# Molecular Analysis of Activation-Induced Cytidine Deaminase Gene in Immunoglobulin-E Deficient Patients

Authors: Sergio Roa; Maria Isidoro-Garcia; Ignacio Davila; Elena Laffond; Felix Lorente; Rogelio Gonzalez-Sarmiento
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Understanding how class switch recombination (CSR) is regulated to
produce immunoglobulin E (IgE) has become fundamental because of the dramatic
increase in the prevalence of IgE-mediated hypersensitivity reactions. CSR
requires the induction of the enzyme AICDA in B cells. Mutations in AICDA have been linked to Hyper-IgM syndrome (HIGM2), which shows absence of switching to IgE as well as to IgG and IgA. Although isolated IgE deficiency is a rare entity, here we show some individuals with normal serum IgM, IgG, and IgA levels that had undetectable total serum IgE levels. We have analyzed theAICDA gene in these individuals to determine if there are mutations in AICDA that could lead to selective IgE deficiency. Conformational sensitive gel electrophoresis (CSGE) and sequencing analysis of AICDA coding sequences demonstrated sequence heterogeneity due to 5923A/G and 7888C/T polymorphisms, but did not reveal any novel
mutation that might explain the selective IgE deficit.


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## Body

## 1. Introduction

The prevalence  of immunoglobulin-E-(IgE-) mediated hypersensitivity reactions, such as allergic asthma, rhinitis,
hay fever, or food allergy, has been dramatically increasing for the  last
decades [1]. Total serum IgE levels tend to be higher in allergic patients
compared with nonallergic individuals, although the diagnostic value of total serum
IgE is limited [2] and the presence of specific IgE is not always equal to disease [3, 4]. The
effectiveness of humoral immune responses depends on the capacity of B-cells to
class switch from IgM to the other downstream isotypes. Class switch
recombination (CSR) is a recombinational process that requires the introduction
of double-stranded DNA breaks into the donor
μ  switch region, that is 5′ to the μ  constant regions, and into a
recipient γ,ε, or α switch region that is 5′ to each of those
constant regions. The antibody repertoire is shaped not only by CSR, but also
by somatic hypermutation (SHM) to create higher affinity antibodies. Both
processes occur in centroblast B cells in the germinal centers of secondary
lymphoid organs [5, 6].  A
major break through in the understanding of how these processes are initiated
was provided by the discovery of the mutagenic enzyme activation-induced
cytidine deaminase (AICDA, also known as AID)  [7–9].Class
switching to IgE requires two signals: the first is delivered by IL-4 and IL-13,
and the second is provided by the interaction of the B-cell surface antigen
CD40 with its ligand CD154 (CD40L) [10], which is transiently expressed on activated T cells and synergizes
with IL-4 to induce AICDA-encoding messenger RNA and AICDA protein [11]. When initiating IgE switching, IL-4 induces the binding of STAT6
to a site in the 5′ region of the AICDA gene, and CD40 activation induces the binding of NF-kB to two sites in the same
region of the AICDA gene [12].
Synergy between IL-4 and CD40 might be required to achieve a threshold level of AICDA expression for CSR to IgE [13].
Several groups have reported an association between serum IgE levels, allergic
disorders, and polymorphisms in the AICDA gene [14–16],
although this association is not completely understood and might vary among
populations [17, 18].Defects in CSR
have been described in hyperimmunoglobulin M (IgM) syndromes, which are primary
immunodeficiencies characterized by normal or elevated serum IgM levels with
the absence of other isotypes [19]. A group of patients with the autosomal recessive form of the hyper-IgM
syndrome (HIGM2) are known to have mutations in the AICDA gene [7, 20].
Since some of these mutations in AICDA are not in its active site, it has been
assumed that they related to the targeting of AICDA. This is born out by the
fact that mutations and deletions in the C-terminal region of AICDA result in
the loss of class switching while SHM persists [21, 22],
whereas mutations in the N-terminal part of AICDA lead to the loss of SHM and
retention of CSR [23]. This
suggests that there may be AICDA associated proteins that are required for the
targeting to switch regions and raises the possibility that different proteins
associate with AICDA to target it to each of the switch regions. One way to
screen for such interactions is to search for mutations in AICDA that lead to selective CSR impairment in clinical groups with
specific immunodeficiencies.Isolated IgE
deficiency is a rare entity and its association to clinical relevant
immunodeficiency is controversial [24–29]. As
opposed to hyper-IgM syndromes, the levels of other isotypes are normal in
individuals with isolated IgE deficiency, suggesting the possibility of a
selective CSR defect to this isotype. In the present study, we had the
opportunity to investigate a rare group of 9 individuals with isolated IgE
deficiency. In an attempt to further understand the contributions of AICDA to
the mechanisms underlying CSR and IgE production, we performed a molecular
characterization of AICDA gene in
these subjects, to assess whether specific defects in AICDA are related to
isolated deficiency of total serum IgE levels.


