C. elegans genome-wide analysis reveals DNA repair pathways that act cooperatively to preserve genome integrity upon ionizing radiation.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions.

Protection of the C. elegans germ cell genome depends on diverse DNA repair pathways during normal proliferation.

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.

Mutational signatures are jointly shaped by DNA damage and repair.

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

Caenorhabditis elegans BUB-3 and SAN-1/MAD3 Spindle Assembly Checkpoint Components Are Required for Genome Stability in Response to Treatment with Ionizing Radiation.

Relatively little is known about the cross-talk between the spindle assembly checkpoint and the DNA damage response, especially in multicellular organisms. We performed a Caenorhabditis elegans forward genetic screen to uncover new genes involved in the repair of DNA damage induced by ionizing radiation. We isolated a mutation, gt2000, which confers hypersensitivity to ionizing radiation and showed that gt2000 introduces a premature stop in bub-3 BUB-3 is a key component of the spindle assembly checkpoint. We provide evidence that BUB-3 acts during development and in the germline; irradiated bub-3(gt2000) larvae are developmentally retarded and form abnormal vulvae. Moreover, bub-3(gt2000) embryos sired from irradiated worms show increased levels of lethality. Both bub-3 and san-1 (the C. elegans homolog of MAD3) deletion alleles confer hypersensitivity to ionizing radiation, consistent with the notion that the spindle assembly checkpoint pathway is required for the DNA damage response. bub-3(gt2000) is moderately sensitive to the cross-linking drug cisplatin but not to ultraviolet light or methyl methanesulfonate. This is consistent with a role in dealing with DNA double-strand breaks and not with base damage. Double mutant analysis revealed that bub-3 does not act within any of the three major pathways involved in the repair of double-strand breaks. Finally, the cdc-20 gain-of-function mutant cdc-20/fzy-1(av15), which is refractory to the cell cycle delay conferred by the spindle checkpoint, showed phenotypes similar to bub-3 and san-1 mutants. We speculate that BUB-3 is involved in the DNA damage response through regulation of cell cycle timing.

Connections between genetics and clinical data: Role of MCP-1, CFTR, and SPINK-1 in the setting of acute, acute recurrent, and chronic pancreatitis.

OBJECTIVES: Acute, acute recurrent, and chronic pancreatitis are inflammatory diseases with multifactorial pathogenic mechanisms. Genetic mutations and polymorphisms have been correlated with pancreatitis. The aim of this study was to investigate the association of cystic fibrosis transmembrane conductance regulator (CFTR) and serine protease inhibitor Kazal type 1 (SPINK-1) gene mutations and monocyte chemoattractant protein 1 (MCP-1) -2518A/G polymorphism with acute pancreatitis (AP), acute recurrent pancreatitis (ARP), and chronic pancreatitis (CP), and to associate genetic backgrounds with clinical phenotype in these three conditions. METHODS: One hundred eighteen AP, 64 ARP, 142 CP patients, and 88 normal controls were enrolled consecutively. We analyzed MCP-1 serum levels using enzyme-linked immunosorbent assay. Polymorphism -2518 of MCP-1 and SPINK-1 N34S gene mutations were determined by PCR-restriction-fragment length polymorphism. Sequence analysis was performed when necessary. Thirty-three CFTR mutations were analyzed in CP and ARP patients using multiplex DNA testing. RESULTS: Serum MCP-1 levels were significantly higher in all patients affected by pancreatic inflammatory diseases. Moreover, we found a significant over-representation of the MCP-1G allele in ARP patients. We found a statistically significant association of CFTR gene mutations with ARP, but not with CP. We did not find a statistically significant association of ARP or CP with the N34S SPINK-1 gene mutation. Interestingly, 39 of 64 ARP patients (61%) carried at least one genetic mutation and/or polymorphism. Five of 64 ARP patients had pancreas divisum and four of these five also carried the G allele. CONCLUSIONS: Analysis of a comprehensive range of potential susceptibility variants is needed to support modeling of the effects of genes and environment in pancreatitis. As such, beyond gene mutations, the context within which those mutations exist must be considered. In pancreatitis the context includes the inflammatory response, clinical features, and exogenous factors.

Usefulness of serum pepsinogens in Helicobacter pylori chronic gastritis: relationship with inflammation, activity, and density of the bacterium.

