefirst evidence for a genetic etiology2,3. Subsequent studies identifiedmutations in the genes encoding IFNgR2 (ref. 4), the interleukin-12p40 (IL-12p40) subunit shared by IL-12 and IL-23 (ref. 5), theIL-12Rb1 subunit shared by the IL-12 and IL-23 receptors6,7, andthe signal transducer and activator of transcription-1 (Stat-1)8. Allelic

heterogeneity at these five disease-associated autosomal gene loci isresponsible for ten known disorders, all of which involve impairedfunction of the IL-12/23-IFNg circuit9–15. Complete Stat-1 deficiencyis associated with a related but more severe syndrome of vulnerabilityto mycobacterial and viral infections due to an impaired cellularresponse to both IFNg and IFNa/b16.

IFNgR2 deficiency is the most infrequent of the inherited forms ofMSMD: only three children with MSMD have been reported, two withcomplete IFNgR2 deficiency4,17 and one with partial IFNgR2 defi-ciency14. By contrast, 22 individuals are known to have completeIFNgR1 deficiency, and 38 are known to have partial IFNgR1deficiency15. Here we report four children with complete IFNgR2deficiency, from three unrelated families. One of these children has anin-frame microdeletion in the gene IFNGR2 such that the encodedprotein does not reach the cell surface normally. The other three

Published online 29 May 2005; doi:10.1038/ng1581

1Laboratory of Human Genetics of Infectious Diseases, University of Paris Rene Descartes INSERM U550, Necker Medical School, 156 rue de Vaugirard, 75015 Paris,France. 2French-Chinese Laboratory of Genomics and Life Sciences, Ruijin Hospital, Shanghai Second Medical University, Shanghai, China. 3Biostatistics andBioinformatics Unit and 4Institute of Medical Genetics, Cardiff University, Cardiff CF14 4XN, UK. 5Department of Immunopathology, Saint Louis Hospital, 75010Paris, France. 6Pediatric Infectious Diseases and Immunology Units, Department of Pediatrics, King Faisal Specialist Hospital & Research Centre, Riyadh, SaudiArabia. 7National Research Institute of Tuberculosis and Lung Diseases, Shaheed Beheshti University of Medical Sciences, Dar-Abad, 19556 Tehran, Iran. 8Laboratoryof Clinical Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. 9Service of Immunology, Hospital de la Santa Creu i Sant Pau,Barcelona, Spain. 10National Cancer Institute and 11National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.12Department of Pediatrics, University of Washington, Seattle, Washington 98109, USA. 13Department of Pediatrics, Klinikum Wels, 4600 Wels, Austria.14Department of Pediatrics, Hospital for Sick Children, 80337 Munchen, Germany. 15Department of Pathology and Immunology, Washington University, Saint Louis,Missouri 63110, USA. 16Pediatric Immunology & Hematology Unit, Necker Hospital, 75015 Paris, France. 17Present address: Servicio de Inmunologia, HospitalGarrahan, Buenos Aires, Argentina. Correspondence should be addressed to J.-L.C. ([email protected]).

6 92 VOLUME 37 [ NUMBER 7 [ JULY 2005 NATURE GENETICS

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children have the same IFNGR2 missense mutation, which gives rise toa new inherited form of complete IFNgR2 deficiency due to theexpression of nonfunctional receptors on the cell surface. The mis-sense mutation also creates a consensus site for N-glycosylation,resulting in IFNgR2 molecules that carry an additional polysaccharidethat is both necessary and sufficient to account for the pathogeniceffect of the mutation. Notably, we found that 13.3% of the genesencoding proteins bearing signal peptides or signal anchors listed inthe Human Gene Mutation Database (HGMD) carried potential gain-of-glycosylation mutations, representing B1.4% of the correspondingpathogenic missense mutations. Our study thus defines a new class ofhuman pathological mutation in which the creation of an N-glycosy-lation site leads to loss of function.

RESULTSIdentification of IFNGR2 gene mutationsWe studied four children with severe mycobacterial disease from threeunrelated families, all consanguineous (individual P1 from Austria,individual P2 from Iran and individuals P3 and P4 from Saudi Arabia;Fig. 1a). The severity of the clinical phenotype and the absence ofdetectable mutations in the gene IFNGR1 led us to study IFNGR2.Individual P1 was homozygous with respect to a new in-frame 27-bpmicrodeletion of nucleotides 663–689 (663del27), predicted to lead tothe deletion of amino acids 222–230 from the protein (Fig. 1b,c).Individuals P2, P3 and P4 were all homozygous with respect to a newmissense mutation at nucleotide position 503 (503C-A; codonchange ACC-AAC), resulting in the amino acid substitutionT168N (Fig. 1b,c). The microdeletion was not found in 50 unrelatedhealthy individuals of European descent, and the missense mutationwas not found in 77 unrelated healthy individuals of Saudi Arabiandescent. No Iranian controls were genotyped. The only other sequencevariant identified in the exons and flanking intronic regions of

IFNGR2 was present in individual P1: a homozygous SNP at position191 (191A-G; codon change CAA-CGA; frequency of 0.496 inEuropeans), resulting in the amino acid substitution Q64R. Theparents of the four children were healthy and were heterozygouswith respect to the corresponding mutations. The healthy sibling ofindividual P1 (individual A.II.2) did not carry the IFNGR2 mutation.These findings suggest that the 663del27 and 503C-A mutations arepathologically relevant.

Complete IFNcR2 deficiencyFresh whole blood cells from individuals with MSMD showed poorsecretion of IL-12 in response to BCG alone, which was not increasedby IFNg, consistent with the affected individuals having a deficiency ofIFNgR1, IFNgR2 or Stat-1 (in the functional sense of the term; datanot shown)18. We then stimulated B-cell lines transformed withEpstein-Barr virus (EBV-B cells) from the affected individuals withIFNg13. Electrophoretic mobility shift assays (EMSAs) identified nog-activated sequence (GAS)-binding proteins in response to IFNg inany of the affected individuals, as was found in a child with completeIFNgR2 deficiency who was homozygous with respect to the 278delAGnull recessive mutation4 (Fig. 2a). Successful complementation of cellsfrom individuals P1 and P3 with a wild-type IFNGR2 allele indicatedthat the individuals had IFNgR2 deficiency (Fig. 2b). We transientlytransfected IFNgR2-deficient EBV-B cells homozygous with respect tothe 278delAG allele4 with wild-type, 278delAG, 663del27 and 503C-A IFNGR2 alleles. Under these conditions of hyperexpression, thewild-type allele, but none of the mutated alleles, successfully comple-mented the deficiency state, confirming that the mutated allelesresulted in loss of function (Fig. 2c). IFNgR2 mutant molecules taggedwith enhanced green fluorescent protein (eGFP) or the His/V5 epitopewere produced but were nonfunctional, whereas wild-type eGFP- orHis/V5-tagged IFNgR2 molecules were produced and functional (data

Figure 1 IFNGR2 genotype and clinical

phenotype of individuals with MSMD.

(a) The three kindreds with IFNgR2 deficiency.

Healthy individuals carry one or two wild-type

copies of IFNGR2 (open symbols). Affected

individuals P1 (663del27) and P2, P3 and P4

(503C-A) are homozygous with respect to their

respective mutations (filled symbols). Family A is

from Austria; family B is from Iran; and family C

is from Saudi Arabia. (b) The 663del27 (left: P1)

and T168N (right: P2, 3 and 4) IFNGR2 alleles

compared with a wild-type (WT) IFNGR2 allele,

as shown by PCR and direct sequencing of

exons 5 and 4, respectively. The amino acids

that are deleted (663del27) or altered (T168N)are indicated in red. The amino acid marked in

green is retained despite a change in the

corresponding codon (663del27). Red boxes

denote two direct repeats, which may promote

mispairing due to slippage during DNA

replication. (c) New mutations in IFNGR2

(663del27 and 503C-A). The IFNGR2

coding region is represented with vertical

bars between exons that are designated by

Roman numerals. The leader sequence

(L, 1–22), extracellular domain (EC, 23–248),

transmembrane domain (TM, 249–272)

and intracellular domain (IC, 273–337) are

indicated. Consensus sites for N-glycosylation are indicated by asterisks. Mutations marked in red cause complete IFNgR2 deficiency with no detectable

expression of IFNgR2 at the cell surface4,17. The mutation marked in blue causes complete IFNgR2 deficiency with detectable surface expression of

IFNgR2. The mutation marked in purple causes partial, as opposed to complete, IFNgR2 deficiency14.

A

P1 P2 P3 P4

WT

R114C 503C→A*

ECNH2 1 L

5′ 1

278delAG 663del27 791delG

1011 3′

* * * * * *TM IC 337 COOH

663del27(P1)

WT

T168N(P2,P3,P4)

B C

I.1 I.2

II.1 II.2 II.1 II.1 II.2

I.1 I.2 I.1 I.2

I II III IV V VI VII

a

b

c

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not shown). These data indicate that the four individuals with MSMDsuffered from autosomal recessive complete IFNgR2 deficiency.

Subcellular distribution of IFNcR2We then investigated the subcellular distribution of the mutantIFNgR2 molecules. We constructed expression vectors that encodedIFNgR2 molecules tagged with eGFP (at the C-terminal cytoplasmictail, unlike the N-terminally–tagged extracellular constructs recentlyreported17). We then transiently transfected IFNgR2-deficient SV40-transformed fibroblasts4 with vectors encoding various eGFP-taggedIFNgR2 molecules and examined them by confocal microscopy. Wild-type IFNgR2 was predominantly located in large vesicles in thecytoplasm, with perinuclear localization typical of the secretory path-way. T168N IFNgR2 molecules had the same distribution (Fig. 3a). Bycontrast, 663del27 IFNgR2 molecules were disseminated in smallercytoplasmic vesicles, suggestive of abnormal trafficking. We thentested whether the IFNgR2 proteins were present in the plasmamembrane. We stained IFNgR2-deficient fibroblasts with DiC16 tolabel the plasma membrane and then expressed constructs encodingvarious eGFP-tagged IFNgR2 molecules in these cells (Fig. 3b). Afraction of wild-type and T168N IFNgR2 molecules colocalized to theplasma membrane, but 663del27 molecules did not. These datastrongly suggest that T168N IFNgR2, like wild-type IFNgR2, wasable to reach the cell surface.

Biochemical properties of IFNcR2After transfecting IFNgR2-deficient SV40-transformed fibroblasts4

with IFNGR2 alleles encoding V5 epitope–tagged wild-type, T168Nor 663del27 IFNgR2 proteins, we detected IFNgR2 proteins in celllysates by western blotting (Fig. 4a). Wild-type IFNgR2 moleculesshowed heterogeneous processing, resulting in proteins of 55–66 kDa.T168N proteins were larger, with molecular weights of 55–77 kDa. Wedetected two main 663del27 molecules, both B50 kDa. We thenbiotinylated the surface proteins expressed by IFNgR2-deficient fibro-blasts4 transfected with various V5-tagged IFNgR2 constructs. Weimmunoprecipitated cell lysates with streptavidin-agarose and analyzedthem by SDS-PAGE followed by western blotting with a V5-specificantibody (Fig. 4b). Both wild-type IFNgR2 (B65 kDa) and theheavier T168N molecules (B75 kDa) were detected among thesurface-expressed biotinylated proteins and, to a lesser extent, amongthe nonbiotinylated cytoplasmic proteins. By contrast, most 663del27molecules were detected in the cytoplasm. These results stronglysuggest that T168N IFNgR2 molecules, but not 663del27molecules, are routed normally to the cell surface, despite having an

abnormally high molecular weight. This defines a new form ofcomplete IFNgR2 deficiency.

T168N creates a new N-glycosylation siteThe T168N mutation results in a change of protein sequence, with anew asparagine residue at position 168 (Thr-Ser-Thr-Ala-Asn-Ser-Thr-Ala) creating a consensus N-glycosylation site (Asn-X-Ser/Thr-X,where X is any amino acid except proline)19. We transfected IFNgR2-deficient fibroblasts4 with the IFNGR2 alleles and analyzed endogly-cosidase-H (Endo-H)- and peptide N-glycosidase-F (PNGase-F)-digested protein extracts from these cells by western blotting with aV5-specific antibody20. Both wild-type and T168N IFNgR2 glycosy-lated proteins were partially resistant to Endo-H (Fig. 5a). By contrast,663del27 molecules were sensitive to Endo-H, indicating an immaturepattern of N-glycosylation. Moreover, the PNGase-F-digested formsof wild-type and T168N IFNgR2 had similar molecular weights(B47 kDa), indicating that the higher molecular weight of T168Nmolecules was due to additional N-glycosylation. Finally, we culturedtransfected fibroblasts with and without tunicamycin, an inhibitor ofN-glycosylation that blocks the assembly of the lipid-linked oligosac-charide precursor21. On western blots, T168N and wild-type proteinshad similar molecular weight distributions after prolonged treatmentwith tunicamycin (48 h at 0.1 mg/ml; Fig. 5b). Therefore, we

