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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.

Mice homozygous for a null glial-cell-line-derived neurotrophic factor (GDNF) allele exhibit the absence of kidneys, ureters and enteric nervous system (ENS). The mutants show defects in the sympathetic and nodose neurons, and dorsal root ganglion. The basal forebrain, cerebellum, striatum, neocortex, hippocampus, and the dopaminergic, catecholaminergic and noradrenergic neurons are normal. This indicates that GDNF is necessary for the development of ENS, kidney, and sympathetic and sensory neurons. The homozygous mice die within two days of birth while the heterozygous mice are normal in size.

Mice containing an inactive glial-cell-line-derived neurotrophic factor (GDNF) gene show defects in kidneys, gastrointestinal enteric innervation and peristalsis. The mutant mice show unilateral abnormally shaped kidneys, cortical cysts, unilateral renal agenesis and bilateral kidney dysgenesis. The homozygous mutants lack myenteric plexus neurons and enteric parasympathetic cholinergic ganglion cells. However, other urogenital organs, dopamine and cortical cholinergic neurons, and brain-stem motor nuclei are normal in the mutant mice.

The class IV POU-domain transcription factors, Brn-3.1 and Brn-3.2, have a specific spatio-temporal pattern of expression and regulate the final differentiation of sensorineural cells during neural development. Deletion of the Brn-3.1 gene causes loss of hair cells in the organ of Corti and sensory epithelia maculae and cristae. There is neuronal degeneration in the vestibular ganglion. Mice homozygous for deletion of the Brn-3.2 coding region have 60-70% fewer retinal ganglion cells than wild-type mice. The Brn-3.2 mutants behave normally while the Brn-3.1 mutants have defective balance.

A cross between tyrosine phosphatase CD45-deficient mice and mice carrying immunoglobulin transgenes specific for hen egg lysozyme (HEL) shows that CD45 positively regulates antigen-receptor signaling. CD45-deficient HEL-specific B cells show diminished signaling in response to HEL. Circulating HEL autoantigen controls negative selection of mature CD45(super +/+) HEL-binding B cells. The autoantigen positively selects CD45(super -/-) HEL-binding B cells. Inherited susceptibility to autoimmune disease is possible due to changes in antigen receptor signaling.

A lack of the signal transducer and activator of transcription 6 (Stat6) gene in mice affects the expression of cell surface markers, differentiation of Th2 helper T-cells, and immunoglobulin class switching to IgE. The absence of Stat6 affects IL-4 mediated functions while the lymphoid cell development is unaffected. IL-4-induced cell proliferation is only slightly affected. However, the mutant mice are viable and exhibit normal development.

The signal transducer and activator of transcription-6 (Stat6) gene plays a crucial role in the biological responses induced by interleukin-4 (IL-4). The expression of CD23 and the major histocompatibility complex class II are unaffected by IL-4 in mutant mice deficient in Stat6. B-cell proliferation, that is stimulated by IL-4 and anti-IgM antibody, is suppressed in the mutants. The formation of Th2 cytokines from T cells and the responses of IgE and IgG1 after nematode infection decrease. The responses compare well with those observed in IL-4 deficient mice and in response to IL-4 antibodies.

Embryos containing a single inactive vascular endothelial growth factor (VEGF) gene show abnormal development of blood vessels. The homozygous VEGF-deficient embryos exhibit more abnormality in the blood vessel development, and more fatality than heterozygous mutants. The haploid-insufficient phenotype is associated with heterozygous VEGF deficiency. VEGF participation in vascular development depends on the ligand dose. The embryonic phenotypes from germline transmission and aggregation of heterozygous mutant embryo stem cells with tetraploid hosts are similar.

The addition of eight amino acids to loop 3 present between helices C and D of interleukin 5 (IL-5) generates a monomer, whose biological activity is similar to that of IL-5. The monomer is formed by deleting residues from the IL-5's C-terminal or N-terminal, and inserting amino acids from the granulocyte-macrophage-colony-stimulating factor into the gap formed. The monomer induces proliferation of the IL-5-responsive TF-1 cell line. A single helical bundle regulates the functions of IL-5.

The leptin receptor variant, Ob-Rb, undergoes abnormal splicing in the C57BL/Ks mice containing a mutant diabetic (db) gene. Abnormal splicing of the protein results in a loss of the cytoplasmic area which is involved in signal transduction. Ob-Rb is expressed in high concentrations in the hypothalamus. Signal transduction by a hypothalamic leptin receptor is probably essential for the leptin's activity in decreasing weight. The leptin receptor, which maps the position of the db gene in mouse genome, has about six alternatively spliced forms.

