Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2–Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.During humoral immune responses, the recombined antibody variable [V(D)J] region genes undergo somatic hypermutation (SHM), which, after selection, greatly increases the affinity of antibodies for the activating antigen. This process occurs in germinal centers (GCs) in the spleen, lymph nodes, and Peyer’s patches (PPs) and entirely depends on activation-induced cytidine deaminase (AID) (1, 2). AID initiates SHM by deamination of cytidine nucleotides in the variable region of antibody genes, converting the cytosine (dC) to uracil (dU) (1, 3, 4). Some AID-induced dUs are excised by the ubiquitous enzyme uracil DNA glycosylase (UNG), resulting in abasic (AP) sites that can be recognized by apurinic/apyrimidinic endonuclease (APE) (4, 5). APE cleaves the DNA backbone at AP sites to form a single-strand break (SSB) with a 3′ OH that can be extended by DNA polymerase (Pol) to replace the excised nucleotide (6). In most cells, DNA Pol β performs this extension with high fidelity, reinserting dC across from the template dG. In contrast, GC B cells undergoing SHM are rapidly proliferating, and some of the dUs are replicated over before they can be excised and are read as dT by replicative polymerases, resulting in dC to dT transition mutations. Unrepaired AP sites encountering replication lead to the nontemplated addition of any base opposite the site, causing transition and transversion mutations. However, it is not clear why dUs and AP sites escape accurate repair by the highly efficient enzymes UNG and APE1 and lead instead to mutations.Instead of removal by UNG, some U:G mismatches created by AID activity are recognized by the mismatch repair proteins Msh2–Msh6, which recruit exonuclease 1 to initiate excision of one strand surrounding the mismatch (7–9). The excised region (estimated at ∼200 nt; ref. 10) is subsequently filled in by DNA Pols, including error-prone translesion Pols, which spreads mutations beyond the initiating AID-induced lesion. The combined, but noncompeting interaction of the UNG and MMR pathways in generating mutations at A:T base pairs (bp) has been described (10–12). This mismatch repair-dependent process has been termed phase II of SHM (3). Pol η and Msh2–Msh6 have been shown to be essential for nearly all mutations at A:T bp (13–15). During repair of the excision patch, additional C:G bp can be mutated by translesion Pols, but mutations at C:G bp due to AID activity can also be repaired back to the original sequence during this step (16).Mammals express two known homologs of AP endonuclease (APE), APE1 and APE2. APE1 is the major APE; it is ubiquitously expressed and essential for early embryonic development in mice and for viability of human cell lines (17–19). APE1 has strong endonuclease activity and weaker 3′-5′ exonuclease (proofreading) and 3′-phosphodiesterase (end-cleaning) activities (20, 21). Recombinant purified human APE2 has much weaker AP endonuclease activity than APE1, but its 3′-5′ exonuclease activity is strong compared with APE1, although it is not processive (20). However, APE2 has been shown to interact with proliferating cell nuclear antigen (PCNA) (22), which can recruit error-prone translesion polymerases (23, 24), and PCNA also increases the processivity of APE2 exonuclease in vitro (25). Both APE1 and APE2 are expressed in splenic B cells activated in culture (26). APE2 is nonessential, but APE2-deficient mice show a slight growth defect, a twofold reduction of peripheral B and T cells (27), and impaired proliferation of B-cell progenitors in the bone marrow (28).In this study we examine SHM in GC B cells isolated from the PPs of unimmunized apex1^+/−, apex2^Y/−, and apex1^+/−apex2^Y/− mice relative to WT mice. [Because the APE2 gene is located on the X chromosome, we used APE2-deficient male mice (apex2^Y/−) in all experiments.] We demonstrate that not only is APE2 important for SHM frequency, as reported (29), but APE2 also contributes to the generation of A:T mutations. The proportion of mutations at A:T bp is reduced in apex2^Y/− mice to the same extent as it is in ung^−/− mice, consistent with APE2 acting as an endonuclease that incises AP sites generated by UNG. Surprisingly, in the absence of both UNG and APE2, mutations at A:T bp are greatly reduced. In addition, we find that expression of APE1 is dramatically reduced in GC B cells, and APE1 haploinsufficiency has very little effect on SHM. We propose a model in which APE2 promotes SHM through inefficient and error-prone repair, whereas APE1, which is known to interact with XRCC1 and Pol β to promote error-free SSB repair (30, 31), is suppressed in GC B cells.     

Serum ferritin level as a marker of disease activity and renal involvement in Egyptian children with juvenile systemic lupus erythematosus

Introduction

Ferritin is an acute-phase reactant that is elevated in several autoimmune disorders. Serum ferritin levels have been correlated with disease activity scores of juvenile systemic lupus erythematosus (JSLE). Furthermore, enhanced levels of ferritin have also been described in lupus nephritis (LN).

Retroperitoneal angioleiomyomatosis

Retroperitoneal intravenous leiomyomatosis is a rare benign tumor that can spread through veins carrying significant morbidity. The challenge of its management lies within the complexity of completely excising the tumor, which if carried out improperly can result in neurological or vascular complications requiring complex reparative surgeries. Here we present the successful resection of a retroperitoneal angio-leiomyoma by combining laparoscopic route, micro-surgical techniques and modern endoscopic tools.Electronic supplementary materialThe online version of this article (10.1007/s13224-020-01404-7) contains supplementary material, which is available to authorized users.