The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

Machine learning-based prediction of rheumatoid arthritis with development of ACPA autoantibodies in the presence of non-HLA genes polymorphisms.

Machine learning (ML) algorithms can handle complex genomic data and identify predictive patterns that may not be apparent through traditional statistical methods. They become popular tools for medical applications including prediction, diagnosis or treatment of complex diseases like rheumatoid arthritis (RA). RA is an autoimmune disease in which genetic factors play a major role. Among the most important genetic factors predisposing to the development of this disease and serving as genetic markers are HLA-DRB and non-HLA genes single nucleotide polymorphisms (SNPs). Another marker of RA is the presence of anticitrullinated peptide antibodies (ACPA) which is correlated with severity of RA. We use genetic data of SNPs in four non-HLA genes (PTPN22, STAT4, TRAF1, CD40 and PADI4) to predict the occurrence of ACPA positive RA in the Polish population. This work is a comprehensive comparative analysis, wherein we assess and juxtapose various ML classifiers. Our evaluation encompasses a range of models, including logistic regression, k-nearest neighbors, naïve Bayes, decision tree, boosted trees, multilayer perceptron, and support vector machines. The top-performing models demonstrated closely matched levels of accuracy, each distinguished by its particular strengths. Among these, we highly recommend the use of a decision tree as the foremost choice, given its exceptional performance and interpretability. The sensitivity and specificity of the ML models is about 70% that are satisfying. In addition, we introduce a novel feature importance estimation method characterized by its transparent interpretability and global optimality. This method allows us to thoroughly explore all conceivable combinations of polymorphisms, enabling us to pinpoint those possessing the highest predictive power. Taken together, these findings suggest that non-HLA SNPs allow to determine the group of individuals more prone to develop RA rheumatoid arthritis and further implement more precise preventive approach.

SNP in PTPN22, PADI4, and STAT4 but Not TRAF1 and CD40 Increase the Risk of Rheumatoid Arthritis in Polish Population.

Single nucleotide polymorphisms in non-HLA genes are involved in the development of rheumatoid arthritis (RA). SNPS in genes: PADI4 (rs2240340), STAT4 (rs7574865), CD40 (rs4810485), PTPN22 (rs2476601), and TRAF1 (rs3761847) have been described as risk factors for the development of autoimmune diseases, including RA. This study aimed to assess the prevalence of polymorphisms of these genes in the Polish population of patients with rheumatoid arthritis as compared to healthy controls. 324 subjects were included in the study: 153 healthy subjects and 181 patients from the Department of Rheumatology, Medical University of Lodz who fulfilled the criteria of rheumatoid arthritis diagnosis. Genotypes were determined by Taqman SNP Genotyping Assay. rs2476601 (G/A, OR = 2.16, CI = 1.27-3.66; A/A, OR = 10.35, CI = 1.27-84.21), rs2240340 (C/T, OR = 4.35, CI = 2.55-7.42; T/T, OR = 2.80, CI = 1.43-4.10) and rs7574865 (G/T, OR = 1.97, CI = 1.21-3.21; T/T, OR = 3.33, CI = 1.01-11.02) were associated with RA in the Polish population. Rs4810485 was also associated with RA, however after Bonferroni's correction was statistically insignificant. We also found an association between minor alleles of rs2476601, rs2240340, and rs7574865 and RA (OR = 2.32, CI = 1.47-3.66; OR = 2.335, CI = 1.64-3.31; OR = 1.88, CI = 1.27-2.79, respectively). Multilocus analysis revealed an association between CGGGT and rare (below 0.02 frequency) haplotypes (OR = 12.28, CI = 2.65-56.91; OR = 3.23, CI = 1.63-6.39). In the Polish population, polymorphisms of the PADI4, PTPN22, and STAT4 genes have been detected, which are also known risk factors for RA in various other populations.

Polymorphisms in DNA Repair Genes and Association with Rheumatoid Arthritis in a Pilot Study on a Central European Population.

