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The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanism: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analysing the three-dimensional structures of protein–DNA complexes, here we show that the binding of arginine residues to narrow minor grooves is a widely used mode for protein–DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a prominent example of this effect. Minor-groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings indicate that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA-binding specificity. Major to minor How sequence-specific DNA-binding proteins can find targets in the midst of vast amounts of non-specific DNA is a long-standing puzzle. A favoured model was that the sequence was read as hydrogen bonds formed between the protein and bases in the major groove of the DNA helix. A new analysis of the three-dimensional structures of protein–DNA complexes suggests that DNA shape is key to recognition. DNA sequence context alters the width of the minor groove of the helix by preferential binding of arginines to electronegative pockets. The positioning of DNA in the nucleosome core particle is an example of this effect. The question of how proteins recognize specific DNA sequences in the face of vastly higher concentrations of non-specific DNA remains unclear. One suggested mechanism involves the formation of hydrogen bonds with specific bases, primarily in the major groove. The comprehensive analysis of the three-dimensional structures of protein–DNA complexes now shows that the binding of arginine residues to narrow minor grooves is a widely used mode for protein–DNA recognition.

In partnership with NHS England, Genomics England’s ambitious plans to embed genomic medicine into routine patient care are well underway. Clare Turnbull and colleagues discuss its progress

Most research on genetic screening and precision oncology is based on individuals of European ancestry. We applied the National Health Service (NHS) England's cancer variant prioritisation workflow to evaluate the performance of these approaches in ethinically and ancestrally diverse populations. The second aim of the study was to assess the representativeness of the 100 000 Genomes Project cancer cohort of the population of England. In this cross-sectional analysis, whole-genome sequencing data from patients with cancer recruited into the 100 000 Genomes Project between February 2015 to December 2018 were analysed. Clinical information, including tumour stage and grade, was gathered from the NHS England National Cancer Registration and Analysis Service. Patients with cancer types with fewer than five individuals, haematological cancers, childhood cancers, unknown primary carcinomas, patients with indeterminate sex, and patients missing somatic mutations in genes were excluded. To assess ethnicity representation in the 100 000 Genomes Project, we calculated the recruitment ratios for self-reported ethnicities for patients with cancer recruited to the 100 000 Genomes Project and patients with cancer in England. We also analysed differences in classification rates for potentially pathogenic variants to assess ancestry-related differences in germline and somatic mutations of different ancestry groups. 14 775 patients with cancer were recruited between February, 2015, and December, 2018, into the 100 000 Genomes Project. There was no evidence of under-representation of diverse ethnic groups in the 100 000 Genomes Project when compared with the national statistics. The recruitment rate ratio for breast cancer was 2·2 (95% CI 1·6–3·0) for Black versus White women in the 100 000 Genomes Project compared with 0·81 (0·79–0·83) for Black versus White women in the national data (fold-change in rate ratios 2·7; 95% CI 2·0–3·7, p<0·0001), suggesting higher representation of Black women in the 100 000 Genomes Project than expected given the ethnicity-specific incidence rates in England. Compared with national rates, the 100 000 Genomes Project also had higher recruitment rates of Black versus White men with prostate cancer (fold-change in rate ratios 3·7; 1·8–7·5, p=0·0004), Black versus White men with bladder cancer (fold change in rate ratios 6·1; 2·0–18·8, p=0·0016), and Asian versus White women with breast cancer (fold change in rate ratios 1·4; 1·2–1·7, p=0·0008). Ancestry had a significant association with the likelihood of carrying a variant classified as a potentially pathogenic (likelihood ratio test p=0·0011). Potentially pathogenic variants were identified in 23 (4·6%) of 500 South Asian (adjusted model odds ratio [OR] 1·88, 95% CI 1·21–2·93, p=0·0052) and 24 (5·3%) of 453 African ancestry patients (OR 2·24, 1·44–3·48, p=0·0003) compared with 263 (2·2%) of 11 955 in European-ancestry patients. However, we found that fewer tumour mutations in actionable genes were identified for patients of non-European ancestry compared with patients of European ancestry when adjusting for sex and cancer type (likelihood ratio test p<0·0001). The was an excess of germline variants classified as potentially pathogenic variants in patients with non-European ancestry, which might impede the diagnostic process. Improved variant prioritisation workflows and more research in diverse groups are needed to ensure equitable implementation of genomics in cancer care. The UK Department of Health and Social Care and the EU's Horizon 2020 Research and Innovation Programme.

