Background Mutation Frequency Research Articles

Determination of a positive response in the Ames Salmonella mutagenicity assay.

Genetic toxicology tests are used to categorize substances as genotoxic and potentially carcinogenic. In general, test results are designated as mutagenic, not mutagenic, or inconclusive and, depending on its potential use and applicable regulations, a mutagenic result can restrict or remove a substance from further development, or assign limits to its use. In these tests, mutation responses form a continuum without a clear delineation between an increase over the background, untreated, mutant frequency and a frequency that would define the test substance as a mutagen and a potential carcinogenic hazard. This situation is illustrated using the Salmonella mutagenicity (Ames) test which is the initial, and often only, test used to characterize substances as mutagenic or nonmutagenic. It has its widest use by industry and regulatory authorities to identify potential carcinogens among chemicals in development. The OECD Test Guideline No. 471 has been adopted by regulatory agencies internationally, and describes the minimum requirements for a negative response, but does not provide a specific approach for evaluating the test data. The most widely used criterion for making yes-or-no mutagenicity decisions is a 2- or 3-fold increase over the background (solvent) mutant frequency. Other approaches rely on formal statistics and/or expert judgment. These approaches and recently proposed modifications are evaluated here. Recommendations are made that are in conformity with the OECD guideline and are based on biological relevance and the biology of the mutagenic response rather than on arbitrary decision points (e.g., ≥2-fold increase or p ≤ .05).

Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring

BackgroundGene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring.ResultsMale gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30–39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10− 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10− 8/bp). It indicated that the gpt MFs in the ENU-treated father’s sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10− 8/bp and significantly higher than that of the control (2 × 10− 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group.ConclusionsDespite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

PacBio sequencing detects genome-wide ultra-low-frequency substitution mutations resulting from exposure to chemical mutagens.

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models-features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single-Molecule, Real-Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error-free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone-sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10-7 mutations per base pair (mut/bp).

Ultra-Accurate Assessment of Pretreatment ABL1 Kinase Domain (KD) Mutations in Patients (pts) with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Using Duplex Sequencing (DS)

Ultra-Accurate Assessment of Pretreatment ABL1 Kinase Domain (KD) Mutations in Patients (pts) with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Using Duplex Sequencing (DS)

Evaluation of U. S. National Toxicology Program (NTP) mouse lymphoma assay data using International Workshop on Genotoxicity Tests (IWGT) and the Organization for Economic Co-Operation and Development (OECD) criteria.

The forward gene mutation mouse lymphoma assay (MLA) is widely used, as part of a regulatory test battery, to identify the genotoxic potential of chemicals. It identifies mutagens capable of inducing a variety of genetic events. During the 1980s and early 1990s, the U.S. National Toxicology Program (NTP) developed a publicly available database (https://tools.niehs.nih.gov/cebs3/ui/) of MLA results. This database is used to define the mutagenic potential of chemicals, to develop structure-activity relationships (SAR), and to draw correlations to animal carcinogenicity findings. New criteria for MLA conduct and data interpretation were subsequently developed by the International Workshop for Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development (OECD). These recommendations are included in a new OECD Test Guideline (TG490). It is essential that early experimental data be re-examined and classified according to the current criteria to build a curated database to better inform chemical-specific evaluations and SAR models. We re-evaluated more than 1900 experiments representing 342 chemicals against the newly defined acceptance criteria for background mutant frequency (MF), cloning efficiency (CE), positive control values (modified for this evaluation due to lack of colony sizing), appropriate dose selection, and data consistency. Only 17% of the evaluated experiments met all acceptance criteria used in this re-evaluation. Results from 211 chemicals were determined to be uninterpretable, 92 were positive, and 39 equivocal. The authors could not classify any responses as negative because colony sizing was not performed for any of these experiments and it is clear, based on many experiment with unacceptably low background and positive control MFs, that mutant colony recovery was often suboptimal. This re-evaluation provides a curated database for the MLA. A similar curation should be done for other widely used genetic toxicology assays, but will be more difficult for certain assays (e.g., in vitro chromosomal aberrations) because important parameters such as level of cytotoxicity were often not evaluated/reported. Environ. Mol. Mutagen. 59:829-841, 2018. © 2018 Wiley Periodicals, Inc.

The development of an in vitro Pig-a assay in L5178Y cells.

A recent flow cytometry-based in vivo mutagenicity assay involves the hemizygous phosphatidylinositol class A (Pig-a) gene. Pig-a forms the catalytic subunit of N-acetylglucosaminyltransferase required for glycophosphatidylinositol (GPI) anchor biosynthesis. Mutations in Pig-a prevent GPI-anchor synthesis resulting in loss of cell-surface GPI-linked proteins. The aim of the current study was to develop and validate an in vitro Pig-a assay in L5178Y mouse lymphoma cells. Ethyl methanesulfonate (EMS)-treated cells (186.24-558.72µg/ml; 24h) were used for method development and antibodies against GPI-linked CD90.2 and stably expressed CD45 were used to determine GPI-status by flow cytometry. Antibody concentration and incubation times were optimised (0.18µg/ml, 30min, 4°C) and Zombie Violet™ (viability marker; 0.5%, 30min, RT) was included. The optimum phenotypic expression period was 8 days. The low background mutation frequency of GPI-deficiency [GPI(-)] in L5178Y cells (0.1%) constitutes a rare event, thus flow cytometry acquisition parameters were optimised; 104 cells were measured at medium flow rate to ensure a CV ≤ 30%. Spiking known numbers of GPI(-) cells into a wild-type population gave high correlation between measured and spiked numbers (R2 0.999). We applied the in vitro Pig-a assay to a selection of well-validated genotoxic and non-genotoxic compounds. EMS, N-ethyl-N-nitrosourea and 4-nitroquinoline-N-oxide dose dependently increased numbers of GPI(-) cells, while etoposide, mitomycin C, and a bacterial-specific mutagen did not. Cycloheximide and sodium chloride were negative. Sanger sequencing revealed Pig-a mutations in the GPI(-) clones. In conclusion, this in vitro Pig-a assay could complement the in vivo version, and follow up weak Ames positives and late-stage human metabolites or impurities.

Quantitative differentiation of whole smoke solution-induced mutagenicity in the mouse lymphoma assay.

In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD10 , BMD50 , BMD100 , and BMD200 , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10−8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

Mutant DNA Quantification by Digital PCR Can Be Confounded by Heating During DNA Fragmentation

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

Limited evidence that cancer susceptibility regions are preferential targets for somatic mutation.

BackgroundGenome wide-association studies have successfully identified several hundred independent loci harboring common cancer susceptibility alleles that are distinct from the more than 110 cancer predisposition genes. The latter are generally characterized by disruptive mutations in coding genes that have been established as ‘drivers’ of cancer in large somatic sequencing studies. We set out to determine whether, similarly, common cancer susceptibility loci map to genes that have altered frequencies of mutation.ResultsIn our analysis of the intervals defined by the cancer susceptibility markers, we observed that cancer susceptibility regions have gene mutation frequencies comparable to background mutation frequencies. Restricting analyses to genes that have been determined to be pleiotropic across cancer types, genes affected by expression quantitative trait loci, or functional genes indicates that most cancer susceptibility genes classified into these subgroups do not display mutation frequencies that deviate from those expected. We observed limited evidence that cancer susceptibility regions that harbor common alleles with small estimated effect sizes are preferential targets for altered somatic mutation frequencies.ConclusionsOur findings suggest a complex interplay between germline susceptibility and somatic mutation, underscoring the cumulative effect of common variants on redundant pathways as opposed to driver genes. Complex biological pathways and networks likely link these genetic features of carcinogenesis, particularly as they relate to distinct polygenic models for each cancer type.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3’ portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

Development and validation of a new transgenic hairless albino mouse as a mutational model for potential assessment of photocarcinogenicity

Short-term phototoxicity testing is useful in selecting test agents for the longer and more expensive photocarcinogenesis safety tests; however, no validated short-term tests have been proven reliable in predicting the outcome of a photocarcinogenesis safety test. A transgenic, hairless, albino (THA) mouse model was developed that carries the gpt and red/gam [Spi−] genes from the gpt delta mouse background and the phenotypes from the SKH-1 mouse background to use as a short-term test in lieu of photocarcinogenesis safety tests. Validation of the THA mouse model was confirmed by exposing groups of male mice to sub-erythemal doses of ultraviolet B (UVB) irradiation for three consecutive days emitted from calibrated overhead, Kodacel-filtered fluorescent lamps and measuring the mutant frequencies (MFs) in the gpt and red/gam (Spi−) genes and types of mutations in the gpt gene. The doses or irradiation were monitored with broad-spectrum dosimeters that were calibrated to a NIST-traceable standard and cumulative CIE-weighted doses were 20.55 and 41.0mJ/cm2 (effective). Mice were sacrificed 14 days after the final UVB exposure and MFs in both the gpt and red/gam genes were evaluated in the epidermis. The exposure of mice to UVB induced significant ten- to twelve-fold increases in the gpt MF and three- to five-fold increases in the Spi− MF over their respective background MF, 26±3×10−6 and 9±1×10−6. The gpt mutation spectra were significantly different between that of the UVB-irradiated and that of non-irradiated mice although the mutation spectra of both groups were dominated by C→T transitions (84% and 66%). In mice exposed to UVB, the C→T transitions occurred almost exclusively at dipyrimidine sites (92%), whereas in non-irradiated control mice, the C→T transitions occurred at CpG sites (86%). These results suggest that the newly developed THA mice are a useful and reliable model for testing UVB-induced mutagenicity in skin tissue. The application of this model for short-term prediction of solar-induced skin carcinogenicity is presently under investigation.

