A conclusion one could draw from the first two chapters is that the role of genes in determining phenotypes is complex and dynamic. That is true. It is an important insight that should be kept in mind when evaluating a genetic question. But, by the same token, complexity does not necessarily mean that rules and order are weak. In this chapter we will explore some of these rules, specifically the question of how gene regulation activates a particular gene in the appropriate developmental context and how molecular signals carry out largescale patterning to organize the structure of the body in early development.
To a degree that still impresses most biologists, there is extensive similarity, or homology, in the genes that control development in humans and in model organisms like fruit flies, nematodes, and zebrafish. These homologies underlie the unity of life. The insights the field has gained from model organisms will help tell the story of genetic control of development in humans.
Part 1: Background and Systems Integration
Overview of Timing and the Processes at Work
The nuclear control of development does not actually begin when the genes of an egg and a sperm fuse at fertilization. The earliest stages of embryonic development are actually controlled by the mother’s genome. Molecular signals like inducers and mRNA coded by the maternal genome are stored in the cytoplasm during egg formation. Very few genes in the new individual are transcribed at first. Genetic influences at this early stage are called maternal effects. Details vary among organisms, but basically the fertilized egg nucleus divides several times to become a ball of cells, the morula. The cells then form a fluid-filled ball, the blastula or in mammals the blastocyst, in which cells are set off to one side. The internal space keeps cells that will become ectoderm and endoderm from interacting prematurely and provides a space for cell movement. In humans the embryo forms the morula as it travels along the fallopian tube, and by 41/2 to 5 days it has formed into a blastocyst and enters the uterine cavity. This blastocyst implants into the uterine wall about 6 days after fertilization.
It is at this blastula stage that the embryo’s own genes first become active and take over control of development. One process that may trigger this transition is the demethylation of various promoters that then bind with transcription factors to initiate transcription. At that point there can be cytoplasmic differences among the blastula cells, blastomeres, due to process like the partitioning of inducers and RNA molecules in the original egg cytoplasm. The resulting transcriptional cascades in different parts of the blastula ultimately contribute to the patterned organization of tissues, organs, and organ systems of the fetus and adult.
The process of early development can be conveniently described in terms of three types of processes: cellular differentiation, pattern formation, and morphogenesis. They are not mutually exclusive. Cellular differentiation is the gradual specialization of cells. Cell specialization is determined by the specific array of genes that are active in the cell lineage as it forms, and it can be described in terms of the cell’s biochemical profile, thus its molecular structure and function. Pattern formation is the establishment of spatial organization of differentiated cells. It is the series of processes like cell signaling and gradient formation that establishes the spatial addresses of cells and tissues with respect to each other. Patterned interactions form the basis for succeeding phases of developmental specialization and the temporal changes in spatial arrangement that occur as the embryo forms. Morphogenesis is literally the “origin of form.” The sculpting of form involves the movement of cells and sheets of cells as well as events like programmed cell death, apoptosis. Our focus will be on the way the genome controls these processes during early development. In humans most of this occurs during the first trimester of development, because, except for the brain, the main thing the embryo does after the first trimester is simply grow larger.
Control of Gene Action–Models From Bacteria
The organization of gene action in bacteria is different from that in eukaryotes in many ways. But insights gained from studies of bacterial gene regulation help us better understand the more complex and flexible eukaryotic processes. For this discussion we will define structural gene as a gene that codes for the synthesis of a polypeptide (although we will not argue if someone wants to include the genes for RNAs like the tRNAs and rRNAs in the mix). Other genetic sequences have a regulatory role, such as serving as binding sites for transcription factors and polymerase. Indeed, the wide array of functions that are being discovered for regions of the genome make it increasingly difficult to define what we really mean by “a gene.” An important characteristic of bacteria is that several genes needed at the same time to carry out a particular activity can be linked under the control of a single set of regulatory genes, a genetic organization called an operon. In eukaryotes, each structural gene has its own individual regulatory gene control system. Bacterial operons give us ideas about the regulatory strategies the more complex eukaryotic cell might employ. Our discussion of operons will, however, be relatively brief and will not cover the full range of specific examples that are now well understood. But it is a convenient way of introducing some useful terminology.
