Replication Errors Research Articles

Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes

Multi-nucleotide variants (MNVs), defined as two or more nearby variants existing on the same haplotype in an individual, are a clinically and biologically important class of genetic variation. However, existing tools typically do not accurately classify MNVs, and understanding of their mutational origins remains limited. Here, we systematically survey MNVs in 125,748 whole exomes and 15,708 whole genomes from the Genome Aggregation Database (gnomAD). We identify 1,792,248 MNVs across the genome with constituent variants falling within 2 bp distance of one another, including 18,756 variants with a novel combined effect on protein sequence. Finally, we estimate the relative impact of known mutational mechanisms - CpG deamination, replication error by polymerase zeta, and polymerase slippage at repeat junctions - on the generation of MNVs. Our results demonstrate the value of haplotype-aware variant annotation, and refine our understanding of genome-wide mutational mechanisms of MNVs.

Environmental Effects on Guanine-Thymine Mispair Tautomerization Explored with Quantum Mechanical/Molecular Mechanical Free Energy Simulations.

DNA bases can adopt energetically unfavorable tautomeric forms that enable the formation of Watson-Crick-like (WC-like) mispairs, which have been proposed to give rise to spontaneous mutations in DNA and misincorporation errors in DNA replication and translation. Previous NMR and computational studies have indicated that the population of WC-like guanine-thymine (G-T) mispairs depends on the environment, such as the local nucleic acid sequence and solvation. To investigate these environmental effects, herein G-T mispair tautomerization processes are studied computationally in aqueous solution, in A-form and B-form DNA duplexes, and within the active site of a DNA polymerase λ variant. The wobble G-T (wG-T), WC-like G-T*, and WC-like G*-T forms are considered, where * indicates the enol tautomer of the base. The minimum free energy paths for the tautomerization from the wG-T to the WC-like G-T* and from the WC-like G-T* to the WC-like G*-T are computed with mixed quantum mechanical/molecular mechanical (QM/MM) free energy simulations. The reaction free energies and free energy barriers are found to be significantly influenced by the environment. The wG-T→G-T* tautomerization is predicted to be endoergic in aqueous solution and the DNA duplexes but slightly exoergic in the polymerase, with Arg517 and Asn513 providing electrostatic stabilization of G-T*. The G-T*→G*-T tautomerization is also predicted to be slightly more thermodynamically favorable in the polymerase relative to these DNA duplexes. These simulations are consistent with an experimentally driven kinetic misincorporation model suggesting that G-T mispair tautomerization occurs in the ajar polymerase conformation or concertedly with the transition from the ajar to the closed polymerase conformation. Furthermore, the order of the associated two proton transfer reactions is predicted to be different in the polymerase than in aqueous solution and the DNA duplexes. These studies highlight the impact of the environment on the thermodynamics, kinetics, and fundamental mechanisms of G-T mispair tautomerization, which plays a role in a wide range of biochemically important processes.

High polymerase ε expression associated with increased CD8+T cells improves survival in patients with non-small cell lung cancer.

DNA replicase polymerase ε (POLE) is critical in proofreading and correcting errors of DNA replication. Low POLE expression plays a pivotal role in accumulation of mutations and onset of cancer, contributing to development and growth of tumor cells. The aim of this study is to reveal the survival, alternative genes and antitumoral immune activities in non-small cell lung cancer (NSCLC) patients with low POLE expression and provide treatment strategies that can increase their survival rates. This study investigated the clinicopathologic parameters, various tumor-infiltrating lymphocytes (TILs), endogenous retrovirus, molecular interactions and in vitro drug screen according to POLE mutation/expression in 168 and 1,019 NSCLC patients from the Konkuk University Medical Center (KUMC) and the Cancer Genome Atlas, respectively. We identified mutations of 75 genes in the sequencing panels, with POLE frame shift p.V1446fs being the most frequent (56.8%) in KUMC based on 170 targeted sequencing panels. Mutant and high expression of POLE correlated with favorable prognosis with increased TILs and tumor mutation burden, compared with wild type and low expression of POLE. We found specific molecular interactions associated with cell cycle and antigen presentation. An in vitro drug screen identified dasatinib that inhibited growth of the NSCLC cell line with low POLE expression. POLE could contribute to the future development of anticancer drugs for patients with NSCLC.

Correlation of pathogenic POLE mutations with clinical benefit to immune checkpoint inhibitor therapy.

3008 Background: Mutations in DNA polymerase epsilon ( POLE) may induce DNA replication errors, increasing neoantigen load and potentially enhancing clinical benefit to immune checkpoint inhibitors (ICI). We present a clinicopathologic analysis of patients (pts) with advanced cancers harboring POLE mutations and their response to ICI therapy at MD Anderson Cancer Center. Methods: We used targeted exome sequencing via CLIA-certified next generation sequencing assays to identify pts with POLE-aberrant tumors and their co-occurring mutations. The pathogenicity of each POLE mutation was annotated utilizing InterVar and ClinVar databases. Chi-square analysis was performed. Results: Tumors from 12,947 pts were analyzed and 448 (3.5%) pts had a mutation or copy number variation in POLE (3.5%), comparable to the TCGA PanCancer Atlas (4.0%). Clinical data were available for 293 pts; the most common cancers were colorectal (14.7%), non-small cell lung (13.7%), cholangiocarcinoma (13.3%) and melanoma (10.2%). There were 267 unique co-mutations, including KRAS (23.0%), ARID1A (21.5%), BRCA2 (18.7%), ATM (18.4%), CDKN2A (17.5%), BRAF (15.3%), EGFR (15.3%), ATRX (12.6%), CREBBP (11.7%), APC (11.3%), ATR (11.0%), BRCA1 (11.0%) and CDK12 (10.4%). POLE variants were annotated in all pts: pathogenic/likely pathogenic (n = 34, 11.6%), benign/likely benign (61, 20.8%), and variant of unknown significance (198, 67.6%). 104 (35.8%) of 293 pts with POLE mutations received PD-1/L1 inhibitors as monotherapy or in combination. 93 (88.4%) of 104 pts were evaluable for response: Radiological CR 4.3% (n = 4), PR 26.9% (n = 25), SD 22.6% (n = 21), PD 46.2% (n = 43), for a clinical benefit rate (CR + PR + SD) of 53.8%. Pathogenic status of POLE mutation was associated with clinical benefit to PD-1/L1 inhibitors (p = 0.04). TMB (p = 0.44), PD-L1 (p = 0.11), and MSI (p = 0.66) status were not associated with pathogenic status. MSI-H status was not over-represented in pts with ICI clinical benefit (p = 0.36). Conclusions: Pathogenic POLE mutations were associated with clinical benefit to ICI therapy. Further studies are warranted to validate POLE mutations as a predictive biomarker. Multiple co-occurring DNA damage response mutations were found, which may contribute to ICI clinical benefit.

MMR deficiency of lung cancer patients.

e21523 Background: DNA Mismatch repair (MMR) plays an essential role in the genetic. The main function of MMR is thought to correct post replication errors since the system could recognizes base exchanges and short DNA insertions or deletions. Somatic mutation of MMR are common detected in lung cancer, and several studies has shown that the presence of MMR mutation may be resistance to EGFR-TKI. Here we retrospectively analyzed the MMR mutation in chinese lung cancer. Methods: We retrospectively analyzed the genetic aberration in 910 Chinese lung patients. All the patients were detected by hybridization capture-based NGS 1021-gene panel. Results: Here is an interesting lung cancer case which was called EGFR T790M, TP53 and MSH2 somatic mutations without any germline mutations, and this patient showed no response to Osimertinib. We retrospectively analyzed the MMR in 910 patients and called 39 patients with MMR mutation.The percence of MLH1, MLH2, MSH3, MSH6, PMS1 and PMS2 mutations rate is as followed: 10.3% (4/39), 33.3% (13/39), 7.7% (3/39), 28.2% (11/39), 5.1% (2/39), and 15.4% (6/39) cases in PMS2 mutation. Also we found that TP53 is the most highly concurrent gene for MRR deficiency the rate is 79.5%(31/39). The rate of EGFR mutation concurrent with MMR deficiency is 27% (10/39). Conclusions: MMR deficiency was found in 4.3% chinese lung cancer and 27% of them concurrent with EGFR. [Table: see text] [Table: see text] [Table: see text]

Genetic Polymorphism of Mismatch Repair Genes and Susceptibility to Prostate Cancer.

