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Electronic supplementary materials
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Supplementary materials and methods
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DNA/ RNA extraction, cDNA synthesis and cell isolation
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Genomic DNA and RNA were extracted from PBMCs using the QIAamp DNA Blood Mini kit, AllPrep
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DNA/RNA/Protein Mini kit, and RNeasy Mini kit (all from Qiagen, Hilden, Germany). cDNA was
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synthesized using reverse transcriptase and the SuperScript VILO cDNA Synthesis kit (Invitrogen, Carlsbad,
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US). PBMCs and neutrophils were isolated from heparinized peripheral blood using Lymphoprep TM (Axis-
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Shield PoC AS, Oslo, Norway), Mono-poly resolving medium (DS Pharma Biomedical Co., Osaka, Japan),
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and a previously described standard technique [1]. B cells and naïve B cells were isolated from whole blood
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using the RosetteSepTMHuman B Cell Enrichment Cocktail (Stemcell Technologies, Vancouver, Canada) and
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the Naïve B Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany).
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Antibodies
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The following antibodies were used for flow cytometric staining: anti-IgD fluorescein isothiocyanate (FITC),
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anti-IgM phycoerythrin-cyano dye 5 (PECy5), anti-IgG allophycocyanin (APC), anti-CD19 APC, and anti-
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CD21 APC (all from BD Pharmingen Inc., San Diego, US); anti-CD38 FITC, anti-IgD FITC, anti-CD27
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phycoerythrin (PE), and anti-CD27 phycoerythrin-cyanine 7 (PC7), anti-CD24 phycoerythrin 5-
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succinimidylester (PC5), anti-CD19 phycoerythrin-Texas Red (ECD) (all from Beckman Coulter, Inc.,
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Indianapolis, US); anti-IgA PE, anti-CD19 APC, anti-CD19 VioBlue, anti-CD19 PacificBlue, anti-CD20
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VioGreen, and anti-CD24 PE (all from Miltenyi Biotec, Bergisch Gladbach, Germany); and anti-CD14 Alexa
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Fluor 700 (BioLegend, San Diego, US). Expression of BTK was determined in monocytes stained with an
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anti-BTK monoclonal antibody [48-2H] and control IgG1 (Dako Japan Inc., Tokyo, Japan) as previously
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reported [2]. Expression of tumor necrosis factor receptor superfamily, member 13B (TNFRSF13B) was
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determined in memory B cells by using a rat anti-TNFRSF13B monoclonal antibody [1A1] (Abcam,
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Cambridge, UK) and control IgG2a (eBioscience, San Diego, US).
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Exome and sequencing analysis
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Variant calling was performed using the Genome Analysis Toolkit (GATK) [3] In the validation study,
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genomic DNA from the patient and his parents was amplified by means of polymerase chain reaction (PCR)
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and PCR products were sequenced using the BigDye Terminator (Applied Biosystems, Foster City, US).
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Sequencing analysis of BTK and TNFRSF13B
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In the validation study, genomic DNA was amplified by PCR using High Fidelity PCR Master (Roche
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Applied Science, Indianapolis, US) or PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Shiga, Japan).
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PCR was performed for 30 cycles at an annealing temperature of 60 °C for 15 to 30 seconds. PCR products
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were sequenced with the forward primer 5’-ACAGCTTCTTTTTCGTTGTTTC-3’ and the reverse primer 5’-
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GAAAGAGGAGAGCTTCTGATTTTG-3’ for exon 11 of BTK, and with the forward primer 5’-
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GTGATTGCCCTTAGCTCCTG-3’ and the reverse primer 5’-AACCCAAGGTCACAAGCTTC-3’ for exon 5
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of TNFRSF13B.
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In vitro class switch recombination
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Naïve B cells (CD19+CD27- cells) were purified to a purity of >90% by using the Naïve B Cell Isolation Kit II
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(Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 1  106 cells/ml B cells or naïve B cells were
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incubated in RPMI 1640 supplemented with 10% fetal bovine serum in 96-well plates at 37°C in 5% CO2 with
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or without LEAFPurified anti-CD40 [G28.5] (1 µg/ml; BioLegend, San Diego, US) and human IL-21 (100
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ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany). The culture supernatants were collected on day 12 and
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the level of immunoglobulin was measured by enzyme-linked immunosorbent assay (ELISA) [4].
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Measurement of T cell receptor excision circles and kappa-deleting recombination excision circles levels
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The levels of TRECs and KRECs were measured in whole blood by real-time PCR as described previously
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[5]. RNase P was used as an internal control.
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Measurement of reactive oxygen species production by neutrophils
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ROS production by neutrophils was quantified using standard chemiluminescence using phorbol 12-myristate
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13-acetate (PMA) and luminol (Sigma-Aldrich) as previously described [1]. In brief, neutrophils (1 × 106
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cells) were suspended in 0.5 ml PBS containing luminol (10 μM) preheated to 37 °C. After a baseline
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measurement was obtained, cells were stimulated with PMA (100 ng/ml), and luminescence signals were
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monitored throughout the reaction.
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Supplementary results
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Supplementary Table 1.
Parameter
Value
Normal range
White blood cells (/μL)
12,800
6,600–13,600
CD3+ T lymphocytes (% of lymphocytes)
43.0
56–87
CD16+CD56+ NK cells (% of lymphocytes)
32.1
1–64
CD19+ B lymphocytes (% of lymphocytes)
14.2
11.1–45.4
IgG (mg/dl)
1,335
792  376.5
IgG1 (mg/dl)
963
234.0–830.6
IgG2 (mg/dl)
337
50.8–224.0
IgG3 (mg/dl)
151
18.7–95.4
IgG4 (mg/dl)
<3.0
0.3–16.5
IgA (mg/dl)
<4.0
56.0  21.7
IgM (mg/dl)
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83.8  37.2
anti-measles-specific IgG antibody (EIA)
4.1
2.0
anti-rubella-specific IgG antibody (EIA)
14.0
2.0
TRECs (copies/μg DNA)
2.0 × 105
KRECs / intronRSS-KDE (copies/μg DNA)
4.6 × 104 / 3.8 × 104
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Supplementary Table legend
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Table I. Laboratory data of patient at 10 months of age
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Age-matched normal range was taken from previous reports [6-9]. EIA, Enzyme immunoassay; TRECs, T cell
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receptor excision circles; KRECs, kappa-deleting recombination excision circles; RSS-KDE, Recombination
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signal sequence-kappa deleting element.
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Supplementary figure legends
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Supplementary Fig 1. Patient (1-year-old) peripheral B cell subpopulation.
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Flow cytometric analyses of patient lymphocyte CD21, CD24, CD38, IgD, and CD27 expression.
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Supplementary Fig 2. Filtering approach for detected mutations by whole-exome sequencing.
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Mutations registered in dbSNP database version137 were excluded, and coding region, splice site, and non-
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synonymous mutations were selected. The number of these variants after each filtering step is shown.
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Genotype and type of amino acid change are shown for selected mutations.
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Supplementary Fig 3. Genetic analysis of TNFRSF13B.
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a, Sanger sequencing of TNFRSF13B exon 5 in the patient and his parents. b, Flow cytometric analysis of
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TNFRSF13B expression. Solid line, healthy donor; dotted line, mother; small dotted line patient; gray, isotype
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control.
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Supplementary Fig 4. B cell signaling and in vitro immunoglobulin production.
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a. Flow cytometric analyses of PLCγ2 phosphorylation in CD19normal (solid line) or CD19low (dotted line)
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stimulated with anti-IgM antibody. gray, unstimulated cells. b. IgG and IgA production from naïve B (healthy
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donors) or B cells (patient) incubated with anti-CD40 antibody and IL-21. Control values represent four
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independent experiments (mean  SE). Open bars, unstimulated cells; solid bars, stimulated cells.
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Supplementary Fig 5. Neutrophil ROS production.
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Kinetics of H2O2 production in neutrophils from healthy donors (open symbols) and patient (closed symbols).
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Arrows indicate time points of stimulation with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-
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Phe (fMLP), and LPS plus fMLP
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Supplementary references
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