Common variable immunodeficiency (CVID) is an heterogeneous syndrome characterized by decreased levels of serum Ig and recurrent bacterial infection. Here, we were interested to study whether a qualitative defect of the affinity Ab maturation process could be combined to the low level of serum Ig in a cohort of 38 CVID patients. For this, we designed a novel and rapid screening test for the detection of hypomutated V gene expressed by memory B cells. This test delineated a subset of 9/38 (23%) CVID patients with an abnormal pattern of Ig V gene mutation. The mean frequency of V gene mutation of this subset was significantly lower (1.74%) compared with other CVID patients (5.46%) and normal donors (6.5%) (p < 0.0001). The mean age of this subgroup was significantly higher than other hypogammaglobulinemic patients with normal levels of V gene mutation (p < 0.02), whereas no difference in the duration of symptoms was noted between the two groups. This suggests that hypomutation characterizes patients who began CVID late in life. Recently, it was shown that non-Ig sequences, such as the intronic BCL-6 gene, could be the target of the somatic hypermutation process in normal memory B cells. Our finding of a normal mutation frequency of the BCL-6 gene in two hypomutated CVID point to a defect of the Ig targeting of hypermutation machinery in these cases.

Common variable immunodeficiency (CVID)3 is the most common symptomatic primary Ab-deficiency syndrome. This entity probably represents a group of syndromes, yet undifferentiated, but always characterized by decreased levels of at least two serum Ig isotypes (IgG and usually IgA) and recurrent pyogenic infections. The clinical presentation of CVID is generally characterized by bacterial infections, predominantly of the upper and lower respiratory tracts and of the gastrointestinal tract. A high incidence of lymphoid and gastrointestinal malignancies, nonmalignant lymphoid hyperplasia, autoimmune disorders, and granulomatous inflammation has been reported (1, 2, 3).

Susceptibility genes for CVID within the MHC class II and III loci have been reported (4, 5), but the molecular defects associated with CVID are unknown, and the heterogeneity of clinical presentations of CVID has hampered genetic and molecular investigations. Although a number of in vitro immunologic abnormalities have been identified in patients with this syndrome (6, 7, 8), none has provided a pattern sufficiently consistent for immunological or clinical classification.

The affinity maturation of T cell-dependent Ab responses results from the accumulation of point mutations in the V region of Ig genes followed by Ag-driven selection of the B lymphocytes expressing high affinity Abs (9, 10). This process takes place in germinal centers where Ag-specific B cells differentiate into memory and/or plasma cells after switching of the heavy chain isotypes (11, 12, 13, 14). In a preliminary report, we have identified two patients with CVID and with a low frequency of Ig VH gene somatic hypermutation (15). In the present study, we investigate a large group of well-characterized patients with CVID and with selective IgA deficiency (IgA-D) because intercurrent sinopulmonary infections have been reported in some of these patients with normal serum IgG levels. Finally, we tried to correlate this defect with selected specific clinical features.

We focused our study on somatic mutations of the V3-23 gene, a member of the VH3 family expressed in 4–10% of B cells (16). Analysis of the pattern of mutation accumulated in the V3-23 gene in normal donors (ND) (15) showed the major individual hot spots of mutation previously reported for this gene (17), e.g., the Ser31, Ser35, and Ala 50 codons. We took advantage of this observation to design a screening test that could detect the presence of circulating IgG memory B cells harboring the V3-23 V gene without mutations on these hot spots. This test avoided extensive sequencing of V3-23-IgG transcripts from the whole cohort of patients. In a second step, we determined accurately the frequency and the pattern of mutation of V3-23-IgG transcripts from selected patients.

Thirty-eight CVID (including patients LE and SO previously reported; 15) and nine selective IgA-D patients recruited from immunology clinics in England, Sweden, and France were studied after informed consent was obtained. A cohort of six CVID patients previously studied (15) and with a normal rate of Ig V gene mutation was added in this study. All patients fulfilled the World Health Organization diagnostic criteria for CVID and IgA-D (3) and were receiving Ig replacement therapy.