Gene action, or turning genes “on” and “off,” is simply another way of describing the regulation of transcription. Typically this involves transcription factors, which are regulatory proteins with at least two active sites. One active site binds to DNA and the other binds to a small effector molecule that can change the conformation of the regulatory protein and, thus, its DNA binding domain. Some regulatory proteins are repressors that inhibit transcription when they bind to DNA. Others are activators that increase the rate at which transcription occurs. Representative molecular interactions are shown in Figure 3-1 for inducible genes and repressible genes. We can see how this works by looking at the relatively simple regulation of bacterial operons.
Figure 3-1. Regulatory proteins for inducible and repressible genes. An active repressor protein will inhibit transcription, but binding with an inducer will alter its structure so it cannot bind to DNA. An active activator protein bound to an inducer will promote transcription, but binding to an inhibitor will shut transcription down. In these examples, the regulatory protein has two binding sites. One region can bind to DNA, and the other can bind to a small effector molecule (inducer, corepressor, or inhibitor). (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
When you think about how a process can be controlled, there are two obvious alternatives. It can be “off” because it is not needed except under specific conditions. When needed, a molecular signal turns it “on.” It is inducible. Or it can be “on” because its product is needed continuously, but it is turned “off” when its product has reached a required level. It is repressible. It is wasteful of precious energetic resources to continue to produce even a necessary product when there is a sufficient supply. During development, probably most genes operate inducibly. They are turned on when their coded enzyme or structural protein is needed by the cell in which they will work. Both approaches are dynamic, in the sense that they are reversible as the need changes.
One of the first bacterial operons to be studied in detail was the inducible lactose, or lac, operon (Figure 3-2). There are three structural genes, lacZ, lacY, and lacA. Several regulatory gene functions are also involved. In this example (Figure 3-3), a regulatory gene, lacI, codes for a repressor protein. By binding to the Operator site (lacO), the repressor protein physically blocks binding or transcription by RNA polymerase. If the Operator gene site is not blocked, the Promoter gene (lacP) can bind with RNA polymerase. Transcription continues until a termination signal is reached. The resulting mRNA is a transcript of several tandem structural genes. For each coding region, it has individual start (AUG) and stop (UAA, UAG, or UGA) codons. The term cistron is sometimes used by geneticists to describe a gene. The word comes from a test for allelism. The mRNA transcript in bacteria is, therefore, polycistronic, in that each transcript carries the information of several functionally-related genes.
Figure 3-2. Organization and function of the bacterial lactose (lac) operon. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 3-3. The lac operon is inducible. The lac repressor is synthesized in an active form, so transcription is inhibited until the appropriate inducer, allolactose, enters the cell. When it binds to the repressor protein, a conformational change causes it to release from the DNA and allow RNA polymerase to transcribe the three genes of this operon. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Since a regulatory gene makes a repressor molecule that is diffusible throughout the cell, its regulatory action is at a distance from the regulatory gene itself. It is said to be transacting, literally “acting across.” The Operator, on the other hand, regulates transcription of the structural genes to which it is directly attached. It is said to be cis-acting.
The lac operon is inducible, because the lacI regulatory gene codes for an active repressor protein. This has DNA binding sites that attach to the operator and inhibit RNA polymerase. Transcription of the operon is, therefore, at a very low level (essentially “off”) until something induces it to begin working. In this case, the inducer is the sugar allolactose, a derivative of lactose, the carbon and energy source that proteins coded by the lac operon process for the bacterial cell. It is not the preferred sugar resource, so the operon is off until its preferred source, glucose, has been depleted.
