Novel Mutations in TACI (TNFRSF13B) Causing Common Variable Immunodeficiency
Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by impaired immunoglobulin production. The disorder is also characterized by co-occurrence of autoimmune, lymphoproliferative, and granulomatous diseases. Mutations in the gene encoding TACI (Transmembrane Activator and CAML Interactor, TNFRSF13B) were previously found to be associated with CVID.
Materials and Methods
We therefore sequenced TNFRSF13B gene in a cohort of 48 Iranian CVID patients. Expression of TACI and binding of A proliferation-inducing ligand (APRIL) were tested by FACS.
We identified one patient with a homozygous G to T substitution in the TNFRSF13B gene at the splice site of intron 1 (c.61+1G>T), which abolished expression of the TACI molecule and binding capacity of APRIL. This represents the second CVID patient in the world with a complete absence of TACI expression. B cell lines from family members carrying the same mutation in a heterozygous form showed a reduced level of TACI expression and APRIL-binding capacity, suggesting a gene dosage effect. In addition, we found the previously recognized C104R and C172Y mutations in a heterozygous form in two patients with CVID and one, novel, heterozygous P42T mutation.
TACI mutations were observed in Iran CVID patients in a similar frequency as in other Caucasian populations. The novel mutations identified in this study support the notion of a crucial role for TACI in B cell differentiation.
KeywordsCommon variable immunodeficiency TACI human immunoglobulins mutation
Common variable immunodeficiency (CVID) is the most frequent symptomatic primary immunodeficiency disorder. It is characterized by low serum levels of IgA, IgG and, in half of the patients, low levels of IgM. The clinical manifestations are due to a reduction of Ig levels resulting in frequent respiratory and gastrointestinal tract infections [1, 2, 3, 4]. Patients with CVID also have an increased incidence of polyclonal lymphocytic infiltration, autoimmunity, enteropathy , and malignancies . In the last few years, several monogenic defects have been suggested to be associated with development of CVID, including mutations in inducible T cell costimulator, encoded on chromosome 2q , transmembrane activator and CAML interactor (TACI) on chromosome 17p [7, 8], B cell-activating factor receptor (BAFF-R) on chromosome 22q , CD19 on chromosome 16p , CD81 on chromosome 11p , and MSH5 on chromosome 6p .
The tumor necrosis factor receptor (TNFR) superfamily member TACI is expressed on B cells and binds two ligands, BAFF and A proliferation-inducing ligand (APRIL). The latter proteins are both members of the TNF family of ligands and are mainly expressed by neutrophils and monocytes .
Mice with a targeted disruption of TACI (TACI−/−) have enlarged spleens and lymph nodes with an increased number of mature B cells [14, 15]. These B cells show an increased proliferation rate and increased Ig production in vitro , and with age, the animals develop autoantibodies . Yet, their serum IgA, IgG, and IgM levels are low in response to thymus-independent antigens . Decreased apoptosis in B cells from TACI−/− mice suggests that TACI normally delivers an apoptotic signal and that it has a regulatory role in B cell development [15, 16].
TACI is encoded by TNFRSF13B, which is mutated in a significant proportion of patients with CVID (7–21%) [8, 17, 18]. In a majority of patients, the IgG levels are low at the time of diagnosis, but IgA is the most affected Ig class . Reduced IgG and IgA levels in individuals with TACI deficiency are probably due to an inefficient class switch recombination in B cells [7, 8].
Multiple mutations, including nonsense mutations (S144X, Y164X, C193X, and S194X), frameshift mutations (c.121delG, c.204insA, c. 298insT, and c.571insG), and missense mutations (W40R, D41H, Y79C, I87N, C104Y, C104R, A149T, G152E, A181E, R202H, and V246F) have previously been observed in TNFRSF13B in CVID patients [7, 8, 18, 19]. Most of these mutations have only been observed in one or two patients; however, some mutations (C104R and A181E) are more common, with various frequencies in different ethnic populations .
The aim of the present study was to determine the prevalence of TNFRSF13B mutations in Iranian patients with CVID.