Total RNA was extracted from 5 × 106 PBMC using the RNA-plus B extraction procedures (Bioprobe Systems, Richmond, CA). Total RNA was reverse transcribed into cDNA using SuperScript II Rnase H-Reverse Transcriptase (Life Technologies, Rockville, MD) and a CγA (5′-GTCCTTGACCAGGCAGCCCAG-3′) primer. After ethanol precipitation, the cDNA was resuspended in 50 μl of water, and PCR was performed with 0.5 U of PFU polymerase (Stratagene, La Jolla, CA) on 1/20 of the cDNA. The following primers were used for amplification: V3-23 leader exon (5′-GGCTGAGCTGGCTTTTTCTTGTGG-3′) and CγB (5′-AAGACCGATGGGCCCTTGGTGG-3′). CγA and CγB primers match equally all γ isotypes. The PCR conditions were 35 cycles at 94°C for 45 s, at 65°C for 1.5 min, and 72°C for 2 min. PCR products were then cloned using the TA cloning kit (Invitrogen, San Diego, CA), and V3-23 positive colonies were sequenced with the dRhodamine dye terminator cycle sequencing kit (Applied Biosystems prism, Foster City, CA) and analyzed with the Applied Biosystems prism 310 genetic analyzer.

The V3-23-Cγ PCR products was amplified with 1.5 U of Gold Taq polymerase using a reaction mixture containing [γ-32P]-labeled 5′ V3-23-framework region (FR) 1 primer (5′-TCCCTGAGACTCTCCTGT-3′) and the CγB primer. Amplification was performed in a 40-μl reaction mixture containing 1.5 U of Gold Taq polymerase (Perkin-Elmer, Norwalk, CT), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 10 mM of each dNTP, and 25 pmol of each primer containing one-fifth of radiolabeled FR1 primer. The PCR conditions were 94°C for 10 min and 30 cycles (94°C for 45 s; 55°C for 1.5 min; 72°C for 2 min). The labeled PCR products were purified from agarose gel by electroelution and, after an ethanol precipitation step, resuspended in 50 μl of water. The radioactivity was counted, and 2000 cpm DNA samples were digested with either AluI or AvaII enzyme. After ethanol precipitation, digested products were resuspended in 10 μl of water and loaded onto a 8% nondenaturant polyacrylamide gel. After electrophoresis, gel was dried and autoradiography was performed.

Purification of blood CD19+ IgM-IgD memory B cells from ND and patients LE and SO was performed as previously described (15). Total genomic DNA was extracted from 104 memory B cells by proteinase K digestion. A 790-bp fragment of the BCL-6 gene located in the first intron was amplified using primers 5′-CCGCTGCTCATGATCATTATTT-3′ (sense) and 5′-TAGACACGATACTTCATCTCAT-3′ (antisense). A first round of amplification (94°C for 5 min for 1 cycle; 55°C for 30 s; 72°C for 1 min for 35 cycles), using 0.5 U of PFU polymerase (Stratagene) was performed. A second step of amplification using the same primers and following the same conditions was performed on 1/100 of the first PCR product. PCR products were then cloned and sequenced.

Patients’ characteristics were analyzed using nonparametric models Wilcoxon and Kruskal Wallis tests. Results of comparison between groups of patients according to the rate of Ig V gene somatic mutation were presented with the 95% confidence limits and associated p value. The nominal significance level for the end points is 5% (two-sided).

V3-23-Cγ transcripts from three normal donors (ND3, ND4, ND5), 38 and 9 patients with CVID and IgA-D, respectively, were amplified using a 5′-V3-23 primer hybridizing to the V3-23 leader exon together with a 3′-Cγ primer. Next, V3-23-Cγ PCR products were amplified by using a 5′-γ-32P FR1 primer hybridizing to the framework region of the V3-23 gene together with a 3′-Cγ primer (Fig. 1,A). This FR1-Cγ PCR product contained, in its germline state, three AluI sites located 44, 56, and 100 bp 3′ from the labeled 5′ end of the fragment (Fig. 1,A). As these three AluI sites contained part of the Ser31, Ser35, and Ala50 codons, which are highly mutated during the Ab affinity maturation process, nucleotide substitution within these codons led to different labeled restriction fragments after AluI digestion of the FR1-Cγ PCR fragment (Fig. 1 A). As a control, we also cut the radiolabeled fragment with AvaII, the site of which is a nonmutated site located in the FR2.