NS

IFN

γ

NS

IFN

γ

NS

– Moc

kW

T27

8delA

G

663d

el27

503C

→A

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

NS

IFN

γ

IFN

α

NS

IFN

γ

IFN

α

NS

IFN

γ

IFN

α

NS

IFN

γ

IFN

α

C+

(WT)

C– (278delAG)

P1

(663

del27

)C–

(278

delA

G)P3

(503

C→A)

Vectors

a b cC+

(WT)Mock

C– C–

WT

P1(663del127)

WT

P3(503C→A)

WT(278delAG)

Mock

Figure 2 Cellular phenotype of individuals with MSMD. (a) GAS probe-binding nuclear proteins from EBV-B cells from a positive control (C+), two affected

individuals (P1 and P3) and a negative control (C–; homozygous with respect to the 278delAG IFNGR2 allele4), with or without (NS) 105 IU ml�1 IFNg and

IFNa, as determined by EMSA. WT, wild-type. (b) GAS probe-binding nuclear proteins from EBV-B cells from a positive control (C+), a negative control (C�)

and two affected individuals (P1 and P3) transiently transfected with a mock vector or with a vector encoding wild-type (WT) IFNgR2, with or without (NS)

105 IU ml�1 of IFNg, as determined by EMSA. (c) GAS probe-binding nuclear proteins from EBV-B IFNgR2-deficient cells (C�), either not transfected (�) or

transiently transfected with a mock vector (mock) or with vectors encoding wild-type (WT), 278delAG, 663del27 and T168N IFNgR2, with or without (NS)

105 IU ml–1 of IFNg, as determined by EMSA.

C+ 663del27 T168Na

b

Figure 3 Subcellular distribution of IFNgR2. (a) Subcellular distribution of

IFNgR2-eGFP (green) in IFNgR2-deficient SV40-transformed fibroblasts

(C�) after transfection with wild-type (C+), 663del27 and T168N IFNgR2

constructs, as shown by indirect immunofluorescence upon confocalmicroscopy. (b) Subcellular colocalization (yellow) of IFNgR2-eGFP (green)

and DiC16 (red) in IFNgR2-deficient SV40-transformed fibroblasts (C�)

after transfection with wild-type (C+), 663del27 and T168N IFNgR2

constructs, as shown by indirect immunofluorescence upon confocal

microscopy (Z-stack slides). Scale bar, 10 mm.


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concluded that T168N IFNgR2 molecules have a higher molecularweight than wild-type molecules owing to an additional N-glycosyla-tion event probably, but not necessarily, involving Asn168.

The additional N-glycosylation is pathogenicWe then investigated whether the T168N mutation exerted its patho-genic effect through additional N-glycosylation specifically at Asn168.We constructed a series of vectors to test the glycosylation consensussite: we replaced Thr168 with an alanine (T168A) or a glutamine(T168Q). We also replaced Thr170 with alanine (T170A) and replacedThr168 and Thr170 with asparagine and alanine, respectively, yieldinga T168N-T170A double mutant. IFNgR2-deficient EBV-B cells4 tran-siently transfected with alleles encoding T168A and T168Q IFNgR2responded normally to IFNg (Fig. 5c). This indicates that Thr168 isnot essential for the function of IFNgR2 and that Gln168 (andprobably Asn168) does not have a deleterious effect on either thefolding or the function of the protein. IFNgR2-deficient EBV-B cellstransfected with T170A or T168N-T170A IFNgR2 also responded wellto IFNg, but cells bearing T168N did not. In the same experiment,western blotting and PNGase-F digestion showed that T168N IFNgR2molecules had higher molecular weight than did wild-type IFNgR2 orthe other mutants, owing to the addition of a new carbohydrate chain(Fig. 5c). These observations indicate unambiguously that Asn168does not exert its deleterious effects directly by altering polypeptide

folding. Rather, it creates a new consensus site for N-glycosylation thatis both necessary and sufficient to account for the pathological effectof the T168N mutation.

Chemical complementation of IFNcR2 deficiency in cell linesWe cultured EBV-B cells from individuals with MSMD and a controlwith tunicamycin for 30 min under conditions in which glycosylationis inhibited but cell viability is preserved and then stimulated them 24h later with IFNg for 30 min. Wild-type cells responded to IFNg inboth the presence and absence of tunicamycin, as measured by EMSA(Fig. 6a). Cells from individual P1, who is homozygous with respectto the 663del27 mutation, and from a negative control4 did notrespond to IFNg in either the presence or absence of tunicamycin. Butcells from individuals P2 and P3, homozygous with respect to the503C-A loss-of-function mutation, responded to IFNg upon treat-ment with tunicamycin, as shown by the GAS binding of Stat-1homodimers (Fig. 6a and data not shown). We obtained a similarresult when we treated cells with PNGase-F (Fig. 6a). We then treatedSV-40–transformed fibroblasts from individual P2 and from a negativecontrol4 with tunicamycin or PNGase-F for 48 h while stimulating thecells with IFNg and measured cell surface HLA-DR induction by flowcytometry. Tunicamycin and PNGase-F treatment fully restored IFNgresponsiveness to fibroblasts from individual P2 but not those fromthe negative control individual (Fig. 6b).

