Is Metal Stabilization of the Leaving Group Required or Can Lysine Facilitate Phosphodiester Bond Cleavage in Nucleic Acids? A Computational Study of EndoV.

Endonuclease V (EndoV) is a single-metal-dependent enzyme that repairs deaminated DNA nucleobases in cells by cleaving the phosphodiester bond, and this enzyme has proven to be a powerful tool in biotechnology and medicine. The catalytic mechanism used by EndoV must be understood to design new disease detection and therapeutic solutions and further exploit the enzyme in interdisciplinary applications. This study has used a mixed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) approach to compare eight distinct catalytic pathways and provides the first proposed mechanism for bacterial EndoV. The calculations demonstrate that mechanisms involving either direct or indirect metal coordination to the leaving group of the substrate previously proposed for other nucleases are unlikely for EndoV, regardless of the general base (histidine, aspartate, and substrate phosphate moiety). Instead, distinct catalytic pathways are characterized for EndoV that involve K139 stabilizing the leaving group, a metal-coordinated water stabilizing the transition structure, and either H214 or a substrate phosphate group activating the water nucleophile. In silico K139A and H214A mutational results support the newly proposed roles of these residues. Although this is a previously unseen combination of general base, general acid, and metal-binding architecture for a one-metal-dependent endonuclease, our proposed catalytic mechanisms are fully consistent with experimental kinetic, structural, and mutational data. In addition to substantiating a growing body of literature, suggesting that one metal is enough to catalyze P-O bond cleavage in nucleic acids, this new fundamental understanding of the catalytic function will promote the exploration of new and improved applications of EndoV.

Colibactin mutational signatures in NTHL1 tumor syndrome and MUTYH associated polyposis patients.

Polyketide synthase (pks) island harboring Escherichia coli are, under the right circumstances, able to produce the genotoxin colibactin. Colibactin is a risk factor for the development of colorectal cancer and associated with mutational signatures SBS88 and ID18. This study explores colibactin-associated mutational signatures in biallelic NTHL1 and MUTYH patients. Targeted Next Generation Sequencing (NGS) was performed on colorectal adenomas and carcinomas of one biallelic NTHL and 12 biallelic MUTYH patients. Additional fecal metagenomics and genome sequencing followed by mutational signature analysis was conducted for the NTHL1 patient. Targeted NGS of the NTHL1 patient showed somatic APC variants fitting SBS88 which was confirmed using WGS. Furthermore, fecal metagenomics revealed pks genes. Also, in 1 out of 11 MUTYH patient a somatic variant was detected fitting SBS88. This report shows that colibactin may influence development of colorectal neoplasms in predisposed patients.

NTHL1 is a recessive cancer susceptibility gene.

In search of novel breast cancer (BC) risk variants, we performed a whole-exome sequencing and variant analysis of 69 Finnish BC patients as well as analysed loss-of-function variants identified in DNA repair genes in the Finns from the Genome Aggregation Database. Additionally, we carried out a validation study of SERPINA3 c.918-1G>C, recently suggested for BC predisposition. We estimated the frequencies of 41 rare candidate variants in 38 genes by genotyping them in 2482-4101 BC patients and in 1273-3985 controls. We further evaluated all coding variants in the candidate genes in a dataset of 18,786 BC patients and 182,927 controls from FinnGen. None of the variants associated significantly with cancer risk in the primary BC series; however, in the FinnGen data, NTHL1 c.244C>T p.(Gln82Ter) associated with BC with a high risk for homozygous (OR = 44.7 [95% CI 6.90-290], P = 6.7 × 10-5) and a low risk for heterozygous women (OR = 1.39 [1.18-1.64], P = 7.8 × 10-5). Furthermore, the results suggested a high risk of colorectal, urinary tract, and basal-cell skin cancer for homozygous individuals, supporting NTHL1 as a recessive multi-tumour susceptibility gene. No significant association with BC risk was detected for SERPINA3 or any other evaluated gene.

NTHL1 Gene Mutations in Polish Polyposis Patients-Weighty Player or Vague Background?