Abstract 5193: Novel candidate oncogenes with mutation hot spots in microsatellite unstable colorectal cancer

Abstract Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). MSI CRCs are prone to repeat mutations due to defective mismatch repair. The high background mutation frequency has discouraged systematic mutations screens in this tumor type. However, these tumors might form a sensitive system for generation and selection of oncogenic mutation hot spots. The aim of this study is to identify novel oncogenes with mutation hot spots that drive MSI CRC tumorigenesis. The exomes of 25 MSI tumors and respective healthy tissues were sequenced as the discovery set. The exome data was searched for mutation hot spots, with recurrent somatic missense mutations in at least two tumors. Potential mutation hot spots were observed in 43 genes and among these were the following known oncogenes: BRAF (V600E), CTNNB1 (T41A) and PIK3CA (H1047R). Novel potential mutation hot spots were identified in 40 genes and these were validated by Sanger sequencing. Mutation hot spots in 33 genes were confirmed and these were further screened in a validation set of 254 MSI CRCs. Fourteen genes displayed hotspot mutations also in the validation set with a total hot spot mutation frequency of 1.1-3.6 %. Many of the validated genes encode for known cancer-related proteins and for proteins with molecular and cellular functions relevant to cancer development and progression. Further work is needed to clarify the functional role of the identified hot spot mutations in MSI CRC tumorigenesis. The novel mutation hotspots may be utilized to develop personalized tumor profiling and therapy. Citation Format: Alexandra E. Gylfe, Sari Tuupanen, Ulrika Hänninen, Johanna Kondelin, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Minna Taipale, Jussi Taipale, Claus Lindbjerg Andersen, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Lauri A. Aaltonen. Novel candidate oncogenes with mutation hot spots in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2014-5193

Abstract 2401: Identification of new target genes in microsatellite unstable colorectal cancer by exome sequencing

Abstract Colorectal cancer (CRC) is the third most common cancer in Western countries. Approximately 15% of CRCs display microsatellite instability (MSI) caused by defective cellular mismatch repair. Most of the MSI CRCs are sporadic and result from biallelic inactivation of the MLH1 gene, which is most often due to hypermethylation of its promoter. Cells displaying MSI accumulate a high number of mutations throughout the genome, especially in short repeat areas called microsatellites. These mutations typically lead to a premature stop codon resulting in a truncated protein product that may inactivate the gene, a mechanism known typical of tumor suppressor genes. The aim of this study is to identify new target genes in MSI CRC. To date, several genes have been proposed as MSI target genes based on high mutation frequency in targeted searches of microsatellites. The availability of new sequencing and bioinformatic technologies has enabled genome-wide investigation of mutations in human cancers. This has led to identification of a vast amount of mutations in cancer, most of which are passenger mutations that do not confer selective growth advantage. The challenge is therefore to distinguish true driver genes from passengers. The high number of mutations in MSI tumors augments this challenge. In this study, 25 sporadic MSI CRCs and their corresponding normal samples have been exome sequenced to identify changes of somatic origin. An analysis and visualization program developed in our group, “RikuRator” (unpublished), will be utilized along with a script to estimate the accurate somatic mutation frequency of each coding mononucleotide repeat. A statistical model based on exome sequencing data will be developed that takes into account the background mutation frequency of microsatellites depending on the repeat length and nucleotide context. The most mutated repeats will be validated by Sanger sequencing in a set of additional MSI CRCs. Functional studies will be carried out to further investigate the pathogenic effect of the new target genes identified. The systematic screening of mutations in microsatellites will improve our understanding on the mutation profile typical of these tumors. With our approach that takes into account the background mutation frequency of microsatellites we aim to identify true driver genes in MSI CRC. A detailed understanding of the molecular background of this tumor type is important for the development of more efficient screening methods and personalized treatments to improve the prognosis of patients diagnosed with MSI type CRC. Citation Format: Johanna Kondelin, Esa Pitkänen, Alexandra E. Gylfe, Kimmo Palin, Heikki Ristolainen, Riku Katainen, Eevi Kaasinen, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Sari Tuupanen, Lauri A. Aaltonen. Identification of new target genes in microsatellite unstable colorectal cancer by exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2401. doi:10.1158/1538-7445.AM2014-2401