MW

C– (278delAG)

C– (278delAG)

Moc

kW

TT16

8N

663d

el27 M

ock

WT

T168N

663d

el27

100

MW

IP-Strept

Flow-through

Biotin – + – + – + – +

100

75

50

100

75

50

77

66

50

a b

+–+–+–+–+–++ ––PNGase-F

45-

50-66-

77-MW

IFN

γ

IFN

γ

IFN

γ

IFN

γ

IFN

γ

IFN

γ

IFN

γ

NS

NS

NS

NS

NS

NS

NS

T168Q

663d

el27

T168N

WT

Moc

kT16

8N

WT

Moc

k66

3del2

7

T168A

T170A

T170A

T168N

-

T168N

WT

Moc

k

C– (278delAG)

0.1 µg/mlNoneTunicamycin

45-

50-

66-77-

MW

C– (278delAG)

37-45-50-

66-75-

MWPNGase-FEndo-H

–– – – – – –

– – – – –+

+ + +++

WT

663d

el27

T168N

C– (278delAG)

a cb

Figure 5 Pattern of IFNgR2 glycosylation. (a) IFNgR2-deficient SV40-transformed fibroblasts (C�) were transfected with wild-type (WT), 663del27 and

T168N IFNgR2 V5-tagged constructs. Whole-cell extracts were digested with Endo-H or PNGase-F for 3 h, resolved by SDS-PAGE and immunoblotted with

an antibody to V5. (b) IFNgR2-deficient SV40-transformed fibroblasts (C�) were transfected with mock, wild-type (WT), T168N and 663del27 IFNgR2

V5-tagged constructs and incubated or not incubated with 0.1 mg ml�1 tunicamycin for 48 h. The cell lysates were then resolved by SDS-PAGE and

immunoblotted with an antibody to V5. (c) IFNgR2-deficient EBV-B cells (C�) were transiently transfected with mock, wild-type (WT), T168N, T168A,

T170A, T168N-T170A and T168Q IFNgR2 V5-tagged constructs. The response to IFNg (105 IU ml�1) was determined by EMSA, using a radioactively

labeled GAS probe (upper panel). The cell lysates were also resolved by SDS-PAGE, treated (+) or not treated (�) with PNGase-F (15,000 IU ml�1 for 3 h)

and immunoblotted with an antibody to V5 (lower panel). MW, molecular weight in kDa.

Figure 4 Biochemical properties of IFNgR2.

(a) IFNgR2-deficient SV40-transformed

fibroblasts (C�) were transformed with wild-type

(WT), T168N and 663del27 IFNgR2 V5-tagged

constructs. The proteins in these cells were

resolved by SDS-PAGE and immunoblotted with

an antibody to V5. (b) IFNgR2-deficient SV40-

transformed fibroblasts (C�) were transformed

with wild-type (WT), T168N and 663del27

IFNgR2 V5-tagged constructs. Cell surfaces were

biotinylated (+) or not (�), and the lysates were

immunoprecipitated with streptavidin; both the

precipitate (IP-Strept) and the flow-through were

analyzed by western blotting with horseradish

peroxidase–conjugated antibody to V5. MW,molecular weight in kDa.


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Chemical complementation of IFNcR2 deficiency in fresh bloodWe treated fresh blood from individuals P2 and P4 carrying theT168N mutation, from a positive control and from individual P1carrying the 663del27 mutation (as a negative control) with tunica-mycin or PNGase-F and stimulated the cells with either live BCG orBCG plus IFNg for 48 h (Fig. 6c,d). We then measured IL-12p40production by ELISA18. Blood cells from individuals P2 and P4, whichdid not respond to endogeneous (BCG stimulation) or exogeneous(BCG plus IFNg stimulation) IFNg in the absence of treatment,responded to BCG and, to a much lesser extent, to BCG plus IFNgwhen treated with tunicamycin or PNGase-F (Fig. 6c,d). We observedresidual production of IL-12p40 in response to BCG in cells fromIFNgR2-deficient individual P1, which defined the IFNg-independentbackground. The low levels of IL-12p40 produced by cells from

individual P1 treated with tunicamycin mayreflect the cytotoxicity of this compound. Weobtained chemical correction of completeIFNgR2 deficiency by tunicamycin andPNGase-F treatment in vitro, in two indepen-dent cell lines from individuals with MSMD(EBV-B cell lines and SV40-transformedfibroblasts), and ex vivo, in fresh wholeblood cells, irrespective of short-term (Stat-1 activation) or long-term (HLA-DR and IL-12p40 induction) effects of IFNg stimulation.

General prevalence of gain-of-glycosylation mutationsEighteen human germline gain-of-glycosyla-tion missense mutations have been previouslyreported, two polymorphic22,23 and sixteenrare and pathogenic24–38. We searched theHGMD39 for potential gain- or loss-of-glycos-ylation missense mutations and identified246 missense mutations (of 20,667; B1.2%)in 158 genes (of 1,325; B11.9%) that createpotential N-glycosylation sites in the corre-sponding proteins, including 13 of the 15missense mutations reported in the litera-ture24,25,27–38 (Supplementary Table 1online). The sixteenth known gain-of-glycos-ylation mutation was an in-frame micro-deletion26. The proportion of reported muta-tions predicted to result in the gain of anN-glycosylation site was significantly higher(P ¼ 7 � 10�12) than expected, whereas theproportion of mutations predicted to resultin the loss of an N-glycosylation site wassignificantly lower (P ¼ 4 � 10�20) thanexpected (Table 1). We repeated the proce-dure for a subset of 577 genes encodingproteins predicted to have either a signalpeptide or a signal anchor (selected on thebasis of in silico prediction40; Table 1 andSupplementary Table 1 online)41. In thissubset of genes, the over-representation ofpotential gain-of-glycosylation mutations waseven more significant (P ¼ 8 � 10�16); theproportion of potential gain-of-glycosylationmutations was B1.4% of all missense muta-tions (142 of 10,047) in B13.3% (77 of 577)

of the corresponding genes. These data therefore suggested that manygenetic diseases may be caused by missense mutations that lead to again of glycosylation. Moreover, losses of glycosylation are correspond-ingly under-represented in the HGMD, implying that whereas not allglycosylation events are crucial to a protein’s function, the gain ofpolysaccharide in an inappropriate context is usually deleterious.

Structural validation of other gain-of-glycosylation mutationsThe potential gain-of-glycosylation mutations in the 158 genes foundin the HGMD are associated with a variety of human inheriteddisorders (Supplementary Table 1 online). Fourteen of the 158genes are associated with a primary immunodeficiency. We selectedsix mutations in five genes encoding proteins that normallyundergo trafficking through the endoplasmic reticulum: Y125N in

P2P1C+

P4P1C+

P4P1C+

P2P1C+

10

100

1,000

10,000

IL-1

2 (p

g/m

l)

10

100

1,000

10,000

IL-1

2 (p

g/m

l)

10

100

1,000

10,000

IL-1

2 (p

g/m

l)

10

100

1,000

10,000

IL-1

2 (p

g/m

l)

PNGase-F

Tunicamycin

Untreated

Untreated

PNGase-F

Tunicamycin

Untreated

HLA-DR

104103102101100

040

8012

016

00

4080

120

160

040

8012

016

0

Fl2-H104103102101100

Fl2-H104103102101100

Fl2-H

PN

Gas

e-F

Tuni

cam

ycin

Unt

reat

ed

C–(278delAG)

P2(T168N)

C+(WT)

Cel

l cou

nt

IFN

γ

IFN

γ

IFN

γ

IFN

γ

IFN

γ

NS

NS

NS

NS

NS

P1

(663

del27

)

P3

(T16

8N)

P2

(T16

8N)C–

(278

delA

G)

C+(W

T)

a c

d

b

Figure 6 Chemical complementation of the cellular phenotype. (a) Response of EBV-B cells to IFNg(105 IU ml�1 for 30 min), as determined by EMSA analysis of GAS probe-binding nuclear proteins

from a positive control (C+), three affected individuals (P1, P2 and P3) and a negative control (C�).