The proto-oncogene Ets-1 is necessary for the survival and differentiation of T and B lymphocytes in the body. Embryonic stem (ES) cells containing mutant inactive Ets-1 genes form T cells which show DNA fragmentation and causes a high rate of cell death. The ES cells form a normal amount of B cells but many of the B cells preferentially differentiate into IgM plasma cells.

Homozygous mice in which the Hoxa10 of the Abdominal B gene is absent are viable. However, the males show bilateral cryptorchidism where the position and development of the testis is abnormal. Although only 40% of the homozygous male mice are sterile when young, the number of sterile males increases with age. The homozygous females show normal ovulation, but, due to death of the preimplantation embryos, almost 80% of the females are sterile. The reproductive tissues of these females did not show histological differences from the wild-type females.

There are two main theories to explain the ageing process. The first assumes that ageing is the product of the selection for a genotype which maximizes early reproductive and survival performance at the expense of senescent stages. Another explanation regards deleterious mutations,which accumulates at the later, and less selected part of life history, as the main cause of ageing. Evidence for both of these hypotheses exist, and the maincause of ageing has not yet been resolved. What is apparent is that ageing is acomplex, polygenic process which is not likely to be halted by genetic intervention.

A cation-sensitive ion channel can be turned into an anion-sensitive channel through site-directed mutagenesis involving three amino acids. The acids, from the MII segment of glycine or gamma-aminobutyric acid receptors, are inserted into the MII segment of the alpha-seven nicotinic receptor. The role of protein geometrical constraints on ion selectivity was underscored by the mutation involving the insertion of an uncharged residue at the amino-terminal end of MII.

A point mutation causes the fibroblast growth factor (FGF) receptor to lose its ability to link with the tyrosine-phosphorylate phospholipase C-gamma (PLC-gamma) or to bring about the hydrolysis of phosphatidylinositol. This point mutation involves the insertion of a phenylalanine residue (Y766F) in place of Tyr 766. However, the Y766F FGF receptor mutant is capable of autophosphorylation. Loss of phosphatidylinositol hydrolysis does not impair FGF-stimulated mitogenesis despite the importance of Tyr 766 phosphorylation for PLC-gamma phosphorylation and for phosphatidylinositol hydrolysis.

Fibroblast growth factor (FGF) receptor with a point mutation will no longer link with phospholipase C-gamma (PLC-gamma). This point mutation involves phenylalanine taking the place of tyrosine at residue 766. In addition, the mutant receptor will no longer regulate the hydrolysis of phosphatidylinositol (PtdIns) of the formation of Ca2+ following FGF stimulation. Hence the point mutation of the FGF receptor can sometimes stop activation of PLC-gamma. Moreover, FGF-induced mitogenesis can happen without Ca2+ mobilization or PtdIns hydrolysis.

The -117 point mutation in the gamma-globulin promoter induces the hereditary persistence of fetal hemoglobin (HPFH) phenotype. Moreover, the suppression of the gamma-globulin gene in mice produces a parallel suppression of the linked human beta-globulin gene. These results, obtained by introducing the point mutation into the gamma-globulin gene through genetic engineering, are important because the abnormal presence of gamma-globulin in adults due to HPFH is known to lesson the severity of two diseases, beta-thalassemia and sickle cell anemia.

The disease maturity-onset diabetes of the young (MODY) is a form of non-insulin-dependent diabetes mellitus and may result from the MODY1 gene mutation. MODY1 is the gene which is responsible for encoding the hepatocyte nuclear factor (HNF)-4 alpha. HNF-4 alpha is a part of the steroid/thyroid hormone receptor family and is responsible for the regulation of the HNF-1 alpha expression. HNF-1 alpha is a transcription factor involved in the control of liver genes in specific tissues.

The insertion of heterochromatin into a euchromatic gene causes position-effect variegation in Drosophila melanogaster. The insertion also results in association of the gene with heterochromatin. Proteins responsible for the formation of heterochromatin affect the interphase nuclear position of a chromosomal region. Heterochromatin and the proteins offer a structural framework for the interphase nucleus. A study uses fluorescent in situ hybridization to interphase diploid nuclei.

The activity of the Drosophila patterning system of anteroposterior subdivision of wing disc affects specification of the wing primordium in early second instar. The dorsoventral (DV) patterning system has no affect. The wingless gene (wg) has an important role in specifying the wing primordium. The gene controls the patterning activities of the DV compartment boundary. The DV system activates the wg and Vestigial proteins, and both the proteins are essential for the wing growth.