The unexpected contamination of normal samples with tumour cells reduces variant detection sensitivity, compromising downstream analyses in canonical tumour-normal analyses. Leveraging whole-genome sequencing data available at Genomics England, we develop a tool for normal sample contamination assessment, which we validate in silico and against minimal residual disease testing. From a systematic review of 771 patients with haematological malignancies and sarcomas, we find contamination across a range of cancer clinical indications and DNA sources, with highest prevalence in saliva samples from acute myeloid leukaemia patients, and sorted CD3+ T-cells from myeloproliferative neoplasms. Further exploration reveals 108 hotspot mutations in genes associated with haematological cancers at risk of being subtracted by standard variant calling pipelines. Our work highlights the importance of contamination assessment for accurate somatic variants detection in research and clinical settings, especially with large-scale sequencing projects being utilised to deliver accurate data from which to make clinical decisions for patient care. Assessing tumour contamination in normal samples is critical for accurate variant calling in cancer samples. Here, the authors develop TINC, a computational method to determine the level of tumour in normal contamination, and demonstrate its application in the Genomics England 100,000 Genomes Project dataset.

Copy number alterations (CNAs) are among the most important genetic events in cancer, but their detection from sequencing data is challenging because of unknown sample purity, tumor ploidy, and general intra-tumor heterogeneity. Here, we present CNAqc, an evolution-inspired method to perform the computational validation of clonal and subclonal CNAs detected from bulk DNA sequencing. CNAqc is validated using single-cell data and simulations, is applied to over 4000 TCGA and PCAWG samples, and is incorporated into the validation process for the clinically accredited bioinformatics pipeline at Genomics England. CNAqc is designed to support automated quality control procedures for tumor somatic data validation.

Testicular germ cell tumours (TGCT), which comprise seminoma and non-seminoma subtypes, are the most common cancers in young men. In this study, we present a comprehensive whole genome sequencing analysis of adult TGCTs. Leveraging samples from participants recruited via the UK National Health Service and data from the Genomics England 100,000 Genomes Project, our results provide an extended description of genomic elements underlying TGCT pathogenesis. This catalogue offers a comprehensive, high-resolution map of copy number alterations, structural variation, and key global genome features, including mutational signatures and analysis of extrachromosomal DNA amplification. This study establishes correlations between genomic alterations and histological diversification, revealing divergent evolutionary trajectories among TGCT subtypes. By reconstructing the chronological order of driver events, we identify a subgroup of adult TGCTs undergoing relatively late whole genome duplication. Additionally, we present evidence that human leukocyte antigen loss is a more prevalent mechanism of immune disruption in seminomas. Collectively, our findings provide valuable insights into the developmental and immune modulatory processes implicated in TGCT pathogenesis and progression. Testicular germ cell tumours (TGCT) are the most common cancers in young men. Here, the authors analyse the genomic landscape of TGCT using data from the Genomics England 100,000 Genomes Project, revealing divergent evolutionary trajectories and the prevalence of human leukocyte antigen loss.