Cells were untreated or treated 24 h earlier with tunicamycin (8 mg ml�1 for 30 min) or with PNGase-F

(3,000 IU ml�1 for 30 min). WT, wild-type. (b) SV40-transformed fibroblasts from a positive control

(C+), an individual with MSMD (P2) and a negative control (C�) were incubated for 48 h in completeculture medium with (black histogram) or without (white histogram) IFNg (105 IU ml�1), tunicamycin

(0.1 mg ml�1) or PNGase-F (750 IU ml�1). The surface expression of HLA-DR molecules was

determined by flow cytometry using a specific antibody. WT, wild-type. (c) Production of IL-12p40 in

whole blood cells from a positive control (C+), an individual (P4) carrying the T168N mutation and an

individual (P1) carrying the 663del27 mutation, after a 48-h stimulation with complete medium

(white), live BCG alone (gray) or live BCG plus IFNg (5,000 IU ml�1; black), with or without

tunicamycin (0.06 mg ml�1), as detected by ELISA. This experiment is representative of two

independent experiments. (d) Production of IL-12p40 in whole blood cells from a positive control

(C+), an individual (P2) carrying the T168N mutation and an individual (P1) carrying the 663del27

mutation, after a 48-h stimulation with complete medium (white), live BCG alone (gray) or live BCG

plus IFNg (5,000 IU ml�1; black), with or without PNGase-F (750 IU ml�1), as detected by ELISA.

This experiment is representative of two independent experiments.


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IL2RG42, the common g chain of cytokine receptors; G111S inCD8A43, the CD8 a chain; G284S in ITGB2 (ref. 44) or CD18;G220S in PRF1 (ref. 45) or perforin; and T147N and T211N inCD40LG46,47 or CD154, the ligand of CD40. We produced V5-taggedmolecules encoded by the corresponding wild-type and mutatedalleles in SV-40 fibroblasts. All six mutant proteins had highermolecular weights than their wild-type counterparts (Fig. 7a anddata not shown), but in the presence of PNGase-F, the mutantproteins had molecular weights similar to those of the wild-type(Fig. 7a). On the basis of binomial computations, all six mutantproteins tested were associated with gains of glycosylation, suggestingthat the overall proportion of bona fide gain-of-glycosylation muta-tions among the 142 predicted by the HGMD search is probably460% with a probability 40.95. This proportion would increase to85% if we were to include the 13 gain-of-glycosylation HGMDmissense mutations documented in the literature, although we cannotexclude the operation of a publication bias in the reporting of thesemutations that could have affected this latter estimate.

Functional validation of other gain-of-glycosylation mutationsWe then considered the mutation T147N in CD40L and the mutationG284S in CD18. We transfected the 293T/17 epithelial cell line withwild-type, T147N, T147N-S149A and T147A CD40L variants, mon-itored the surface expression of CD40L by flow cytometry using aspecific antibody and assessed CD40L function using a soluble CD40molecule. All CD40L molecules were detected by the specificantibody, but only wild-type, T147N-S149A and T147A moleculeswere recognized by the soluble CD40 (Fig. 7b). We then transfectedCD18-deficient EBV-B cells (carrying the G284S mutation inCD18; ref. 44) with mock, wild-type, G284S and G284A CD18variants, monitored the surface expression of CD18 by flow cytometryusing a specific antibody and assessed CD18 function using a CD11a-specific antibody (both CD18 and CD11a are required to form asurface-expressed heterodimer). Both CD18 and CD11a weredetected upon transfection with constructs encoding wild-type andG284A CD18 but not with constructs encoding G284S CD18(Fig. 7c). We further showed that G284S molecules, unlike wild-type and G284A CD18 molecules, were Endo-H–sensitive, implyingthat the mutant proteins were retained in the cytoplasm (data notshown). In conclusion, these data strongly suggest that a sizeableproportion of missense mutations causing human inherited disease(B1%) constitute gains of glycosylation and that these mutations

may exert their pathological effects through the creation of newglycosylation sites.

DISCUSSIONHere we report a new form of complete IFNgR2 deficiency, char-acterized by surface-expressed nonfunctional receptors. The T168NIFNgR2 mutation results in a protein carrying an N-linked carbohy-drate moiety attached at Asn168. This polysaccharide is both necessaryand sufficient to account for the pathological effect of the T168Nmutation. Despite this glycosylation, the T168N IFNgR2 moleculeshave the same intracellular localization as wild-type molecules. Similarcomplete deficiencies due to nonfunctional receptors expressed at thecell surface have been reported in other individuals with inheriteddefects of the IL12/23-IFNg axis involving the other two knownreceptor defects: complete deficiencies of IFNgR1 (ref. 11) and IL-12Rb1 (ref. 9). Surface-expressed nonfunctional IFNgR1 and IL-12Rb1 molecules have been associated with missense mutations andin-frame small and large deletions; the mutant molecules fail to bindtheir natural ligands, IFNg and IL-12/2, respectively9,11. By contrast,the mechanism by which the T168N-associated neoglycosylation ofIFNgR2 affects IFNg signaling is still unknown.

The T168N mutation in IFNgR2 is the first reported germlinemutation for which a causal relationship has been unequivocallyestablished between the gain of glycosylation and the loss of function.Six other previously described missense mutations associated with aprimary immunodeficiency are also characterized by gains of glyco-sylation42–47 (Supplementary Note online). Such mutations are notconfined to primary immunodeficiencies, as indicated by the 16pathogenic mutations in 11 genes previously shown to involve gainsof glycosylation24–38 (Supplementary Note online). Moreover, among577 genes bearing missense mutations and encoding proteins thatmigrate through the secretory pathway, up to 77 (B13.3%) may besubject to potential gains of glycosylation (corresponding to B1.4% ofpathogenic missense mutations found in the 577 genes; Table 1 andSupplementary Table 1 online). Some trans-membrane proteins thatlack a signal peptide or signal anchor41, such as the cystic fibrosistransmembrane conductance regulator, may even be subject to a gain-of-glycosylation mutation34,48,49. Gain-of-glycosylation mutationsmay therefore affect many thousands of individuals worldwide.

These calculations are crude (and possibly conservative) estimatesbecause the variable amino acids present in N-glycosylation sites,and perhaps also some more spatially remote residues, may affect

Table 1 Predicted gains and losses of consensus N-glycosylation sites associated with missense mutations in the HGMD

Total missense mutations

Predicted gain-of-glycosylation

missense mutations (%)

Predicted loss-of-glycosylation

missense mutations (%)

1,325 577 748 1,325 577 748 1,325 577 748

Documented in HGMD 20,667 10,047 10,620 246 (1.19%) 142 (1.41%) 104 (0.98%) 164 (0.79%) 82 (0.82%) 82 (0.77%)

Possible in silico 5,949,671 2,418,935 3,530,736 45,299 (0.76%) 17,410 (0.72%) 27,889 (0.79%) 94,194 (1.58%) 47,430 (1.95%) 46,764 (1.32%)

P value – – – 7 � 10�12 8 � 10�16 0.04 4 � 10�20 9 � 10�17 5 � 10�7

A total of 20,667 pathogenic missense mutations affecting 1,325 genes have been logged in the HGMD (May 2004 release). Of these genes, 577 encode proteins that are predictedto enter the endoplasmic reticulum and that are therefore likely to be exposed to the N-glycosylation machinery (based on the in silico identification of a signal peptide with acleavage site or of a signal anchor without a cleavage site) with a combined probability of 0.75 or above. The remaining 748 genes encode proteins that are not predicted to enterthe secretory pathway, based on the lack of in silico detectable signal peptide or signal anchor. Of the sets of 1,325, 577 and 748 genes, mutations in 158, 77 and 81 genes,respectively, potentially cause gains of glycosylation. The numbers of documented pathogenic missense mutations, including those that lead to a potential gain or loss of N-glycosylation (based on the creation or removal of a consensus site for N-glycosylation), with the corresponding total number of possible missense mutations (created in silico) foreach of these three groups are compared. In silico modeling yielded the expected proportions of gain- and loss-of-glycosylation mutations, under the assumption that these mutationsoccur at random. w2 statistics were used to compare the observed proportions with those expected (P values are indicated). Missense mutations of the initiation and terminationcodons are not included in the table, but similar results were obtained when such mutations were included in the analysis (data not shown).


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glycosylation efficiency19. Moreover, muta-tions other than missense mutations, suchas in-frame microdeletions and microinser-tions, may also yield gains of glycosylation, asin the case of the C1 inhibitor mutation in anindividual with hereditary angioneuroticedema (K273del)26. New O-glycosylation sites could also arise frommissense mutations that introduce a serine or a threonine50. It istherefore difficult to determine accurately the proportion of patholo-gical mutations that may exert their deleterious effects through gainsof N- or O-glycosylation. Nevertheless, our results suggest that anunexpectedly large proportion of human pathogenic missense muta-tions may involve gains of glycosylation and that a substantial fractionof these are pathogenic precisely because they involve gains ofglycosylation. Our study opens the door to the exploration of a newfield involving the chemical treatment of human inherited disorders,by specifically targeting gain-of-glycosylation mutations.

METHODSAffected individuals. We studied four children from three unrelated families

with severe mycobacterial disease. These families were consanguineous and

originated from three different countries (Austria, Iran and Saudi Arabia;

Fig. 1a). The first child (P1) developed disseminated Mycobacterium avium

infection at the age of 3 y, which responded to antibiotics with which he has

been treated for 7 y. The second child (P2) developed disseminated BCG

vaccine infection at the age of one month; he is now 2.5 y old and is still being

treated with antibiotics. The third and fourth children (P3 and P4) developed

disseminated disease caused by Mycobacterium fortuitum and cytomegalovirus;

the first died at 6 y of age and the second is still alive at 5 y of age (Fig. 1a). This

study was approved by the French institutional review committee (CCPPRB),

and informed consent was obtained from all families.

DNA and RNA extraction, cDNA synthesis and PCR amplification. We

extracted genomic DNA and total RNA from EBV-B cell lines with Trizol

(Gibco-BRL)9. We reverse-transcribed RNA in the presence of oligo(dT) with

Superscript II reverse transcriptase (Invitrogen Corporation). We amplified

IFNGR1 and IFNGR2 cDNAs using appropriate pairs of primers (PCR

conditions available on request).

Cell culture and transfection. We cultured EBV-B cells, SV40-transformed

fibroblasts (SV40 cells) and 293T/17 epithelial cells in RPMI 1640 medium

supplemented with 10% heat-inactivated bovine fetal serum (Gibco-BRL). Two

days before transfection, we plated 3 � 105 SV40-transformed fibroblasts in

35-mm dishes (Nunc). We transformed SV40-transformed fibroblasts and

293T/17 cells by lipofection (LipofectAMINE plus Reagent; Invitrogen) in

accordance with the manufacturer’s instructions. We transfected aliquots of 107

EBV-B cells by electroporation with a pulse at 300 V, 900 mF, R p in 400 ml of

complete medium. We tested electroporated cells by EMSA the following day.

104103102101100104103102101100

FL1-HFL1-H104103102101100

FL1-H104103102101100

FL1-H

020

4060

800

2040

6080

020

4060

800

2040

6080

020

4060

80

010

2030

4050

010

2030

4050

010

2030

4050

010

2030

4050

010

2030

4050

FluorescenceFluorescence

CD18–/–G284A

CD18–/–G284S

CD18–/–

CD18–/–

WT

Mock

MockWT

Mock

S149A

T147N

T147A

T147N

WT

Cel

l cou

nt

Cel

l cou

nt

PNGase-F

CD18CD11a

Mulg-CD40Ab

Untreated

30-

45-

66-

30-

45-

66-

T168

N

T211

N

T147

N

G11

1S

Y125

N

Moc

k

G21

0SW

T

WT

WT

WT

WT

MWT168

N

T211

N

T147

N

G11

1S

Y125

N

Moc

k

G21

0SW

T

WT

WT

WT

WT

MW

IL2RG CD8A CD40LG PRFI IFNGR2 IL2RG CD40LG PRF1 IFNGR2CD8Aa

b c

Figure 7 Other gain-of-glycosylation mutations.

