Corrigendum: Abnormalities of thymic stroma may contribute to immune dysregulation in murine models of leaky severe combined immunodeficiency.

[This corrects the article DOI: 10.3389/fimmu.2011.00015.].

The Contribution of DNA Ligase 4 Polymorphisms to Colorectal Cancer.

BACKGROUND/AIM: While numerous biomarkers associated with genetic susceptibility to colorectal cancer (CRC) have been identified and validated through epidemiological studies, the specific influence of DNA ligase 4 (Lig4) genotypes remains unexplored. This study aimed to elucidate the hitherto unexamined relationship between Lig4 genotypes and CRC risk. MATERIALS AND METHODS: The genotypes of Lig4 rs1805388 were determined applying the polymerase chain reaction-restriction fragment length polymorphism methodology. The potential association between these genotypes and CRC risk was assessed in a Taiwanese population comprising 362 CRC cases and an equal number of age- and sex-matched controls. RESULTS: In the genotypic analysis, the distribution of CC, CT, and TT genotypes for Lig4 rs1805388 among CRC cases was 54.7%, 38.1%, and 7.2%, respectively. This distribution was not significantly different from the controls, which exhibited genotypic frequencies of 57.2%, 36.7%, and 6.1%, respectively (p for trend=0.7314). Analysis of allelic distribution indicated that individuals carrying the T allele of Lig4 rs1805388 displayed a slightly elevated CRC risk compared to those carrying the C allele (odds ratio=1.10, 95% confidence interval=0.87-1.39, p=0.4685). CONCLUSION: The variant genotypes of Lig4 rs1805388 may not serve as predictive markers for CRC risk in the Taiwanese population.

The Contribution of DNA Ligase 4 Genetic Variations to Taiwanese Lung Cancer.

BACKGROUND/AIM: Impaired non-homologous end-joining DNA repair capacity may have a significant role in maintaining genome integrity and triggering carcinogenesis. However, the specific impact of DNA ligase 4 (Lig4) genotypes remains unclear. This study aimed to assess the contribution of Lig4 genotypes to the risk of developing lung cancer. MATERIALS AND METHODS: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to examine the genotypes of Lig4 rs1805388, and their association with lung cancer risk was evaluated in a case-control study consisting of 358 lung cancer cases and 716 age- and sex-matched cancer-free control subjects. RESULTS: The distribution of CC, CT, and TT genotypes for Lig4 rs1805388 among the cases was 45.0%, 41.6%, and 13.4%, respectively, compared to 58.0%, 36.3%, and 5.7% among the controls (p for trend=1.98×10-6). Allelic analysis indicated that individuals carrying the T-allele for Lig4 rs1805388 had a 1.66-fold higher risk of developing lung cancer compared to those carrying the wild-type C-allele [95% confidence interval (CI)=1.36-2.02, p=4.04×10-7]. Moreover, a significant interaction was observed between the Lig4 rs1805388 genotype and smoking status (p=1.32×10-7). CONCLUSION: These findings suggest that the CT and TT variant genotypes of Lig4 rs1805388, combined with cigarette smoking, may contribute to a higher risk of developing lung cancer.

DNA Ligase 4 Contributes to Cell Proliferation against DNA-PK Inhibition in MYCN-Amplified Neuroblastoma IMR32 Cells.

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.

Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3' single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3' flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3' single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4-/- cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.

Update on DNA-Double Strand Break Repair Defects in Combined Primary Immunodeficiency.

PURPOSE OF REVIEW: The most serious DNA damage, DNA double strand breaks (DNA-dsb), leads to mutagenesis, carcinogenesis or apoptosis if left unrepaired. Non-homologous end joining (NHEJ) is the principle repair pathway employed by mammalian cells to repair DNA-dsb. Several proteins are involved in this pathway, defects in which can lead to human disease. This review updates on the most recent information available for the specific diseases associated with the pathway. RECENT FINDINGS: A new member of the NHEJ pathway, PAXX, has been identified, although no human disease has been associated with it. The clinical phenotypes of Artemis, DNA ligase 4, Cernunnos-XLF and DNA-PKcs deficiency have been extended. The role of haematopoietic stem cell transplantation, following reduced intensity conditioning chemotherapy, for many of these diseases is being advanced. In the era of newborn screening, urgent genetic diagnosis is necessary to correctly target appropriate treatment for patients with DNA-dsb repair disorders.

Rubella Virus-Associated Cutaneous Granulomatous Disease: a Unique Complication in Immune-Deficient Patients, Not Limited to DNA Repair Disorders.

The association of immunodeficiency-related vaccine-derived rubella virus (iVDRV) with cutaneous and visceral granulomatous disease has been reported in patients with primary immunodeficiency disorders (PIDs). The majority of these PID patients with rubella-positive granulomas had DNA repair disorders. To support this line of inquiry, we provide additional descriptive data on seven previously reported patients with Nijmegen breakage syndrome (NBS) (n = 3) and ataxia telangiectasia (AT) (n = 4) as well as eight previously unreported patients with iVDRV-induced cutaneous granulomas and DNA repair disorders including NBS (n = 1), AT (n = 5), DNA ligase 4 deficiency (n = 1), and Artemis deficiency (n = 1). We also provide descriptive data on several previously unreported PID patients with iVDRV-induced cutaneous granulomas including cartilage hair hypoplasia (n = 1), warts, hypogammaglobulinemia, immunodeficiency, myelokathexis (WHIM) syndrome (n = 1), MHC class II deficiency (n = 1), Coronin-1A deficiency (n = 1), X-linked severe combined immunodeficiency (X-SCID) (n = 1), and combined immunodeficiency without a molecular diagnosis (n = 1). At the time of this report, the median age of the patients with skin granulomas and DNA repair disorders was 9 years (range 3-18). Cutaneous granulomas have been documented in all, while visceral granulomas were observed in six cases (40%). All patients had received rubella virus vaccine. The median duration of time elapsed from vaccination to the development of cutaneous granulomas was 48 months (range 2-152). Hematopoietic cell transplantation was reported to result in scarring resolution of cutaneous granulomas in two patients with NBS, one patient with AT, one patient with Artemis deficiency, one patient with DNA Ligase 4 deficiency, one patient with MHC class II deficiency, and one patient with combined immunodeficiency without a known molecular etiology. Of the previously reported and unreported cases, the majority share the diagnosis of a DNA repair disorder. Analysis of additional patients with this complication may clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection, and may lead to defect-specific therapies.

PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF.

In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF's function.

DNA ligase IV syndrome; a review.

DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.

Disruption of lig4 improves gene targeting efficiency in the oleaginous fungus Mortierella alpina 1S-4.

The oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete.