Besides water fleas (Cladocera) (Federici and Hazard ; Bergoin et al. The genomic diversity and phylogenetic relationship in the. control may decrease the risk of developing these complications, diabetes remains a very significant cause of social relationship was by Borch-Johnsen et al., who found that T1D children were breast-fed for shorter . The INS gene, located on chromosome 11p, has been designated as IDDM2. .. At the end of the. Ethical, Legal and Societal Challenges of Human Genome Editing Matthias Braun. terminating its life, it is prevented from existing as a human subject to whom the The relationships between “instrumentalization” and violations of human.
The role of molecular epidemiology in detecting developmental toxicants is discussed, as well as the difficulties in the detection of complex genotype-environment interactions. He was fortunate to choose several traits, each of which was controlled by a single genetic locus. The alleles at each locus, when inherited, acted in either a dominant or recessive manner, and their action was not significantly influenced by other genes or by environmental factors under his conditions of testing.
Consequently, he observed precise and interpretable mathematical ratios for the phenotypes of the progeny in each breeding experiment. Traits of phenotype that show such easily interpretable patterns of inheritance are called simple, or Mendelian, traits, and these generally are governed by a single genetic locus. However, the relationship between genotype and phenotype is almost always very complex.
Even when scientists consider one particular gene and know its particular allelic form, its effect on phenotype is often subject to either or both of two variables: Such traits display a multifactoral pattern of inheritance also called complex or non-Mendelian inheritance and are termed complex traits or multiplex phenotypes for a recent review, see Lander and Schork Multifactorial inheritance is much more common than simple inheritance.
Such traits entail the interaction of two or more genes a polygenic trait. The genes can contribute to the phenotypic trait in a quantitative and additive manner e.
Still more complex patterns of inheritance can be traced to multiple genes acting in nonadditive manners. Segregated alleles might be neither dominant nor recessive. Finally, a gene might show incomplete penetrance only some members of the population show the trait or variable expressivity members of the population vary in the extent of the trait or both.
Other traits are modifiable by the environment. Such traits are not at all unusual and might overlap with polygenic traits. Studies in model organisms, such as the fruit fly Drosophila melanogaster, have long shown that a gene's effect on a trait can be modified by such extrinsic factors as temperature, chemicals, nutrition, and crowding.
Although multifactorial inheritance is a nuisance to geneticists, it describes most human heritable diseases and virtually all susceptibilities. Humans and experimental animals are notoriously heterogeneous in their responses to drugs or environmental pollutants.
The favored explanation at present is that the heterogeneity reflects a combination of the heterogeneous exposure circumstances extrinisic conditions and heterogeneous genotypes for susceptibility intrinsic conditions. Examples of exposure plus susceptibility would be the age of onset of lung cancer in cigarette smokers or the likelihood of asthma induced by urban pollution. The gene-environment relationship is further confounded in developmental toxicology by the need to consider the genotype of both the mother and the embryo or fetus, how and where a toxicant is metabolized, and the developmental stage at which a toxicant crosses the placenta.
Gene -environment interactions are obviously relevant to the fields of molecular epidemiology and developmental toxicology. Whatever the frequency, alleles are now defined in the most general way, namely, as different nucleotide sequences of the same gene—that is, as changes of one or more bases adenine, thymine, cytosine, and guanine relative to the reference DNA base sequence.
However, finding such a difference does not in itself reveal much about an effect on phenotype. If the sequence difference occurs in a coding region of the gene, protein activity or stability might be affected.
If the change is synonymous i. If a DNA sequence change occurs in the transcribed region of the gene but not in the coding region, it might affect the reading frame, splicing, mRNA stability, translation efficiency, or transcriptional regulation. If outside the transcription region of the gene, the change still might affect the time, place, and level of expression of the gene, although not the protein's sequence. Additional work has to be done to identify the effect of the particular DNA change on protein function or level.
Such a polymorphism might have no effect on protein activity or amount. It would just be a marker of that lineage of organisms. It might even have a negative selective effect. Modern sequencing methods have greatly increased the capacity of researchers to detect alleles. For a particular gene sequence, any two unrelated people within a population are likely to have sequence differences.
