Immunoglobulin Heavy Chain Class Switch Research Articles

SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

Mouse Models of c-myc Deregulation Driven by IgH Locus Enhancers as Models of B-Cell Lymphomagenesis.

Chromosomal translocations linking various oncogenes to transcriptional enhancers of the immunoglobulin heavy chain (IgH) locus are often implicated as the cause of B-cell malignancies. Two major IgH transcriptional enhancers have been reported so far. The Eμ enhancer located upstream of the Cμ gene controls early events in B-cell maturation such as VDJ recombination. The 3' regulatory region (3'RR) located downstream from the Cα gene controls late events in B-cell maturation such as IgH transcription, somatic hypermutation, and class switch recombination. Convincing demonstrations of the essential contributions of both Eμ and 3'RR in B-cell lymphomagenesis have been provided by transgenic and knock-in animal models which bring the oncogene c-myc under Eμ/3'RR transcriptional control. This short review summarizes the different mouse models so far available and their interests/limitations for progress in our understanding of human c-myc-induced B-cell lymphomagenesis.

Mucosa-associated lymphoid tissue (MALT) lymphoma developing in ectopic mediastinal thyroid tissue: a case report

BackgroundNormally located in the neck, ectopic mediastinal thyroid tissue consists of very rare ectopic thyroid tissue that does not connect to the thyroid gland. A patient with mucosa-associated lymphoid tissue (MALT) lymphoma that has developed in mediastinal thyroid tissue, to our best knowledge, has not been previously reported.Case presentationA 67-year-old woman presented with a superior mediastinal mass that was revealed by chest computed tomography (CT), an optional examination she hoped, during a medical checkup. Contrast-enhanced CT scan performed in our hospital for close examination confirmed the presence of a 2 × 1.3 cm poorly enhanced mass anterior to the trachea during the arterial phase. Magnetic resonance imaging depicted low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. I-131 meta-iodobenzylguanidine did not accumulate in the mass. Serum levels of interleukin-2 receptor, catecholamine, and anti-acetylcholine receptor antibody were within the normal range. The mass was resected through a transverse neck incision for the diagnosis and treatment. The histopathological diagnosis of the specimen was ectopic mediastinal thyroid tissue associated with MALT lymphoma and chronic thyroiditis. Immunoglobulin heavy chain class switch recombination was identified. Fine-needle aspiration biopsy of the cervical thyroid showed chronic thyroiditis but not lymphoma. The patient’s postoperative thyroid function was normal. To date, the patient’s recovery has been uneventful, and she is being monitored without further treatment.ConclusionWe treated the patient with MALT lymphoma that developed in ectopic mediastinal thyroid tissue. This novel case illustrates a new differential diagnosis associated with ectopic mediastinal thyroid tissue.

Fundamental roles of chromatin loop extrusion in antibody class switching

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cμconstant region exons (CHs) with one of 6 CHs lying 100-200kb downstream1. Each CH is flanked upstream by an I-promoter and long repetitive switch (S) region1. Cytokines/activators induce Activation-Induced Cytidine Deaminase (AID)2 and I-promoter transcription, with 3’IgH regulatory region (3’IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3’IgHRR interactions3-8. Transcription through donor Sμ and an activated downstream acceptor S region targets AID-generated deamination lesions at, potentially, any of 100s of individual S region deamination motifs9-11. General DNA repair pathways convert these lesions to DSBs and join an Sμ upstream DSB-end to an acceptor S region downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here, we report that chromatin loop extrusion underlies the "unprecedented" mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3’IgHRR enhancers with the 200kb upstream Sμ to generate a CSR center ("CSRC"). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic sub-domains that directionally align a downstream S region with Sμ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (“CBE”)-based impediment in the extrusion path. CBE insertion also inactivates upstream S region CSR, while converting adjacent downstream sequences into an ectopic S region by, respectively, inhibiting or promoting their dynamic alignment with Sμ in the CSRC. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

Aberrant expression of interleukin-10 and activation-induced cytidine deaminase in B cells from patients with Behçet's disease.

