Novel Mutations in a Japanese Patient with CD19 Deficiency

ORIGINAL ARTICLE Novel mutations in a Japanese patient with CD19 deficiency

H Kanegane1,5, K Agematsu2,5, T Futatani1, MM Sira1, K Suga3, T Sekiguchi3, MC van Zelm4 and T Miyawaki1 1Department of Pediatrics, Graduate School of Medicine, University of Toyama, Toyama, Japan; 2Department of Infection and Host Defense, Shinshu University Graduate School of Medicine, Matsumoto, Japan; 3Department of Pediatrics, Takamatsu Red Cross Hospital, Takamatsu, Japan and 4Department of Immunology, University Medical Center, Erasmus MC, Rotterdam, The Netherlands

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by recurrent bacterial infections, hypogammaglobulinemia and low to normal numbers of circulating B cells. Mutations in the ICOS, TACI and CD19 genes have recently been identified in o10% of CVID patients. We, herein, describe two novel CD19 gene disruptions in an 8-year-old Japanese boy, who had been clinically diagnosed as having CVID at the age of 5 years. Flow-cytometric analysis demonstrated absence of CD19 and reduced CD21 expression on CD20-postive peripheral blood B cells. Mutation analysis of CD19 revealed a mutation in the splice acceptor site of intron 5 (IVS5-1G4T) of the maternal allele, resulting in skipping of exon 6, and a truncated protein product. The paternal allele was disrupted by a gross deletion encompassing at least the ATP2A1, CD19 and NFATC2IP genes. The patient had a small number of IgDÀ CD27 þ memory B cells, in which somatic mutation were detected. His B cells showed substantial proliferation upon stimulation, but reduced IgG and IgA production in vitro. These findings extend the mutation spectrum of the CD19 deficiency to four, and confirm the homogeneity of the CD19 deficiency as a unique type of CVID. Genes and Immunity (2007) 8, 663–670; doi:10.1038/sj.gene.6364431; published online 20 September 2007

Keywords: CD19; common variable immunodeficiency; hypogammaglobulinemia

Introduction expressed on the surface of B lymphocytes, and may play a pivotal role in B-cell differentiation and activa- Common variable immunodeficiency (CVID) is the most tion.12 On mature B cells, the protein complex consisting common primary immunodeficiency that requires clin- of CD19, CD21, CD81 and CD225, signals in conjunction ical attention with an incidence of 1:10 000 to 1:50 000.1 with the B-cell antigen receptor upon antigen encounter, It is a type of primary antibody–deficiency syndrome, and is necessary to decrease the threshold for receptor- which is characterized by susceptibility to bacterial dependent signaling.12,13 infections, hypogammaglobulinemia and low to normal Recently, four hypogammaglobulinemic patients from numbers of circulating B cells.2 CVID is a heterogeneous two unrelated families were shown to have defective group of disorders, and diagnosis is mainly made CD19, resulting from mutations in the CD19 gene.11 Due by means of exclusion from other immunodeficiency to an impaired antibody response and memory B-cell diseases, such as X-linked agammaglobulinemia,3,4 hyper formation, these patients developed an immunodefi- -IgM syndrome5 or X-linked lymphoproliferative ciency in the first decade of life. We, herein, describe a syndrome.6,7 However, in several CVID patients, muta- new case of CD19 deficiency with two novel mutations in tions in genes involved in B-cell differentiation are the CD19 gene. In spite of two novel mutations, the thought to be affected. Recent studies demonstrated that immunological and clinical phenotypes are in line with in some CVID patients genetic defects in the ICOS, TACI previous observations. The CD19 deficiency appears to and CD19 genes underlie the antibody deficiency.8–11 be homogeneous and well-defined antibody deficiency. Whereas TACI is affected in B8% of CVID patients,8,9 mutations in ICOS and CD19 are thought to be very rare.10,11 CD19 is a member of the Ig superfamily Results Identification of the CD19 defects Correspondence: Dr H Kanegane, Department of Pediatrics, The exclusion of known genetically identifiable immuno- Graduate School of Medicine, University of Toyama, 2630 Sugitani, deficiencies is necessary for diagnosis of CVID.14 In light Toyama 930-0194, Japan. of this current notion, we attempted to immunologically E-mail: [email protected] 5These authors contributed equally to this work. and genetically re-evaluate a young male CVID patient. Received 19 July 2007; revised 10 August 2007; accepted 10 August The patient had normal numbers of CD20-positive 2007; published online 20 September 2007 peripheral blood B cells, but two-color flow-cytometric Japanese CD19 deficiency H Kanegane et al 664 analysis revealed the lack of CD19 membrane expression only one signal for ATP2A1 and NFATC2IP genes (Figure 1a). This raised the possibility that the patient (Figures 3d and f). Thus, the patient carries an allele had a CD19 deficiency. Furthermore, the level of CD19 that contains a gross deletion of at least 68.5 kbp expression in the mother was lower than that in the including the ATP2A1, CD19 and NFATC2IP genes. control, suggesting that the mother might be a carrier. In Because no additional material could be obtained from addition, expression of CD19-complex member CD21 the father, it remains unclear whether the gross genomic was also slightly reduced on the patient’s B cells as deletion in the patient was a de novo event or inherited compared with that of a healthy control (Figure 1b). from his father. Mutation analysis of the patient’s CD19 gene disclosed a seemingly homozygous mutation at the splice acceptor Memory B-cell sub-population site of intron 5 (IVS5-1G4T) (Figure 2a). CD19 transcript Similar to most CVID patients, the previously described analysis showed that the mutation caused skipping CD19-deficient patients show decreased numbers of of exon 6, and coupling of exons 5–7 (Figure 2b). As a IgDÀCD27 þ Ig class-switched memory B cells.11,15,16 result, the frame of exon 7 is shifted, leading to Substantial numbers of Ig class-switched memory B cells a truncated protein with four altered amino acids and a were detectable, though reduced as compared to healthy premature stop codon at position 320, prior to all controls, in peripheral blood of our patient, suggesting conserved intracellular tyrosine residues (Figure 2c). some potential to undergo class-switch recombination As expected, the mother was heterozygous for (Figure 4a). mutated allele (Figure 2a). The father, however, did not To examine whether B cells of the patient were capable