A gene sequence is taken to include all regulatory and transcribed regions of the DNA. Before this synthesis, the base change often results in a mismatch in the DNA double helix, and a number of mismatch repair enzymes remove such errors Snow However, out of every million or more DNA sites that become damaged, an error occasionally escapes uncorrected.
Unrepaired mutations are thought to occur naturally at frequencies of once per bases per generation. Because humans have such a large genome, roughly 75 new mutations accumulate per human individual per lifetime. Most of these are probably not deleterious.
Some are deleterious, however, and the deleterious mutation rate in humans nonsynonymous amino acid changes affecting activity has recently been estimated to be at least 1. It is likely that the human population is full of genetic variation, and this variation must be considered and appraised in any evaluation of an individual's susceptibility to developmental toxicants. The study of the genomes of organisms, which is called genomics, includes areas of research determining the genetic and physical maps of genomes, the DNA sequences of genomes, the functions of genes and proteins, the cis-regulatory elements of genes, and the time, place, and conditions of expression of genes.
A prominent part of genomics has become the managing of the massive amount of gathered information a field referred to as bioinformatics and the analysis of data with regard to, for example, aspects of the organization of the genome, the comparison of genomes of different organisms, and the global patterns of expression of genes.
The immediate goal was then, as it is now, to complete the accurate sequencing of the approximately 3. In the longer term, a goal is to identify all human genes. In an organism such as yeast, which is favorable for the identification of genes by mutational genetic analysis, more than half the genes had gone undetected until the genome sequence became available Brown and Botstein The lack of detection was in part due to large redundant regions of the yeast genome.
In vertebrates, mutational genetic analysis is much more difficult, and redundancy might be more widespread.
Therefore, initial gene identification by sequencing is the approach of choice. A gene is initially identified as an open reading frame ORFwhich can be discerned directly by looking at the sequence, or it is initially identified as an expressed sequence tag EST site, a sequence complementary to a known piece of transcribed RNA see below.
Thereafter, the goal is to identify each gene as a sequence encoding a full-length RNA and a protein of known function. The functions of nontranscribed regions, such as the numerous large cis-regulatory regions setting conditions for gene expression, will have to be elucidated as well. This task is still more difficult, currently involving a number of approaches, including the construction of transgenic animal lines carrying portions of the regulatory region in conjunction with a reporter gene e.
Department of Energy DOE at 22 specialized genome research centers in the United States and in many university, national, and private-sector laboratories.
At least 14 other countries also have programs for analyzing the genomes of various organisms—ranging from microbes and economically important plants and animals to humans.
The explosion of genomics information has occurred sooner than the most daring scientists would have predicted. Following the first complete genome sequence, that of Haemophilus influenzae inseven more genomes were completed in the next 18 months, namely, four more eubacterial genomes, two archaebacterial genomes, and one unicellular eukaryote genome—that of the yeast Saccharomyces cerevisiae.
In Decemberthe genome of the first multicellular eukaryote, Caenorhabditis elegans, was completed kilobases of DNA sequence and 19, genes identified at least as ORFs. As of the end ofmore than 30, human genes had been partially identified, located, and sequenced.
Human chromosome 22 has been sequenced and is projected to contain at least genes Dunham et al. In the mouse, at least 14, genes have been described.
The fruit fly Drosophila melanogaster sequence was completed in Adams et al. Bynumerous nonhuman genomes will be sequenced as well, including the mouse Mus musculus, the zebrafish Danio rerio, the silkworm Bombyx mori, the rat, dog, cat, chicken, rice, corn, wheat, barley, cotton, the plant Arabidopsis thaliana, and probably also the cow, sheep, pig, and horse.
The sequencing of the mouse genome is running well ahead of schedule. New technologies, resources, and applications have become increasingly available to researchers of many diverse scientific fields, including cancer research, drug discovery, medical genetics, and environmental genetics, and their availability should also accelerate numerous major advances in developmental toxicology in the next decade, as discussed later in this chapter.
Functional Genomics and Microarray Technology From the outset, it was expected that the completion of sequencing of the human genome would mark but a first step in the HGP. The information about the sequence and location of genes in the genome will greatly facilitate further studies—not only of human genetic variability but also of functional genomics.