In contrast, a repressible operon controls a pathway that is needed continuously by the cell. The tryptophan, or trp, operon is a good example (Figure 3-4). It codes for enzymes that catalyze the production of tryptophan, an amino acid. The essence of the process is that when tryptophan levels are low, the operon is transcribed normally. But when tryptophan levels are high, the tryptophan participates in a feedback reaction to inhibit transcription temporarily. It is a waste of energetic resources to continue producing it. When sufficient product is present, the operon is repressed. Feedback inhibition is an important element in many regulatory pathways. An example of such a mechanism is illustrated in Figure 3-5. When the concentration of an end product of a biochemical pathway is high, it can bind with an inhibitor site on an earlier enzyme to inactivate it temporarily.
Figure 3-4. The tryptophan (trp) operon is repressible. At high-end product levels, tryptophan binds to the trp repressor protein and inhibits further transcription. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 3-5. An example of feedback inhibition of enzyme activity. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Control of Gene Action in Eukaryotes
Eukaryotes have a much more complex cell structure than that found in prokaryotes. Most are multicellular with a range of cell types reflecting the division of labor for specialized structures and functions. This requires a higher degree of flexibility in genetic regulation to allow gene products to be produced in the various combinations needed by different cell types. In eukaryotes, transcription factors regulate the binding of the transcription complex to the core promoter, and they initiate the elongation phase of RNA transcription. There are three classes of transcription in eukaryotes characterized by their promoters, and different RNA polymerases transcribe each one. RNA polymerase I transcribes rRNA in the nucleolus region of the nucleus. RNA polymerase II transcribes mRNA throughout the genome. RNA polymerase III transcribes tRNA and some other types of small RNA.
General transcription factors are needed to bind RNA polymerase to DNA at any type of promoter region. Some promoters have short upstream sequences (i.e., a string of nucleotides located before the beginning of a coding sequence) that bind upstream factors that increase the efficiency of transcription initiation. Inducible or regulatory transcription factors have a regulatory role and are produced at specific times in each cell type. They bind at short DNA sequences called response elements and affect the patterns of transcription sequentially through development. Not surprisingly, transcription factors make up a large family of genes. About 10% of the proteins coded for in the human genome have DNA binding domains, so most of these are likely to be transcription factors. This makes them perhaps the largest family of proteins coded by the human genome.
Regulatory transcription factors can either activate or repress transcription (Figure 3-6). Indeed, transcription in eukaryotes has many possible influences. In addition to activator and repressor proteins that might themselves be affected by binding with small regulatory molecules, transcription can be inhibited by methylation. The degree of DNA compaction, i.e., its looping, supercoiling, and bonding with nucleosomes, is also an important variable in the process. At this point, however, we will focus on the role of transcription factors. DNA structure will be discussed further in Chapter 4.
Figure 3-6. Regulatory transcription factors can act by either increasing or decreasing the rate of transcription. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Since the mechanisms of transcriptional regulation are so central to the expression of genetic information, it should be no surprise that structural similarities can be seen in families of transcription proteins from even distantly-related organisms. Proteins have domains with specific functions. One transcription factor domain has characteristics that enable it to bind with DNA, while one or more other domains might bind with small effector molecules or other proteins. In Figure 3-7, several common domain structures, or motifs, are shown. Many transcription factors have regions of α-helix coiling. The width of the α-helix fits well into the major groove in DNA. The helix-turn-helix and the helix-loop-helix structures can form hydrogen bonds with nucleotides in the major groove, thus allowing each transcription factor to bind to a specific DNA region. In a similar way, a zinc finger and a leucine zipper bind specific nucleotides in the DNA major groove. The helix-loop-helix and the leucine zipper often cause pairs of transcription factors to bond, forming a protein dimer. A homodimer is where the two factors are the same; a heterodimer is made up of two different transcription factors. This contributes to the wide diversity of regulatory structures that can be produced.
Figure 3-7. Some of the common structural motifs of transcription factors include: (a) helix-turn-helix; (b) helix-loop-helix; (c) zinc finger; and (d) leucine zipper. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
The promoter sequence in eukaryotic genes is, therefore, more complex than the promoter in prokaryotes (Figure 3-8). Regulatory transcription factors that serve as activator proteins will bind with an enhancer sequence to increase the rate of transcription. This is up regulation. When a repressor protein binds to a silencer sequence, the rate of transcription is down regulated. Enhancer and silencer elements are often found within about 100 bp from the initiation start site, but they can be located much further away and still have a strong effect due to conformational changes in DNA folding.