carry a mutated allele (Figure 2a). This raised the of undergoing somatic hypermutation, VH5-Cm tran- question whether the second allele of the patient was scripts were studied. Six out of 13 individual clones disrupted by a de novo mutation, or it contained a gross revealed somatic hypermutations (Figure 4b). The deletion including the mutation site. In order to confirm frequency of somatic hypermutations was 0.94% (normal the latter hypothesis, DNA fluorescence in situ hybridi- adults 1.8%70.4, and cord blood 0.07%70.01), and was zation analysis using a CD19-specific probe was per- indicative of the potential to form an antigen-selected formed. Although two signals for the CD19 gene were B-cell receptor repertoire. detected in the metaphases from a healthy control (Figure 3a), only one signal was detected in cells from B-cell antigen response the patient (Figure 3b). In addition, fluorescence in situ In agreement with previous observations,11 our clinical hybridization analysis was performed for the neighbor- and immunological findings of the patient suggested that ing genes ATP2A1 and NFATC2IP. The control samples the CD19 deficiency appeared to result in an impaired revealed two signals for ATP2A1 and NFATC2IP genes antigen response by B cells. To quantify the B-cell (Figures 3c and e), whereas the patient samples revealed response in our patient, we employed two in vitro

Control Patient Mother 1000 1000 1000 0.2 18.2 15.2 0.2 1.1 11.4 CD20 (FITC)

0.1 0.1 0.1

0.1 1000 0.11000 0.1 1000 CD19 (PE)

Control Patient Mother 1000 1000 1000 CD20 (ECD) 3 3 0.1 0.1 0.1

0.1 1000 0.1 1000 0.1 1000 CD21 (FITC) Figure 1 Flow-cytometric analysis of B cells. The peripheral blood B cells of a healthy control, the patient and his mother were evaluated by two-color flow cytometry employing FITC-conjugated anti-CD20 and PE-conjugated anti-CD19 monoclonal Abs (a), and ECD-conjugated anti-CD20 and FITC-conjugated anti-CD21 monoclonal Abs (b). Abs, antibodies; FITC, fluorescein isothiocyanate.

