A Correction to this paper has been published: https://doi.org/10.1038/s41586-021-03379-5.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes(1-8). Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease(9-13) than in normal liver(13-16), which enables positive selection to shape the genomic landscape(9-13). Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1(S22W) hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes(17-19), and GPAM, which produces storage triacylglycerol from free fatty acids(20,21), also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Background: Inflammatory bowel disease (IBD) needs early interventions and an individual specialist–patient relationship. Distance from a tertiary IBD center might affect patient’s disease course and outcome. We investigated whether the patient-to-specialist distance has an impact on the disease course using the well-defined patient collective of the Swiss Inflammatory Bowel Disease Cohort Study (SIBDCS). Methods: Patient’s home address at diagnosis (postal zip code) was extracted from the SIBDCS database. Distance between each zip code and the nearest located IBD specialist center was calculated and classified into the following three sections based on proximity: <10 km (group 1); 10–35 km (group 2); >35 km (group 3). Results: Our study included in total 408 IBD patients [234 Crohn’s disease (CD), 154 ulcerative colitis (UC), 20 IBD unclassified (IBDU)]. Median age was lowest in group 2 at diagnosis (G1: 28 years; G2: 21 years, G3: 26 years, p < 0.01). The diagnostic delay did not differ between groups. CD patients in group 1 were treated more often with anti-tumor necrosis factor (TNF) agents (72% versus 56%, p = 0.04) and 5-aminosalicylates (44% versus 28%, p = 0.04) than in group 3. UC/IBDU patients in group 1 were treated more often with corticosteroids than patients in group 3 (83% versus 58%, p < 0.01). The occurrence of IBD-related surgeries did not differ between groups. Conclusions: Patient-to-specialist distance might affect drug treatment. However, disease course and the need for IBD-related surgery does not seem to be associated with a longer distance to specialist care in Switzerland.

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100–1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2–3 weeks would be a more typical turnaround time.

All normal somatic cells are thought to acquire mutations. However, characterisation of the patterns and consequences of somatic mutation in normal tissues is limited. Uterine endometrium is a dynamic tissue that undergoes cyclical shedding and reconstitution and is lined by a gland-forming epithelium. Whole genome sequencing of normal endometrial glands showed that most are clonal cell populations derived from a recent common ancestor with mutation burdens differing from other normal cell types and manyfold lower than endometrial cancers. Mutational signatures found ubiquitously account for most mutations. Many, in some women potentially all, endometrial glands are colonised by cell clones carrying driver mutations in cancer genes, often with multiple drivers. Total and driver mutation burdens increase with age but are also influenced by other factors including body mass index and parity. Clones with drivers often originate during early decades of life. The somatic mutational landscapes of normal cells differ between cell types and are revealing the procession of neoplastic change leading to cancer.

Chronic liver disease is associated with metabolic dysregulation, liver failure and hepatocellular carcinoma. We analysed somatic mutations from 1202 genomes across 32 liver samples, including normal controls, alcohol-related and non-alcoholic fatty liver disease. Five of 27 patients with liver disease carried hotspot driver mutations in FOXO1, the major transcription factor downstream of insulin signalling. FOXO1 mutations were independently acquired by up to 5 distinct clones within the same patient’s sample, and impaired insulin-mediated nuclear export of FOXO1. GPAM, which produces storage triacylglycerol from dietary calories, also had significant excess of mutations, similarly exhibiting convergent evolution within biopsies. Telomeres were shorter in diseased than normal liver, with attrition more pronounced in larger clones. Multiple independent acquisitions of drivers within one small liver sample imply that such mutations could affect hundreds of grams of tissue across the whole organ, potentially contributing to systemic metabolic dysfunction.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.

Background Many inflammatory bowel disease (IBD) patients follow a restrictive diet due to perceived positive effects on their symptoms. We assessed the prevalence of vegetarian (VD) and gluten-free diets (GFDs) in IBD patients, the reasons for following such a diet, and whether nutrition has an impact on disease activity and microbiota composition. Methods We included 1254 patients from the Swiss Inflammatory Bowel Disease Cohort Study with prospective acquisition of clinical data and psychosocial, disease-related and lifestyle factors between 2006 and 2015. Dietary habits were assessed through a self-report questionnaire. In 92 patients, we analysed intestinal mucosa-associated microbial composition using high-throughput sequencing. Results Overall, 4.1% (n = 52) of the patients reported following a VD and 4.7% (n = 54) a GFD. No differences regarding disease activity, fistula, hospitalization or surgery rates were observed. Patients on a VD or GFD had significantly higher levels of post-traumatic stress symptoms. Furthermore, GFD patients had significantly higher anxiety and depression symptom levels. The gut microbiota composition in IBD patients following a VD or GFD was significantly different compared to that of omnivores. Conclusions Although we did not identify a relevant impact of a specific diet on the course of the disease, there was a significant association with lower psychological well-being in VD and GFD patients.

BACKGROUND & AIMS: Swallowed topical-acting corticosteroids are recommended as first-line therapy for eosinophilic esophagitis (EoE). Asthma medications not optimized for esophageal delivery are sometimes effective, although given off-label. We performed a randomized, placebo-controlled trial to assess the effectiveness and tolerability of a budesonide orodispersible tablet (BOT), which allows the drug to be delivered to the esophagus in adults with active EoE. METHODS: We performed a double-blind, parallel study of 88 adults with active EoE in Europe. Patients were randomly assigned to groups that received BOT (1 mg twice daily; n = 59) or placebo (n = 29) for 6 weeks. The primary end point was complete remission, based on clinical and histologic factors, including dysphagia and odynophagia severity <= 2 on a scale of 0-10 on each of the 7 days before the end of the double-blind phase and a peak eosinophil count <5 eosinophils/high power field. Patients who did not achieve complete remission at the end of the 6-week double-blind phase were offered 6 weeks of open-label treatment with BOT (1 mg twice daily). RESULTS: At 6 weeks, 58% of patients given BOT were in complete remission compared with no patients given placebo (P < .0001). The secondary end point of histologic remission was achieved by 93% of patients given BOT vs no patients given placebo (P < .0001). After 12 weeks, 85% of patients had achieved remission. Six-week and 12-week BOT administration were safe and well tolerated; 5% of patients who received BOT developed symptomatic, mild candida, which was easily treated with an oral antifungal agent. CONCLUSIONS: In a randomized trial of adults with active EoE, we found that budesonide oral tablets were significantly more effective than placebo in inducing clinical and histologic remission. Eudra-CT number 2014-001485-99;

Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.

BackgroundThe frequency of upper gastrointestinal [GI] tract involvement in Crohn`s disease [CD] has been reported with a large variation. Risk factors and disease course of patients with upper GI tract involvement remain largely elusive.MethodsData on CD patients in the Swiss Inflammatory Bowel Disease Cohort were analysed. Patients with upper GI tract involvement were compared with controls. Logistic regression models for prediction of upper GI tract involvement and Cox proportional hazard models for occurrence of complications were computed.ResultsWe included 1638 CD patients, of whom 107 [6.5%] presented with upper GI tract involvement at the time of diagnosis and 214 [13.1%] at any time. Prevalence of such involvement at diagnosis increased over time [5.1% for 1955-95 versus 11.3% for 2009-16]. In a multivariate logistic regression model, male sex and diagnosis between 2009 and 2016 [versus before 1995] were independent predictors for presence of upper GI tract involvement at CD diagnosis (odds ratio [OR] 1.600, p = 0.021 and OR 2.686, p < 0.001, respectively), whereas adult age was a negative predictor [OR 0.388, p = 0.001]. Patients with upper GI tract involvement showed a disease course similar to control patients (hazard ratio [HR] for any complications 0.887, (95% confidence interval [CI] 0.409-1.920), and a trend towards occurrence of fewer intestinal fistulas [log-rank test p = 0.054].ConclusionsPrevalence of upper GI tract involvement has been increasing over the past decades. Male sex and young age at diagnosis were identified as the main predictive factors for such involvement at CD diagnosis. Involvement of upper GI tract did not result in a worse outcome.

Abstract Transcription networks consist of hundreds of transcription factors with thousands of often overlapping target genes. While we can reliably measure gene expression changes, we still understand relatively little why expression changes the way it does. How does a coordinated response emerge in such complex networks and how many input signals are necessary to achieve it? Here, we unravel the regulatory program of gene expression in Escherichia coli central carbon metabolism with more than 30 known transcription factors. Using a library of fluorescent transcriptional reporters, we comprehensively quantify the activity of central metabolic promoters in 26 environmental conditions. The expression patterns were dominated by growth rate‐dependent global regulation for most central metabolic promoters in concert with highly condition‐specific activation for only few promoters. Using an approximate mathematical description of promoter activity, we dissect the contribution of global and specific transcriptional regulation. About 70% of the total variance in promoter activity across conditions was explained by global transcriptional regulation. Correlating the remaining specific transcriptional regulation of each promoter with the cell's metabolome response across the same conditions identified potential regulatory metabolites. Remarkably, cyclic AMP, fructose‐1,6‐bisphosphate, and fructose‐1‐phosphate alone explained most of the specific transcriptional regulation through their interaction with the two major transcription factors Crp and Cra. Thus, a surprisingly simple regulatory program that relies on global transcriptional regulation and input from few intracellular metabolites appears to be sufficient to coordinate E. coli central metabolism and explain about 90% of the experimentally observed transcription changes in 100 genes.

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to biosynthesize defined phosphoproteins. Here we describe a rapid approach for directly discovering aminoacyl-tRNA synthetase-tRNA pairs that selectively incorporate non-natural amino acids into proteins; our method uses parallel positive selections combined with deep sequencing and statistical analysis and enables the direct, scalable discovery of aminoacyl-tRNA synthetase-tRNA pairs with mutually orthogonal substrate specificity. By combining a method to biosynthesize phosphothreonine in cells with this selection approach, we discover a phosphothreonyl-tRNA synthetase-tRNA(CUA) pair and create an entirely biosynthetic route to incorporating phosphothreonine in proteins. We biosynthesize several phosphoproteins and demonstrate phosphoprotein structure determination and synthetic protein kinase activation.

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.