Discovery and saturation analysis of cancer genes across 21 tumour types

SummaryWhile a few cancer genes are mutated in a high proportion of tumors of a given type (>20%), most are mutated at intermediate frequencies (2–20%). To explore the feasibility of creating a comprehensive catalog of cancer genes, we analyzed somatic point mutations in exome sequence from 4,742 tumor-normal pairs across 21 cancer types. We found that large-scale genomic analysis can identify nearly all known cancer genes in these tumor types. Our analysis also identified 33 genes not previously known to be significantly mutated, including genes related to proliferation, apoptosis, genome stability, chromatin regulation, immune evasion, RNA processing and protein homeostasis. Down-sampling analysis indicates that larger sample sizes will reveal many more genes, mutated at clinically important frequencies. We estimate that near-saturation may be achieved with 600–5000 samples per tumor type, depending on background mutation rate. The results help guide the next stage of cancer genomics.

Action Spectrum Analysis of UVR Genotoxicity for Skin: The Border Wavelengths between UVA and UVB Can Bring Serious Mutation Loads to Skin

UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.

Adjusting for Background Mutation Frequency Biases Improves the Identification of Cancer Driver Genes

A common goal of tumor sequencing projects is finding genes whose mutations are selected for during tumor development. This is accomplished by choosing genes that have more non-synonymous mutations than expected from an estimated background mutation frequency. While this background frequency is unknown, it can be estimated using both the observed synonymous mutation frequency and the non-synonymous to synonymous mutation ratio. The synonymous mutation frequency can be determined across all genes or in a gene-specific manner. This choice introduces an interesting trade-off. A gene-specific frequency adjusts for an underlying mutation bias, but is difficult to estimate given missing synonymous mutation counts. Using a genome-wide synonymous frequency is more robust, but is less suited for adjusting biases. Studying four evaluation criteria for identifying genes with high non-synonymous mutation burden (reflecting preferential selection of expressed genes, genes with mutations in conserved bases, genes with many protein interactions, and genes that show loss of heterozygosity), we find that the gene-specific synonymous frequency is superior in the gene expression and protein interaction tests. In conclusion, the use of the gene-specific synonymous mutation frequency is well suited for assessing a gene's non-synonymous mutation burden.

Abstract 1789A: Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors.

Abstract Next-Generation DNA Sequencing now allows us to sequence billions of nucleotides in a single experiment and to detect germ-line mutations and driver mutations in human cancer. However, a major limitation is the error rate of approximately 1% to 0.1%. These scattered errors limit its utility in sequencing genetically heterogeneous mixtures such as human cancers or viruses. We have developed a method termed "Duplex Sequencing” that allows for the detection of rare mutations in DNA with unprecedented accuracy. This approach greatly lowers errors by independently sequencing each of the two strands of single DNA molecules. As the two strands are complementary, true mutations are scored only if they are present at the same position and are complementary. Moreover, small deletions and insertions can be easily identified. In contrast, sequencing errors, which occur from PCR - amplification of DNA containing damaged nucleotides, are present in only one strand and thus are not scored as mutations. To demonstrate the power of duplex sequencing we measured the background error frequency in human mitochondrial DNA obtained from cortical regions of the brain of aged normal humans; an overall mutation frequency was 3 X 10-5. These mutations are not random, but rather, show an inordinately large frequency of mutations in the D-loop of the mitochondrial genome. The spectrum of mutations is strikingly different from that reported in the literature using next-generation sequencing. Prior reports have indicated a high frequency of G->T; however, we find that these are likely technical artifacts arising from oxidative DNA damage. In contrast, by Duplex Sequencing, we find a predominance of G->A and T->C single base substitutions. The G->A and T->C mutations could result from cytidine deamination or errors during replication by the mitochondrial DNA polymerase gamma. In order to establish the background mutation frequency in nuclear DNA by Duplex Sequencing we first captured a small portion of the genome, the exons of the replicative DNA polymerases. Sequencing captured DNA from normal human tissues yielded a background mutation frequency of 10-6 to 1O-7 indicating that the error rate of duplex sequencing is less than one to ten mutations in ten million nucleotides. Current effort is to sequence nuclear DNA from different human cancers and the corresponding normal tissues. This result should indicate the distribution of subclonal mutations in human tumors. Subclonal mutations could delineate tumor cell lineage, account for the geographic heterogeneity of mutations in human tumors and serve as a biomarker for the presence of drug resistant cells prior to or during chemotherapy. Citation Format: Lawrence A. Loeb, Edward J. Fox, Scott R. Kennedy, Jessica Kuong, Michael W. Schmitt, Jesse J. Salk. Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1789A. doi:10.1158/1538-7445.AM2013-1789A

A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated–naphthoylated DEAE–cellulose, resulting in a low background mutation frequency (∼1×10−4). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.