Multiple polyposes are heterogeneous diseases with different underlying molecular backgrounds, sharing a common symptom: the presence of transforming into cancerous intestinal polyps. Recent reports have indicated biallelic mutations in the NTHL1 gene, which is involved in base excision repair (BER), as predisposing to an elevated risk of colorectal cancer (CRC). We aimed to evaluate the significance of the p.Q82* truncating variant in predisposition to intestinal polyposis by assessing its frequency in polyposis patients. We genotyped 644 Polish patients and 634 control DNA samples using high-resolution melting analysis (HRM) and Sanger sequencing. We found the p.Q82* variant in four polyposis patients; in three, it was homozygous (OR = 6.90, p value = 0.202). Moreover, the p.R92C mutation was detected in one patient. We also looked more closely at the disease course in patients carrying NTHL1 mutations. Two homozygous patients also presented other neoplasia. In the family case, we noticed the earlier presence of polyps in the proband and early hepatoblastoma in his brother. We cannot univocally confirm the relationship of p.Q82* with an increased risk of CRC. However, homozygous p.Q82* was more frequent by 10-fold in patients without other mutations identified, which makes NTHL1 gene screening in this group reasonable.

Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.

Six case reports of NTHL1-associated tumor syndrome further support it as a multi-tumor predisposition syndrome.

NTHL1-associated tumor syndrome (NATS) is an autosomal recessive condition characterized by an increased risk for colorectal polyposis and colorectal cancer (CRC). Only 46 case reports have been previously published. In a retrospective review, we analyzed the clinical histories of six patients found to have NATS after genetic counseling and testing. NATS appears to be associated with an increased risk for colorectal polyposis, CRC, female breast cancer, meningiomas, and endometrial cancer. Although research is limited, prior publications have reported a multi-tumor predisposition for individuals with biallelic pathogenic or likely pathogenic variants in NTHL1. Additional data are necessary to further define the cancer risks so affected individuals can be appropriately managed.

Bowhead NEIL1: molecular cloning, characterization, and enzymatic properties.

Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and removes bases, primarily oxidized pyrimidines, which have been damaged by endogenous oxidation or exogenous mutagenic agents. NEIL1 functions through a combined glycosylase/AP (apurinic/apyrimidinic)-lyase activity, whereby it cleaves the N-glycosylic bond between the DNA backbone and the damaged base via its glycosylase activity and hydrolysis of the DNA backbone through beta-delta elimination due to its AP-lyase activity. In our study we investigated our hypothesis proposing that the cancer resistance of the bowhead whale can be associated with a better DNA repair with NEIL1 being upregulated or more active. Here, we report the molecular cloning and characterization of three transcript variants of bowhead whale NEIL1 of which two were homologous to human transcripts. In addition, a novel NEIL1 transcript variant was found. A differential expression of NEIL mRNA was detected in bowhead eye, liver, kidney, and muscle. The A-to-I editing of NEIL1 mRNA was shown to be conserved in the bowhead and two adenosines in the 242Lys codon were subjected to editing. A mass spectroscopy analysis of liver and eye tissue failed to demonstrate the existence of a NEIL1 isoform originating from RNA editing. Recombinant bowhead and human NEIL1 were expressed in E. coli and assayed for enzymatic activity. Both bowhead and human recombinant NEIL1 catalyzed, with similar efficiency, the removal of a 5-hydroxyuracil lesion in a DNA bubble structure. Hence, these results do not support our hypothesis but do not refute the hypothesis either.

Concurrent Reduced Expression of Contiguous PKD1, TSC2 and NTHL1 Leading to Kidney Diseases and Multiple Diverse Renal Cancers.

BACKGROUND/AIM: Several cases of concurrent reduction of expression of polycystin 1 (PKD1) and Tuberous Sclerosis Complex 2 (TSC2) that are contiguous in chromosome 16p13 have been previously reported. This study newly addresses the concurrent reduction of expression of PKD1, TSC2 and NTHL1, which is adjacent to TSC2 and is a tumor suppressor gene. MATERIALS AND METHODS: We investigated the mRNA expression levels of PKD1, TSC2, PKD2, TSC1 and NTHL1 in blood and renal cell carcinoma (RCC) tissues in a proband with autosomal dominant polycystic kidney disease (ADPKD), tuberous sclerosis complex (TSC) and multiple pathologically diverse RCCs, including clear cell, papillary and chromophobe types. Additionally, we investigated germline variants in blood using whole exome sequencing (WES) in the proband and her four siblings. RESULTS: mRNA expression levels of PKD1, TSC2 and NTHL1 were reduced in the proband's blood and RCCs, compared with control groups. WES identified one novel variant with amino acid changes in the PKD1 exon in the three subjects with ADPKD, including the proband. Moreover, two variants in the TSC2 intron specific to the proband were also identified. CONCLUSION: In this study, we report a novel pathogenic variant in the PKD1 exon which likely led to ADPKD, and two variants in the TSC2 intron, which might have led to reduction in the expression of both TSC2 and NTHL1, consequently leading to TSC and multiple pathologically diverse RCCs.

