Electronic supplementary materials
Supplementary materials and methods
DNA/ RNA extraction, cDNA synthesis and cell isolation
Genomic DNA and RNA were extracted from PBMCs using the QIAamp DNA Blood Mini kit, AllPrep DNA/RNA/Protein Mini kit, and RNeasy Mini kit (all from Qiagen, Hilden, Germany). cDNA was synthesized using reverse transcriptase and the SuperScript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, US).PBMCs and neutrophils were isolated from heparinized peripheral blood using LymphoprepTM (Axis-Shield PoC AS, Oslo, Norway), Mono-poly resolving medium (DS Pharma Biomedical Co., Osaka, Japan), and a previously described standard technique[1]. B cells and naïve B cells were isolated from whole blood using the RosetteSepTMHuman B Cell Enrichment Cocktail (Stemcell Technologies, Vancouver, Canada) and the Naïve B Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany).
Antibodies
The following antibodies were used for flow cytometric staining: anti-IgD fluorescein isothiocyanate (FITC), anti-IgM phycoerythrin-cyano dye 5 (PECy5), anti-IgG allophycocyanin (APC), anti-CD19 APC, and anti-CD21 APC (all from BD Pharmingen Inc., San Diego, US); anti-CD38 FITC, anti-IgD FITC, anti-CD27 phycoerythrin (PE), and anti-CD27 phycoerythrin-cyanine 7 (PC7), anti-CD24 phycoerythrin 5-succinimidylester (PC5), anti-CD19 phycoerythrin-Texas Red (ECD) (all from Beckman Coulter, Inc., Indianapolis, US); anti-IgA PE, anti-CD19 APC, anti-CD19 VioBlue, anti-CD19 PacificBlue, anti-CD20 VioGreen, and anti-CD24 PE (all from Miltenyi Biotec, Bergisch Gladbach, Germany); and anti-CD14 Alexa Fluor 700 (BioLegend, San Diego, US).Expression of BTK was determined in monocytes stained with an anti-BTK monoclonal antibody [48-2H] and control IgG1 (Dako Japan Inc., Tokyo, Japan) as previously reported [2]. Expression of tumor necrosis factor receptor superfamily, member 13B (TNFRSF13B) was determinedin memory B cells by usinga rat anti-TNFRSF13B monoclonal antibody [1A1] (Abcam, Cambridge, UK) and control IgG2a (eBioscience, San Diego, US).
Exome and sequencing analysis
Variant calling was performed using the Genome Analysis Toolkit (GATK)[3] In the validation study, genomic DNA from the patient and his parents was amplified by means of polymerase chain reaction(PCR) and PCR products were sequenced using the BigDye Terminator (Applied Biosystems, Foster City, US).
Sequencing analysis of BTK and TNFRSF13B
In the validation study, genomic DNA was amplified by PCR using High Fidelity PCR Master (Roche Applied Science, Indianapolis, US) orPrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Shiga, Japan).PCR was performed for 30 cycles at an annealing temperature of 60 °C for 15 to 30 seconds.PCR products were sequenced with the forward primer 5’-ACAGCTTCTTTTTCGTTGTTTC-3’ and the reverse primer 5’-GAAAGAGGAGAGCTTCTGATTTTG-3’ for exon 11 of BTK, and with the forward primer 5’-GTGATTGCCCTTAGCTCCTG-3’ and the reverse primer 5’-AACCCAAGGTCACAAGCTTC-3’ for exon 5 of TNFRSF13B.
In vitro class switch recombination
Naïve B cells (CD19+CD27- cells) were purified to a purity of >90% by using the Naïve B Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). A total of 1 106 cells/ml B cells or naïve B cells were incubated in RPMI 1640 supplemented with 10% fetal bovine serum in 96-well plates at 37°C in 5% CO2 with or without LEAFPurified anti-CD40 [G28.5] (1 µg/ml; BioLegend, San Diego, US) and human IL-21 (100 ng/ml; Miltenyi Biotec, Bergisch Gladbach, Germany). The culture supernatants were collected on day 12 and the level of immunoglobulin was measured by enzyme-linked immunosorbent assay (ELISA)[4].
Measurement of T cell receptor excision circlesand kappa-deleting recombination excision circles levels
The levels of TRECs and KRECs were measured in whole blood by real-time PCR as described previously[5]. RNase P was used as an internal control.
Measurement of reactive oxygen species production by neutrophils
ROS production by neutrophils was quantified using standard chemiluminescence using phorbol 12-myristate 13-acetate (PMA) and luminol (Sigma-Aldrich)as previously described[1].In brief, neutrophils (1 × 106cells) were suspended in 0.5 ml PBS containing luminol (10 μM) preheated to 37 °C. After a baseline measurement was obtained, cells were stimulated with PMA (100 ng/ml), and luminescence signals were monitored throughout the reaction.
Supplementary results
Supplementary Table 1.
Parameter / Value / Normal rangeWhite 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) / 49 / 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
SupplementaryTable legend
TableI.Laboratory data of patient at 10 months of age
Age-matched normal range was taken from previous reports[6-9].EIA, Enzyme immunoassay; TRECs, T cell receptor excision circles; KRECs, kappa-deleting recombination excision circles; RSS-KDE, Recombination signal sequence-kappa deleting element.
Supplementaryfigure legends
SupplementaryFig 1. Patient (1-year-old) peripheral B cell subpopulation.
Flow cytometric analyses of patient lymphocyte CD21, CD24, CD38, IgD, and CD27 expression.
SupplementaryFig2. Filtering approach for detected mutations by whole-exome sequencing.
Mutations registered in dbSNP database version137 were excluded, and coding region, splice site, and non-synonymous mutations were selected. The number of these variants after each filtering step is shown. Genotype and type of amino acid change are shown for selected mutations.
SupplementaryFig 3. Genetic analysis of TNFRSF13B.
a, Sanger sequencing of TNFRSF13B exon 5 in the patient and his parents. b, Flow cytometric analysis of TNFRSF13B expression. Solid line, healthy donor; dotted line, mother; small dotted line patient; gray, isotype control.
Supplementary Fig 4.B cell signaling andin vitro immunoglobulin production.
a.Flow cytometric analyses of PLCγ2 phosphorylation in CD19normal (solid line) or CD19low (dotted line) stimulated with anti-IgM antibody. gray, unstimulated cells. b. IgG and IgA production from naïve B (healthy donors) or B cells (patient) incubated with anti-CD40 antibody and IL-21. Control values represent four independent experiments (mean SE). Open bars, unstimulated cells; solid bars, stimulated cells.
Supplementary Fig5. Neutrophil ROS production.
Kinetics of H2O2 production in neutrophils from healthy donors (open symbols) and patient (closed symbols). Arrows indicate time points of stimulation with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and LPS plus fMLP
Supplementary references
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