We sought to study the relationship between serum pepsinogens and different histopathologic features of Helicobacter pylori-related chronic gastritis. One hundred forty-nine consecutive dyspeptic patients underwent endoscopy with biopsies; serum pepsinogens I and II were measured by immunoassay. Serum levels of pepsinogens (sPG) were significantly correlated with H. pylori density both of the corpus (sPGI: r = 0.32, P < .001; sPGII: r = 0.56, P < .001) and antrum (sPGI: r = 0.41, P < .001; sPGII: r = 0.43, P < .001) as well as with chronic inflammation (sPGI: r = 0.26, P < .001; sPGII: r = 0.49, P < .001) and activity (sPGI: r = 0.38, P < .001; sPGII: r = 0.50, P < .001) in the antrum. Only sPGII was correlated with chronic inflammation (r = 0.44, P < .001) and activity (r = 0.40, P < .001) in the corpus. SPGI was inversely correlated with atrophy (r = -0.33, P < .001) and intestinal metaplasia (r = -0.37, P < .001) in the corpus. sPGII levels could be considered as markers of gastric inflammation all over in the stomach. sPGI levels are inversely related to atrophic body gastritis.

Influence of antisecretory treatment with proton pump inhibitors on serum pepsinogen I levels.

It has been reported in literature that serum pepsinogen levels rise during omeprazole and lansoprazole administration. However, the influence of pantoprazole and esomeprazole on serum pepsinogens levels is still to be assessed. The aim of this study was to evaluate the influence of proton pump inhibitor (PPI) therapy on pepsinogen I (PGI) levels. PGI and gastrin (G17) levels (EIA; Biohit, Helsinki, Finland) in 126 consecutive patients (M 57; F 69, mean age 53, range 15-91), with upper gastrointestinal symptoms at baseline condition and after 2 months of PPI treatment, were evaluated. Patients underwent a therapy schedule based on: omeprazole 20 mg b.i.d. (20 patients), pantoprazole 40 mg b.i.d. (27 patients), esomeprazole 40 mg b.i.d. (29 patients), lansoprazole 30 mg b.i.d. (21 patients) and rabeprazole 20 mg b.i.d. (26 patients) for 2 months. A significant increase in serum PGI (sPGI) levels was found after a 2-month treatment for all five different PPIs: omeprazole, pantoprazole, esomeprazole, lansoprazole and rabeprazole (P < 0.05). The effect of rabeprazole on sPGI was less pronounced as compared with other PPIs, whereas esomeprazole achieved superior sPGI levels, with no overall statistically significant difference among the five groups (P > 0.05). However, a comparison within a single group of PPIs showed a statistical significance when the esomeprazole group was compared with the rabeprazole group (P = 0.007). sPGI levels are significantly influenced by antisecretory therapy, rising under PPI treatment. Moreover, a statistically significant difference in sPGI levels between the rabeprazole and esomeprazole groups has been demonstrated.

Clinical usefulness of serum pepsinogen II in the management of Helicobacter pylori infection.

BACKGROUND: Serum pepsinogen II (sPGII) levels are known to increase during Helicobacter pylori infection. AIM: To assess H. pylori infection and success of H. pylori therapy by means of sPGII levels. METHODS: sPGII levels were determined in 156 H. pylori-positive and 157 H. pylori-negative consecutive patients with dyspeptic symptoms. Additionally, sPGII determination was performed in 70 H. pylori-positive patients 2 months after H. pylori eradication therapy. In 29 of these 70 patients, gastroscopy was performed to evaluate the effect of H. pylori therapy on gastric activity. RESULTS: H. pylori-positive subjects demonstrated a significantly higher mean of sPGII levels than H. pylori-negative subjects (16.8 +/- 7.4 vs. 8.6 +/- 3.7 microg/l; p < 0.001). The best sPGII cut-off for predicting H. pylori infection was 9.93 microg/l (sensitivity 83%, specificity 73%). The best cut-off values to evaluate success of therapy were: sPGII of 9.47 microg/l, a sPGII variation level (difference between baseline and after therapy) of 4.54 microg/l, and a sPGII Deltavalue (sPGII variation divided by sPGII before therapy) of 25% (sensitivity 93%, specificity 91%). CONCLUSIONS: sPGII levels may be used as a reliable marker of H. pylori infection in the initial diagnosis as well as to evaluate H. pylori eradication and subsequent changes in gastric inflammation.