Distinct Mechanisms of Target Search by Endonuclease VIII-like DNA Glycosylases.

Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA non-specifically and then slides along the DNA contour by one-dimensional diffusion. Opposite to the processive mechanism is distributive search, when an enzyme binds, samples and releases DNA without significant lateral movement. Many DNA glycosylases, the repair enzymes that excise damaged bases from DNA, use processive search to find their cognate lesions. Here, using a method based on correlated cleavage of multiply damaged oligonucleotide substrates we investigate the mechanism of lesion search by three structurally related DNA glycosylases-bacterial endonuclease VIII (Nei) and its mammalian homologs NEIL1 and NEIL2. Similarly to another homologous enzyme, bacterial formamidopyrimidine-DNA glycosylase, NEIL1 seems to use a processive mode to locate its targets. However, the processivity of Nei was notably lower, and NEIL2 exhibited almost fully distributive action on all types of substrates. Although one-dimensional diffusion is often regarded as a universal search mechanism, our results indicate that even proteins sharing a common fold may be quite different in the ways they locate their targets in DNA.

TRIM26 Maintains Cell Survival in Response to Oxidative Stress through Regulating DNA Glycosylase Stability.

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.

Incorporation of transition to transversion ratio and nonsense mutations, improves the estimation of the number of synonymous and non-synonymous sites in codons.

A common approach to estimate the strength and direction of selection acting on protein coding sequences is to calculate the dN/dS ratio. The method to calculate dN/dS has been widely used by many researchers and many critical reviews have been made on its application after the proposition by Nei and Gojobori in 1986. However, the method is still evolving considering the non-uniform substitution rates and pretermination codons. In our study of SNPs in 586 genes across 156 Escherichia coli strains, synonymous polymorphism in 2-fold degenerate codons were higher in comparison to that in 4-fold degenerate codons, which could be attributed to the difference between transition (Ti) and transversion (Tv) substitution rates where the average rate of a transition is four times more than that of a transversion in general. We considered both the Ti/Tv ratio, and nonsense mutation in pretermination codons, to improve estimates of synonymous (S) and non-synonymous (NS) sites. The accuracy of estimating dN/dS has been improved by considering the Ti/Tv ratio and nonsense substitutions in pretermination codons. We showed that applying the modified approach based on Ti/Tv ratio and pretermination codons results in higher values of dN/dS in 29 common genes of equal reading-frames between E. coli and Salmonella enterica. This study emphasizes the robustness of amino acid composition with varying codon degeneracy, as well as the pretermination codons when calculating dN/dS values.

Development of an Endonuclease V-Assisted Analytical Method for Sequencing Analysis of Deoxyinosine in DNA.

Deoxyinosine (dI) is a highly mutagenic lesion that preferentially pairs with deoxycytidine during replication, which may induce A to G transition and ultimately contribute to carcinogenesis. Therefore, finding the site of dI modification in DNA is of great value for both basic research and clinical applications. Herein, we developed a novel method to sequence the dI modification site in DNA, which utilizes endonuclease V (EndoV)-dependent deamination repair to specifically label the modification site with biotin-14-dATP that allows the affinity enrichment of dI-bearing DNA for sequencing. We have achieved efficient determination of the location of the modified nucleotide in dI-bearing plasmid DNA with the assistance of EndoV-dependent deamination repair. We have also successfully applied this approach to locate the dI modification sites in the mitochondrial DNA of human cells. Our method should be generally applicable for genome-wide sequencing analysis of dI modifications in living organisms.

Polymorphic variant Asp239Tyr of human DNA glycosylase NTHL1 is inactive for removal of a variety of oxidatively-induced DNA base lesions from genomic DNA.

Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant's role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant.

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry.

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix-two-turn-helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 - a large insert in its N-terminal domain absent from other DNA glycosylases - is unstructured in solution. It was suggested that helix-two-turn-helix DNA glycosylases undergo open-close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein-protein interactions.

Mycobacterium tuberculosis Endonuclease VIII 2 (Nei2) forms a prereplicative BER complex with DnaN: Identification, characterization, and disruption of complex formation.

Mycobacterium tuberculosis Nei2 (Rv3297) is a BER glycosylase that removes oxidized base lesions from ssDNA and replication fork-mimicking substrates. We show that Endonuclease VIII 2 (Nei2) forms a BER complex with the ß-clamp (DnaN, Rv0002) with a KD of 170 nM. The Nei2-ß-clamp interactions enhance Nei2's activities up to several folds. SEC analysis shows that one molecule of Nei2 binds to a single ß-clamp dimer. Nei2 interacts with subsites I and II of the ß-clamp via a noncanonical 223 QGCRRCGTLIAY239 Clamp Interacting Protein (CIP) motif in the C-terminal zinc-finger domain, which was previously shown by us to be dispensable for intrinsic Nei2 activity. The 12-mer peptide alone exhibited a KD of 10.28 nM, suggesting that the motif is a key mediator of Nei2-ß-clamp interactions. Finally, we identified inhibitors of Nei2-ß-clamp interactions using rational methods, in vitro disruption, and SPR assays after querying a database of natural products. We found that Tubulosine, Fumitremorgin C, Toyocamycin, and Aleuritic acid exhibit IC50 values of 94.47, 83.49, 109.7, and 71.49 µM, respectively. They act by disrupting Nei2-ß-clamp interactions and do not affect intrinsic Nei2 activity. Among other things, the present study gives insights into the role of Nei2 in bacterial prereplicative BER.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA.

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3' to inosine in single-strand RNA, at a low reaction temperature of 20-25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.

Prevalence and Characterization of Biallelic and Monoallelic NTHL1 and MSH3 Variant Carriers From a Pan-Cancer Patient Population.

NTHL1 and MSH3 have been implicated as autosomal recessive cancer predisposition genes. Although individuals with biallelic NTHL1 and MSH3 pathogenic variants (PVs) have increased cancer and polyposis risk, risks for monoallelic carriers are uncertain. We sought to assess the prevalence and characterize NTHL1 and MSH3 from a large pan-cancer patient population. MATERIALS AND METHODS: Patients with pan-cancer (n = 11,081) underwent matched tumor-normal sequencing with consent for germline analysis. Medical records and tumors were reviewed and analyzed. Prevalence of PVs was compared with reference controls (Genome Aggregation Database). RESULTS: NTHL1-PVs were identified in 40 patients including 39 monoallelic carriers (39/11,081 = 0.35%) and one with biallelic variants (1/11,081 = 0.009%) and a diagnosis of isolated early-onset breast cancer. NTHL1-associated mutational signature 30 was identified in the tumors of the biallelic patient and two carriers. Colonic polyposis was not identified in any NTHL1 patient. MSH3-PVs were identified in 13 patients, including 12 monoallelic carriers (12/11,081 = 0.11%) and one with biallelic MSH3 variants (1/11,081 = 0.009%) and diagnoses of later-onset cancers, attenuated polyposis, and abnormal MSH3-protein expression. Of the 12 MSH3 carriers, two had early-onset cancer diagnoses with tumor loss of heterozygosity of the wild-type MSH3 allele. Ancestry-specific burden tests demonstrated that NTHL1 and MSH3 prevalence was not significantly different in this pan-cancer population versus controls. CONCLUSION: NTHL1 and MSH3 germline alterations were not enriched in this pan-cancer patient population. However, tumor-specific findings, such as mutational signature 30 and loss of heterozygosity of the wild-type allele, suggest the potential contribution of monoallelic variants to tumorigenesis in a subset of patients.

Endonuclease V Regulates Atherosclerosis Through C-C Motif Chemokine Ligand 2-Mediated Monocyte Infiltration.

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine-to-inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine-to-inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine-ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E-deficient (ApoE-/-) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE-/- mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.