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VAN NIEUWENHOVE ET AL 3J ALLERGY CLIN IMMUNOLVOLUME nnn, NUMBER nnVariant effect prediction on protein structureIn vitro protein transportMutations were analyzed by using the available cryo-EM structure (ProteinData Bank identifier 2wwb). All structural visualization was done by usingYASARA Structure (version 18.4.2424). Images were generated by usingRay-traced screenshots.Protein translocation experiments were performed as previouslydescribed.25,26 For a detailed description, please refer to the Supplemental Data.Western blottingFresh PBMCs and primary fibroblasts were solubilized in lysis buffercontaining 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mMegtazic acid, 1% Triton-X, phosphatase inhibitor (PhosSTOP [Roche, Basel,Switzerland]), and protease inhibitor (Pierce Protease Inhibitor[ThermoFisher Scientific, Waltham, Mass]). A quantity of 50 mg of lysate,which was determined by using a Bradford Protein Assay (Bio-Rad, Hercules,Calif), was separated on a 4% to 12% bis-Tris acrylamide gel with3-(N-morpholino)propanesulfonic acid buffer (NuPAGE Precast Gel System[Thermo Fisher Scientific]) before blotting on polyvinylidene fluoridemembrane (GE Healthcare). After blocking, the membranes were incubatedwith specific primary antibodies: rabbit anti-Sec61a1 (Abcam, Cambridge,United Kingdom, Ab183046), rabbit anti-Actin (Sigma-Aldrich, St Louis,Mo, A2103), mouse anti–glyceraldehyde-3-phosphate dehydrogenase(Thermo Fisher Scientific, MA5-15738), and mouse anti-Vinculin (Sigma,V9264). Proteins were revealed by using ECL Prime (GE Healthcare,Chicago, Ill) or Western Lightning Plus-ECL (Perkin Elmer, Waltham,Mass) with the G:box Chemi-XRQ and quantified by using ImageJ software.RNA isolation and quantificationTotal RNA was isolated by using TRIzol reagent (Ambion, Thermo FisherScientific), except for CD341 cells, in which case the ReliaPrep RNA CellMiniprep System (Promega, Madison, Wis) was used. ComplementaryDNAwas synthesized by using the GoScriptTM Reverse Transcription System(Promega). Quantitative PCR was performed on a StepOnePlus real-time PCRsystem (ABI) with Fast SYBR Green Master Mix (Applied Biosystems, FosterCity, Calif) supplemented with gene-specific primers (see Table E1 in this article’s Online Repository at www.jacionline.org). Experiments were performed in duplicate and repeated thrice. For DDIT3 and HSPA5, TaqManGene Expression Assays were used (ThermoFisher Scientific) and expressionwas normalized to 18S.Confocal microscopyHL-60 cells were differentiated by using 1% dimethyl sulfoxide (DMSO)and allowed to attach to coverslips coated with poly-L-lysine (0.1%) (Sigma)by a 20-minute incubation in PBS at room temperature. After fixation with 4%paraformaldehyde (or with methanol for anti–protein disulphide isomerase),cells were permeabilized with 0.1% Triton X-100 in PBS and incubated inblocking buffer (PBS 1 2% BSA 1 10% donkey serum 1 0.1% TritonX-100). The specific primary antibodies used include rabbit anti-GM130(G7295, Sigma) for Golgi detection, rabbit anti-protein disulphideisomerase (3501S, Cell Signaling Technology, Danvers, Mass) for ER detection, and 4’,6-diamino-2-phenylindole (D1306; Molecular Probes, Eugene,Ore). The secondary antibody used was donkey anti-rabbit Alexa Fluor 555(A31572; Molecular Probes). Images were acquired with the Nikon A1Rconfocal unit mounted on an Eclipse Ti inverted microscope.Calcium fluxAfter labeling with Zombie Aqua (Biolegend, San Diego, Calif, catalog No.4231), cells were loaded with 1 mM FuraRed AM (Thermo Fisher Scientific,catalog No. F3020) in HBSS and incubated in calcium-free buffer (150 mMNaCl, 6 mM KCl, 10 mM HEPES, and 1.2 mM MgCl2 [pH 7.4]). Followingacquisition of background signals, 1 mM thapsigargin was added