As noted above, the latter is the comprehensive analysis of gene expression and gene-product function. In the cases of yeast and C. Some of this functional analysis can go forward even before a genome is sequenced. In the case of humans, the study of ESTs has been an important step of such analysis. The information is entered in a database. These sequences represent genes expressed in the human. For example, more than 1 million human ESTs are now available, representing greater than 50, genes.
The most comprehensive libraries are prepared from a wide range of tissues and times of development in an effort to include all expressed genes. Unfortunately for developmental toxicologists, although the initial sources of RNA included placenta, they were underrepresented in the variety of early embryonic tissue.
New methods have become available to obtain full-length cDNAs from transcripts, and these will be more useful than fragments. A further step of analysis of genome function is the determination of the time, place, and conditions of expression of each gene. Until recently, this analysis has been done one gene at a time. DNA microarray techniques recently have made possible the description of simultaneous changes of thousands of genes as cells and tissues undergo development or various changes of environmental conditions.
The technology is now suitable for simultaneously comparing the amounts of thousands of kinds of mRNA in two tissues or cell samples e. To do the comparison, thousands of different DNA sequences e. The expense and time of producing such slides are modest enough that a hundred or so can be prepared, each serving for the analysis of one comparison condition e.
In the procedure of Brown and Botsteinthe tissue samples for comparison are separately extracted and the mRNAs are labeled with different fluorescent dye molecules, say green for the control and red for the treated tissue see Figure The slide is then read in a fluorescence microscope to see if each particular DNA spot has bound more of the green or rRNA. The ratio of red to green tells whether a particular gene is expressed more or less than normal under the treatment condition.
Yellow is seen when equal red and green mRNA has hybridized. This technique has been applied recently to human cells cultured in the presence or absence of serum. Indeed, hundreds of genes changed expression, including many genes encoding stress-related proteins seen in wound healing Iyer et al. The technique also has been applied to yeast cells progressing along the sporulation pathway Chu et al.
Recently, it has been used to discover the response of a single kind of cell to two signaling ligands, each acting through a different receptor tyrosine kinase Fambrough et al. In all cases, the expression of hundreds of genes changed. Viewing global patterns reveals that each gene does not seem to behave individually; instead, concerted expression of large batteries of genes seems to occur under various conditions.
These results give credence to the value of analysis of the global patterns of gene expression. Previous studies of individual genes might have missed large-scale patterns. However, much remains to be done in the interpretation of the manifold changes of gene expression. As mentioned above, hundreds of gene expression changes are observed even with seemingly modest changes in a cell's circumstances, such as its ploidy.
As shown in the upper left, mRNA is prepared separately from two kinds of cells, such as normal and tumorous, and each mRNA is converted more In the analysis of toxicant effects, it is expected that cells or organisms could be treated with toxicants of unknown mechanism of action, and the changes of gene expression could be profiled by the DNA microarray method.
If enough were known about the function and interaction of proteins encoded by the genes undergoing changes of expression, sound deductions might be made about the mechanism of action of the toxicant.
In the future, it is expected that DNA microarray methods will allow rapid and detailed characterization of a cell or organism's response to a toxicant.
As more information is collected, different toxicants can be grouped by their similarities of effect, and the analysis of toxicant action can be pursued on a more systematic basis.
The DNA microarray method is already in use to compare normal cells and cancer cells.
Many of the questions of interpretation of gene expression differences are being explored in that case as well. Although it is preferrable to have a large set of cloned and sequenced DNAs representing different identified genes for the microarray, as is the case for yeast, it is not necessary. ESTs have been useful already for human and mouse studies, as in the serum study mentioned above Iyer et al. If expression of a particular sequence, known only by its ESTis found to change greatly in the test condition, it might then qualify as interesting enough to deserve full-length cloning, sequencing, and further analysis of function.
Vast amounts of data accumulate in such comparisons e. The multidimensional data sets have challenged applied mathematicians to find means to express them in ways useful to biologists e.
Yet, larger data sets loom on the horizon e.
Could WA be the genetic testing ground for 'synthetic mice' to end mice?
As described below, the demand is great for managers and analysts of these data sets. Although the various microarray techniques promise to reveal exciting new information about where, when, and under what conditions the genes of the genome are transcribed, this approach will not provide information concerning the translation and post-translational modification of proteins encoded by these mRNAs—that is, information about when and where the proteins are present and active.
Protein function is almost always the immediate cause of cell function. To provide such functional information is the goal of proteomics.
A large amount of 2D-gel information is stored in the Proteomics database see Appendix B for the Internet address. Plans have been made to identify every protein spot on a 2D gel, because the nanogram amount of protein in a spot is sufficient to determine a partial amino acid sequence by tandem mass spectrometry Yates The partial sequence can then be looked up in the genome database and the protein identified.
When proteins are modified by phosphorylation, acylation, glycosylation, farnesylation, limited proteolysis, or any of the other 30 or so covalent post-translational alterations, their migration on a 2D gel changes, thus allowing a correlation to be made with their activity or inactivity in the tissue.
Finally, many proteins are required to associate with other proteins in order to achieve activity, and there are various nondenaturing gel-electrophoresis methods to detect such associations. Current and future efforts can be expected to produce new technological advances for the analysis of proteins and their functions.
In developmental toxicology, the combination of genomics and proteomics offers the possibility of assessing developmental toxicants not only for their capacity to alter gene expression but also for their capacity to alter protein function. Applications of Genomic Technologies Researchers have recently made use of the genomic technologies to identify and sequence genes with a role in disease etiology.
It is probable that these genomic technologies will be applied to the study of the effects of chemicals on development in the near future. Two models already exist that demonstrate the application of new genomic technologies: The goal of CGAP is to provide a complete catalogue of all genes whose expression changes in cancer cells relative to normal cells for all types of cancer Pennisi In the past, a major barrier to the analysis of cancer cells has been the mixture of cell types normal and cancerous present in a typical tumor.
We separately considered coronary artery disease identified by coronary artery calcification on CT and coronary stenosis by coronary angiography.
Coronary calcification can be an earlier manifestation of coronary artery disease and is not equal to the degree of coronary stenosis produced by atherosclerosis that consists of different plaque components especially cholesterol[ 29 ]. Although the two conditions were examined separately, the results indicate that ADAMTS7 is associated with both conditions suggesting separate but complementary processes. The strongest relationship and consistent across two different studies was for rs ADAMTS7 expression was upregulated and was associated with calcification of radial arteries of patients with chronic kidney disease and uremia[ 30 ].
The data suggests that upregulation of this metalloproteinase mediates vascular smooth muscle calcification[ 30 ]. COMP or thrombospondin-5 is abundant in the vasculature[ 11 ]. The amount of CAC is a reflection of the underlying burden of atherosclerosis in coronary arteries and the presence of CAC is a prognostic marker for adverse outcome from coronary artery disease[ 29 ]. Coronary artery atherosclerosis The significant relationship of ADAMTS7 to coronary atherosclerosis is part of its general effect on atherosclerosis.
Matrix metalloproteinases can have a profound effect on the vessel wall[ 33 ]. Metalloproteases play various roles in atherosclerotic plaque formation including vascular smooth muscle cell migration and plaque instability[ 34 ].
In addition, the rs was associated with less severe CAD as reflected in lower Gensini and lower Sullivan scores which quantitate the extent and severity of CAD[ 18 ]. Genetic changes in ADAMTS7 are presumably translated into changes in protein secretion and increases in circulating levels. Plasma ADAMTS7 levels are significantly higher in patients with more severe coronary artery disease Plasma ADAMTS7 levels were significantly and positively correlated with the severity of coronary artery disease as reflected by the Syntax score even in multivariate analysis[ 35 ].
Genotyping for rs polymorphism show that the AA genotype is an independent risk factor for CV mortality compared with reference genotype GG[ 28 ].
Similar data was identified in a another cohort as survival was best for GG genotype, worst for the AA genotype and intermediate for the AG genotype[ 18 ]. Prognosis is in part determined by the nature of the coronary plaque and its probability of progression or rupture. The interaction of genes and environment have produced the interesting observation that Allelic variations at rs that are associated with reduced ADAMTS7 expression confers CAD protection which is stronger in never-smokers than in cigarette smokers[ 36 ].
The mechanism by which ADAMTS7 produced coronary atherosclerotic narrowing is not fully delineated but there are several explanations.
ADAMTS7 localizes to smooth muscle cells in human coronary artery disease lesions, with a subcellular localization at the cytoplasm and cell membrane, where it co-localized with podosomes[ 15 ].