Population sequencing data reveal a compendium of mutational processes in the human germ line

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of LINE repeats. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors.

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. They are not thought to occur in the same cell at the same time. By destabilizing p300, β-HPV type 8 protein E6 (β-HPV8 E6) attenuates both repair pathways. However, β-HPV8 E6 delays rather than abrogates DSB repair. Thus, β-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that β-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an β-HPV8 E6 mutant demonstrate that these phenotypes are the result of β-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. β-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that β-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ.

Attribution of Cancer Origins to Endogenous, Exogenous, and Actionable Mutational Processes

SUMMARYTumor genomes are suffused with mutations, each caused by specific mutational processes that can leave tell-tale signatures revealing their aetiology. However, knowledge of these underlying mutational processes acting on our DNA does not on its own convey the causation of cancer: many mutations are “passenger” mutations that do not affect progression. One must determine which mutations are causative, which are not, and to what degree. Here we directly tie the cancer effect size—a metric of the proliferation and survival benefit conferred by molecular variants—to the mutational processes that generated molecular variation within individual tumors, quantifying the contribution of each mutagenic process to the formation of each whole-exome sequenced tumor. Furthermore, we extend this result at the individual level to demonstrate the specific mutational processes that are most responsible for tumors of 23 major cancer types. Melanomas and lung cancers are attributable to the actionable, preventable, exogenous mutational processes of ultraviolet light and tobacco exposure, whereas gliomas and prostate adenocarcinoma tumors are largely attributable to endogenous processes associated with aging. Other potentially actionable mutational processes, such as mutations associated with pathogen exposure and APOBEC activity, account for a large proportion of the cancer effect within head and neck, bladder, cervical, and breast cancers. These molecular attributions complement and expand longstanding epidemiological approaches that inform cancer prevention by revealing the burden of cancer caused by either endogenous or exogenous, non-actionable or actionable processes.

BRAF Exon 15 Mutations in Papillary Carcinoma and Adjacent Thyroid Parenchyma: A Search for the Early Molecular Events Associated with Tumor Development

BRAF exon 15 mutations are the most common molecular alterations found in papillary thyroid carcinoma (PTC). To date, there is no information regarding BRAF alterations in the thyroid parenchyma surrounding the tumor. To explore the early events associated with the development of PTC, we used massively parallel sequencing to investigate BRAF exon 15 in 30 PTCs and in 100 samples from the thyroid parenchyma surrounding the tumor. BRAF p.V600E was identified in 19/30 PTCs (63.3%). BRAF p.V600E mutations were identified in the tissue adjacent the PTC only in samples containing psammoma bodies. The other samples were either BRAF wild type (WT) or carried BRAF non p.V600E mutations. Specifically, BRAF p.G593D, -p.A598T, -p.V600M, -p.R603Q, -p.S607F, and -p.S607P were identified in 4 of 36 (11.1%) samples with follicular cell atypia, in 2 of 16 (12.5%) with follicular cell hyperplasia, and in 1 of 33 (3.0%) histologically normal samples—Only in tissue surrounding BRAF p.V600E mutated PTCs. These mutations are predicted to affect protein function in silico but, in vitro, have kinase activity and BRAF phosphorylation levels similar to BRAF WT. No BRAF exon 15 mutations were identified in samples adjacent to PTCs that were BRAF WT. A mutagenic process affecting BRAF exon 15 occurs in a subset of thyroid glands that develop BRAF p.V600E mutated PTCs.

Population sequencing data reveal a compendium of mutational processes in human germline

Mechanistic processes underlying human germline mutations remain largely unknown. Variation in mutation rate and spectra along the genome is informative about the biological mechanisms. We statistically decompose this variation into separate processes using a blind source separation technique. The analysis of a large-scale whole genome sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. Seven of these processes lend themselves to a biological interpretation. One process is driven by bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes independently track direction of replication fork and replication timing. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions. We also demonstrate that a recently discovered mutagenic process specific to oocytes can be localized solely from population sequencing data. This process is spread across all chromosomes and is highly asymmetric with respect to the direction of transcription, suggesting a major role of DNA damage.