Despite extensive studies, the pathogenesis of Behçet's disease (BD) remains unclear. In particular, the roles of B cells in patients with BD have not been elucidated. Activation-induced cytidine deaminase (AID) is a critical enzyme for immunoglobulin (Ig) heavy chain class switching and somatic hypermutation in B cells and the abnormal expression of AID in various immune conditions has previously been studied. B10 cells, an interleukin (IL)-10-secreting subset of regulatory B cells, function to downregulate inflammation and autoimmunity. Thus, in the present study, the relevance of B cells in patients with BD was investigated. The plasma levels of IL-10 and IgA and the proportions of cluster of differentiation (CD)43+ B cells, excluding naïve B cells, were measured in 16 patients with BD and 16 age- and sex-matched healthy controls (HCs). Additionally, the mRNA levels of IL-10 and AID were assessed in B cells from fresh peripheral blood samples of the BD patients and HCs. The plasma level of IL-10 in patients with BD did not differ significantly from that in HCs. Similarly, there was no significant difference in the plasma level of IgA, although a slight increase was observed in patients with BD compared with that in HCs. There were no differences in CD43+CD19+ B cell numbers between patients with BD and HCs. However, IL-10 mRNA levels were significantly reduced (P<0.05), while AID mRNA levels were significantly increased (P<0.01) in the B cells of patients with BD compared with those in HCs. These results provide insight into the role of B cells in patients with BD.

CD11b regulates antibody class switching via induction of AID

The integrin CD11b, which is encoded by the integrin subunit alpha M (ITGAM), is primarily expressed on the surface of innate immune cells. Genetic variations in ITGAM are among the strongest risk factors for systemic lupus erythematosus, an autoimmune disease characterized by the presence of autoantibodies. However, the regulatory function of CD11b in the antibody responses remains unclear. Here, we report the induction of CD11b in activated B2 B cells and define its unexpected role in immunoglobulin heavy chain class switch recombination (CSR). LPS-activated B cells lacking CD11b yielded fewer IgG subtypes such as IgG1 and IgG2a in vitro, and immunization-dependent CSR and affinity maturation of antibodies were severely impaired in CD11b-deficient mice. Notably, we observed the reduced expression of activation-induced cytidine deaminase (AID), an enzyme that initiates CSR and somatic hypermutation, and ectopic expression of AID was sufficient to rescue the defective CSR of CD11b-deficient B cells. LPS-induced phosphorylation of NF-κB p65 and IκBα was attenuated in CD11b-deficient B cells, and hyperactivation of IκB kinase 2 restored the defective AID expression and CSR, which implied that CD11b regulates the NF-κB-dependent induction of AID. Overall, our experimental evidence emphasized the function of CD11b in antibody responses and the role of CD11b as a vital regulator of CSR.

Hotspots for Vitamin-Steroid-Thyroid Hormone Response Elements Within Switch Regions of Immunoglobulin Heavy Chain Loci Predict a Direct Influence of Vitamins and Hormones on B Cell Class Switch Recombination.

Vitamin A deficiencies are common throughout the world and have a significant negative influence on immune protection against viral infections. Mouse models demonstrate that the production of IgA, a first line of defense against viruses at mucosal sites, is inhibited in the context of vitamin A deficiency. In vitro, the addition of vitamin A to activated B cells can enhance IgA expression, but downregulate IgE. Previous reports have demonstrated that vitamin A modifies cytokine patterns, and in so doing may influence antibody isotype expression by an indirect mechanism. However, we have now discovered hundreds of potential response elements among Sμ, Sɛ, and Sα switch sites within immunoglobulin heavy chain loci. These hotspots appear in both mouse and human loci and include targets for vitamin receptors and related proteins (e.g., estrogen receptors) in the nuclear receptor superfamily. Full response elements with direct repeats are relatively infrequent or absent in Sγ regions although half-sites are present. Based on these results, we pose a hypothesis that nuclear receptors have a direct effect on the immunoglobulin heavy chain class switch recombination event. We propose that vitamin A may alter S site accessibility to activation-induced deaminase and nonhomologous end-joining machinery, thereby influencing the isotype switch, antibody production, and protection against viral infections at mucosal sites.