Figure 3-8. Examples of how a regulatory transcription factor influences transcription. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Instead of binding to the RNA polymerase directly, most regulatory transcription factors work indirectly by influencing other proteins that bind with the polymerase. Two such intermediaries are TFIID and mediator. TFIID is a protein complex that works as a general transcription factor. It binds to a sequence called the TATA box in the core promoter of eukaryotes (Figure 2-18) and then binds with other transcription factors that facilitate binding RNA polymerase II to structural gene promoters. Mediator is also a protein complex that binds with other general transcription factors and with RNA polymerase. Depending on these binding interactions, mediator can either lead to up regulation or down regulation of transcription. Additional information on transcriptional regulation will be introduced in other chapters. An especially important example will be the interaction of steroid hormones with their specific regulatory transcription factor, the steroid receptor.
Cellular Differentiation
From the examples presented here, it is clear that eukaryotes call upon a range of mechanisms to influence the transcription rate of each gene. In some cases, an effector molecule like a hormone can activate a transcription factor (Figure 3-9). In other cases, protein-protein interactions or phosphorylation can turn a gene on. An activator protein might bind at several different genes that have the same response element sequence, and the product of one gene might affect the regulatory signals of other genes. These interactions can cause a cascade of transcriptional activities that is different from one cell type to another. Cellular differentiation is essentially the process by which cells become different. That difference is reflected in the profile of proteins that affect its composition and function at any given time during development.
Figure 3-9. The activity of regulatory transcription factors can be influenced by binding to small effector molecules, such as certain types of hormones, or by protein-protein interactions or phosphorylation. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Cell-to-cell communication is a critical component of the cellular differentiation process since many of the signals that determine the appropriate transcriptional responses come from outside the cell. In most cases, the extracellular signal is a molecule that cannot pass through the cell membrane. Instead, it binds to a receptor in the membrane and initiates a chain of events by way of a secondary cascade inside the cell. When an activated membrane receptor initiates a response pathway inside the cell, the process is called signal transduction. The signal is literally transduced or “carried across” the membrane. A molecule that is produced intracellularly in response to signal transduction is called a second messenger. Thus, in order to respond, the target cell must be preprogrammed to detect the presence of the extracellular signal.
First, a signaling molecule binds to a specific protein receptor on the outer membrane. Either directly through protein kinase activity associated with the receptor or indirectly through a G protein, it activates a target protein in the cytosol. A protein kinase is an enzyme that affects activity by adding a phosphate group to an amino acid of another protein. G proteins have the ability to bind guanine nucleotides. The inactive form is a trimer bound to guanine diphosphate (GDP). The active receptor replaces this with guanine triphosphate (GTP), which then causes the G protein to dissociate into a monomer carrying GTP or a dimer. One of these acts upon a target protein that causes production of a second messenger.
An example of this chain of events is the cAMP response element-binding (CREB) protein (Figure 3-10). Cyclic-AMP (cAMP) plays the role of an intracellular messenger in many pathways. In this case, an activated membrane receptor activates adenylate cyclase (also denoted, adenylyl cyclase) that catalyzes the production of cAMP from ATP. The cAMP second messenger then activates protein kinase A, which phosphorylates the CREB protein in the nucleus to form a dimer. This dimer is an active regulatory transcription factor that initiates transcription at genes that have a cAMP response element (CRE) upstream of the core promoter of a target gene.
Figure 3-10. Signal transduction is illustrated by the sequence of events involving the CREB protein. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
These events are, of course, also accompanied by cell division. The control of cell division rate shares many of the general mechanisms described here. But we will focus upon that important process separately in Chapter 4. By cascades of molecular interaction within both the cytosol and nucleus and through signal transduction in response to extracellular signals, transcription of genes in a cell lineage ultimately leads to the formation of each of the differentiated cell types of the adult body.
