Genetic Risk Variants for Class Switching Recombination Defects in Ataxia-Telangiectasia Patients

Background Ataxia-telangiectasia (A-T) is a rare autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. A-T patients manifest considerable variability in clinical and immunological features, suggesting the presence of genetic modifying factors. A striking heterogeneity has been observed in class switching recombination (CSR) in A-T patients which cannot be explained by the severity of ATM mutations. Methods To investigate the cause of variable CSR in A-T patients, we applied whole-exome sequencing (WES) in 20 A-T patients consisting of 10 cases with CSR defect (CSR-D) and 10 controls with normal CSR (CSR-N). Comparative analyses on modifier variants found in the exomes of these two groups of patients were performed. Results For the first time, we identified some variants in the exomes of the CSR-D group that were significantly associated with antigen processing and presentation pathway. Moreover, in this group of patients, the variants in four genes involved in DNA double-strand breaks (DSB) repair signaling, in particular, XRCC3 were observed, suggesting an association with CSR defect. Conclusion Additional impact of certain variants, along with ATM mutations, may explain the heterogeneity in CSR defect phenotype among A-T patients. It can be concluded that genetic modulators play an important role in the course of A-T disease and its clinical severity.

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4−/− cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.

Generation of vFLIP Transgenic Mice: A Model to Study KSHV-Associated Lymphomagenesis

Primary effusion lymphoma (PEL) is a distinct subtype of aggressive non-Hodgkin’s lymphoma (NHL), specifically associated with infection by Kaposi’s sarcoma-associated herpesvirus (KSHV) and occurring more frequently in HIV-infected individuals. Several in vitro observations suggest that vFLIP, a viral protein expressed during latency, is an important viral oncogene. It is essential for the survival of KSHV-infected PEL cells, mainly by constitutively activating the NF-kB pathway. In order to assess the role of vFLIP in the pathogenesis of PEL, we knocked a cDNA encoding vFLIP, preceded by a loxP-flanked neoR-Stop cassette and followed by Frt-flanked IRES-eGFP sequences, into the ubiquitously expressed ROSA26 locus. A specifically restricted expression of the transgene in CD19+ B-cells has been achieved by crossing the ROSA26. vFLIP knock-in mice with mice expressing cre recombinase under the control of the CD19 promoter. These mice have also been crossed with transgenic mice expressing KSHV LANA, which is considered to also be a viral oncogene, to assess a potential synergistic effect of these two KSHV latent proteins in the lymphomagenic process of PEL. vFLIP expression in the CD19+ B-cells results in splenomegaly, with an increase in both T and B-cells, and with a relative increase of the T versus B-cell ratio. Although primary follicles were enlarged, the expression of vFLIP in the CD19+ B-cells results in lack of germinal center formation in the spleen, lymph nodes and intestine, and in partially impaired class-switching recombination. vFLIP transgenic mice had an increased number of plasmablast-like cells expressing lambda light chain, reminiscent of a phenomenon seen in KSHV-associated multicentric Castleman’s disease (MCD). These results indicate that by constitutively activating the NF-κB pathway in pre-germinal center B-cells expressing CD19, the normal B-cell differentiation is impaired, and provide clues about possible aberrant differentiation in PEL and MCD.