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Detection of true IgE-expressing mouse B lineage cells.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

Reprint of "Functional overlaps between XLF and the ATM-dependent DNA double strand break response".

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.

DNA Ligase I Is Not Essential for Mammalian Cell Viability

SUMMARYOf the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

Functional overlaps between XLF and the ATM-dependent DNA double strand break response

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.

Transgenes of the Mouse Immunoglobulin Heavy Chain Locus, Lacking Distal Elements in the 3′ Regulatory Region, Are Impaired for Class Switch Recombination

The immunoglobulin heavy (H) chain class switch is mediated by a deletional recombination event between µ and γ, α, or ε constant region genes. This recombination event is upregulated during immune responses by a regulatory region that lies 3′ of the constant region genes. We study switch recombination using a transgene of the entire murine H chain constant region locus. We isolated two lines of mice in which the H chain transgenes were truncated at their 3′ ends. The truncation in both transgenic lines results in deletion of the 3′-most enhancer (HS4) and a region with insulator-like structure and activities. Even though both truncated transgenes express the µ H chain gene well, they undergo very low or undetectable switch recombination to transgenic γ and α constant region genes. For both transgenic lines, germline transcription of some H chain constant regions genes is severely impaired. However, the germline transcription of the γ1 and γ2a genes is at wild type levels for the transgenic line with the larger truncation, but at reduced levels for the transgenic line with the smaller truncation. The dramatic reduction in class switch recombination for all H chain genes and the varied reduction in germline transcription for some H chain genes could be caused by (i) insertion site effects or (ii) deletion of enhancer elements for class switch recombination and transcription, or (iii) a combination of both effects.

Gonadal steroids and humoral immunity

Humoral immune responses are sexually dimorphic. Female individuals generally exhibit more-robust antibody responses to vaccines and, in the clinical setting as well as in experimental models, are more likely than male individuals to produce autoreactive antibodies of pathogenic potential. A number of differences between the sexes might account for these observations, including differences in the dosage of specific X-chromosome and Y-chromosomal genes, increased exposure of female individuals to antigenic stimulation in childbearing, and differences in circulating concentrations of gonadal steroid hormones. The role of gonadal steroids in modulating such humoral immune responses has been studied for nearly a century, but advances in our knowledge of B-lymphocyte development and function, the mechanisms of immune tolerance, and the molecular basis of gonadal steroid hormone action are now yielding new understanding of the influence of gonadal steroid hormones on the humoral immune system. This Review examines how oestrogens and androgens modulate B-lymphocyte development and function, focusing on the areas of B-cell production in the bone marrow, the maintenance of immune tolerance for self antigens, and the processes of immunoglobulin heavy chain gene somatic hypermutation and class switch recombination during maturation of cells involved in humoral immune responses.

Activation of Aicda gene transcription by Pax5 in plasmacytoma cells

Activation-induced deaminase (AID) is an enzyme responsible for somatic hypermutation and immunoglobulin heavy chain class switch recombination. Because AID causes double-stranded breaks in DNA, its expression is highly regulated and is normally restricted to germinal-center B cells. Dysregulated AID expression can lead to cancer as a result of AID-mediated chromosomal translocations. Many transcription factors including paired box protein 5 (Pax5) have been implicated in regulating the expression of Aicda, the gene encoding AID. In this study, we demonstrate that exogenous expression of Pax5 in a murine plasmacytoma cell line, 558LμM, leads to robust activation of endogenous Aicda transcription. Pax5 is known to initiate transcription through both its N-terminal-paired DNA-binding domain and its C-terminal-activation domain. Through mutational analysis, we demonstrate that Pax5 regulates Aicda transcription through its C-terminal-activation domain. Together, our work describes a novel system that will be useful for determining how Pax5 regulates Aicda transcription.