Materials and Methods
Forty-eight Iranian patients with sporadic CVID, referred to the Children’s Medical Center Hospital in Tehran or the Immunodeficiency Unit at the Karolinska University Hospital Huddinge in Stockholm, were included in the study as were ethnically matched blood donor controls (n = 244). All patients and controls were unrelated Iranian Caucasians. The diagnosis of CVID was made using the European Society for Immunodeficiency criteria (www.esid.org). The rate of consanguinity among the studied Iranian CVID patients is 58% (28 of 48; 23 first cousins and five second cousins). Local ethical permissions were obtained from the Karolinska Institute and the Tehran University of Medical Science for use of all samples collected in the study.
Serum Immunoglobulin Levels
Serum levels of IgG, IgA, and IgM were measured by nephelometry.
Sequence Analysis of TNFRSF13B
Polymerase chain reaction (PCR) amplification and full sequencing of TNFRSF13B was performed as previously described . Briefly, 50 ng of genomic DNA was used in PCR employing exon-specific primers (sequence of the primers are available in ) using the following conditions: 95°C 2 min for one cycle followed by 95°C 15 s, 65°C 30 s, 68°C 1 min for 30 cycles, and a final extension of 72°C for 10 min. An annealing temperature of 67°C was used for exons 4 and 5. The PCR products were purified using a gel extraction kit (Qiagen, Stockholm, Sweden). The purified amplicons were sequenced at the Macrogen Company (Seoul, South Korea) and analyzed using Lasergene (DNAStar, Madison, WI, USA).
Analysis of Mutations of TNFRSF13B in Population-based Controls
Frequencies of the G to T substitution at the first nucleotide of intron 1 (c.61 + 1G > T), P42T, C104R, C172Y, and A181E variants were analyzed by matrix-assisted laser desorption/ionization-time of flight analysis in a single nucleotide polymorphism (SNP)-based assay as described previously [8, 20]. Amplification and detection primers for the tested mutations were designed using the SpectroDesigner software (Sequenom, San Diego, CA, USA) and are available upon request. The PCR temperature profile started with 15 min of denaturation at 95°C followed by 45 cycles of 94°C for 30 s, 60°C for 15 s, and 72°C for 15 s. A final elongation step of 72°C for 5 min ended the program.
Primer extension was carried out in a total volume of 9 μl after salt removal. About 10 nl of the samples were spotted onto Maldimatrix-containing SpectroCHIPS (Sequenom Inc.) using a nanodispenser (Robodesign). The SpectroCHIPS were analyzed using an Autoflex MassARRAY mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Finally, the data were analyzed independently by two persons using the SpectroTyper software (Sequenom Inc.).
TACI Expression and Binding of Flag-APRIL
Staining of TACI on Epstein–Barr virus (EBV) cell lines or peripheral B cells was performed as described previously [8, 18]. Peripheral B cells or EBV-transformed cells were stained either with a biotin-labeled polyclonal goat anti-human TACI antibody (Peprotech, London, UK), followed by Streptavidin PE (BD Biosciences, Heidelberg, Germany) or PE-labeled monoclonal rat anti-human-TACI antibody (1A1, Abcam, Cambridge, UK), together with CD19-PC7 (J4.119; Beckman Coulter, Marseille, France), anti-IgM Cy5 (Dianova, Hamburg, Germany), and anti-CD27 fluorescein isothiocyanate (BD Biosciences). At least 104 cells, gated according to their forward and sideward scatters, were collected using a FACSCalibur (Becton Dickinson, Mountain View, CA, USA) and analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA). Dead cells were excluded by forward/side scatter electronic gating.
For binding of flag-APRIL, 106 EBV-transformed cells were incubated with 100 ng of Flag-ACRP-hAPRIL (Alexis Biochemicals, Lausen, Switzerland)  in the presence of 0.1 μl heparin (Liquemin, Roche Pharma) and detected with the monoclonal mouse anti-Flag antibody M2 (Sigma, Seelze, Germany) and PE-labeled goat anti-mouse-antibodies (Caltag, Hamburg, Germany), or the biotinylated monoclonal mouse anti-Flag antibody M2 (Sigma, Seelze, Germany) and Streptavidin PE. The specificity of antibody staining and Flag-ACRP-hAPRIL binding to EBV-transformed B cells was assessed by simultaneous staining of an EBV-transformed B cell line known to carry a TNFRSF13B null mutation (S144X) .
Total RNA Preparation, cDNA Synthesis, and RT-PCR
Total RNA was extracted from peripheral blood mononuclear cell, and cDNA was prepared using a first-strand cDNA synthesis kit (Amersham Biosciences, UK) according to the manufacturer’s instruction. First-strand cDNA was synthesized using 1.5 μg of total RNA in a 15-μl reaction mixture consisting of bulk first-strand reaction mix 5 μl, 200 uM dithiothreitol 1 μl, and Not I-d (T)18 primer 0.2 μg. The mixture was incubated at 37°C for 1 h. PCR amplification was performed using two pair of primers, the first pair of primers cover exons 1–3 (AGCATCCTGAGTAATGAGTGG as sense and CCTCTGTGCTCCAATCCTT as antisense); the second pair of primers cover exons 3–5 (GACAGCACCCTAAGCAATG as sense and CCGACCTCCTGCTCTATCT as antisense). Briefly, 25-μl reaction mixture was prepared using 2.5 μl of 10× PCR buffer, 1.5 μl of 25 mM MgCl2, 1.5 μl dNTPs (10 mM), 10 pmol of each primer, and 1 unit of Go-Taq DNA polymerase (Promega). The PCRs were run under the following conditions: 95°C 15 s, 63°C 30 s, and 72°C 1 min 30 s for 30 cycles. The PCR product was finally visualized by running agarose gel electrophoresis containing ethidium bromide. To assure the specificity of primers, the PCR products from agarose gel slices were purified using QIAquick Gel extraction Kit (QIAGEN) and sequenced (Macrogen, Seoul, Korea). The β actin gene was used as a control using the following primers: 5′-GATGATGATATCGCCGCGCT-3′ and 5′-TGGGTCATCTTCTCGCGGTT-3′ .
Analysis was carried out using the Stata statistical program, and the frequencies of the variants were compared using Fisher’s exact and chi-square test as appropriate.
Sequence Analysis of TNFRSF13B in Iranian CVID Patients
Sequence Analysis of TNFRSF13B Gene in Iranian Common Variable Immunodeficiency Patients
Prediction on functionc
c.61 + 1G > T (ho)
One CVID patient (P2) was heterozygous with respect to a novel TACI mutation in the extracellular CRD1 domain (P42T), and one patient (P4) was heterozygous for a recently described mutation in the transmembrane region (C172Y) . One patient carried the well-known heterozygous C104R missense mutation in the CRD2 region. Several additional, previously noted variants (P97P, P251L, and S277S), not associated with development of CVID, were also found in the Iranian CVID patients at frequencies comparable to those in other Caucasian populations (data not shown).
Clinical and Immunological Manifestations of the Iranian CVID Patients with TNFRSF13B Mutations
Clinical and Immunological Manifestations in Iranian Common Variable Immunodeficiency Patients Carrying Mutations in Transmembrane Activator and CAML Interactor
c.61 + 1G > T
Otitis, sinusitis, pneumonia, chronic diarrhea, urinary tract infection, epididymitis, asthma, nasal polyps, anemia, ulcerative colitis (?)
Chronic otitis, sinusitis, pneumonia, liver granulomas, thrombocytopenia, chronic diarrhea, splenomegaly, clubbing of finger, bronchiectasis
Otitis media, chronic diarrhea, pneumonia, skin infections
Otitis media, sinusitis, pneumonia, bronchiectasis, chronic diarrhea, cirrhosis hepato/splenomegaly, chronic active hepatitis
TACI Mutations in Iranian Controls
A total of 244 healthy Iranian controls were analyzed for mutations in the TNFRSF13B gene. In this screening, no splice site mutation in intron 1 was found. One (0.4%) of the healthy controls was heterozygous for the P42T allele, and two (0.8%) were heterozygous for the C104R allele. The C172Y mutation was not observed in the healthy control group (Table I).
TACI Expression in Individuals with Splice Site Mutations (c.61 + 1G > T)
Prediction on Function of TACI Protein Affected by Mutations
By in silico analysis using Polyphen , the three missense mutations in the Iranian CVID patient group were predicted to be probably (C172Y, C104R) or possibly (P42T) damaging (Table I). Using the GeneRunner software (version 3.05, 1994 Hastings Software, Inc., http://www.generunner.com), hydrophobicity of the P42T protein was predicted to be changed (from hydrophobic to hydrophilic); whereas, the hydrophobicity of both C104R and C172Y was unaltered.
The frequency of TACI mutations in our Iranian CVID patients was similar (8.3%) to that noted in patients from other Caucasians populations [17, 18], which is significantly higher (p = 0.003) than in healthy Iranian controls. The novel mutation in the splice site of intron 1 (c.61 + 1G > T), when in a homozygous form, eliminates expression of TACI and abolishes the binding of APRIL on B cells. Hence, it has a profound influence on TACI function and the development of CVID. In the older sister with the same homozygous mutation, the decreased immunoglobulin levels also support the notion of a significant influence of this mutation on B cell function. The heterozygous family members are healthy. However, B cell lines from these individuals showed a reduced level of TACI expression and APRIL binding, suggesting that this mutation may also affect TACI function even in a heterozygous form. The P42T mutation is a new variant with the same frequency as in controls (Table I). The C104R mutation has previously been described as a disease-associated mutation, but in the present study, its frequency did not differ significantly from normal controls. The C172Y mutation recently described as a potential risk factor for development of CVID  was not found in healthy Iranian donors. However, further analysis of a larger sample size of patients and controls is required to determine whether heterozygous c.61 + 1G > T, P42T, and C172Y mutations actually do constitute risk factors for development of CVID.
Structural characteristics can serve as reasonably reliable predictors of the effect of amino acid substitutions. As previously noted, most disease-associated/causing mutations and deleterious non-synonymous SNPs have an effect on protein stability rather than functionality , and hydrophobic core stability parameters are the best predictors for the effect of a mutation on the protein . Proline is a helix-breaking amino acid  and, thus, the P42T mutation will probably lead to an alteration of the structure of TACI. Based on the Swiss-Prot database, there is a disulfide bond between amino acids number 34 and 47 in the TACI molecule. Thus, the P42T mutation is likely to change non-covalent and van der Waals bonding forces and, consequently, the folding of the receptor. It also introduces a hydrophilic propensity into the hydrophobic core of the protein, possibly destabilizing its folded structure.
The cysteine at position 104 forms a disulfide bond with the cysteine at position 93 and participates in the maintenance of the spatial structure of the CRD2 domain of TACI, and is localized close to the APRIL ligand-binding domain . The C104R mutation may impair the function of TACI both in homozygous and heterozygous states [18, 29]. Homozygous C104R mutations have previously been identified exclusively in CVID patients; whereas, heterozygous mutations have been identified in both CVID patients and healthy individuals [8, 18]. However, C104R heterozygosity is clearly associated with an increased risk for CVID  and may be associated with a low number of IgD−CD27+ B cells, benign lymphoproliferation, and autoimmune complications . Recently, three individuals (relatives of CVID patients) with normal immunoglobulin levels, carrying homozygous C104R mutations, have been described , suggesting that even in a homozygous form, this variant may still show incomplete penetrance.
Tyrosine is a hydrophilic amino acid with a higher positive charge than cysteine. As the transmembrane region has a hydrophobic propensity, the C172Y mutation probably damages the structure and function of the receptor, even though the cysteine at position 172 is not linked by an intra-chain disulfide bond (based on the Swiss-Prot databases entry O14836).
We have previously reported that heterozygous A181E mutations in TNFRSF13B constitute risk factors for the development of CVID, but not for IgAD . In that study, the A181E mutation was observed both in normal and immunodeficient individuals, supporting the notion of an incomplete penetrance of the described TNFRSF13B mutations. However, the A181E mutation was present neither in the Iranian CVID patients nor in the ethnically matched controls (data not shown).
The splice site mutation found in one of our Iranian CVID patients is the second in the world who has no expression of TACI, the first being a patient with a homozygous S144X mutation . As is the case in our patient, he had a sibling carrying the same mutation in a homozygous form, but with a much milder phenotype suggesting the existence of, as yet unidentified, “modifier” genes.
The work was supported by the Swedish Research Council, EU (EUROPAD), the Swedish Cancer Society, and funds from the Karolinska Institutet. The authors have no conflicting financial interests.
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