Author Affiliations: Axolotls have a remarkable ability to re-grow legs, making them useful models for limb regeneration. Getty Axolotls have a remarkable ability to re-grow legs, making them useful models [...], CRISPR provides window into axolotl limb replacement. CRISPR provides window into axolotl limb replacement.

Author Affiliations: Gene drives face an uphill struggle Genetic tools that aim to spread specific mutations through wild populations to eliminate diseases will need to overcome numerous sources of resistance. [...], CRISPR-based technologies could fight spread of diseases, if they can overcome resistance. CRISPR-based technologies could fight spread of diseases, if they can overcome resistance

Author Affiliations: This Lipizzan stallion is among the many modern breeds to have roots in the East. Spanische Hofreitschule Wien This Lipizzan stallion is among the many modern breeds to [...], Most modern horse breeds are descended from oriental stallions imported less than 700 years ago. Most modern horse breeds are descended from oriental stallions imported less than 700 years ago.

A mutation in the CC-chemokine receptor 5 (CCR-5) prevents HIV infection despite repeated exposure to the infection. Homozygous mutants of the gene are found in HIV resistant people. HIV-infected individuals have a lower frequency of heterozygous CCR-5 mutants. This indicates a partial protection from HIV infection in individuals with a single mutant gene. Individuals without functional CCR-5 are immunologically well.

Mutant mice lacking neuropeptide Y (NPY) show normal feeding and inhibition by leptin, but are more prone to seizures. The mutant mice maintain normal body weight despite the absence of NPY. Leptin causes an immediate and significant suppression of food intake. The lack of defects in feeding shows that other peptides control feeding in the absence of NPY. The tendency of the mutants to have seizures shows that NPY is involved in the suppression of hippocampal glutamate secretion.

A rare consequence of bone-marrow transplantation gave scientists the opportunity to observe the evolution of one cancer in two patients. Seven years after donating bone marrow and white blood cells [...]

Identification of a gene mutation in an alcohol-intolerant (ANT) rat may improve understanding of alcohol's physiological effect. The mutation, discovered by Esa R. Korpi and colleagues, consists of one amino-acid alteration in one subunit of a gamma-aminobutyric acid (GABA) receptor. ANT rats have a heightened susceptibility to alcohol possibly because the mutation leads to benzodiazepine-related motor impairment and abnormal reaction to GABA receptors.

Compounds are tested to determine if they are carcinogenic (cancer causing) to humans by exposing rodents to the maximum dose tolerated, and observing if cancer develops in the rodents' lifetime. These tests are slow and expensive. Compounds exist that directly cause changes, or mutations, in the DNA, the genetic information of cells, leading to cancer development. Other compounds directly cause mutations, but also cause more frequent cell division, which increases the chance that a mutation will occur. Some compounds do not directly cause mutations, but at the maximum tolerated dose, increase the frequency of cell division, which may lead to cancer development; these compounds may not be harmful at lower doses. Some compounds cause mutations in rodent DNA, but not in human DNA. Therefore, testing the maximum tolerated dose in rodents is not an effective way to determine carcinogenicity. Another strategy for testing is presented. Rodents can be used for short-term testing of the possible carcinogenic effects of compounds, but other tests should be done in tissue culture (outside of the body) to further examine the effects. (Consumer Summary produced by Reliance Medical Information, Inc.)

Males are more susceptible to certain mitochondrial diseases and mutations as compared to females. The maternally inherited Leber's hereditary optic neuropathy affects males more seriously and also has an earlier age of onset in males. Inherited mitochondrial diseases and somatic mitochondrial DNA are related to reduced sperm motility and poor male fertility. The detrimental effects of the increased equilibrium frequency of germline mitochondrial mutations depend on their correlated fitness effects when in females. The causes of male-female asymmetry in the mitochondrial diseases are discussed.

Mutations in the N-ethylmaleimide-sensitive fusion protein (NSF) cause temperature-sensitive paralysis in comatose mutants of Drosophila. NSF acts at the yeast vacoular vesicles at a pre-docking stage. It helps in folding the Botulinum neurotoxin substrates into a manner adequate for vesicle docking and fusion. The NSF takes more time to act than the synaptic vesicle recycling.

The expansion of microsatellites, small tandem nucleotide repeats sequences in the genomes of eukaryotes, initially occurs slowly during evolution. Analysis of the eta-globin pseudogene region in primates indicates that the region is just one nucleotide away from the formation of two types of simple repeat sequences. In hominoids, substitution of a G to an A occurs along the lineage leading to the common ancestor of the African apes and humans, and produces a tetranucleotide repeat. Point mutations produce repeat units for microsatellite expansion.