Mismatch repair (MMR) is one of the DNA repair systems that correct mispaired bases during DNA replication errors. Polymorphisms in genes can increase susceptibility to the development of prostate cancer (PCa). In this study, we investigated mutL homolog 1 (MLH1) -93G>A (rs1800734) and mutS homolog 3 (MSH3) (rs26279) polymorphisms with the risk of PCa. In this study of Iranian population, 175 histopathologically confirmed (PCa) patients and 230 benign prostate hyperplasia (BPH) as the controls were recruited. The genotypes of MLH1 and MSH3 were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. There was no significant difference of MLH1 (P = 0.4) and MSH3 (P?=?0.5) genotype distributions among PCa cases and controls. And also patients with PCa were not significant differences compared to those without in stage of cancer, grade of tumor, perineural invasion, and vascular invasion. Our results did not show adequate evidence for any significant association of MLH1 and MSH3 polymorphisms and PCa .

Established and Novel Mechanisms Leading to de novo Genomic Rearrangements in the Human Germline

During gametogenesis, the human genome can acquire various de novo rearrangements. Most constitutional genomic rearrangements are created through 1 of the 4 well-known mechanisms, i.e., nonallelic homologous recombination, erroneous repair after double-strand DNA breaks, replication errors, and retrotransposition. However, recent studies have identified 2 types of extremely complex rearrangements that cannot be simply explained by these mechanisms. The first type consists of chaotic structural changes in 1 or a few chromosomes that result from “chromoanagenesis (an umbrella term that covers chromothripsis, chromoanasynthesis, and chromoplexy).” The other type is large independent rearrangements in multiple chromosomes indicative of “transient multifocal genomic crisis.” Germline chromoanagenesis (chromothripsis) likely occurs predominantly during spermatogenesis or postzygotic embryogenesis, while multifocal genomic crisis appears to be limited to a specific time window during oogenesis and early embryogenesis or during spermatogenesis. This review article introduces the current understanding of the molecular basis of de novo rearrangements in the germline.

Regulation of ultraviolet radiation-induced cutaneous DNA damage by type I interferons

Abstract Type I interferons (IFNs) are cytokines, that are important regulators of immune responses and are downregulated in human cancers, including skin cancer. Solar ultraviolet (UV) radiation is a proven environmental carcinogen, and exposure to solar radiation contributes to the high prevalence of skin cancer. The carcinogenic effects of UV light can be attributed to the formation of cyclobutane pyrimidine dimers (CPD) and errors in repair and replication of DNA. It is believed that type I IFNs reduce cellular proliferation and allow DNA repair in various diseases. Interferon receptor 1 (IFNAR1) is critical for signaling of type I IFNs. We hypothesized that type I IFNs (IFNα/β) will repair UVB induced DNA damage in mice. To test our hypothesis, shaved mice lacking IFNAR1 and wild type mice on a C57BL/6 background were irradiated with single dose of UVB radiation (100 mJ/cm2). Skin samples were examined for CPD by immunofluorescence and ELISA. We found that mice lacking IFNAR1 had more CPD as evident by ELISA and histology. In order to determine the cell type, which produces type I IFNs in skin after UVB exposure, single cell suspensions were prepared from irradiated skin samples and stained for type I IFNs using flow cytometry. We found that type I IFNs were produced by inflammatory monocytes (CD45+CD11b+Ly6C+). We also found that DNA repair gene XPA was downregulated in the mice lacking IFNAR1. In conclusion, our studies show that mice lacking the IFNAR1 had decreased repair of UVB induced CPD in the skin. Our ongoing studies will elucidate whether type I IFNs regulate development of UVB induced skin tumors.

How defective spermatogenesis affects sperm DNA integrity.

Spermatogenesis is the essential process to maintain and promote male fertility. It is extraordinarily complex with many regulatory elements and numerous steps. The process involves several cell types, regulatory molecules, repair mechanisms and epigenetic regulators. Evidence has shown that fertility can be negatively impacted by reduced sperm DNA integrity. Sources of sperm DNA damage include replication errors and causes of DNA fragmentation which include abortive apoptosis, defective maturation and oxidative stress. This review outlines the process of spermatogenesis, spermatogonial regulation and sperm differentiation; additionally, DNA damage and currently studied DNA repair mechanisms in spermatozoon are also covered.

Impact of DNA End Structure and Cellular Growth Phase on DNA Repair by Nonhomologous End‐Joining (NHEJ) in Saccharomyces cerevisiae

DNA within the cell is constantly bombarded by endogenous sources of damage (replication errors, cellular metabolism) and exogenous sources (radiation, chemical exposure), resulting in the formation of more than 100,000 DNA lesions per cell daily. There are several potential consequences of this damage, including stimulation of a cell stress response, changes in gene transcription, induction of controlled cell death (apoptosis), and the activation of DNA repair pathways. Mutations causing impaired DNA repair are associated with multiple human genetic disorders, which frequently lead to an increased cancer risk. A double‐strand break (DSB) is the most deleterious type of DNA damage, as it promotes mutations, chromosomal rearrangements, and other genomic changes that can lead to cell death. The two primary pathways that repair DSBs are Homologous Recombination (HR) and Nonhomologous End‐joining (NHEJ). This project is focused on the NHEJ pathway in Saccharomyces cerevisiae, which is mediated by three main protein complexes: Yku70‐Yku80 (Yku), Mre11‐Rad50‐Xrs2 (Mrx), and Dnl4‐Lif1‐Nej1 (Dnl). Our experiments use plasmid NHEJ assays to measure the efficiency and accuracy of DSB repair in mutant strains of yeast deficient in DNA repair. This is done by transforming plasmids containing a DSB in one of two selectable markers into cells and utilizing the NHEJ pathway to repair the plasmid. The results are then quantified by measuring cellular growth on selective media to determine the degree of reduced repair efficiency. Preliminary results indicated that the Mrx DNA end‐bridging complex is situationally required for efficient NHEJ, while the Yku and Dnl complexes are always required. The requirement for Mrx was dependent on the cell growth phase, as it was lower in cultures with high levels of G1 cells. Mrx was always required for efficient repair of ends with 3′ single‐stranded overhangs, but not for DSBs with 5′ single‐stranded overhangs. Our hypothesis is that Mrx may play a protector role at DSB ends containing 3′ overhangs, shielding them from nucleases such as Exo1 or Sgs1‐Dna2 that may preferentially target this end structure. In addition, using an improved version of the plasmid assays, we have identified three new S. cerevisiae genes that affect NHEJ repair efficiency. Current experiments are investigating the role of chromatin remodeling proteins Arp5, Arp6, and Arp8, along with DNA end‐processing proteins, to assess their role in NHEJ repair.

Exo1‐independent MMR at euchromatin is error‐prone and involves Pol ζ and Rev1

Genomes are under a constant attack by exogenous and endogenous agents that cause various forms of DNA damage. If the DNA damage is left unrepaired, it leads to a mutation. The mutation might alter the cell’s phenotype and trigger a disease. DNA repair processes and the high‐fidelity DNA replication play an important role in maintaining genome integrity. DNA mismatch repair (MMR) is a key DNA repair process that corrects DNA replication errors that have escaped proofreading. MMR can occur in an error‐free and error‐prone manner. Both error‐free and error‐prone MMR mechanisms have a strong impact on human health. Previous research has established that the exonuclease Exo1 is a non‐essential MMR factor that participates in MMR by removing the DNA mismatch in a 5′→3′ hydrolytic reaction. We have found that the Exo1‐independent MMR at euchromatin is error prone and occurs in a DNA polymerase (Pol) ζ‐ and DNA polymerase Rev1‐dependent manner. Surprisingly, the Pol ζ‐ and Rev1‐dependent error‐prone MMR does not involve PCNA K164 residue and ubiquitin ligase Siz1, factors that cooperate with Pol ζ in other DNA transactions. Our results have suggested that a novel error prone MMR mechanism is activated in response to the loss of a non‐essential MMR factor.Support or Funding InformationNational Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM095758 Spontaneous CAN1 mutation rates in euchromatic CAN1 gene Genotype CAN1 mutation rate Absolute rate (×10−8) 95% CI (×10−8) Relative rate Wild‐type 19 16–24 1 exo1Δ 120n,q 100–190 6 pol30‐K164R 24 18–31 1 msh2Δ 600p 450–800 32 pol30‐K164R msh2Δ 550p 400–650 29 pol30‐K164R exo1Δ 120q 100–190 6 siz1Δ 29 17–42 1.5 exo1Δ siz1Δ 120n 73–160 6 The mutant strains are isogenic to E134 (wild type). CI represents 95% confidence interval. The mutation rates marked n,p,q do not statistically differ from each other. p=0.5, 0.06, 0.1 respectively.

Exploring the Interaction of Msh2‐Msh6 with the Holliday Junction Through ATPase Activity

DNA mismatch repair systems (MMR) are important in reducing post replicative errors and preventing recombination between homologous sequences. Mismatch repair is highly conserved in most organisms and is initiated by the MutS homodimer in prokaryotes. In eukaryotes, small insertion/deletion loops (IDL), and single base pair mismatches are recognized by the MutS homolog Msh2‐Msh6. Prior research in our lab and by others (Marsischky G. T., Lee S., Griffith J., and Kolodner R. D. (1999) J.Biol.Chem. 274: 7200–7206 doi:10.1074/jbc.274.11.7200) determined the binding affinity of the junction with Msh2‐Msh6 with a Kd = 12.9 ± 1.3 nM, which is similar to the binding affinity for a G:T duplex, suggesting that the Msh2‐Msh6 Holliday Junction interaction is specific. Our investigation aims to understand the Msh2‐Msh6‐junction binding interaction by studying nucleotide utilization upon binding and determining any conformational changes associated with complex formation and ATP hydrolysis. We use the well‐characterized J3 Holliday junction with 17 base pair arms to characterize binding. Malachite green assays measuring inorganic phosphate were performed to obtain the rate of ATP hydrolysis in the presence of Msh2‐Msh6, J3 junction, and other DNA substrates. Msh2‐Msh6 alone has a Kcat = 0.27s−1 in the presence of 100mM NaCl, with the Kcat weakly increasing with a G:T mismatch 34‐mer duplex and the J3 junction to 0.47s−1and 0.6s−1 respectively. We will also report on junction conformational changes associated with protein binding and how protein affinity for the junction is modulated by nucleotide binding.Support or Funding InformationASBMB Undergraduate Research Grant.

Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide.

DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our understanding of chemical mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered DNA polymerases offers a potential basis for site-specific detection of DNA adducts, but the availability of effective artificial nucleotides that insert opposite DNA adducts is extremely limited, and furthermore, there has been no report of a quantitative strategy for determining how much DNA alkylation occurs in a sequence of interest. In this work, we synthesized an artificial nucleotide triphosphate that is selectively inserted opposite O6-carboxymethyl-guanine DNA by an engineered polymerase and is required for DNA synthesis past the adduct. We characterized the mechanism of this enzymatic process and demonstrated that the artificial nucleotide is a marker for the presence and location in the genome of O6-carboxymethyl-guanine. Finally, we established a mass spectrometric method for quantifying the incorporated artificial nucleotide and obtained a linear relationship with the amount of O6-carboxymethyl-guanine in the target sequence. In this work, we present a strategy to identify, locate, and quantify a mutagenic DNA adduct, advancing tools for linking DNA alkylation to mutagenesis and for detecting DNA adducts in genes as potential diagnostic biomarkers for cancer prevention.

Efficiency of alpha lattice design in crop evaluation trials

In large field experiments, the precision of randomized complete block (RCB) design is reduced because of theheterogeneity in field condition. Alpha lattice design (ALD) provides better control on experimental variability among the experimental units under field conditions. Each replication is divided into incomplete blocks and that blocks with in replication error will be eliminated from total error, so that the precision will be increased. The data from an experiment conducted in ALD with 22 entries, three replications and each replication divided in to two blocks was used for this study. The results indicated that RCBD should be replaced by alpha lattice when more than 10 treatments were compared.