(a) Five of the six mutations identified

from the HGMD, associated with primary

immunodeficiencies and affecting genes

encoding proteins with a peptide signal or a

peptide anchor, constituted potential gain-of-

glycosylation mutations and were tested in this

experiment: Y125N in the cytokine common gchain (encoded by IL2RG), G111S in CD8a(encoded by CD8A), G220S in perforin (encoded

by PRF1), and T147N and T211N in CD154, the

CD40 ligand (encoded by CD40LG). V5-tagged

molecules encoded by the corresponding wild-

type (WT) and mutated alleles were produced in

control SV40-transformed fibroblasts, and themolecular weights (MW, in kDa) of the proteins

were determined by SDS-PAGE and western

blotting with a V5-specific antibody. Alternatively,

the cell lysates were treated with PNGase-F

(15,000 IU ml�1 for 3 h) before SDS-PAGE and

western blotting. (b) Surface expression of

CD40L (encoded by CD40LG) in the 293T/17

cell line transiently transfected with mock, wild-

type (WT), T147N, T147A and T147N-S149A

CD40L constructs, as detected 48 h after

transfection by flow cytometry using a CD40L-

specific antibody (Ab) and a soluble CD40 muIg

fusion protein (black histogram), compared with

the isotypic control (white histogram). (c) Surface

expression of CD18 (encoded by ITGB2) and

CD11a (coexpressed with CD18 on the cell

surface) in control EBV-B cells and CD18-

deficient EBV-B cells transiently transfected withmock, wild-type (WT), G284S and G284A CD18

constructs, as detected 48 h after transfection by

flow cytometry using CD18-specific and CD11a-

specific antibodies (plain line), compared with

the isotypic control (dotted line).


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We used transfected cells for flow cytometry, western blotting or confocal

microscopy within 2 d.

Cytokines, enzymes, inhibitors and antibodies. We used recombinant non-

glycosylated human IFNg (Imukin), recombinant IFNa2b (R&D Systems),

antibody to V5 (1:5,000; Invitrogen), Endo-H (New England Biolabs), PNGase-F

(New England Biolabs), tunicamycin (Sigma), streptavidin-agarose (Invitro-

gen), human CD40 muIg fusion protein (COGER), mouse antibody to human

CD40L (BD Pharmingen), mouse antibody to HLA-DR conjugated to phy-

coerythrin (Becton Dickinson), mouse antibody to human CD18 conjugated to

fluorescein isothiocyanate (Immunotech), mouse antibody to human CD11a

conjugated to fluorescein isothiocyanate (Immunotech), antibody to mouse Ig

labeled with horseradish peroxidase (1:10,000; Amersham Biosciences) and

antibody to mouse Ig labeled with Alexa 488 (Molecular Probes).

Expression plasmids. We amplified cDNA fragments encoding human IFNg-

R2, CD40L, CD18, IL-2Rg, CD8a and perforin with AmpliTaq DNA poly-

merase (Applied Biosystems). We digested PCR products with Acc65I-EcoRI for

IFNg-R2 amplification and inserted them into pcDNA3 (Invitrogen). Alter-

natively, we digested them with BglII-XmaI and inserted them into peGFP-N1

(Clontech) or tagged them in the V5/His Tag pcDNA3 vector using a

directional topoisomerase-based method. For direct mutagenesis, we used the

Stratagene kit in accordance with the manufacturer’s instructions.

Cell surface biotinylation. Two days after transfection, we labeled cells by

incubation for 30 min at 4 1C in phosphate buffered saline (PBS; pH 8.0) with

or without sulpho-NHS-LC-Biotin (Pierce). We then washed them twice in

PBS. The cross-linking reaction was stopped by adding 50 mM NH4Cl in PBS.

We then collected cells, centrifuged them in a microtube (Eppendorf) and

washed them three times with PBS. We lysed biotinylated SV40-transformed

fibroblasts with the appropriate buffer for immunoprecipitation (as recom-

mended by the manufacturer) and incubated them overnight with streptavidin-

agarose. We analyzed immunoprecipitates by SDS-PAGE and chemilumines-

cence with an antibody to V5.

EMSA. We analyzed EBV-B cells by EMSA as previously described16. We

annealed 50 mg of sense/antisense GAS or ISRE oligonucleotide probes by

boiling them for 5 min at 100 1C and then leaving them at room temperature

for 1 h. We then stored the mixture at �20 1C. The annealed probe (0.1 mg) was

radioactively labeled with P32-dATP by incubation with the Klenow fragment of

DNA polymerase I (USB). We purified the radioactively labeled probe on a G25

column and collected and tested fractions by scintillation counting. We lysed

cells in membrane lysis buffer, centrifuged the nuclear extracts and dissolved

the pellet in nuclear lysis buffer. We collected supernatants and incubated them

with the radioactively labeled probe and poly-dIdC in a loading buffer for

30 min. We then loaded the nuclear extracts (10 mg) onto a 5% acrylamide gel

and separated them in 0.5� Tris-buffered EDTA. We dried the gel and placed it

against a phosphor screen (Molecular Dynamics).

Endo-H and PNGase-F digestions and inhibition of glycosylation. Endo-H

and PNGase-F specifically cleave N-linked carbohydrates. Endo-H cleaves the

immature oligosaccharide core added in the endoplasmic reticulum but not the

carbohydrate moieties matured in the Golgi apparatus20. PNGase-F cleaves

the carbohydrate chain between the innermost N-acetylglucosamine and aspar-

agine residues of both immature and mature oligosaccharides. Two days after

transfection, we lysed SV40-transformed fibroblasts and digested them with

Endo-H or PNGase-F in the appropriate buffer (in accordance with the

manufacturer’s instructions). We carried out digestions for 3 h or overnight

and analyzed the products by SDS-PAGE and chemiluminescence. To block N-

glycosylation, we incubated cells with 8 mg ml�1 tunicamycin for 30 min, 18 h

before EMSA analysis. After transfection, we treated fibroblasts with PNGase-F

(750 UI ml�1) or tunicamycin (0.1 mg ml�1) for 48 h, with or without IFNg,

and analyzed them by flow cytometry.

