Paz Polak

Mutational heterogeneity in cancer and the search for new cancer-associated genes.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour–normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Whole-genome sequence variation, population structure and demographic history of the Dutch population

Whole-genome sequencing enables complete characterization of genetic variation, but geographic clustering of rare alleles demands many diverse populations be studied. Here we describe the Genome of the Netherlands (GoNL) Project, in which we sequenced the whole genomes of 250 Dutch parent-offspring families and constructed a haplotype map of 20.4 million single-nucleotide variants and 1.2 million insertions and deletions. The intermediate coverage (∼13×) and trio design enabled extensive characterization of structural variation, including midsize events (30–500 bp) previously poorly catalogued and de novo mutations. We demonstrate that the quality of the haplotypes boosts imputation accuracy in independent samples, especially for lower frequency alleles. Population genetic analyses demonstrate fine-scale structure across the country and support multiple ancient migrations, consistent with historical changes in sea level and flooding. The GoNL Project illustrates how single-population whole-genome sequencing can provide detailed characterization of genetic variation and may guide the design of future population studies.

Genome sequencing reveals insights into physiology and longevity of the naked mole rat

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the ‘queen’, who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat’s exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

Cell-of-origin chromatin organization shapes the mutational landscape of cancer

Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.

Genome-wide patterns and properties of de novo mutations in humans

Mutations create variation in the population, fuel evolution and cause genetic diseases. Current knowledge about de novo mutations is incomplete and mostly indirect. Here we analyze 11,020 de novo mutations from the whole genomes of 250 families. We show that de novo mutations in the offspring of older fathers are not only more numerous but also occur more frequently in early-replicating, genic regions. Functional regions exhibit higher mutation rates due to CpG dinucleotides and show signatures of transcription-coupled repair, whereas mutation clusters with a unique signature point to a new mutational mechanism. Mutation and recombination rates independently associate with nucleotide diversity, and regional variation in human-chimpanzee divergence is only partly explained by heterogeneity in mutation rate. Finally, we provide a genome-wide mutation rate map for medical and population genetics applications. Our results provide new insights and refine long-standing hypotheses about human mutagenesis.

Differential Relationship of DNA Replication Timing to Different Forms of Human Mutation and Variation

Human genetic variation is distributed nonrandomly across the genome, though the principles governing its distribution are only partially known. DNA replication creates opportunities for mutation, and the timing of DNA replication correlates with the density of SNPs across the human genome. To enable deeper investigation of how DNA replication timing relates to human mutation and variation, we generated a high-resolution map of the human genomes replication timing program and analyzed its relationship to point mutations, copy number variations, and the meiotic recombination hotspots utilized by males and females. DNA replication timing associated with point mutations far more strongly than predicted from earlier analyses and showed a stronger relationship to transversion than transition mutations. Structural mutations arising from recombination-based mechanisms and recombination hotspots used more extensively by females were enriched in early-replicating parts of the genome, though these relationships appeared to relate more strongly to the genomic distribution of causative sequence features. These results indicate differential and sex-specific relationship of DNA replication timing to different forms of mutation and recombination.

Comprehensive assessment of cancer missense mutation clustering in protein structures

Significance Tumor sequencing efforts have enabled the identification of cancer genes based on an excess of mutations in the gene or clustering of mutations along the (one-dimensional) DNA sequence of the gene. Here, we show that this approach can be extended to identify cancer genes based on clustering of mutations relative to the 3D structure of the protein product. By analyzing the PanCancer compendium of somatic mutations in nearly 5,000 tumors, we identified known cancer genes and previously unidentified candidates based on clustering of missense mutations in protein structures or at interfaces with binding partners. In addition, we found that 3D clustering is present in both oncoproteins and tumor suppressors—contrary to the view that such clustering is a hallmark of oncoproteins. Large-scale tumor sequencing projects enabled the identification of many new cancer gene candidates through computational approaches. Here, we describe a general method to detect cancer genes based on significant 3D clustering of mutations relative to the structure of the encoded protein products. The approach can also be used to search for proteins with an enrichment of mutations at binding interfaces with a protein, nucleic acid, or small molecule partner. We applied this approach to systematically analyze the PanCancer compendium of somatic mutations from 4,742 tumors relative to all known 3D structures of human proteins in the Protein Data Bank. We detected significant 3D clustering of missense mutations in several previously known oncoproteins including HRAS, EGFR, and PIK3CA. Although clustering of missense mutations is often regarded as a hallmark of oncoproteins, we observed that a number of tumor suppressors, including FBXW7, VHL, and STK11, also showed such clustering. Beside these known cases, we also identified significant 3D clustering of missense mutations in NUF2, which encodes a component of the kinetochore, that could affect chromosome segregation and lead to aneuploidy. Analysis of interaction interfaces revealed enrichment of mutations in the interfaces between FBXW7-CCNE1, HRAS-RASA1, CUL4B-CAND1, OGT-HCFC1, PPP2R1A-PPP2R5C/PPP2R2A, DICER1-Mg2+, MAX-DNA, SRSF2-RNA, and others. Together, our results indicate that systematic consideration of 3D structure can assist in the identification of cancer genes and in the understanding of the functional role of their mutations.

Reduced local mutation density in regulatory DNA of cancer genomes is linked to DNA repair.

Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in components of the nucleotide excision repair system. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation.

Somatic ERCC2 mutations are associated with a distinct genomic signature in urothelial tumors

Alterations in DNA repair pathways are common in tumors and can result in characteristic mutational signatures; however, a specific mutational signature associated with somatic alterations in the nucleotide- excision repair (NER) pathway has not yet been identified. Here we examine the mutational processes operating in urothelial cancer, a tumor type in which the core NER gene ERCC2 is significantly mutated. Analysis of three independent urothelial tumor cohorts demonstrates a strong association between somatic ERCC2 mutations and the activity of a mutational signature characterized by a broad spectrum of base changes. In addition, we note an association between the activity of this signature and smoking that is independent of ERCC2 mutation status, providing genomic evidence of tobacco-related mutagenesis in urothelial cancer. Together, these analyses identify an NER-related mutational signature and highlight the related roles of DNA damage and subsequent DNA repair in shaping tumor mutational landscape.

A mutational signature reveals alterations underlying deficient homologous recombination repair in breast cancer

Biallelic inactivation of BRCA1 or BRCA2 is associated with a pattern of genome-wide mutations known as signature 3. By analyzing ∼1,000 breast cancer samples, we confirmed this association and established that germline nonsense and frameshift variants in PALB2, but not in ATM or CHEK2, can also give rise to the same signature. We were able to accurately classify missense BRCA1 or BRCA2 variants known to impair homologous recombination (HR) on the basis of this signature. Finally, we show that epigenetic silencing of RAD51C and BRCA1 by promoter methylation is strongly associated with signature 3 and, in our data set, was highly enriched in basal-like breast cancers in young individuals of African descent.