Introduction
AID-deficiency results from deleterious mutations in
AICDA encoding activation-induced cytidine deaminase (AID) and causes hyper-IgM syndrome type 2 (HIGM2) in humans [
1]. AID is essential for initiating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes in B cells [
2]. Hence, defects in AID result in abolished CSR and inadequate generation of the antibody isotypes IgG, IgA and IgE. In addition, affinity maturation of antibodies is impaired due to the lack of SHM in immunoglobulin genes [
3].
On a molecular level, AID deaminates deoxycytidine (dC) to deoxyuridine (dU) in distinct motives within the variable (V) or switch (S) regions of immunoglobulin genes, thereby initiating SHM or CSR, respectively [
4]. In SHM, AID particularly targets WRCY/RGYW hotspot motives in V regions (where R = purine, Y = pyrimidine, and W = A or T) [
5]. Additionally, the S region targeted in CSR is enriched in the AGCT sequence, which is a palindromic version of the above-mentioned SHM hotspot motives. During CSR, deamination at both strands is followed by the removal of dU by either the uracil DNA-glycosylase (UNG) or components of the mismatch repair (MMR) pathway leading to double-strand breaks (DSB) and subsequently joining of the VDJ-segment with α, γ or ε constant region exons [
6]. During SHM, following the initial deamination several downstream error-prone DNA-repair pathways may be engaged that further diversify the mutational pattern. Up to now, five different molecular pathways are known that process AID-initiated dU in V regions and differentially operate on the pattern of SHM [
6,
7].
Most patients with AID-deficiency carry homozygous or compound-heterozygous
AICDA mutations, which are inherited as an autosomal-recessive trait (AR) and affect both CSR and SHM [
8]. Most of these variants are complete loss-of-expression and/or –function and mainly affect the nuclear localization signal or the cytidine deaminase domain itself. However, some patients only carry heterozygous
AICDA mutations, which transmit the disease in an autosomal-dominant (AD) pattern. These distinct AD mutations (e.g., V186X and R190X) truncate the C-terminal nuclear export signal of AID, which result in an accumulation in the nucleus of mutated AID proteins that are unable to promote CSR but only partially affect SHM [
9‐
12]. Hence, other mechanisms than abolished enzymatic activity must account for defective CSR in these patients, and involvement of several co-factors interacting with AID has been indicated by these observations [
11,
13,
14].
AID-deficient patients suffer from recurrent infections most often affecting the respiratory tract [
15]. Furthermore, autoimmune manifestations and/or lymphoproliferation may develop in AR-AID but not AD-AID patients, which both display defects in the peripheral B cell tolerance checkpoint [
16,
17]. However, B cell tolerance is further breached in AR-AID patients as evidenced by exaggerated germinal center (GC) reactions, increased T follicular helper (T
FH) cells and secretion of antinuclear antibodies (ANAs). In addition, patients with UNG-deficiency, which impairs CSR but not SHM, did not display broken B cell tolerance nor enhanced GC reactions, suggesting that SHM but not CSR regulates both features in humans [
16].
Herein, we further refine this observation by describing two siblings with HIGM2 due to a novel homozygous AICDA mutation (AID-ΔE4a) that leads to the production of truncated AID proteins and results in impaired CSR but selectively impinges on SHM targeting, suggesting a rather qualitative impairment of SHM in these patients.
Material and Methods
Sample Preparation, Flow Cytometry and Cell Culture
Peripheral blood mononuclear cells (PBMCs) were purified with Ficoll density gradient centrifugation. For flow cytometry or cell sorting, PBMCs were stained in 1X PBS 0.5% BSA with appropriate antibodies at 4 °C for 30 min. Flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed with FlowJo version 10 (Tree Star). For assessing in vitro immunoglobulin CSR, CD19+CD27−IgM+ naïve B cells were sorted on a FACSAria III (BD Biosciences) and stimulated with CD40L (5 µg/mL; Biolegend) and IL-21 (100 ng/mL; Preprotech). After 5 days, expression of IgG and IgA was assessed on CD19+CD27+ B cells using flow cytometry.
Immunohistochemistry
Excised tonsil and adenoid tissues were fixed in 4% paraformaldehyde and embedded in paraffin after dehydration in alcohol. Immunohistochemistry was performed using standard protocols.
DNA Sequencing
Whole-exome sequencing (WES) was performed by a commercial provider using the SureSelect Human All Exon 50 Mb kit on a Illumina HiSeq 2500 system followed by an in-house analysis work-flow (CeGaT, Tübingen, Germany).
Sanger sequencing: The following primers were used for the amplification (AID Exon 4/5 FW CCCCGAGGAAATGAGAAAAT, AID Exon 4/5 REV GCAGAGATATTTCATCGTGTGTG) and sequencing (AID Ex4 REV Seq AGAGGGCTCTGAATGGTGAAAC) of the AICDA intron 3/exon 4 junction. PCR was carried out on genomic DNA obtained from PBMCs using GoTaq DNA Polymerase (Madison, WI, USA). For the analysis of exon/intron splicing, RNA obtained from CD40L/IL-21 stimulated naïve B cells was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). The following primers were used in RT-PCR for the amplification of the exon 3/4 junction of AICDA: FW TACTTCTGTGAGGACCGCAA, REV CATACAGGGGCAAAAGGATG. Sanger sequencing of purified PCR products was performed by a commercial provider (MWG eurofins, Martinsried, Germany).
Single-Cell PCR and Immunoglobulin Repertoire Sequencing
Single CD19
+CD27
+IgM
+ non-switched memory (NSM) B cells were sorted on a FACSAria III into 96-well PCR plates and immediately frozen on dry ice. RT-PCR amplification and sequencing of the variable and µ constant region of the immunoglobulin heavy chain (IgH) gene were performed as described before [
16,
18]. IgH sequences derived from NSM B cells of previously described patients (AD-AID-12, AD-AID-13 and AD-AID-18 as AD-AID patients, AID01, AID04, AID05, AID06 and AID17 as AR-AID patients [
16,
19]) were obtained using the same experimental approach and served as disease controls.
Immunoglobulin Repertoire Analysis
After manual quality control of sequencing chromatograms, IgH sequences were aligned to germline sequences and processed using IMGT/HighV-QUEST with standard settings [
20]. The output files were further analyzed using ARGalaxy [
21]: Sequences covering the complete IgH sequence from CDR1 to CDR3 were assessed for SHM and associated hotspot motives. The location, predicted mutation pathways and effect on amino acid sequence were assessed for each mutation, and the mutational frequency was calculated for each IgH sequence. Corrected mutation numbers and frequencies as well as corrected A over T ratios were computed as described [
7].
Statistical Analysis
Using GraphPad Prism® 9, unpaired two-sided Student’s t-test was applied for analysis of continuous variables. Depending on the sample size, Fisher’s exact t-test or Chi-square with Yates’ correction was used for categorical variables.
Discussion
Herein we report the functional impact of a novel
AICDA intronic splice site mutation identified in two AR-AID patients on CSR, SHM and GC responses. This
AICDA mutation disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant (AID-ΔE4a) when present in a homozygous state. This new AID variant abrogated CSR but only partially affected SHM as previously observed in AD-AID patients [
10]. However, unlike AD-AID deficiency, AID-ΔE4a patients displayed enhanced GC responses and secreted autoantibodies previously associated with the complete loss of SHM activity [
16,
29]. Interestingly, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns, suggesting that qualitative alterations rather than merely reduced enzymatic activity account for the impaired SHM process in AID-ΔE4a patients.
The AID-ΔE4a variant has already been detected as an alternative splicing product in tonsil B cells from healthy donors as well as CLL B cells [
25]. There are apparent discrepancies regarding the functional outcome of the AID-ΔE4a variant [
25,
27,
28]. Conflicting results between different experimental
in vitro systems and
in vivo analyses were also obtained when assessing the enzymatic function of other AID variants [
9]. The AID-ΔE4a splice variant has been reported to display proper subcellular localization but a pGFP* reversion assay in murine 70Z/3 cells suggested increased SHM activity [
25]. In contrast, van Maldegem et al
., using the same assay in NIH-3T3 cells, argued for a technical artifact, which seems to elicit this phenomenon [
25,
27,
28]. Furthermore, they demonstrated that AID-ΔE4a was not able to deaminate cytidine in an oligonucleotide substrate
in vitro and therefore should be regarded as catalytically inactive [
27,
28]. The analysis of AID-ΔE4a patients reveals that SHM is only partially affected by this variant
in vivo but present compared to patients expressing AID loss-of-function variants. The amount of SHM associated with the AID-ΔE4a variant was in the range of AD-AID patients known to possess residual SHM activity [
10,
16].
In contrast to AR-AID patients with
AICDA mutations that directly impinge on cytidine deaminase activity or even abolish AID protein expression and who suffer from defective SHM and CSR [
15], both functional
in vitro studies and our
in vivo analysis of two HIGM2 patients, who solely expressed the AID-ΔE4a variant, revealed the inability of this AID splicing product to catalyze CSR [
25]. Beside the absence of class-switched memory B cells, both patients also showed alterations within their naïve B cell compartment with increased frequencies of transitional B cells. Increasing evidence from mouse models and human studies indicated that AID is also expressed in developing B cells of the bone marrow and functions in central B cell tolerance [
18,
19,
30,
31]. Hence, the alterations within the transitional B cell compartment could be a result of defective AID-function during B cell development in the bone marrow. Alternatively, elevated BAFF levels that are present in AID-deficient patients might augment the generation of transitional B cells [
16].
Defective CSR with residual SHM activity is a characteristic feature of AD-AID patients harboring heterozygous
AICDA mutations, which affect the last 8–12 amino acid C-terminal nuclear export signal domain encoded by exon 5 [
10]. The AID-ΔE4a variant does not affect this C-terminal AID domain but lacks a sequence of 10 highly conserved amino acids encoded at the beginning of exon 4, which does not directly affect the cytidine deaminase domain. Hence, conformational alterations that indirectly entail decreased deaminase activity and/or impaired cytoplasmatic-nuclear shuttling might account for the impaired function of the AID-ΔE4a variant. However, the observation that CSR and SHM are differentially affected and mutational targeting and the mutational pattern are selectively distorted—as seen by lost preference for CDRs, reduced targeting of WRCY/RGYW but not WA/TW hotspots and altered strand targeting—may argue against these considerations. Indeed, these observations may point to a selective qualitative targeting defect of AID rather than a globally decreased enzymatic activity of the AID-ΔE4a variant. Several co-factors have been described, which are essential for proper AID function and impaired interaction with these co-factors has been suggested to account for impaired function of known AID variants [
11,
13,
14,
32]. Indeed, the dominant negative effect of the C-terminal AID variants in AD-AID patients is ascribed to depletion of CSR-specific co-factors from the wild-type allele [
13]. Furthermore, CTNNBL1 interacts with AID, thereby facilitating AID shuttling to the nucleus. Mutations in both CTNNBL1 and AID have been described to interfere with this interaction and impair CSR and SHM in humans [
33]. In line with this, the ten amino acids missing in the AID-ΔE4a variant form a highly conserved alpha helix, which seems to be essential for interaction with other AID co-factors. In detail, the RNA-binding protein ROD1 (
PTBP3) serves as a guiding system for AID and is required for AID-targeting to immunoglobulin loci [
22]. ROD1 interacts with AID via a highly conserved loop, comprising the amino acid residues 140–151 [
22]. Intriguingly, the amino acid residues 143–152 lacking in the AID-ΔE4a variant exactly match the essential ROD1 interaction site. Hence, the abolished CSR and distorted SHM pattern observed in the two HIGM2 patients expressing the AID-ΔE4a variant may result from the inability of their truncated AID to bind ROD1. We did not find any other potential disease causing mutations in known hyper-IgM-syndrome-related genes. However, we cannot completely rule out the presence of other noncoding variants or small copy number variants that might have been missed by whole-exome sequencing and additionally modify the immunological phenotype of these patients.
AR-AID patients who lack SHM secreted IgM autoantibodies associated with increased T
FH cells, whereas AD-AID or UNG-deficient patients with preserved SHM activity did not display autoantibodies or elevated T
FH frequencies [
16]. This suggested that SHM but not CSR is essential for maintaining B cell tolerance in humans [
16]. Additionally, SHM but not CSR is also involved in controlling GC reactions since mice harboring distinct
AICDA mutations that abrogate SHM but not CSR develop GC hyperplasia [
29]. It was, therefore, not expected to observe increased GC activity and IgM autoantibodies in the AID-ΔE4a patients whose ability to exert SHM is still preserved with SHM frequencies almost similar to AD-AID patients. Several explanations may account for this observation: first, the slightly lower frequency of SHM in the AID-ΔE4a patients compared to AD-AID patients could provoke follicular hyperplasia and/or autoimmunity. Second, the defective targeting of SHM to hotspot motives in AID-ΔE4a patients may result in B cell receptors (BCRs) that are less effective in binding and clearing antigens from follicular dendritic cells in GCs [
17]. SHM processes that have been suggested to redeem autoreactive BCRs encoded by distinct V gene segments with inherent self-reactivity as well as defects in SHM targeting might also impair reversion of self-reactivity and activation of these autoreactive B cells [
34]. Remarkably, giant GC as well as increased T
FH and T
PH cells resolved in the AID-ΔE4a patients after initiation of IRT, suggesting that provision of correctly mutated antibodies besides preventing severe infections may also dampen immune dysregulation in these patients.
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