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Institut National de la Santé et de la Recherche Médicale Unit 474;
Département d’Information Hospitalière;
Service d’Immunologie Clinique, Hôpital Henri Mondor, Créteil, France;
§
Center for Biotechnology, Karokinska Institute, Huddinge, Sweden;
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Department of Clinical Immunology, Royal Free Hospital, Hampsted, London, United Kingdom;
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Department of Immunology, Oxford Radcliffe Hospital, John Radcliffe Site, Oxford, United Kingdom; and
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Service d’hématologie;
**
Institut National de la Santé et de la Recherche Médicale Unit 429, Hôpital Necker-Enfants Malades, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
Cloning and sequencing of V3-23-C
transcripts
Total RNA was extracted from 5 x 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 CA (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
CB (5'-AAGACCGATGGGCCCTTGGTGG-3'). CA and CB primers match
equally all 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.
Screening for V3-23 IgG transcripts without mutations in hot spot consensus sequences
The V3-23-C PCR products was amplified with 1.5 U of Gold
Taq polymerase using a reaction mixture containing
[-32P]-labeled 5' V3-23-framework region
(FR) 1 primer (5'-TCCCTGAGACTCTCCTGT-3') and the CB 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.
Analysis of BCL-6 gene mutations in purified memory B cells
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.
Statistical analysis
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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V3-23-C 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'-C primer. Next, V3-23-C PCR products were amplified by
using a 5'--32P FR1 primer hybridizing to the
framework region of the V3-23 gene together with a 3'-C primer (Fig. 1
A). This FR1-C 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. 1A). 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-C
PCR fragment (Fig. 1A). As a control, we also cut the
radiolabeled fragment with AvaII, the site of which is a
nonmutated site located in the FR2.
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B. The first pattern consisted in a single
44-bp fragment and was obtained with B cells from two CVID cases (Fig. 1B, patients LE and C10), suggesting that the majority of
V3-23-C 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. 1B, 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. 1B, patients HU and C11).
The majority of sequences from three CVID cases (Fig. 1 B,
C) and from the nine IgA-D samples were not digested with
AluI. This fourth pattern was similar to that of ND3 (Fig. 1B), 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-C 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.
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transcripts
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).
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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-C 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.
Analysis of the mutation pattern of V3-23 transcripts from CVID selected cases
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-C 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.
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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 x 10-3 for
ND, 1.7 x 10-3 for patient LE, and
2.5 x 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.
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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%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-C
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 x 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-, 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yves Levy, Service d’Immunologie Clinique Hôpital Henri Mondor, 51, Avenue du Marechal de Lattre de Tasigny, 94010 Créteil, Cedex 10, France.
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.
Received for publication March 7, 2000. Accepted for publication July 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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light chain sequences of mouse antibody. Nature 228:1045.[Medline]
, but proliferate normally upon activation by mitogens. Clin. Exp. Immunol. 78:334.[Medline]
polymorphisms associated with common variable immunodeficiency: role in the pathogenesis of granulomatous disease. J. Immunol. 159:6236.[Abstract]
genes with no or little somatic mutation throughout life. Eur. J. Immunol. 23:3272.[Medline]
genes and their hypermutation. Eur. J. Immunol. 23:3248.[Medline]
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