Breast cancer is the most frequently diagnosed cancer in women. Survival is generally considered favourable, yet some patients remain at risk of early death. We aimed to assess whether comprehensive whole-genome sequencing (WGS) linked to mortality data could add prognostic value to existing clinical measures and identify patients who might respond to targeted therapeutics. In this integrative, retrospective analysis, we analysed 2445 breast cancer tumours (any stage and molecular subtype) collected from 2403 patients recruited through 13 National Health Service Genomic Medicine Centres or hospitals in England affiliated to the 100 000 Genomes Project (100kGP) between 2012 and 2018. We linked 2208 (90%) cases with clinical data; mortality data were obtained for 1188 patients. Following high-depth WGS of tumour and matched normal DNA, we performed comprehensive WGS profiling seeking driver mutations, mutational signatures, and compound algorithmic scores for homologous recombination repair deficiency (HRD), mismatch repair deficiency, and tumour mutational burden. Data from 1803 additional patients with breast cancer from three independent cohorts were used to validate various findings. To evaluate the prognostic value of WGS features, we performed univariable and multivariable Cox regression on data from patients with stage I–III, ER-positive, HER2-negative breast cancer with a cancer-specific mortality endpoint (around 5-year follow-up). Among 2445 tumours in the 100kGP breast cancer cohort, we observed genomic characteristics with immediate personalised medicine potential in 656 (26·8%), including features reporting HRD (298 [12·2%] total cases and 76 [6·3%] ER-positive, HER2-negative cases), highly individualised driver events, mutations underpinning resistance to endocrine therapy, and mutational signatures indicating therapeutic vulnerabilities. 373 (15·2%) cases had WGS features with potential for translational research, including compromised base excision repair and non-homologous end-joining dependency. Structural variation burden (hazard ratio 3·9 [95 CI% 2·4–6·2]; p<0·0001), high levels of APOBEC signatures (2·5 [1·6–4·1]; p<0·0001), and TP53 drivers (3·9 [2·4–6·2]; p<0·0001) were independently prognostic of customary clinical measures (age at diagnosis, stage, and grade) in patients with ER-positive, HER2-negative breast cancer. We developed a prognosticator for ER-positive, HER2-negative breast cancer capable of identifying patients who require either increased intervention or therapy de-escalation, validating the framework in the independent Swedish Cancerome Analysis Network-Breast (SCAN-B) dataset. We show that breast cancer genomes are rich in predictive and prognostic value. We propose a two-step model for effective clinical application. First, the identification of candidates for targeted therapies or clinical trials using highly individualised genomic markers. Second, for patients without such features, the implementation of enhanced prognostication using genomic features alongside existing clinical decision-making factors. National Institute of Health Research, Breast Cancer Research Foundation, Dr Josef Steiner Cancer Research Award 2019, Basser Gray Prime Award 2020, Cancer Research UK, Sir Jeffrey Cheah Early Career Fellowship, the Mats Paulsson Foundation, the Fru Berta Kamprads Foundation, and the Swedish Research Council.

Carboxypeptidase E is a peptide processing enzyme, involved in cleaving numerous peptide precursors, including neuropeptides and hormones involved in appetite control and glucose metabolism. Exome sequencing of a morbidly obese female from a consanguineous family revealed homozygosity for a truncating mutation of the CPE gene (c.76_98del; p.E26RfsX68). Analysis detected no CPE expression in whole blood-derived RNA from the proband, consistent with nonsense-mediated decay. The morbid obesity, intellectual disability, abnormal glucose homeostasis and hypogonadotrophic hypogonadism seen in this individual recapitulates phenotypes in the previously described fat/fat and Cpe knockout mouse models, evidencing the importance of this peptide/hormone-processing enzyme in regulating body weight, metabolism, and brain and reproductive function in humans.