Rheumatoid arthritis (RA) is a chronic, multifactorial autoimmune disease characterized by chronic arthritis, a tendency to develop joint deformities, and involvement of extra-articular tissues. The risk of malignant neoplasms among patients with RA is the subject of ongoing research due to the autoimmune pathogenesis that underlies RA, the common etiology of rheumatic disease and malignancies, and the use of immunomodulatory therapy, which can alter immune system function and thus increase the risk of malignant neoplasms. This risk can also be increased by impaired DNA repair efficiency in individuals with RA, as reported in our recent study. Impaired DNA repair may reflect the variability in the genes that encode DNA repair proteins. The aim of our study was to evaluate the genetic variation in RA within the genes of the DNA damage repair system through base excision repair (BER), nucleotide excision repair (NER), and the double strand break repair system by homologous recombination (HR) and non-homologous end joining (NHEJ). We genotyped a total of 28 polymorphisms in 19 genes encoding DNA repair-related proteins in 100 age- and sex-matched RA patients and healthy subjects from Central Europe (Poland). Polymorphism genotypes were determined using the Taq-man SNP Genotyping Assay. We found an association between the RA occurrence and rs25487/XRCC1, rs7180135/RAD51, rs1801321/RAD51, rs963917/RAD51B, rs963918/RAD51B, rs2735383/NBS1, rs132774/XRCC6, rs207906/XRCC5, and rs861539/XRCC3 polymorphisms. Our results suggest that polymorphisms of DNA damage repair genes may play a role in RA pathogenesis and may be considered as potential markers of RA.

Increased Sensitivity of PBMCs Isolated from Patients with Rheumatoid Arthritis to DNA Damaging Agents Is Connected with Inefficient DNA Repair.

Rheumatoid arthritis (RA) is a systemic, inflammatory disease of the joints and surrounding tissues. RA manifests itself with severe joint pain, articular inflammation, and oxidative stress. RA is associated with certain types of cancer. We have assumed that RA patients' increased susceptibility to cancer may be linked with genomic instability induced by impaired DNA repair and sensitivity to DNA damaging agents. The aim of this work was to analyze the sensitivity of peripheral blood mononuclear cells (PBMCs) isolated from RA patients to DNA damaging agents: tert-butyl hydroperoxide (TBH), bleomycin, ultraviolet (UV) radiation, and methyl methanesulfonate (MMS) and calculate the repair efficiency. TBH induce oxidative DNA lesions repaired mainly by base excision repair (BER). Bleomycin induced mainly DNA double-strand breaks repaired by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). We included 20 rheumatoid arthritis patients and 20 healthy controls and used an alkaline version of the comet assay with modification to measure sensitivity to DNA damaging agents and DNA repair efficiency. We found an increased number of DNA breaks and alkali-labile sites in the RA patients compared to those in the controls. Exposure to DNA damaging agents evoked the same increased damage in both groups, but we observed statistically higher PMBC sensitivity to TBH, MMS, bleomycin as well as UV. Examination of the repair kinetics of both groups revealed that the DNA lesions induced by TBH and bleomycin were more efficiently repaired in the controls than in the patients. These data suggest impaired DNA repair in RA patients, which may accelerate PBMC aging and/or lead to higher cancer incidence among RA patients.

Humoral and cellular immunity in children with Mycoplasma pneumoniae infection: a 1-year prospective study.

To determine whether children have persistent abnormalities in cellular and humoral immunity development after acute Mycoplasma pneumoniae infection, serum immunoglobulin G (IgG), IgA, IgM, and IgE levels and lymphocyte phenotypes were determined. There were no changes in the levels of IgG, IgM, IgA, or CD4+ or CD19+ lymphocytes that were measured in M. pneumoniae-positive patients after 3 months or after 12 months, but there were increases in these in M. pneumoniae-negative patients. Serum IgE increased in M. pneumoniae-positive patients. We have shown alterations in immunity development after M. pneumoniae infection.

Genetic evidence that mycobacterial FtsZ and FtsW proteins interact, and colocalize to the division site in Mycobacterium smegmatis.

We provide genetic evidence to show that the Mycobacterium tuberculosis FtsZ and FtsW proteins interact, and that these interactions are biologically relevant. Furthermore, we show by fluorescence microscopy that Mycobacterium smegmatis FtsW is part of its septasomal complex and colocalizes with FtsZ to the midcell sites. Colocalization experiments reveal that approximately 27% of the cells with septal Z-rings contain FtsW whereas 93% of the cells with FtsW bands are associated with FtsZ indicating that FtsW is late recruit to the septum, as in Escherichia coli. Our results suggest that mycobacterial FtsZ can localize to the septum independent of FtsW, and that interactions of FtsW with FtsZ are critical for the formation of productive FtsZ-rings and the cell division process in mycobacteria.

Molecular characterization of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Poland.

A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.