Pleuripotency and Developmental Plasticity
The ability of a cell to differentiate into various cell types is unlimited in the fertilized egg and early cells, such as those in the morula of the human embryo. At this point, cell fates are unrestricted, since all cell types of the developing body plan must trace their lineage from them as the embryo grows. The nuclei are said to be totipotent, i.e., “totally potent, or totally competent” in developmental opportunity. If the embryo is divided into two or more separate groups of cells at this stage, as in monozygotic (identical) twins, each subgroup of cells has the capacity to yield all cell types in the adult body. But developmental plasticity is gradually lost as the individual develops (Figure 3-11). Many nonmammalian organisms lose totipotency earlier than in mammals, and plants tend to retain it much longer. In the blastocyst stage of human development, when the individual’s own genome begins to control cellular differentiation, the embryonic stem cells have become pluripotent. Each is capable of differentiating into many different cell types, but not into all of them.
Figure 3-11. The ability of stem cells to differentiate into other cell types is gradually restricted during development. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Totipotency and pluripotency are significant for several reasons. If one or a few cells die in early development or are removed from the embryo, their developmental role can be taken over by the remaining cells. Cells removed from an embryo at this time are totipotent or pluripotent stem cells that can divide in cell culture and potentially differentiate into a range of specialized cell types for therapeutic applications. But as embryonic development continues, cells lose even more plasticity. In the adult there are still some stems cells that retain the ability to differentiate into a limited range of types. Hematopoietic stem cells in the bone marrow, for example, are multipotent. But many cell types become fully restricted. Unipotent stem cells are only capable of replacing cells of the same type.
Not all organisms share the same limitations of plasticity. In some the cell fate is determined very early. If a few cells in the Drosophila blastoderm are destroyed with a hot needle, a corresponding part of the adult body is missing. The cell lineage in the nematode, Caenorhabditis elegans, has now been mapped in such detail that the steps of cellular differentiation are known for every cell in the body (Figure 3-12). As mentioned earlier, plants are at the other end of the spectrum. Their cells seem to retain a greater degree of plasticity longer than do most animal cells. Plant cells also have a greater potential to revert from a specialized type into a totipotent one, but this potential can also be generated in mammals as seen for example in cloning an animal like Dolly, the sheep.
Figure 3-12. A brief summary of the cell lineage of the nematode, caenorhabditis elegans, which has now been mapped for each cell of its body. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Pattern Formation
It is not enough that cells have the capacity to specialize into the range of cell types found in the body. They must do so in the correct place, relative to other cell types. Pattern formation is the result of processes that specify the spatial “address” of cells and determine the pathway toward specialization that they will follow. We will illustrate this concept by exploring how the diffusion gradient of an effector molecule, a morphogen, can signal positional information within a developmental field (Figure 3-13). Positional information can also be signaled by direct cell-to-cell or cell-to-extracellular matrix contact by means of cell adhesion molecules and other signaling mechanisms.
Figure 3-13. Three different mechanisms by which positional information can be communicated within a cell population. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
A morphogen is a small diffusible molecule whose concentration can be detected by cells within a limited region of tissue, the developmental field. If the diffusible morphogen is synthesized in one region, sometimes called the source, its concentration declines as a function of distance from the source. Such a concentration gradient can become stable over a small distance if the morphogen is inactivated or destroyed at another point, the sink. Concentration of the morphogen along the gradient communicates a cell’s relative position within the field. In one well-studied example, the anterior-posterior body axis of Drosophila is established by diffusible proteins, such as bicoid, produced by maternal mRNAs deposited in the developing oocyte (Figure 3-14).
Figure 3-14. Immunostaining of bicoid mRNA and protein illustrates a morphogen gradient in the early Drosophila egg. The bicoid mRNA is trapped near the anterior end of the developing oocyte. (b, c: Christiane Nüsslein-Volhard, Development, Supplement 1, 1991. © The Company of Biologists Limited.)