and acquisition was immediately resumed for 10 minutes by using BD FACSCanto II. FuraRed was recorded as the ratio of emission at 450/50 nm following excitationat 405 nm and emission at 670 nm following excitation at 488 nm. Thecollected data (FACSDiva software, BD Biosciences, San Jose, Calif) wereanalyzed as mean fluorescence ratio by using the Kinetics platform providedin FlowJo version 10.6.0 software (Tree Star Inc, Ashland, Ore).Differentiation of CD341 cells from peripheral bloodCD341 cells were isolated from PBMCs by using the CD34 MicroBead KitUltraPure (Miltenyi) and were differentiated by using SCF, FLT3L, TPO, IL-3,and G-CSF. For a detailed description, please refer to the Supplemental Data.Flow cytometryThawed PBMCs were stained in Brilliant Stain Buffer (BD Biosciences) forlive-dead and surface markers by using anti-human antibodies. Data werecollected on BD Symphony (BD Biosciences) and analyzed by using FlowJo forMac version 10.5 (Tree Star Inc). Primary neutrophils were isolated by using theEasySep Direct Human Neutrophil Isolation Kit (StemCell Technologies,Vancouver, British Columbia, Canada) and incubated with Fc block (MiltenyiBiotec, Bergisch Gladbach, Germany) before staining with mAbs. Flowcytometric analysis was performed with a FACS BD LSR Fortessa X20 (BDBiosciences). Analysis was performed with FlowJo software (LLC). For detailson the antibodies used, refer to the Supplemental Data.Single-cell sequencingSingle-cell RNA sequencing libraries were generated by using a commercial droplet platform (Chromium Controller system [10x Genomics, Pleasanton, Calif]). Briefly, the EasySep RBC Depletion Reagent (StemCellTechnologies) was used to deplete erythrocytes from whole bone marrowsamples collected in heparinized tubes. The cell count and viability of the cellswere assessed by using a LUNA dual fluorescence cell counter (LogosBiosystems, South Korea), and as per the 10x Genomics recommendations(Single-Cell 3’ Reagent Kits Version 2 User Guide; CG00052 Rev B), weaimed at a target recovery of 6,000 cells per sample. After the cell count andquality control, the samples were immediately loaded individually onto theChromium Controller. Single-cell RNA sequencing libraries were prepared byusing the manufacturer recommendations, and at the different check points thelibrary quality was assessed by using Qubit (ThermoFisher) and Bioanalyzer(Agilent, Santa Clara, Calif). With a sequencing coverage target of 50,000reads per cell, single-cell libraries were sequenced on the Illumina HiSeq4000platform by using paired-end sequencing workflow with 10x version 3 readparameters (28-8-0-91 cycles). For single-cell sequencing analysis, pleaserefer to the Supplemental Data provided.Annexin V and propidium iodide stainingPBMCs were treated for 24 hours with DMSO (vehicle control),thapsigargin (1 mM), brefeldin A (4 mg/mL), or tunicamycin (10 mg/mL).After the PBMCs had been stained with Zombie Aqua, cells were stained withallophycocyanin-annexin V (Invitrogen, Thermo Fisher Scientific) andpropidium iodide (00-6990) according to the manufacturer’s instructions,followed by acquisition using the BD FACSCanto II system.Lentiviral transductionHuman SEC61A1 (NM 013336.4) was cloned into the pWPXL backboneby using an In-Fusion HD Cloning Kit (Takara Bio USA, Mountainview, Calif).HEK293T cells were transfected with lentiviral vectors and second-generationpackaging plasmids by using X-tremeGEne HP Transfection Reagent (SigmaAldrich). Confluent HL60 cells were resuspended in filtered undiluted viruscontaining medium with 5 mg/mL of polybrene for 16 hours. TransducedHL-60 cells were sorted with BD FACSAria II gating on live GFP1 cells.Differentiation of HL60 cellsHL-60 cells were plated in T75 flasks and treated with 1% DMSO for 6 dayswith a refreshment of media on day 4. Differentiated cells were stained with

4 VAN NIEUWENHOVE ET ALZombie Aqua followed by surface staining with the antibodies anti-CD11ballophycocyanin-eFluor 780 (ICRF44, eBioscience, San Diego, Calif) andanti-CD16 PerCP (3G8, BioLegend). Cells were acquired by using the BDFACSCanto II system. For cytospin preparations, single-cell suspensions werespun on a glass slide and stained with hematoxylin and eosin. Cells werevisualized on a Leica DM 2000 microscope (Leica Microsystems, Wetzlar,Germany) for manual counting by 3 independent researchers.StatisticsStatistical analyses were performed by using a (un)paired t test or asindicated.Data sharing statementFor original data please contact Stephanie Humblet-Baron (E-mail address:stephanie.humbletbaron@kuleuven.be). The 10X Genomics RNA sequencingdata on bone marrow samples are available at the Gene Expression Omnibusunder accession number GSE137496.RESULTSIdentification of a de novo mutation in SEC61A1 in apatient with SCNThe index patient is a 19-year-old female who was born at termas the only child to nonconsanguineous parents of EuropeanBelgian descent. Her birth weight was 2750 g (–1.8 SD) (Fig 1, A).At the age of 4 months she presented with a retroauricularStaphylococcus aureus abscess and subsequently suffered fromrecurrent otitis, bronchitis, sinusitis, and tonsillitis thatnecessitated a tonsillectomy at age 5 years. At the age of 1year, she was treated for varicella that was complicated bybacterial superinfection treated with oral antibiotics. Aside fromher infectious susceptibility, she presented with oral aphthosis,problematic wound healing, gingivitis, and an episode of limitedverrucae plantaris. She was diagnosed with SCN when herneutrophil levels were analyzed during hospitalization forenteritis at the age of 2years. At 7 years of age, she developedpneumonia with Streptococcus viridans complicated byempyema. Aside from prophylactic antibiotics, she beganreceiving subcutaneous granulocyte colony-stimulating factor(G-CSF) at times of infection from the age of 7 years. Followingan Escherichia coli abscess in her facial region at the age of 15years and septicemia secondary to a gluteal E coli abscess ayear later, she began receiving daily G-CSF. When G-CSFtherapy was tapered to every 2 days, she developed a refractoryperianal abscess with Proteus mirabilis that cleared only aftersurgical drainage, intravenous antibiotic treatment, andlong-term administration of G-CSF at a rate of 12 mg/kg perday. Her neutrophil counts were repeatedly below 0.5 3109/Lbut responsive to G-CSF treatment (see Fig E1 in this article’sOnline Repository at www.jacionline.org). Before any initiationof G-CSF treatment at age 3 years, her complete blood countshowed a total leukocyte level of 4.79 3 109/L with a neutrophillevel of 0.048 3 109/L, lymphocyte level of 2.108 3 109/L,monocyte level of 1.916 3 109/L, eosinophil level of0.287 3 109/L, and basophil level of 0.096 3 x109/L. The resultof testing for anti-granulocyte antibodies was negative, andfurther laboratory work-up revealed hypergammaglobulinemiabefore the initiation of daily G-CSF (in particular, IgG2 andIgA [see Fig E1]), mild thrombocytopenia (a nadir of 104[reference range 150-450 3 109/L]), and discrete intermittentanemia (a nadir of 10.2 [reference range 12.0-16.0g/dL]). UnderJ ALLERGY CLIN IMMUNOLnnn 2020daily G-CSF treatment her total leukocyte numbers increased to9.19 3 109/L (reference range 4.0-10.0), with a myelocyte level0%]), neutrophil levelof 0.1 3 109/L (1% [reference percentage of 3.4 3 109/L (37% [reference range 38%-77%]), lymphocytelevel of 2.2 3 109/L (24% [reference range 20%-50%]), basophil1%]), relativelevel of 0.2 3 109/L (2% [reference percentage monocytosis with a monocyte level of 