Waldenström’s macroglobulinemia harbors a unique proteome where Ku70 is severely underexpressed as compared with other B-lymphoproliferative disorders

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70—a component of the non homologous end-joining pathway—might be relevant in disease pathophysiology.

Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8.

IgA and IgG Hypogammaglobulinemia Does Not Predict for Recurrent Infection Risk and Persists Despite Therapeutic Response in Patients with Waldentrom's Macroglobulinemia.

Abstract 1947Poster Board I-970Recurrent infections are commonly observed among patients with WM, and may be related to the presence of IgA and IgG hypogammaglobulinemia. The etiology for this finding remains unclear, and has been speculated to be on the basis of tumor-induced suppression. We therefore evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 untreated WM patients and addressed the associated clinicopathological findings, and impact of therapy. The median age of these patients was 60, median IgM was 2,910, and median bone marrow (BM) infiltration was 40%. Of these patients, 131 (63.3%) and 120 (58.0%) patients demonstrated decreased serum IgA and IgG levels respectively, while 102 (49.3%) of these patients were abnormally low for both. BM infiltration, serum IgM levels, complete blood counts, absolute lymphocyte counts, b2-microglobulin, or the WM International Prognostic Scoring System score had no impact on the odds ratio of having IgA or IgG, or both IgA or IgG hypogammaglobulinemia by logistic regression analysis. The presence of adenopathy and/or splenomegaly was surprisingly associated with a lower incidence of hypogammaglobulinemia (p≤0.03). The presence of IgA, IgG or both IgA and IgG hypogammaglobulinemia did not predict for the occurrence of recurring infections, which were nearly all respiratory in nature and consisted of sinus (n=53; 25.85%), bronchial (n=16; 7.80%), unspecified upper respiratory tract (n=14; 6.83%), and pneumonic (n=7; 3.41%) infections. Lower IgA and IgG levels were however associated with disease progression in watch and wait patients. To understand the impact of WM directed therapeutic intervention on uninvolved immunoglobulin levels, we analyzed changes in IgA and IgG levels in a cohort of 93 patients who underwent treatment for WM. With a median follow-up of 12 months, no significant recovery in the median IgA and IgG levels was observed with any therapy during the course of follow-up, including in those patients who had follow-up in excess of 1 (n=46), 2 (n=25), and 3 (n=8) or more years post-therapy, and in those achieving a major remission including complete response. Lastly, we sequenced 8 genes (AICDA; BTK; CD40; CD154; NEMO, TACI, SH2D1A, UNG) implicated in immunoglobulin deficiency in 19 WM patients with IgA and/or IgG hypogammaglobulinemia. We observed an intronic variation at position c.1056-6T>C in 2 patients, and a hemizygous missense mutation at c.337G>A in another patient for NEMO, as well as a heterozygous missense mutation at c.425A>T in the highly conserved catalytic site of UNG for one patient. The results of these studies demonstrate that IgA and IgG hypogammaglobulinemia is common, and does not predict for recurrent infection risk in WM. Moreover, IgA and IgG hypogammaglobulinemia persists despite therapeutic intervention and response. These studies highlight the importance for further investigations into the IgA and IgG hypogammaglobulinemia of WM, as well as the signaling pathways involved in B-cell differentiation and immunoglobulin heavy chain class switching in the pathogenesis of WM. Disclosures:No relevant conflicts of interest to declare.

Gene conversion causing human inherited disease: Evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair

A variety of DNA sequence motifs including inverted repeats, minisatellites, and the chi recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the chi-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome.