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Author(s): Tim H. H. Coorens [sup.1] , Luiza Moore [sup.1] [sup.2] , Philip S. Robinson [sup.1] [sup.3] , Rashesh Sanghvi [sup.1] , Joseph Christopher [sup.1] [sup.3] [sup.4] , James Hewinson [...], Starting from the zygote, all cells in the human body continuously acquire mutations. Mutations shared between different cells imply a common progenitor and are thus naturally occurring markers for lineage tracing.sup.1,2. Here we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissections. Reconstructed embryonic progenitors in the same generation of a phylogeny often contribute to different extents to the adult body. The degree of this asymmetry varies between individuals, with ratios between the two reconstructed daughter cells of the zygote ranging from 60:40 to 93:7. Asymmetries pervade subsequent generations and can differ between tissues in the same individual. The phylogenies resolve the spatial embryonic patterning of tissues, revealing contiguous patches of, on average, 301 crypts in the adult colonic epithelium derived from a most recent embryonic cell and also a spatial effect in brain development. Using data from ten additional men, we investigated the developmental split between soma and germline, with results suggesting an extraembryonic contribution to primordial germ cells. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals. Somatic mutations obtained from laser microdissected biopsies of human tissues are used to reconstruct the developmental phylogenies of these tissues back to the zygote.

Author(s): Seongyeol Park [sup.1] [sup.2] , Nanda Maya Mali [sup.3] , Ryul Kim [sup.1] , Jeong-Woo Choi [sup.3] [sup.4] , Junehawk Lee [sup.5] , Joonoh Lim [sup.1] , Jung Min [...], Cellular dynamics and fate decision in early human embryogenesis remain largely unknown owing to the challenges of performing studies in human embryos.sup.1. Here, we explored whole-genomes of 334 single-cell colonies and targeted deep sequences of 379 bulk tissues obtained from various anatomical locations of seven recently deceased adult human donors. Using somatic mutations as an intrinsic barcode, we reconstructed early cellular phylogenies that demonstrate (1) an endogenous mutational rate that is higher in the first cell division but decreases to approximately one per cell per cell division later in life; (2) universal unequal contribution of early cells to embryo proper, resulting from early cellular bottlenecks that stochastically set aside epiblast cells within the embryo; (3) examples of varying degrees of early clonal imbalances between tissues on the left and right sides of the body, different germ layers and specific anatomical parts and organs; (4) emergence of a few ancestral cells that will substantially contribute to adult cell pools in blood and liver; and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach also provides insights into the age-related mutational processes and loss of sex chromosomes in normal somatic cells. In sum, this study provides a foundation for future studies to complete cellular phylogenies in human embryogenesis. Adult human tissues from diverse sites around the body are used to reconstruct cellular phylogenies from early development, using somatic mutations as an internal barcode.

Author(s): Georg K. A. Hochberg [sup.1] , Yang Liu [sup.2] , Erik G. Marklund [sup.3] , Brian P. H. Metzger [sup.1] , Arthur Laganowsky [sup.2] , Joseph W. Thornton [sup.1] [...], Most proteins assemble into multisubunit complexes.sup.1. The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work.sup.2. In many complexes, however, multimerization does not enable any known function.sup.3. An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function.sup.3-7. Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous. Accumulation of hydrophobic residues at the interface between monomers may favour the maintenance of multimeric protein states during evolution, even if multimerization confers no functional advantage.

Author(s): Henry Lee-Six [sup.1] , Sigurgeir Olafsson [sup.1] , Peter Ellis [sup.1] , Robert J. Osborne [sup.1] , Mathijs A. Sanders [sup.1] [sup.2] , Luiza Moore [sup.1] , Nikitas Georgakopoulos [...], The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer.sup.1. However, our understanding of the earliest phases of colorectal neoplastic changes--which may occur in morphologically normal tissue--is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer. Genome sequencing of hundreds of normal colonic crypts from 42 individuals sheds light on mutational processes and driver mutations in normal colorectal epithelial cells.

Author(s): Simon F. Brunner [sup.1] , Nicola D. Roberts [sup.1] , Luke A. Wylie [sup.1] , Luiza Moore [sup.1] , Sarah J. Aitken [sup.2] [sup.3] , Susan E. Davies [sup.3] [...], 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 genes.sup.1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures.sup.8, 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. Whole-genome sequencing of liver microdissections from five healthy individuals and nine with cirrhosis demonstrates the effects of liver disease on the genome, including increased rates of mutation, complex structural variation and different mutational signatures.

Author(s): Ashley Acevedo [sup.1] , Leonid Brodsky [sup.2] , Raul Andino [sup.1] Author Affiliations: (1) Department of Microbiology and Immunology, University of California, San Francisco, USA (2) Tauber Bioinformatics Research [...], A new approach to accurately determine mutation frequencies with RNA virus populations called circular sequencing (CirSeq) has been developed; a study of the genetic composition of populations of poliovirus shows the fitness landscape for each nucleotide variant in an evolving RNA virus. Evolving RNA viruses sequenced Raul Andino and colleagues report a new approach to the determination of mutation frequencies within RNA virus populations. The new technique, circular sequencing (CirSeq), uses circularized genomic RNA fragments to generate tandem repeats that then serve as substrates for next-generation sequencing. The authors use CirSeq to track the genetic composition of populations of poliovirus replicating in human cells in culture and uncover the mutational landscape of the population. The extent of change in mutation frequency for each variant over the course of passage in cell culture is a measure of the relative fitness of each variant, thereby delineating the fitness landscape for an evolving RNA virus. RNA viruses exist as genetically diverse populations.sup.1. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses.sup.2 and hence their pathogenesis.sup.3. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure-function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.

A deficiency of functional dystrophin protein in muscle cells causes muscular dystrophy (MD). More than 50% of missense mutations that trigger the disease occur in the N-terminal actin binding domain (N-ABD or ABD1). We examined the effect of four disease-causing mutations--L54R, A168D, A171P, and Y231 N--on the structural and biophysical properties of isolated N-ABD. Our results indicate that N-ABD is a monomeric, well-folded [alpha]-helical protein in solution, as is evident from its a-helical circular dichroism spectrum, blue shift of the native state tryptophan fluorescence, well-dispersed amide crosspeaks in 2D NMR [sup.15]N-[sup.1]H HSQC fingerprint region, and rotational correlation time calculated from NMR longitudinal ([T.sub.1]) and transverse ([T.sub.2]) relaxation experiments. Compared to WT, three mutants--L54R, A168D, and A171P--show a decreased [alpha]-helicity and do not show a cooperative sigmoidal melt with temperature, indicating that these mutations exist in a wide range of conformations or in a 'molten globule' state. In contrast, Y231N has an a-helical content similar to w-r and shows a cooperative sigmoidal temperature melt but with a decreased stability. All four mutants experience serious misfolding and aggregation. FT-IR, circular dichroism, increase in thioflavin T fluorescence, and the congo red spectral shift and birefringence show that these aggregates contain intermolecular cross-[beta] structure similar to that found in amyloid diseases. These results indicate that disease-causing mutants affect N-ABD structure by decreasing its thermodynamic stability and increasing its misfolding, thereby decreasing the net functional dystrophin concentration. actin binding domain | Becket muscular dystrophy | calponin homology domain | Duchenne muscular dystrophy | protein aggregation doi/ 10.1073/pnas.1008818107

Cancer-specific mutations in the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) p110[alpha] occur in diverse tumors in frequencies that can exceed 30%. The majority of these mutations map to one of three hot spots in the gene, and the rest are distributed over much of the PI3K coding sequence. Most of the cancer-specific mutations induce a gain of function that results in oncogenicity, elevated lipid kinase activity and constitutive signaling through the kinases Akt and TOR. The location of the mutations on a model structure of p110[alpha] indicates several distinct mechanisms for the gain of function. The mutated p110[alpha] proteins are promising cancer targets. Although identification of mutant-specific small-molecule inhibitors seems technically challenging, the therapeutic benefits from such inhibitors could be extremely important.

To study the mechanisms by which missense mutations in [alpha]-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing [alpha]-tropomyosin with one of two disease-causing mutations, [Asp.sup.175] Asn or [Glu.sup.180] Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-[alpha]-tropomyosin antibody, ARG1. In transgenic rats with either [alpha]-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. [Ca.sup.2+] sensitivity of cardiac skinned-fiber preparations from animals with mutation [Asp.sup.175] Asn, but not [Glu.sup.180] Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous [Ca.sup.2+] waves were detected only in cardiomyocytes from animals with mutation [Asp.sup.175] Asn, suggesting an increase in intracellular [Ca.sup.2+] concentration compensating for the reduced [Ca.sup.2+] sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation [Asp.sup.175] Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by [alpha]-tropomyosin mutation [Asp.sup.175] Asn in familial hypertrophic cardiomyopathy on the basis of changes in [Ca.sup.2+] handling as a sensitive mechanism to compensate for alterations in sarcomeric structure. familial hypertrophic cardiomynpathy; animal model; calcium transient; cardiac skinned-fiber preparation

The sterility and inviability of species hybrids is thought to evolve by the accumulation of genes that cause generally recessive, incompatible epistatic interactions between species. Most analyses of the loci involved in such hybrid incompatibilities have suffered from low genetic resolution. Here I present a fine-resolution genetic screen that allows systematic counting, mapping, and characterizing of a large number of hybrid incompatibility loci in a model genetic system. Using small autosomal deletions from D. melanogaster and a hybrid rescue mutation from D. simulans, I measured the viability of hybrid males that are simultaneously hemizygous for a small region of the D. simulans autosomal genome and hemizygous for the D. melanogaster X chromosome. These hybrid males are exposed to the full effects of any recessive-recessive epistatic incompatibilities present in these regions. A screen of ~70% of the D. simulans autosomal genome reveals 20 hybrid-lethal and 20 hybrid-semilethal regions that are incompatible with the D. melanogaster X. In further crosses, I confirm the epistatic nature of hybrid lethality by showing that all of the incompatibilities are rescued when the D. melanogaster X is replaced with a D. simulans X. Combined with information from previous studies, these results show that the number of recessive incompatibilities is approximately eightfold larger than the number of dominant ones. Finally, I estimate that a total of ~191 hybrid-lethal incompatibilities separate D. melanogaster and D. simulans, indicating extensive functional divergence between these species' genomes.

Using selectable genes as proof of principle, a new high-through-put genotype-based mutation screen in mouse embryonic stem (ES) cells was developed [Chen et al. (2002) Nat. Genet. 24, 314-317]. If expanded to nonselectable genes, this approach would allow one to proceed quickly from sequence to whole-animal phenotypes. Here data are presented showing that a screen of a cryopreserved library of clonal, germ line competent, N-ethyl-N-nitrosurea (ENU) mutagenized ES cells can identify a large series of allelic mutations in Smad2 and Smad4, two nonselectable genes of the transforming growth factor [beta] superfamily of signaling molecules. Whole animal phenotypic analyses of some of these alleles provided evidence for novel developmental processes mediated by these components of transforming growth factor [beta] signaling, demonstrating the utility of non-null alleles created by chemical mutagens. The accurately assessed mutation load of the ES cell library indicates that it is a valuable resource for developing mouse lines for genetic and functional studies. This methodology can conceptually be applied for the generation of an allelic series of subtle mutations at any locus of interest in the mouse.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue. somatic mutation | clonal expansion | single cell | aging | cancer

Candida albicans mutants lacking the cell wall biosynthesis kinase 1 form large aggregates and are unable to undergo morphological differentiation. Results show that the mutants exhibit altered expression pattern of many cell wall associated proteins.