## 2. Materials and Methods

### 2.1. Subjects

This study was performed
in 9 patients with serum IgE levels below 2 kU/1 selected from a total of 643
patients that were referred during two consecutive years to the Allergy
Department of the University Hospital of Salamanca (Spain)  for an allergic evaluation.
All of them gave informed written consent and the study was performed following
the recommendations of the Ethical Committee of the University Hospital of
Salamanca. Total serum IgE levels were measured in all patients by fluorescent
enzyme immunoassay (ImmunoCAP total IgE, Phadia, Uppsala, Sweden), which
exhibits a measuring range for undiluted serum of 2–5000 kU/l. Since
IgE levels vary greatly in patients younger than 15 years, only patients older
than 18 years were selected. IgE deficiency was defined as a total serum IgE
level below 2 kU/l. Skin prick testing was performed following the European
Academy of Allergology and Clinical Immunology (EAACI) recommendations [30] with a battery of common aeroallergens that included D.
pteronisynuss, D. farinae, L. destructor, T. putrescentiae, A. siro, G. domesticus, E. maynei, mix of grasses, mix of
trees, P. judaica, C. album, A. vulgaris, P. lanceolata, O. europaea, A. alternata, C. herbarum, P. notatum, A. fumigatus, dog, cat, hamster, horse, rabbit dander, and cockroach
(ALK-Abelló, Madrid, Spain). Histamine (10 mg/mL) was used as positive control
and saline was used as negative control. Before skin testing, antihistamines
were discontinued according to published guidelines. Skin tests were considered
positive if at least one wheal reaction of more than 3 mm of diameter after
subtraction of the negative control was observed. IgM, IgG, and IgA levels were
measured in all patients with IgE level below 2 kU/l, by nephelometry (Dade Behring Inc,
Deerfield, Ill, USA).


### 2.2. DNA Extraction, PCR Primers Design, and Amplification Reactions

Genomic DNA was extracted from peripheral
blood lymphocytes using a standard phenol-chloroform protocol. Exons 1–5 and intronic
flanking sequences of theAICDA gene
were amplified using polymerase chain reaction (PCR). 3′ ends of all primers
were designed to be located at least 45 base pair (bp) away from the splice
junctions or the stop codon (Table 1). Amplifications were performed using 250 ng of DNA template, 1 μL of each 10 μM primer and 21 μL of 2xPCR Master Mix
(Promega, Madison, Wis, USA) in a final volume of 25 μL in an GeneAmp PCR System9700 automated thermocycler (PE Applied
Biosystems, Foster City, Calif, USA) with the same conditions: 4 minutes of
denaturation at 94C°, followed by 30 cycles with a denaturation step of 1 minute
at 94C°, an annealing step of 1 minute at 55C°, an extension step of 1 minute
at 72C°, and a final 10 minutes extension at 72C°. Amplification of the samples
was checked by electrophoresis in 2% agarose gels.Table 1
AICDA primers, PCR conditions, and size of the fragments used in the
study.
ExonPrimer designationPrimer sequenceAnnealingProduct size
(bp)Temperature
(C°)Position*1hAID1F5′-GCCGTTGGGGTACCTGGTGG-3′55−179280hAID1R5′-ATGAGAGAAAGGGATAGCTA-3′+1002hAID2F5′-AGCCCAAGTAATGACTTCCTTA-3′55+5661321hAID2R5′-ACCATCAGCAGGTGGCTCTAA-3′+59813hAID3F5′-GACTAAGGCTACCAGAGCCG-3′55+7221440hAID3R5′-GCCCACTTCTTCCCCTCGAG-3′+76604hAID4F5′-GTGAATGGCTCAGAGACAAGG-3′55+7716364hAID4R5′-ATCAGATGAAAACTGAGAGTGA-3′+80795hAID5F5′-GTTACAAAGCCATCCACTCAG-3′55+8334237hAID5R5′-GAGAAGACTTGAAGGACTG-3′+8570*Position of primers is according to GENBANK sequence
Accession Number AB040430.1.