Somatic Mutations Render Human Exome and Pathogen DNA more Similar

Immunotherapy has recently shown important clinical successes in a substantial number of oncology indications. Additionally, the tumor somatic mutation load has been shown to associate with response to these therapeutic agents, and specific mutational signatures are hypothesized to improve this association, including signatures related to pathogen insults. We sought to study in silico the validity of these observations and addressed three questions. First, we investigated whether somatic mutations typically involved in cancer may increase, in a statistically meaningful manner, the similarity between common pathogens and the human exome. Our study shows that specific common mutagenic processes like those resulting from exposure to ultraviolet light (in melanoma) or smoking (in lung cancer) induce, in the upper range of biologically plausible frequencies, peptides in the cancer exome that are statistically more similar to pathogen peptides than the normal exome. Second, we investigated whether this increased similarity is due to the specificities of the mutagenic process or uniformly random mutations at equal rate would trigger the same effect. For certain pathogens the increased similarity is more pronounced for specific mutagenic processes than for uniformly random mutations and for other pathogens the effects cannot be distinguished. Finally, we investigated whether specific mutational processes result in amino-acid changes with functional relevance that are more likely to be immunogenic. We showed that functional tolerance to mutagenic processes across species generally suggests more resilience to natural processes than to denovo mutagenesis. These results support the idea that recognition of pathogen sequences as well as differential functional tolerance to mutagenic processes may play an important role in the immune recognition process involved in tumor infiltration by lymphocytes.

Widespread impact of DNA replication on mutational mechanisms in cancer

ABSTRACTDNA replication plays an important role in mutagenesis, yet little is known about how it interacts with other mutagenic processes. Here, we use somatic mutation signatures – each representing a mutagenic process – derived from 3056 patients spanning 19 cancer types to quantify the asymmetry of mutational signatures around replication origins and between early and late replicating regions. We observe that 22 out of 29 mutational signatures are significantly impacted by DNA replication. The distinct associations of different signatures with replication timing and direction around origins shed new light on several mutagenic processes, for example suggesting that oxidative damage to the nucleotide pool substantially contributes to the mutational landscape of esophageal adenocarcinoma. Together, our results indicate an involvement of DNA replication and associated damage repair in most mutagenic processes.

Endogenous Hot Spots ofDe NovoTelomere Addition in the Yeast Genome Contain Proximal Enhancers That Bind Cdc13

DNA double-strand breaks (DSBs) pose a threat to genome stability and are repaired through multiple mechanisms. Rarely, telomerase, the enzyme that maintains telomeres, acts upon a DSB in a mutagenic process termed telomere healing. The probability of telomere addition is increased at specific genomic sequences termed sites of repair-associated telomere addition (SiRTAs). By monitoring repair of an induced DSB, we show that SiRTAs on chromosomes V and IX share a bipartite structure in which a core sequence (Core) is directly targeted by telomerase, while a proximal sequence (Stim) enhances the probability ofde novotelomere formation. The Stim and Core sequences are sufficient to confer a high frequency of telomere addition to an ectopic site. Cdc13, a single-stranded DNA binding protein that recruits telomerase to endogenous telomeres, is known to stimulatede novotelomere addition when artificially recruited to an induced DSB. Here we show that the ability of the Stim sequence to enhancede novotelomere addition correlates with its ability to bind Cdc13, indicating that natural sites at which telomere addition occurs at high frequency require binding by Cdc13 to a sequence 20 to 100 bp internal from the site at which telomerase acts to initiatede novotelomere addition.

CrispRVariants: precisely charting the mutation spectrum in genome engineering experiments

CRISPR-Cas9 and related technologies efficiently alter genomic DNA at targeted positions and have far-reaching implications for functional screening and therapeutic gene editing. Understanding and unlocking this potential requires accurate evaluation of editing efficiency. We show that methodological decisions for analyzing sequencing data can significantly affect mutagenesis efficiency estimates and we provide a comprehensive R-based toolkit, CrispRVariants and accompanying web tool CrispRVariantsLite, that resolves and localizes individual mutant alleles with respect to the endonuclease cut site. CrispRVariants-enabled analyses of newly generated and existing genome editing datasets underscore how careful consideration of the full variant spectrum gives insight toward effective guide and amplicon design as well as the mutagenic process.