Flow cytometry and confocal microscopy. We monitored expression of

CD18, CD40, CD154 (CD40L) and HLA-DR on EBV-B cells, 293T cells and

SV-40-fibroblasts by flow cytometry as previously described9. For confocal

microscopy, transfected SV40-transformed cells were fixed in 3.7% parafor-

maldehyde and neutralized by incubation with 50 mM NaCl in PBS. We then

washed the cells in PBS and labeled an aliquot with DiC16. We mounted the

cells in Mowiol, covered them with a coverslip and examined them using a

confocal laser scanning microscope (Leica LSCM). We used the following

filters: eGFP: excitation, 488 nm; emission, 510 nm. We acquired Z stacks of

0.5 mm with a 63� objective with an NA of 1.32 and a pinhole setting of 1. All

scanned images thus acquired were transferred to a Leica NT workstation.

Electrophoresis and western blotting. We carried out SDS-PAGE with 10%

polyacrylamide gels (separating gel buffer: 0.375 M Tris (pH 8.8), 0.01% SDS;

stacking gel buffer: 0.5 M Tris (pH 6.8), 0.01% SDS; Laemmli buffer: 25.5 mM

Tris (pH 8.3), 0.18 M glycine, 0.13% SDS)8. We washed samples in PBS and

boiled them for 5 min at 100 1C in stacking gel buffer with 20% glycerol, 4%

SDS, 1.4 M b-mercaptoethanol and bromophenol blue. We then subjected

them to SDS-PAGE and transferred the resulting bands to PVDF membrane

(0.2 mm Bio-Rad) by 2 h at 1.5 mA cm�2 in transfer buffer (25.5 mM Tris,

0.18 M glycine and 20% methanol). We blocked the membrane in 5% bovine

serum albumin in PBS for 1 h, rapidly washed it in 0.035% Tween in PBS and

incubated it overnight with an appropriate first antibody diluted in 0.028%

Tween, 1% bovine serum albumin in PBS. We then washed the membrane five

times in 0.035% Tween in PBS and incubated it with horseradish peroxidase–

conjugated secondary antibody for 30 min in 0.028% Tween, 1% bovine serum

albumin in PBS. We detected bound antibody with ECL western blotting

detection reagents (Amersham Biosciences) and chemiluminescence on Bio-

Max MR film (Kodak).

Whole-blood cultures and activation by BCG. We collected venous blood

samples into heparinized tubes, diluted them 1:2 in RPMI 1640 medium

(GibcoBRL) and supplemented the suspension with 100 U ml�1 penicillin and

100 mg ml�1 of streptomycin (GibcoBRL). We dispensed 6-ml diluted blood

samples into four wells (1.5 ml per well) of a 24plate (Nunc). We then

incubated the plate in a two-stage procedure for 48–72 h at 37 1C under an

atmosphere of 5% CO2 and 95% air and under four different conditions of

activation: with medium alone, with live BCG (Mycobacterium bovis BCG,

Pasteur substrain) at a multiplicity of infection of 20 BCG per leukocyte and

with BCG plus IFNg (5,000 IU ml�1; Imukin, Boehringer Ingelheim) as

described elsewhere18. The first incubation stage was completed after 48 h of

culture; we collected 450 ml of supernatant from each culture well and froze it at

�801C. After 72 h, the end of the second incubation stage, we recovered the

remaining volume of each well, centrifuged it at 1800g for 10 min and stored

the supernatant frozen at �80 1C until analysis. We treated whole blood cells

with tunicamycin (8 mg ml�1) for 30 min at 37 1C and then washed them twice

before stimulating them with BCG and IFNg.

Cytokine ELISA. We analyzed cytokine concentrations by ELISA, using the

human Quantikine IL-12p40 kits from R&D Systems and the human Pelikin or

Pelipair IFNg kit from Sanquin, in accordance with the manufacturers’ guide-

lines. We used these kits with matched antibody pairs. We determined

absorbance using an automated MR5000 ELISA Reader (Thermolab Systems).

Quantitative analysis involved a nonlinear four-parameter logistic (4PL)

calibration model. In-house software based on the Microsoft Excel application

language was developed for this purpose. Intermediate results for each cytokine

are expressed in pg ml�1, as previously described18.

Computational analysis. To assess the relevance of this new type of mutation,

we searched the HGMD39 for potential gain- and loss-of-glycosylation muta-

tions. We counted those missense mutations in the HGMD that either create or

destroy N-glycosylation sites (consensus sequence Asn-X-Ser/Thr-X, where X is

any amino acid except proline). We ignored amino acid substitutions that

involved consensus N-glycosylation sites but were not predicted to abolish

them. We introduced single base-pair changes individually in silico into each

position of every codon for all cDNA ‘reference sequences’ using software

designed for this purpose; we counted the total number of possible changes

leading to amino acid substitutions, as well as the total number of changes

leading to either the gain or loss of an N-glycosylation site. In silico modeling

yielded the expected proportions of gain- and loss-of-N-glycosylation muta-

tions, under the assumption that these mutations occur at random. We

compared the observed proportions with those expected using w2 statistics


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(P values are indicated). Genes encoding proteins that carry either a signal

peptide or signal anchor were selected on the basis of in silico predictions40. The

probability of bearing a signal peptide (a hydrophobic segment with a cleavage

site) was summed with that of bearing a signal anchor (a hydrophobic segment

with no cleavage site), and genes with a combined probability of 0.75 or above

were selected.

URLs. The NetNGlyc server is available at http://www.cbs.dtu.dk/services/

NetNGlyc/. The HGMD is available at http://www.hgmd.org/. The Center

for Biological Sequence Analysis is available at http://www.cbs.dtu.dk/. The

website of the Laboratory of Human Genetics of Infectious Diseases is

http://www.hgid.net/.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank P. Stenson for provision of the HGMD data and C. Antignac,D. Cotton, C. Eidenschenk, J. Jaeken, C. Lamaze, P. de Lonlay, S. Lyonnet,A. Puel, K. Tedin, V. Tolyan, M. Vihinen and all members of the HGIDlaboratory for discussions. The laboratory was partially supported by grantsfrom the Schlumberger Foundation, the BNP-Paribas Foundation and theEuropean Union.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 31 January; accepted 25 April 2005

Published online at http://www.nature.com/naturegenetics/

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Gains of Glycosylation Comprise an Unexpectedly Large

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