Embryonic polarity of invertebrates, amphibians and fish is specified largely by maternal determinants, which fixes cell fates early in development. In contrast, amniote embryos remain plastic and can form multiple individuals until gastrulation. How is their polarity determined? In the chick embryo, the earliest known factor is cVg1 (homologous to mammalian growth differentiation factor 1, GDF1), a transforming growth factor beta (TGFβ) signal expressed posteriorly before gastrulation. A molecular screen to find upstream regulators of cVg1 in normal embryos and in embryos manipulated to form twins now uncovers the transcription factor Pitx2 as a candidate. We show that Pitx2 is essential for axis formation, and that it acts as a direct regulator of cVg1 expression by binding to enhancers within neighbouring genes. Pitx2, Vg1/GDF1 and Nodal are also key actors in left–right asymmetry, suggesting that the same ancient polarity determination mechanism has been co-opted to different functions during evolution. In warm-blooded animals, including chickens and humans, a single embryo can give rise to several separate individuals (identical twins). Some species of armadillos routinely give birth to quadruplets in this way—and in experiments, up to eight identical chick embryos can be produced by cutting one embryo into smaller pieces (a type of ‘experimental twinning’). This ability of a developing embryo to subdivide into separate individuals ends when the embryo starts to form its first midline structure, called the ‘primitive streak’. This is the first line of symmetry and defines where the head–tail axis will later develop. The steps that establish the axes of the embryo in birds and mammals, and the factors that prevent further splitting of the embryo to form twins after this point, are only just beginning to be understood. In chick embryos, the production of a protein called cVg1 is the first known step and precedes the development of a line of symmetry. A similar protein is produced in mammalian embryos and both proteins are members of an important family of signalling proteins. Now, Torlopp, Khan et al. have used a combination of techniques to search for other proteins that that control the production of the cVg1 protein. Genes that are active in the region of the embryo that will express cVg1 later in development were identified, both in normal embryos and during the process of experimental twinning. This search revealed Pitx2 as a protein that acts to switch on the expression of the gene that encodes cVg1. When the Pitx2 protein is removed, the embryonic axis forms from the opposite side. Next, Torlopp, Khan et al. searched the chicken genome to identify stretches of DNA around the cVg1 gene where proteins that regulate gene expression might bind. Six potential sites were found, including four to which Pitx2 can bind. Further experiments confirmed that two of these regulatory sequences encourage the expression of the cVg1 gene at its correct position in the embryo. Pitx2 and related proteins were known to be involved with the development of left–right symmetry later in development; the findings of Torlopp, Khan et al. reveal, unexpectedly, that these proteins are also involved in first establishing the position at which the midline of the embryo will arise. It remains unclear what prevents most embryos from forming twins. But Torlopp, Khan et al.'s findings could help to explain some strange observations, made long ago, about left–right asymmetry in identical twins. For example, they could help explain why one of the twins in an identical twin pair is more likely to be left-handed than an individual in the general population, and why the direction of whorls of hair on the back of the head is often mirrored between identical twins.

The largest whole genome sequencing (WGS) endeavour involving cancer and rare diseases was initiated in the UK in 2015 and ran for 5 years. Despite its rarity, sarcoma ranked third overall among the number of patients' samples sent for sequencing. Herein, we recount the lessons learned by a specialist sarcoma centre that recruited close to 1000 patients to the project, so that we and others may learn from our experience. WGS data was generated from 597 patients, but samples from the remaining approximately 400 patients were not sequenced. This was largely accounted for by unsuitability due to extensive necrosis, secondary to neoadjuvant radiotherapy or chemotherapy, or being placed in formalin. The number of informative genomes produced was reduced further by a PCR amplification step. We showed that this loss of genomic data could be mitigated by sequencing whole genomes from needle core biopsies. Storage of resection specimens at 4 °C for up to 96 h overcame the challenge of freezing tissue out of hours including weekends. Removing access to formalin increased compliance to these storage arrangements. With over 70 different sarcoma subtypes described, WGS was a useful tool for refining diagnoses and identifying novel alterations. Genomes from 350 of the cohort of 597 patients were analysed in this study. Overall, diagnoses were modified for 3% of patients following review of the WGS findings. Continued refinement of the variant‐calling bioinformatic pipelines is required as not all alterations were identified when validated against histology and standard of care diagnostic tests. Further research is necessary to evaluate the impact of germline mutations in patients with sarcoma, and sarcomas with evidence of hypermutation. Despite 50% of the WGS exhibiting domain 1 alterations, the number of patients with sarcoma who were eligible for clinical trials remains small, highlighting the need to revaluate clinical trial design.