2.4 3 109/L (26%[reference range 2.0%-10.0%]), and relative eosinophilia withan eosinophil level of 1.0 3 109/L (10% [reference range 6%]). Bone marrow analysis before G-CSF treatment showeda maturation arrest in granulopoiesis at the level of thepromyelocytes and myelocytes (Fig 1, B).Whole exome sequencing was performed after negative geneticscreening of SBDS, GATA2, and CXCR4 through Sangersequencing and negative genetic screening of known genes forSCN (ELANE, G6PC3, GFI1, HAX1, LAMTOR2, SLC37A4,USB1, VPS13B, SBDS, VPS45, WAS, and JAGN1) and inborn errors of immunity through custom-designed gene panels. Throughfiltering for rare de novo or recessive variants, we identified anovel heterozygous mutation in SEC61A1 (NM 013336),c.A275G:p.Gln92Arg, that was not registered in any publicdatabase and had functional scores predicted as damaging byPolymorphism Phenotyping, Sorting Intolerant fromTolerant, and Combined Annotation-Dependent Depletion(CADD 24.500; mutation significance cut-off 6.099) analyses(Fig 1, C). To date, mutations in SEC61A1 have been reportedin 2 kindreds with ADTKD (c.T200G:p.Val67Gly andc.A553G:p.Thr185Ala)21 and 2 kindreds with common variableimmune deficiency (CVID) with incomplete penetrance(c.T254A:p.Val85Asp and c.G1325T:p.Glu381*)22 (Fig 1, Dand see Table E2 in this article’s Online Repository at www.jacionline.org). Although neutropenia was observed in thep.V67G kindred in addition to the well-described late-onsetADTKD and congenital anemia, it was not further investigated.21In light of our patient, the cosegregation of SCN in the p.V67Gkindred may also be attributable to mutant SEC61A1. Conversely,we investigated the possibility of subclinical renal dysfunction inour patient. Ultrasound imaging revealed small bilateral kidneys(right kidney, 9.6 cm [–3.2 SD]; left kidney 9.8 cm [–2.5 SD]) thatwere morphologically normal. Her urine sediment analysis resultsand kidney function remained normal. However, her uric acidlevels were slightly elevated over the past year (6.6 mg/dL[reference range 2.6-6.0 mg/dL]). Otherwise, no evidence forextramedullary involvement was found. In view of our currentfindings, we conclude that mutations in SEC61A1 can give riseto a clinical spectrum of disease including SCN.Biochemical impact of SEC61A1 mutationsFirst, we investigated the structural consequences of theresulting mutations. Both of the neutropenia-associated mutationsaffect the well-conserved amino acid residues Gln92 (Q92) andVal67 (V67) (Fig 1, E). The heterotrimeric Sec61 translocon complex comprises Sec61b, Sec61g, and Sec61a1, with the lattercontaining 10 transmembrane helices (TMs) that form the centralchannel of Sec61. In the closed conformation, a so-called plugdomain between TM1 and TM2 interacts with the center of transmembrane helix bundle, thereby blocking and stabilizing thepore.21 It is believed that signal peptides of nascent proteins intercalate between TM2 and TM7, with displacement of TM2 andsubsequent opening of the ‘‘lateral gate’’ of the Sec61 complex

J ALLERGY CLIN IMMUNOLVOLUME nnn, NUMBER nnVAN NIEUWENHOVE ET AL 5FIG 1. Autosomal dominant mutations in SEC61A1 cause SCN. A, Pedigree of a family with segregation ofthe p.Q92R mutant allele. The index patient is denoted by a black symbol. B, May-Gr unwald Giemsa stainingof representative bone marrow smears from the index patient. Promyelocytes are marked by the symbol %,and myelocytes are marked by the symbol *. Magnification, 3500. C, Sequencing profiles demonstratingthe heterozygous SEC61A1 c.A275G:p.Gln92Arg mutation in the kindred. D, Table of all reported mutationsin SEC61A1 with associated phenotypes. E, Multiple alignment to display conservation exported by usingHomoloGene (National Center for Biotechnology Information). F-H, Illustration of mutated residues in greenobtained using available cryo-EM structure (Protein Data Bank identifier 2wwb), with lumenal view in (F)and cytosolic view in (H). The plug domain is visible in red, the Sec61b protein is visible in blue, and theSec61g subunit is visible in orange.comprising these 2 TMs. Utilizing the available cryo-EM structure (Protein Data Bank identifier 2wwb),27 we determined thatboth Q92 and V85 are located in TM2 (Fig 1, F-H). In the closedconformation, Q92 points into the core to form a hydrogen bondwith a neighboring residue, whereas in the open conformation, itforms a hydrogen bond with the translocating peptide. V85 islocated closer to the core of the helix bundle. Both Q92R andV85D introduce a buried charge in the hydrophobic core of theTM bundle without the presence of a counter charge and are therefore predicted to severely destabilize the helix bundle thermodynamically. V67 is situated in the middle of an a-helical part of theplug domain, and mutating this residue to a glycine is likely destabilizing (Fig 1, F-H). Glycine mutations notoriously destabilizea-helices thermodynamically, especially when occurring at internal positions.28 Thus, this mutation potentially affects the proteintranslocation regulatory function of the plug domain.Additionally, in the closed conformation, the wild-type (WT)side chain of V67 forms hydrophobic interactions with TM5and TM10 that are completely removed by the truncation toglycine, further decreasing the interaction energy between theplug domain and the 2 transmembrane helices. Mapping the electrostatic potential on the molecular surface of Sec61 revealed thatthe WT is positively charged, maintaining a constant distributionof the potential around the internal surface of the channel (see FigE2, A in this article’s Online Repository at www.jacionline.org).Although V67G does not disturb the electrostatic potential (seeFig E2, B), V85D increases the negative charge at the bottom ofthe channel (see Fig E2, C), in contrast to the Q92R, which insteadraises the positive charge on the inside of the channel (see Fig E2,D). These opposing alterations may directly disturb the conformational changes of the Sec61 complex or affect its ability to interactwith or translocate newly synthesized polypeptide chains. Using

6 VAN NIEUWENHOVE ET ALJ ALLERGY CLIN IMMUNOLnnn 2020FIG 2. Mutations in SEC61A1 affect protein stability and disrupt ER/cytosol calcium homeostasis. A,Sec61a1 expression in healthy controls (HCs) and the patient (with P’ indicating the patient’s status as ofApril 2019 and P’’ indicating the patients status as of June 2018). B, Quantification of (A) normalized tothe average of the HCs. C, SEC61A1 mRNA expression in PBMCs (n 5 3) and primary fibroblasts from 2different passages (n 5 4), normalized to the average of the HCs. Unpaired t test. D, HL-60 cells transducedwith SEC61A1–green fluorescent protein (GFP) stained with anti–protein disulphide isomerase (PDI) (left)and anti-GM130 (right) (n 5 2). E, Ratio of cells with colocalization of Sec61a1 and GM130 over the totalnumber of cells from (D), normalized to WT. F, Calcium flux measurements in transduced HL-60 cells.Mean of 3 independent experiments with duplicates. G, Amplitude of calcium efflux following thapsigargin

J ALLERGY CLIN IMMUNOLVOLUME nnn, NUMBER nnthese structural predictions, we noted that pathogenic mutationscluster in the pore-forming region.In support of the predicted impact of

and Immunology, Laboratory of Molecular Immunology, Rega Institute for Medical . Allergy, Asthma & Immunology. This is an open accessarticle under the CC BY-NC- . CHOP: CCAAT/enhancer-binding protein homologous protein CVID: Common variable immune deficiency DMSO: Dimethyl sulfoxide ER: Endoplasmic reticulum G-CSF: Granulocyte colony .