### 2.3. Mutation Detection by CSGE (conformational Sensitive Gel Electrophoresis)

Mutation detection enhancement (MDE) gels (Cambrex, Rockland, Me, USA) were used to analyze PCR
products for the presence of mutations in theAICDA gene. 10 μL of the PCR product
were combined with 2 μL of 6xTriple dye
loading buffer (Cambrex, Rockland, Me, USA). The samples were heated at 95C°
for 5 minutes, slowly cooled to 37C° and run on 0.5xMDE gels containing
0.6xTris-Borate-EDTA (TBE) buffer (1xTBE = 0.089 M Tris-Borate, 0.002 M EDTA, and
pH 8.3). Gels were run overnight in 0.6xTBE at 20 V/cm for 12–18 hours,
depending on the size of the PCR product. MDE gels were stained using PlusOne DNA
silver staining kit (Amersham Biosciences, Uppsala, Sweden) according to the
manufacturer’s instructions. Heteroduplex bands were visualized on a
transilluminator and documented.


### 2.4. Sequencing

Whenever a heteroduplex was detected,
nucleotide sequence information was determined by direct sequencing of the PCR
products. The genotyping of intron 2 (5923A/G) and exon 4 (7888C/T)
polymorphisms were performed by direct sequencing in all cases. The same
primers designed for PCR amplification were used for the sequencing analysis of
the PCR products (Table1). PCRs fragments were purified with MicroSpin S-300
HR Columns (Amersham Biosciences, Piscataway, Nj, USA) and sequenced using
d-Rhodamine Dye terminator cycle sequencing kit and analysed with ABI Prism
377 genetic analyser (Applied Biosystems, Inc).


## 2.1. Subjects

This study was performed
in 9 patients with serum IgE levels below 2 kU/1 selected from a total of 643
patients that were referred during two consecutive years to the Allergy
Department of the University Hospital of Salamanca (Spain)  for an allergic evaluation.
All of them gave informed written consent and the study was performed following
the recommendations of the Ethical Committee of the University Hospital of
Salamanca. Total serum IgE levels were measured in all patients by fluorescent
enzyme immunoassay (ImmunoCAP total IgE, Phadia, Uppsala, Sweden), which
exhibits a measuring range for undiluted serum of 2–5000 kU/l. Since
IgE levels vary greatly in patients younger than 15 years, only patients older
than 18 years were selected. IgE deficiency was defined as a total serum IgE
level below 2 kU/l. Skin prick testing was performed following the European
Academy of Allergology and Clinical Immunology (EAACI) recommendations [30] with a battery of common aeroallergens that included D.
pteronisynuss, D. farinae, L. destructor, T. putrescentiae, A. siro, G. domesticus, E. maynei, mix of grasses, mix of
trees, P. judaica, C. album, A. vulgaris, P. lanceolata, O. europaea, A. alternata, C. herbarum, P. notatum, A. fumigatus, dog, cat, hamster, horse, rabbit dander, and cockroach
(ALK-Abelló, Madrid, Spain). Histamine (10 mg/mL) was used as positive control
and saline was used as negative control. Before skin testing, antihistamines
were discontinued according to published guidelines. Skin tests were considered
positive if at least one wheal reaction of more than 3 mm of diameter after
subtraction of the negative control was observed. IgM, IgG, and IgA levels were
measured in all patients with IgE level below 2 kU/l, by nephelometry (Dade Behring Inc,
Deerfield, Ill, USA).


## 2.2. DNA Extraction, PCR Primers Design, and Amplification Reactions

Genomic DNA was extracted from peripheral
blood lymphocytes using a standard phenol-chloroform protocol. Exons 1–5 and intronic
flanking sequences of theAICDA gene
were amplified using polymerase chain reaction (PCR). 3′ ends of all primers
were designed to be located at least 45 base pair (bp) away from the splice
junctions or the stop codon (Table 1). Amplifications were performed using 250 ng of DNA template, 1 μL of each 10 μM primer and 21 μL of 2xPCR Master Mix
(Promega, Madison, Wis, USA) in a final volume of 25 μL in an GeneAmp PCR System9700 automated thermocycler (PE Applied
Biosystems, Foster City, Calif, USA) with the same conditions: 4 minutes of
denaturation at 94C°, followed by 30 cycles with a denaturation step of 1 minute
at 94C°, an annealing step of 1 minute at 55C°, an extension step of 1 minute
at 72C°, and a final 10 minutes extension at 72C°. Amplification of the samples
was checked by electrophoresis in 2% agarose gels.Table 1
AICDA primers, PCR conditions, and size of the fragments used in the
study.
ExonPrimer designationPrimer sequenceAnnealingProduct size
(bp)Temperature
(C°)Position*1hAID1F5′-GCCGTTGGGGTACCTGGTGG-3′55−179280hAID1R5′-ATGAGAGAAAGGGATAGCTA-3′+1002hAID2F5′-AGCCCAAGTAATGACTTCCTTA-3′55+5661321hAID2R5′-ACCATCAGCAGGTGGCTCTAA-3′+59813hAID3F5′-GACTAAGGCTACCAGAGCCG-3′55+7221440hAID3R5′-GCCCACTTCTTCCCCTCGAG-3′+76604hAID4F5′-GTGAATGGCTCAGAGACAAGG-3′55+7716364hAID4R5′-ATCAGATGAAAACTGAGAGTGA-3′+80795hAID5F5′-GTTACAAAGCCATCCACTCAG-3′55+8334237hAID5R5′-GAGAAGACTTGAAGGACTG-3′+8570*Position of primers is according to GENBANK sequence
Accession Number AB040430.1.


## 2.3. Mutation Detection by CSGE (conformational Sensitive Gel Electrophoresis)

Mutation detection enhancement (MDE) gels (Cambrex, Rockland, Me, USA) were used to analyze PCR
products for the presence of mutations in theAICDA gene. 10 μL of the PCR product
were combined with 2 μL of 6xTriple dye
loading buffer (Cambrex, Rockland, Me, USA). The samples were heated at 95C°
for 5 minutes, slowly cooled to 37C° and run on 0.5xMDE gels containing
0.6xTris-Borate-EDTA (TBE) buffer (1xTBE = 0.089 M Tris-Borate, 0.002 M EDTA, and
pH 8.3). Gels were run overnight in 0.6xTBE at 20 V/cm for 12–18 hours,
depending on the size of the PCR product. MDE gels were stained using PlusOne DNA
silver staining kit (Amersham Biosciences, Uppsala, Sweden) according to the
manufacturer’s instructions. Heteroduplex bands were visualized on a
transilluminator and documented.


## 2.4. Sequencing

Whenever a heteroduplex was detected,
nucleotide sequence information was determined by direct sequencing of the PCR
products. The genotyping of intron 2 (5923A/G) and exon 4 (7888C/T)
polymorphisms were performed by direct sequencing in all cases. The same
primers designed for PCR amplification were used for the sequencing analysis of
the PCR products (Table1). PCRs fragments were purified with MicroSpin S-300
HR Columns (Amersham Biosciences, Piscataway, Nj, USA) and sequenced using
d-Rhodamine Dye terminator cycle sequencing kit and analysed with ABI Prism
377 genetic analyser (Applied Biosystems, Inc).


## 3. Results

From a total of 643 patients that underwent determination of total
serum IgE, 9 patients had IgE levels below 2 kU/l (1.4%). Strikingly, total
serum IgM, IgG, and IgA levels were normal in all of them (Table2). None of
these patients had personal history of allergy or atopy and all of them had
negative skin tests. No previous personal or familial history suggesting
immunodeficiency was recorded in any of them. Nevertheless, an increase in
nonallergic reactive airway diseases (i.e., nonallergic asthma) and autoimmunity
(i.e., hyperthyroidism and dermatomyositis) was observed.Table 2
Phenotype characteristics of patients with IgE
hypogammaglobulinemia.
No.Sex/AgeClinical diagnosisIgEIgGIgAIgMHistory of immunodeficiency/(kU/l)(mg/dl)(mg/dl)(mg/dl)autoimmunity1F/57Normal<213001822147No/No2F/37Non allergic asthma<2735156226No/No3F/65Normal<295422163No/No4F/28Contact dermatitis<2700108157No/No5F/57Normal<2121024584No/hyperthyroidism6M/44Chronic urticaria<285925769No/No7F/59Non allergic asthma<2108024273No/No8M/48Non allergic asthma<280920058No/No9F/57Normal<21540353234No/dermatomyositisF: female, M: male.MDE gel electrophoresis of PCR products spanning exons 1, 3, and 5
fromAICDA gene revealed germ-line configurations. As expected, due to
5923A/G and 7888C/T polymorphisms [16],
several heteroduplex patterns were detected in PCR products spanning exons 2
and 4, respectively. Sequence analysis of both regions in all patients
identified different alleles for the polymorphic sites (Figure 1) but did not
reveal any novel mutation.Sequencing analysis of regions 2 and 4 from humanAICDA gene, where genetic variants were
detected by heteroduplex. Panels
show sequences of the different alleles corresponding to 5923A/G polymorphism
at the 3′-flanking region of (a) exon 2 and 7888C/T polymorphism at (b) exon 4
of the AICDA gene. Arrows indicate
the polymorphic sites.
(a)(b)Distributions of
genotypes of the 5923A/G and 7888C/T polymorphisms were consistent with
Hardy-Weinberg equilibrium. The distribution of genotypes of these
polymorphisms in the 9 patients is shown in Table3. We have studied a small
number of patients because of the low prevalence of IgE deficiency. Although
this is an important limitation to the study of this condition, the genotype
frequencies of the synonymous 7888C/T polymorphism at exon 4 (Reference SNP Identification
rs2028373 [31]) did not deviate from expected values previously reported [16, 18]. In
the case of the AICDA intronic
5923A/G polymorphism, genotype frequencies were similar to those reported in
NCBI single nucleotide polymorphism database, according to Reference SNP
Identification rs2518144 [31]. The diplotype distribution of both polymorphisms was
heterogeneous. No specific haplotype was detected in this population.Table 3
Genotype distribution of 5923A/G and 7888C/T polymorphisms fromAICDA gene in our population.
Patient5923A/G7888C/T1GGTT2AGCT3AGTT4GGCT5GGTT6AACT7AGCC8GGCT9GGTT


## 4. Discussion

The level of serum IgE is usually 10,000 to 50,000-fold lower than
the level of serum IgG and its production is tightly regulated. The overall
role of IgE in immunity is not completely understood, but it seems to be
involved in the defence against parasitic infections. Reports about IgE
deficiency are limited and their association with clinically relevant
immunodeficiency is still controversial [24–29]. We
have evaluated the clinical characteristics of a group of patients with serum
IgE levels below 2 kU/l, but normal IgM, IgG, and IgA serum levels (Table 2). In
our setting of an outpatient allergy clinic, only 9 out of 643 (1.4%) patients
that underwent IgE determination had serum total IgE levels under the detection
threshold (2 kU/l). There is limited information about prevalence of IgE
deficits. Levy et al. [32] found an incidence of 16.5%, but their threshold was 10 kU/l, and
Smith et al. [29] found 10.5% of patients with IgE levels below 2.5 kU/l. Our
incidence is lower probably due to the lower threshold that we used. Although
controversial, there are some reports on local IgE production [33] and therefore we cannot absolutely discard the possibility of local
CSR to IgE in tissue. Nevertheless we believed that this fact is highly
improbable because it has been shown that patients with local production of
specific IgE did not have undetectable total IgE levels and they suffered from
perennial rhinitis, usually with seasonal symptoms [34].None of the patients with IgE deficiency included in our series
showed personal or familial clinical data of immunodeficiency but three
patients presented nonallergic asthma, and autoimmune disorders were present in
two patients. This is in agreement with the study of Smith et al. [29], which found a higher prevalence of both conditions in patients
with serum IgE levels below 2.5 kU/l, suggesting the existence of common
genetic factors that may predispose to both IgE deficiency and autoimmunity.
Other authors have proposed that deregulation of molecules and signals that
play a key role in B-cell activation and terminal differentiation could be
involved in initiating or maintaining autoimmunity [35].AICDA plays an essential role in CSR, which is impaired in both AICDA-deficient
mice [8] and in patients with HIGM2 syndrome [7, 20]. An
association between serum IgE levels and AICDA has been previously reported,
and several studies have shown the association between polymorphisms in the AICDA gene and allergic disorders [14–16],
although this association is not completely understood and might vary among
populations [17, 18].
Serum IgE levels have an inherited non-Mendelian component, although there is
one report that showed an autosomal dominant pattern with a variable degree of
penetrance in patients with IgE deficiency [28]. Exploration of genetic regulation in IgE responses, based on both
candidate gene approaches and linkage studies has led to the report of several
sets of molecular and cellular interactions that are essential for IgE
synthesis [36]. Nevertheless, no study of susceptibility loci has been performed
on patients with undetectable serum IgE levels. Here, we have examined whether
mutations in the AICDA gene might
explain the phenotype of our cohort of patients with isolated IgE deficiency
and affect the specificity of switching towards IgE production. We have
identified two previously described polymorphisms (intron 2 5923A/G and exon 4
7888C/T) in the AICDA gene, but no
defects in the AICDA coding or
flanking regions account for the IgE deficit in our cohort. Previous reports and
public databases on these polymorphisms did not show any evident association
with AICDA expression levels,
splicing or function. We cannot rule out that mutations in the promoter region,
epigenetic changes or posttranslational modifications in AICDA could be
involved in the decrease of IgE levels. In addition, other factors involved in
immunoglobulin class switching besides AICDA might be implicated. The
characterization of such factors would be important in understanding the
specific targeting of AICDA to the εswitch region and the
molecular basis of isolated IgE deficiency.


## 5. Conclusions

The exploration of various types of clinical
immunodeficiencies provides a unique opportunity to better understand the
molecular basis of immunoglobulin class switch recombination. Here, we
evaluated the clinical characteristics of a cohort of patients with isolated
IgE deficiency, which exhibited a higher prevalence of nonallergic asthma and
autoimmunity disorders. We analyzedAICDA,
the master gene required for CSR, in these patients to see if there were
specific mutations that would result in impaired CSR and explain their
undetectable serum IgE levels. Two previously described
polymorphisms (intron 2 5923A/G and exon 4 7888C/T), but no mutation, were
detected in AICDA gene. Further
search for alternative causes of isolated IgE deficiency could open new ways to
understand specific CSR and IgE production.



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*Source: 146715-2009-02-25.xml*