Genes and Immunity Japanese CD19 deficiency H Kanegane et al 665

Intron 5 Exon 6 Control

Exon 5 Exon 6 Exon 7 AG GT Control

Exon 5 Exon 6 Exon 7

Patient

Patient Exon 5 Exon 6 Exon 7 AT GT

Exon 5 Exon 7

Mother

exon 5 exon 6 Control H L Q R A L V L R ..catcttcaaagagccctggtcctgagg.. ..catcttcaaagagattcttcaaagtga.. H L Q R D S S K * Patient Father exon 5 exon 7

Figure 2 Mutation analysis of the CD19 gene. (a) Electropherograms of the sequences representing the region around the splice acceptor site of exon 6 from a healthy control, the patient and his parents. The patient showed a seemingly homozygous mutation at an invariant position of the splice acceptor site of exon 6 (IVS5-1G4T; indicated by arrows). His mother was heterozygous for the mutation, whereas his father did not carry the mutation. (b) CD19 transcript analysis showed the skipping of exon 6 in B cells of the patients. (c) The coupling of exons 5–exon 7 results in a frame shift and a premature stop codon at position 320 of the protein. assays. First, proliferation of isolated B cells was studied membrane CD19 expression. Furthermore, the lack of all by [3H]thymidine incorporation. The patient’s B cells intracellular domains responsible for signal transduction showed comparable proliferation upon stimulation with makes any expressed protein non-functional.17 Staphylococcus aureus Cowan I þ IL-2 or CpG to B cells The other mutated allele contained a gross deletion from a healthy control (Figure 5a). In vitro Ig production that involved ATP2A1, CD19, NFATC2IP and potentially showed reduced IgA and IgG production of patient’s more genes. In addition to the CD19 gene deletion, the B cells after stimulation with CD40L and CD40L þ IL-10, patient lacks one allele of the ATP2A1 and NFATC2IP whereas IgM production was similar to a healthy control. genes. Mutations in the ATP2A1 gene that encodes SERCA1 are associated with Brody disease, an inherited disorder of skeletal muscle function.18 The patient did not show clinical symptoms suggestive of Brody disease. Discussion Furthermore, mutations in ATP2A1 are associated with In this study we describe a CD19-deficient patient, who an autosomal recessive inheritance, making it likely that was compound heterozygous for two novel mutations. the other allele compensates for the loss. The exact Similarly to two previously identified disrupted CD19 function of the protein encoded by NFAT2CIP is not alleles,11 these mutations lead to loss of CD19 membrane known. However, similar to ATP2A1, it is likely that the expression, and result in an antibody–deficiency syn- loss of one allele does not have clinical consequences. drome, characterized by recurrent (mainly) bacterial The clinical presentation of the patient was very infections. similar to the other four reported CD19-deficient The mutation in the splice acceptor site of exon 6 patients,11 making it a rather homogeneous disorder. (IVS5-1G4T) results in skipping of exon 6, placing exon All patients presented with clinical symptoms of anti- 7 out-of frame after exon 5, which leads to a premature body deficiency in the first decade of life and showed stop codon at the intracellular amino acid 320. The reduced serum Ig levels. In addition, all patients show an resulting protein product is almost similar to the product impaired antibody response after vaccination. Whereas resulting from the 972insA mutation identified before.11 normal numbers of peripheral blood B cells are found, In line with this, the patient’s B cells completely lack they all show a marked decrease in memory B cells. In

Genes and Immunity Japanese CD19 deficiency H Kanegane et al 666

Figure 3 The patient is heterozygous for a gross deletion involving the CD19, ATP2A1 and NFATC2IP gene. FISH analysis with CD19-(a and b), ATP2A1-(c and d) and NFATC2IP-specific probes (e and f) and the chromosome 16-specific D16Z3 probe in a healthy control (a, c and e) and the patient (b, e and f). In both a healthy control and the patient, two signals per cell were found with D16Z3 (green signals; indicated by arrowheads). Whereas the control also showed two signals for the CD19, ATP2A1 and NFATC2IP probes, only one signal was found for each of these in the patient (red signals; indicated by arrows). FISH, fluorescence in situ hybridization.

spite of decreased number, these memory B cells do history of immunodeficiency. At 5 years of age, he show molecular signs of antigen-dependent differentia- presented with pyelonephritis, bronchitis and gastritis, tion. Finally, considering the thrombocytopenia in our and was admitted to the Takamatsu Red Cross Hospital. patient, the CD19 deficiency might be associated with the He showed hypogammaglobulinemia (IgG 249 mg dlÀ1, development of autoimmune disease. IgA 10 mg dlÀ1, IgM 18 mg dlÀ1, IgD o0.6 mg dlÀ1 and We, herein, described the fifth case in the world of a IgE 9.0 U mlÀ1), and the immunological tests demon- patient with CD19 deficiency, resulting from two novel strated CD3 62.7% (1775 mlÀ1), CD4 37.6% (1064 mlÀ1), mutations in the CD19 gene. The clinical and immuno- CD8 27.6% (781 mlÀ1) and CD20 19.0% (538 mlÀ1)in logical characteristics of the patient were similar to four peripheral blood lymphocytes. Antibodies against recently identified cases of CD19 deficiency. These measles, rubella, tetanus and pertussis toxin were not observations support the idea that CD19 deficiency is a detectable, although the patient had been vaccinated. distinct and unique subset of CVID. Our finding and the Since the patient had normal numbers of B cells in his recent observation of a CD21 deficiency19 suggest that blood, he was clinically diagnosed to have CVID, and defects in antigen recognition, resulting from mutations thus was treated with intravenous immunoglobulin- in CD19-complex members (CD19, CD21, CD81 and replacement therapy. In addition, he had also been CD225) might be identified in more patients that suffer recently diagnosed with a mild thrombocytopenia that from an antibody deficiency. did not require medical attention. The patient is now doing well with intravenous immunoglobulin-replacement therapy. Patients, materials and methods Patient’s report Flow-cytometric analysis of B cells The patient was an 8-year-old Japanese boy born to Heparinized venous blood was obtained from the patient non-consanguineous healthy parents who had no family and his parents after obtaining the written informed

Genes and Immunity Japanese CD19 deficiency H Kanegane et al 667 Control Patient 1000 1000 11.5 12.1 7.6 5.4 CD27 (PE)

0.1 72.4 0.1 67.5

0.11000 0.1 1000 IgD (FITC)

CDR1 CDR2

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR

------(7/13)

------I------N--- (1/13)

------R------(2/13)

------W------C------W-D-S--Y-N------H------(1/13)

------Y-I (1/13)

------QA------W------C------W-D-S--Y-N------H------(1/13)

Figure 4 Analysis of memory B cells. (a) B-cell sub-populations. A three-color analysis was carried to analyze the relative distribution of naive and memory B cells in peripheral blood using CD27 and IgD within the CD20-positive B-cell compartments of a healthy control and the patient. (b) Analysis of the somatic hypermutations in the patient’s B cells. The upper sequence is the germline gene of VH5 with the amino- acid translation.

400

300

200

100 cpm 0 30000 B cell only B+DC+CD40L B+DC+CD40L+IL-10

120

20000 80 IgA IgG 40 10000 0 B cell only B+DC+CD40L B+DC+CD40L+IL-10 H]thymidine incorporation 3 [ 400 0 SAC+IL-2 CpG 300

200 IgM

100

0 B cell only B+DC+CD40L B+DC+CD40L+IL-10 Figure 5 Functional characteristics of the patient’s B cells. (a) Proliferation of B cells after stimulation with SAC and IL-2 or CpG, as assessed by [3H]thymidine incorporation. The results are expressed as the mean of triplicate experiments. Open and closed bars indicate the patient and the normal control, respectively. (b) IgG, IgA and IgM production by B cells after stimulation with various stimuli. The spots were enumerated in each experiment. Open and closed bars indicate the patient and the normal control, respectively. SAC, Staphylococcus aureus Cowan I.

Genes and Immunity Japanese CD19 deficiency H Kanegane et al 668 consent. Peripheral blood B cells were assessed by two- genomic probes (7045 and 6017 bp) were also prepared color flow-cytometric analysis employing CD19-PE, by LA-PCR Kit with primers encompassing exons CD20-ECD (phycoerythrin-Texas Red), and CD21-fluor- 10 and 14 (50-GGTTCAAGTGGCTCACCTTGTTACCA escein isothiocyanate (FITC) mAbs (all from Beckman AC-30 and 50-GGTTCAAGTGGCTCACCTTGTTACCA Coulter, Marseille, France) and CD20-FITC mAb (DAKO AC-30) and exons 3 and 7 (50-CTTGCAGCTGCCATAGT Japan, Kyoto, Japan). In addition, the naive and memory TAGTTAGGACA-30 and 50-CAATTCAGACTTCTAGGC B-cell sub-populations were evaluated by three-color TCCAGAAGTG-30), respectively. For fluorescence in situ analysis with a combination of FITC goat anti-IgD Ab hybridization analysis, denatured metaphase spreads (Southern Biotechnology Associates Inc., Birmingham, were hybridized with the probes labeled with digox- AL, USA), CD27-PE mAb (Becton Dickinson, Mountain igenin-11-dUTP using nick translation kit (Roche Diag- View, CA, USA), and CD20-ECD mAb as described nostics KK, Tokyo, Japan). The Spectrum Green-labeled previously.20 chromosome 16 probe (D16Z3, Vysis, Tokyo, Japan) was used as a control. The CD19-, ATP2A1- and NFATC2IP- Mutation analysis of the CD19 gene specific probes were detected with anti-digoxigenin Peripheral blood mononuclear cells (PBMC) were col- rhodamine (Roche Diagnostics KK) providing a red signal, whereas D16Z3 was detected by a green signal. lected after Ficoll–Hypaque gradient centrifugation of 0 total blood. DNA was extracted from PBMC using a Metaphases were counterstained with 4 ,6-diamino-2- QIAamp Blood Kit (Qiagen GMBH, Hilden, Germany), phenylindole dihydrochloride, and the images of the and was subjected to PCR. All of the coding exons and hybridization were captured under fluorescence micro- splice sites of the CD19 gene were amplified, and a scopy (Olympus Optical Co., Tokyo, Japan). At least 20 sequencing reaction was carried out with the BigDye metaphases were observed in each setting. Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an automated ABI PRISM 310 B-cell proliferation assay DNA Sequencer (Applied Biosystems). The PCR primers B cells were isolated from PBMC using a magnetic cell used for amplification of the CD19 gene are listed in sorter (MACS; Miltenyi Biotech GMBH, Bergisch Glad- Table 1. RNA was extracted from PBMC using a TRIzol bach, Germany) by negative selection according to the Reagent (Invitrogen, Carlsbad, CA, USA), and was experiment procedure. Re-analysis showed that more subjected to reverse transcription PCR. A fragment of than 97% of the purified cells were CD20 positive. The the CD19 cDNA was amplified from the first-strand purified B cells were cultured in the presence of 0.01% cDNA with primer pairs encompassing exon 6 (50- Staphylococcus aureus Cowan I (Sigma-Aldrich Inc., AGGGGCCTAAGTCATTGCTG-30 and 50-TGCCCA St Louis, MO, USA) and 50 U mlÀ1 recombinant human AGGTTGGAGTCGTT-30) and sequenced to verify the (rh) IL-2 (Takeda Co., Osaka, Japan), or 5 mgmlÀ1 CpG mutation found in the genomic DNA. (InvivoGen, San Diego, CA, USA) at a final cell density of 2.5 Â 105 mlÀ1 in a volume of 200 ml per well. The cells Fluorescence in situ hybridization analysis of the CD19 gene were cultured in 96-well round-bottomed plates for 3 1 PBMC stimulated in culture with phytohemagglutinin days at 37 C in a humidified atmosphere with 5% CO2. m 3 (PHA) for 72 h were treated with a 0.075 M KCl solution The cells were pulsed with 0.5 Ci (18.5 Bq) [ H]thymi- and fixed with Carnoy’s solution (3:1 methanol and dine. After 18 h of incubation, the cells were harvested by acetic acid) for metaphase preparation. A CD19-specific an automatic cell harvester (Packard, Meriden, CT, USA), 3 genomic probe (11 517 bp) was prepared by LA-PCR Kit and [ H]TdR incorporation was measured on a liquid (Takara, Kyoto, Japan) with primer pairs encompassing scintillation analyzer (Packard). exons 1 and 15 (50-CTGTTCTCAAAGACATTCTGGTG CAGG-30 and 50-TGCTTAGAACATAGTAAGGTCGGG Ig production assay CACT-30). In addition, probes were designed to label the Ig production was assessed by an ELISPOT assay as ATP2A1 gene, which is located 30 kbp upstream of CD19, described elsewhere.21,22 First, the dendritic cells were and the NFATC2IP gene, which is located 14 kbp down- derived from in vitro cultures of the human cord blood stream of CD19. The ATP2A1- and NFATC2IP-specific CD34 þ cells. Briefly, the CD34 þ cells were isolated from