The four AluI restriction fragment patterns obtained are shown in Fig. 1,B. The first pattern consisted in a single 44-bp fragment and was obtained with B cells from two CVID cases (Fig. 1,B, patients LE and C10), suggesting that the majority of V3-23-Cγ transcripts harbored an unmutated Ser31 hot spot. These findings were perfectly in accordance with our previous observation, showing that the Ser31 hot spot was unmutated in 90% of V3-23 sequences from patient LE (15). The second pattern is illustrated by the presence of a predominant 44-bp band and a faint 56-bp fragment and was found in seven CVID cases (Fig. 1,B, patients D and E). This pattern suggested a high proportion of B cell clones with unmutated Ser31 codon. The third pattern consisted in a predominant 56-bp fragment and was observed in 26 CVID cases (Fig. 1,B, patients HU and C11). The majority of sequences from three CVID cases (Fig. 1B, C) and from the nine IgA-D samples were not digested with AluI. This fourth pattern was similar to that of ND3 (Fig. 1 B), where the majority of sequences were mutated within hot spots (15).

The results obtained are shown in Table I. Among the 38 CVID patients, nine cases (including patients LE and SO) expressed an AluI digestion profile that suggests that the majority of V3-23-Cγ transcripts did not harbor somatic mutations within the Ser31 codon. The rate and the pattern of mutations of IgG transcripts from these selected cases were then established by direct sequencing.

We found that 100% of individual sequences obtained from three controls (ND3, ND4, and ND5) were highly mutated with an average mutation frequency of 6.5% (range 4.5–7.9%) (Table II). The frequency of mutations of the V3-23 gene in the nine patients (including cases LE and SO previously studied (15)) selected as hypomutated cases varied from 0.1 to 3.3% (Table II). Interestingly, in case C10, the mutation level was dramatically reduced to 0.1%. In case E, the rate of mutation was 0.8%, similar to that of patient LE. The rate of mutation of the remaining cases was also significantly lower from controls with 1.5% (ES), 2.1% (H), 2.4% (W2), 3.1% (B), and 3.3% (D). In addition, 32% (30/92) of the total sequences from these patients were unmutated (less than one mutation). In cases C10, E, H, and ES, this proportion was high with 7/9, 6/14, 5/14, and 6/16 unmutated IgG sequences, respectively (Table II).

To confirm that this group of patients can be distinguished from other CVID patients, we determined the rate of mutation in six CVID and one IgA-D patient not selected as hypomutated with the FR1-Cγ screening test. In this group, the frequency of V3-23 gene mutation varied from 4% to 7.2%. The mutation level was slightly decreased in two CVID cases (A, 4% and I, 4.2%), whereas in the four remaining CVID cases, the mutation rate was within the normal ranges (C, 4.8%; C2, 5.5%; HU, 7.1%; and BR, 7.2%). The rate of mutation of the IgA-D case P was 6.2%. Furthermore, in this group, only 6% of the whole sequences (6/87) was found unmutated.

Finally, the average frequency of mutation of the group of patients selected as hypomutated with the FR1-Cγ screening test was 1.74%, differing significantly from NDs (mean rate 6.5%), and from other CVID patients (mean 5.46%, p < 0.0001). These results strongly suggest that a subgroup of patients characterized by a severe defect in the process of Ab somatic mutation represented a subgroup within the CVID syndrome.

A comparison of the frequency distribution of mutations accumulated within the V3-23 transcripts showed that, in hypomutated patients, the majority of mutated sequences harbored less than 10 mutations compared with 20–30 mutations in NDs (Fig. 2). As shown in Table II, the CDR/FR targeting ratio of mutations was broadly similar in the two groups of CVID patients and in NDs, except in case C10, where the ratio was not calculated because the number of mutations was too low. Overall, 72% (95/132) of sequences from CVID patients with hypomutation were unmutated within the Ser31 hot spot compared with 44% (38/87) and 10% (3/35) for other CVID cases and NDs, respectively (p < 0.001). These data confirmed well the results of the FR1-Cγ screening test. The comparison of the proportion of sequences harboring 0, 1, 2, or 3 mutated hot spots showed major differences between hypomutated patients and NDs: 54% of sequences from the subgroup of patients with hypomutation were unmutated on the three hot spot motifs compared with 13% and <5% of sequences from other CVID patients and controls, respectively (Fig. 3). The ratio of replacement vs silent mutations (R/S) argued against obvious abnormalities in the antigenic selection process in patients either with a normal or low mutation frequency (Table II). Interestingly, in the hypomutated case E, the high R/S ratio in the complementarity-determining region (CDR) suggests a strong Ag-driven selection, although the small number of mutations accumulated makes it difficult to draw a conclusion.

Recent data showed that the somatic hypermutation machinery may affect a non-Ig sequence, such as the first intron of the BCL-6 gene in malignant and normal memory B cells (18, 19, 20, 21, 22). To approach the elements involved in the defect of Ig V gene somatic mutation, we studied the rate of BCL-6 gene mutation. A fragment of 790 bp of the first intron of the BCL-6 gene was amplified from memory CD19+ IgM-IgD-purified B cells from ND3 and patients LE and SO. In all cases, more than one-third of sequences were mutated (range 47–60%) (Table III). The frequency of mutation was 1.3 × 10−3 for ND, 1.7 × 10−3 for patient LE, and 2.5 × 10−3 in case SO, which was at least 10-fold higher the mutation frequency expected as a result of PCR error (15). Three, five, and six sequences from ND3, LE, and SO, respectively, exhibited more than one mutation. These results indicate that the somatic hypermutation mechanism may target non-Ig sequences in memory B cells from CVID patients characterized with Ig V gene hypomutation.

For statistical reasons, patients LE and SO previously reported were added in this analysis. All cases with hypomutation were sporadic cases. The mean age of the nine patients of this subgroup was 55 years (range: 31–70) compared with 36 years (range: 5–67) for the group of patients with a normal pattern of mutation (p < 0.02). The mean age at onset of symptoms was 31.5 years for hypomutated patients and 21.5 years for other CVID cases (p = 0.08). No significant differences were noted in the duration of evolution (p = 0.2) and the duration of IV Ig treatment between the two groups. No specific clinical features distinguished hypomutated patients from other CVID cases. The serum Ig level at diagnosis in the group of patients with hypomutation varied from 0.5–3.6 g/L for IgG, <0.02–0.18 g/L for IgA, and 0.06–1 g/L for IgM. Two cases had autoimmunity with vitiligo (ES) and thrombopenic purpura (C10), and no patients had a granulomatous form of the disease. No specific infectious complications were noted in these patients. The percentage of circulating B cells in these cases varied within normal range (5–15%).

In this study, we have analyzed the pattern and frequency of somatic mutation of a well represented member of the VH3 gene family, the V3-23 gene, expressed by circulating IgG memory B cells from a cohort of 38 patients with CVID. For this study, two procedures were used. Initially all patients were screened for the presence of V3-23-Cγ transcripts unmutated in the AGC Ser31 codon previously shown as mutated in 90% of sequences from three healthy controls (Ref. 15 , and this report). In the second step, the frequency of V3-23 gene mutation of selected patients was assessed by direct sequencing. These two approaches were concordant because the screening of patients allowed us to discriminate a group of seven new cases characterized by a dramatic reduction in the frequency of Ig V gene somatic mutation. Taking these cases together with patients LE and SO previously described as hypomutated, the average rate of mutation in this subgroup of patients was 1.74% and differed significantly from the group of CVID patients nonselected as hypomutated with the screening assay (mean, 5.46%) and from healthy controls (mean, 6.5%). Our results suggest that the mutation process is functional in patients with selective IgA deficiency, a minor form of the more complex CVID syndrome. This observation reflects the heterogeneity of this syndrome and points to a selective switch defect in patients with IgA-D. Thus, this study allowed to delineate a distinct group of 20% of patients among the CVID syndrome in whom hypogammaglobulinemia is clearly associated to qualitative abnormalities of the Ig affinity maturation process.

Comparison of the pattern of mutations also showed differences between the group of patients with hypomutation and other CVID cases and controls. A high proportion of sequences from hypomutated patients was totally devoid of mutations (mean 30%), whereas nearly all sequences from the remaining CVID patients and controls were mutated. In four cases with hypomutation in the present cohort, as high as 75% of sequences were unmutated, which was similar to that previously observed in patients LE and SO (15). Moreover, when mutated, the majority of sequences from these patients harbored less than 10 mutations compared with 20–30 mutations in controls, indicating that the frequency of mutation accumulated in these sequences remained still lower than healthy donors.

Analysis of the CDR clustering of mutation and the R/S ratio did not show obvious differences between patients and controls, arguing against obvious abnormalities in the antigenic selection process. These mutations fit well the hot spots of mutation situated within the larger consensus sequence RGYW (23). Precise analysis of mutations showed a higher level of transitions over transversions. However, although the CDR-clustering of mutations largely reflects the process of antigenic selection, AGC codons encoding serine represent the major target of the mutational process (24), mainly when situated within RGYW motifs (23). The finding that 72% of sequences from the group with hypomutation harbored unmutated AGC Ser31 triplet confirmed the specificity of the FR1-Cγ test. Interestingly, this test also distinguished CVID patients having a rate of mutation within normal values from ND and IgA-D patients. Indeed, the large majority of samples from this group of patients exhibited a predominant 56 bp, reflecting a low level of mutation within the Ser35 hot spot, despite a global rate of mutation ranged within normal values. Thus, this simple assay might be useful for the rapid screening of samples and avoids extensive sequencing. However, one limitation of this test based on RNA analysis could be the presence of clonally related sequences in patients with a low number of circulating B cells. This was the case of patient HU in whom we found an intermediate AluI digestion pattern discordant with a high rate of mutation. The explanation for this discrepancy came from the analysis of sequences showing the over representation of a B cell clone with unmutated Ser35 hot spot.

The mechanisms underlying the Ig hypermutation process are not fully understood. The finding that germline and mutated IgG sequences can coexist in some patients suggests that the different steps leading to the mutational process can partially function in these cases. Recent data showed that the somatic hypermutation machinery may affect a non-Ig sequence, such as the first intron region of the BCL-6 gene in malignant and normal memory B cells (18, 19, 20, 21, 22). We show here that one-third of the amplified BCL-6 intronic sequences from memory CD19+ IgM-IgD- from ND3 were mutated with an overall frequency of 1.3 × 10−3, which is in accordance with results reported by other groups (25, 26, 27, 28, 29). Interestingly, the frequency and the global rate of mutation of 5′ BCL-6 intronic sequences amplified from memory B cells from cases LE and SO were identical with normal control. These results suggest that the targeting of the hypermutation machinery to the Ig sequences could be hampered in these patients.

Several studies have attempted to delineate different subgroups within the CVID. It has been reported that production of cytokines, such as IL-2, IL-4, IL-10, and IFN-γ, are impaired in some patients (25, 26, 27). Reduced expression of the CD40 ligand was shown in a subset of patients (28). Mononuclear cells from patients with chronic inflammatory complications, particularly granulomas, produce high levels of TNF in vitro (30), and this same subset is associated with a particular TNF genetic polymorphism (31). We found here that the mean age of the group of patients with hypomutation was significantly higher than other CVID cases, whereas no difference in the duration of symptoms was noted between the two groups. Although the number of patients studied here is too low for a valid statistical analysis, we found that the mean age at onset of symptoms of the group of patients with normal pattern of mutation is similar to that recently reported (32), whereas it was higher in the subgroup of patients with Ig V gene hypomutation. This suggests that hypomutation could characterize patients who begin disease late rather than patients with long-term evolution. This is in accordance with the observation that no variation in the rate of somatic mutation is found throughout life (33, 34) or in older healthy individuals (35). Moreover, the observation of a dramatic reduction in the rate of mutation in one patient with a short period of disease duration (<3 years, patient ES) argues also in favor of a distinct subgroup of CVID patients.

We showed here that a distinct entity could be recognized among the CVID syndrome. The low number of hypomutated patients, the long period of disease evolution, and the influence of Ig IV treatment make it difficult to draw definitive conclusions on any clinical correlations. However, individualization of these patients may have clinical implications because the Ab qualitative defect may progressively worsen their susceptibility to infections. Moreover, this defect may explain why in some patients the rate of infections is not absolutely correlated to the serum Ab levels. Finally, long-term clinical and immunological survey of this group of patients, longitudinal study of the rate of VH gene mutation, may provide further clues to the natural history of the disease and on the complex regulation of the mechanism of Ab maturation.

We thank P. Olak for manuscript typing.

1

This work was supported by grants from the Délégation à la Recherche Clinique de l’Assistance Publique des Hôpitaux de Paris (CRC 97160), Biomed 2 CT 98 3007, and Novartis.

3

Abbreviations used in this paper: CVID, common variable immunodeficiency; IgA-D, IgA deficiency; ND, normal donor; FR, framework region; R/S, replacement vs silence mutations; CDR, complementarity-determining region.

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