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Health Status as Result of Analyses of Parameters of Hydrogen Bonds Among Water Molecules in Human Body. Entropy in Living Organisms

Mankind has always been excited about longevity. Biophysical studies indicate that the water in the human body “has departed” from the water at the time of origination of life (Ignatov, 2010; Mosin, Ignatov, 2011). The more rapid the life processes are, the faster the states of orderliness are observed, i.e. entropy in the living organism decreases. Yet this leads to difficulties in compensating entropy with the entropy of the environment, which is associated with metabolism and energy. Such organisms, like mammals, can live up to 100 years. In trees the processes are slower, the states of orderliness are harder to obtain, and entropy decreases more slowly than in animals.There are trees that live for more than 1000 years. For example, a turtle’s life processes are slower than those of mammals and faster than those of trees. It can live up to 300 years. We can define a specific time for each living creature, and their time to a certain degree differs from the time of the environment. This time correlates with parameters of the vital activity of living organisms.Aging is associated with accumulation of errors in DNA replication. The free radicals have an adverse affect on this process. Aging is a non-linear process. The rate of aging increases with time.This paper shows that mountain and melt water is among the most important factors for health and longevity. Other factors are – hereditary, gender, body weight, food, movement, psychological status, family relationships. Keywords : entropy, longevity, spectral analyses, NES, DNES. DOI: 10.7176/JMPB/64-03 Publication date: February 29 th 2020

Proofreading of single nucleotide insertion/deletion replication errors analyzed by MALDI-TOF mass spectrometry assay

Small nucleotide insertion/deletion (indel) errors are one of the common replication errors in DNA synthesis. The most frequent occurrence of indel error was thought to be due to repeated sequences being prone to slippage during DNA replication. Proofreading and DNA mismatch repair are important factors in indel error correction to maintain the high fidelity of genetic information transactions. We employed a MALDI-TOF mass spectrometry (MS) analysis to measure the efficiency of Klenow polymerase (KF) proofreading of indel errors. Herein, a non-labeled and non-radio-isotopic oligonucleotide primer is annealed to a template DNA forming a single nucleotide indel error and was proofread by KF in the presence of a combination of different deoxyribonucleotide triphosphates and/or dideoxyribonucleotide triphosphates. The proofreading products were identified by the KF modified mass change of the primer. We examined proofreading of DNAs containing indel errors at various positions of the primer-template junction. We found that indel errors located 1–5-nucleotides (nt) from the primer terminus can be proofread efficiently, while insertion/deletions at 6-nt from the 3’ end are partially corrected and extended. Indels located 7–9-nt from the primer terminus escape proofreading and are elongated by polymerase. The possible underlying mechanisms of these observations are discussed in the context of the polymerase and primer-template junction interactions via a structure analysis.

Repeated Richardson extrapolation and static hedging of barrier options under the CEV model

AbstractThis paper proposes an accelerated static replication approach for continuous European‐style barrier options by employing the repeated Richardson extrapolation technique with the Romberg sequence. This approach is developed under the constant elasticity of variance (CEV) model of Cox (1975) and Cox and Ross (1976) using the framework offered by Derman, Ergener, and Kani (1995; DEK) and its modified method of Chung et al. (2010, 2013a, 2013b) and Tsai (2014). The numerical results indicate that our method could significantly reduce replication errors for European knock‐out call options and may be superior to the imposition of the theta‐matching condition on the DEK method.

Características histopatológicas del carcinoma colorrectal con inestabilidad microsatelital (IMS)

El carcinoma colorrectal es una de las neoplasias con mayor incidencia a nivel mundial. Algunos de los tipos de cáncer colorrectal tienen componente hereditario y asociación con defectos en la reparación génica a nivel replicacional por mutaciones en los genes encargados (MLH1, MSH2, MSH6 y PMS2) llamándose esto la inestabilidad microsatélite. Esta situación se acompaña de mayor predisposición a desarrollar carcinoma colorrectal y carcinomas extracolónicos que incluyen cáncer de estómago, endometrio, ovarios, tracto urinario, entre otros. Es importante, entonces, evaluar la presencia de la inestabilidad microsatélite a nivel histopatológico conociendo que hay algunos hallazgos que hacen sospechar la presencia de estas alteraciones genéticas, tales como la presencia de linfocitos intraepiteliales, infiltrado inflamatorio Crohn-like, el subtipo histológico y la localización. El objetivo de este artículo es revisar en detalle algunas características de las variables mencionadas, así como de resaltar la importancia de otras variables histopatológicas con impacto pronóstico.

Autosomal-dominant adult neuronal ceroid lipofuscinosis caused by duplication in DNAJC5 initially missed by Sanger and whole-exome sequencing.

Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.