Table 1 PCR primers for amplification of the CD19 gene

Exon(s) Sense primer sequence Antisense primer sequence

1 AGGAGGCAAGTGTTGTGAGT GCCACAACTCAAGTTTCCAG 2 TGCTCTATAACCTGGTGTGA TGCCCACCTAGCTCCGAGTT 3 TTTCACCTCTTCAGTTTCCC TTTCACCTCTTCAGTTTCCC 4 GGGGAAACTGAAGAGGTGAA TTCCACTTCTTCCCAGTACC 5 TCTCAGGATTGGGACGTAAG TGGGAAGATGGAGTAGGGT 6 CCTGTCTCCTTTGTTGAACC CCAAGGAGTTCTTCACAGAG 7 CTTCTGAAGGCCTGGACAGA ACAGCGGATTGGAGGGATGA 8 AGACCCAGAGTCCATTCGCA CCACTATTCGGGCCACAGAC 9–10 TCTGTGGCCCGAATAGTGGA GGATCCCCTTCTAGGAGTGT 11–12 AATTGCAGCGCTGTGACACT TGAGAGGAGTCACAGGATTG 13 CCTCAGTCTTCCTCCTTACC AGGCTGGAAGGCAAATGTCAG 14 ACTTTGCCTTCCAGCCTACT GTGTGAATCTTGGGGACTTG

Genes and Immunity Japanese CD19 deficiency H Kanegane et al 669 the human cord blood cells through positive cell excellent technical assistance. This study was supported selection using MACS. The CD34 þ cells were then by a Grant-in-Aid for scientific research from the cultured in the presence of 100 ng mlÀ1 rhGM-CSF Ministry of Education, Culture, Sports and Technology, (R&D Systems Inc., Minneapolis, MN, USA), 2.5 ng mlÀ1 Japan, and grants from the Ministry of Health, Labour rhTNF-a (R&D Systems Inc.), and 25 ng mlÀ1 rh stem cell and Welfare, Japan. MCvZ was supported by Grant 349 factor (R&D Systems Inc.) in a RPMI-1640 medium from the foundation ‘Sophia Kinderziekenhuis Fonds’. supplemented with 10% fetal calf serum, gentamicin and The authors have no financial conflict of interest. penicillin G sodium. The dendritic cells were harvested after 12–14 days of culture. B cells isolated from peripheral blood of the patient and a healthy control were cultured at 1 Â105 mlÀ1 alone or in the presence of References 1 Â105 mlÀ1 irradiated (3400 rad) dendritic cells and 3.75 Â 104 mlÀ1 irradiated (7000 rad) CD40 ligand-trans- 1 Primary immunodeficiency diseases. Report of an IUIS Scientific Committee. International Union of Immunological fected L cells (a gift from Dr Yong-Jun Liu, DNAX, Palo Societies. Clin Exp Immunol 1999; 118 (Suppl 1): 1–28. Alto, CA, USA) in a final volume of 200 ml. To each well, 2 Hammarstrom L, Smith CIE. Genetic approach to common rhIL-10 (R&D Systems Inc.) was added at a concentration variable immunodeficiency and IgA deficiency. In: Ochs HD, of 200 ng mlÀ1. The cells were used on the sixth day for Smith CIE, Puck JM (eds). Primary Immunodeficiency Diseases, the ELISPOT assay. 2nd edn. Oxford University Press: New York, USA, 2007, IgG, IgA and IgM secretion of the B cells was detected pp 313–325. by an ELISPOT assay after 6 days of culture using the 3 Kanegane H, Tsukada S, Iwata T, Futatani T, Nomura K, Millipore Multiscreen Assay System (Millipore Corp., Yamamoto J et al. Detection of Bruton’s tyrosine kinase mutations Billerica, MA, USA). The plates were precoated by 100 ml in hypogammaglobulinaemic males registered as common per well of a goat anti-human capture antibody (South- variable immunodeficiency (CVID) in the Japanese Immunode- ficiency Registry. Clin Exp Immunol 2000; 120: 512–517. ern Biotechnology Associates Inc.) at a concentration of 4 Weston SA, Prasad ML, Mullighan CG, Chapel H, Benson EM. À1 10 mgml in phosphate-buffered saline overnight at 41C Assessment of male CVID patients for mutations in the Btk Cultured B cells were added to the plate after washing in gene: how many have been misdiagnosed? 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