Morphogenesis
In addition to positional information signaling the events of differentiation, it can also specify the pattern of cell death that helps shape the body. Body form is also determined by the movement of cells and sheets of cells that define positional and signaling interactions. Morphogenesis is the process of shaping body form.
The earliest morphogenetic event in the embryo is gastrulation, in which a portion of the outer layer of the hollow blastocyst folds inward to produce a two-layer embryo with an outer ectoderm and inner endoderm. At about the same time, out pockets of tissue from this inner layer become a middle layer, the mesoderm. All tissues of the adult derive from one or other of these three layers (Table 3-1).
Table 3-1. Derivatives of the Three Embryonic Layers
The next critical morphogenetic event in mammals is neurulation in which a fold occurs in the ectoderm. This defines the dorsal midline and produces the dorsal hollow neural tube, the first step in organogenesis. Many later developmental events are organized around this axis. The establishment of body axes at an early stage of embryonic development is the foundation of embryonic organization. The genes responsible for such fundamental steps are surprisingly well conserved across a wide taxonomic spectrum. But like many key genetic mechanisms, they were first discovered in Drosophila.
The Organization of Embryonic Development
The organization of embryonic development in Drosophila involves two sets of genes. Segmentation genes divide the body into a series of similar segments, and the homeotic genes define the way in which each of these segments will develop. Homeotic mutations in Drosophila have strange phenotypes in which the normal specification of one body part is altered into that of another, such as legs growing in the normal position of antennae (Figure 3-15).
Figure 3-15. The antennapedia mutation is a homeotic mutation that changes the fate of cells in a segment that would normally yield an antenna. Instead, a leg forms. (© F.R. Turner, Indiana University/Visuals Unlimited.)
Studies of the genetic makeup of homeotic genes led to the discovery of a regulatory sequence, the homeobox, which has now been identified in many genes that establish key aspects of body organization in animals as structurally diverse as mammals, insects, nematodes, and the simplest animals, sponges. Indeed the same homeobox is even found in many genes that regulate plant development.
Homeotic genes code for proteins that act as DNA transcription factors (Figure 3-16). In each of them, the homeobox is a conserved 180 bp consensus sequence that codes for a 60 amino acid region called the homeodomain(remember that each DNA triplet corresponds to 1 amino acid). The homeodomain contains α helices that bind in the major groove of DNA at sites within transcription enhancer elements. A transcriptional activation domain in the homeotic protein then increases the rate of transcription in the genes it regulates. This can lead to a cascade of further transcriptional activation in cell signaling pathways that causes each segment to take on its prescribed morphological characteristics.
Figure 3-16. A homeotic gene, shown in tan and orange, contains a 180-bp homeobox, which codes for a DNA-binding protein region called the homeodomain. This binds at an enhancer genetic regulatory element and activates transcription. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
The homeotic genes that control segment specialization in Drosophila are arranged in two clusters on the third chromosome (Figure 3-17). The antennapedia complex includes genes that control the fate of segments in the head and anterior thorax. The bithorax complex controls segment specification in the posterior thorax and abdomen. The anterior to posterior order of the genes in each complex almost perfectly parallels the order of body segments that they control.
Figure 3-17. The direct correspondence between the order of homeotic genes of two complexes, the antennapedia complex and the bithorax complex, and the body regions in which they are expressed. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
In other organisms, the homeotic gene clusters are called Hox complexes, a contraction of “homeobox.” The general Organization of these clusters is largely conserved among organisms, but vertebrates have an increased number of Hox complexes. All invertebrates have one Hox complex, as described in Drosophila. But vertebrates have at least four copies that may be located on different chromosomes. One hypothesis is that this increase in Hoxgene number allows the creation of greater complexity in cell types. Some genes within these duplicates have lost their ability to function and new homeotic genes are present. In mice, there are four Hox clusters (Figure 3-18). As originally seen in Drosophila, there is a correlation between the genetic order of the adjacent Hox genes and the anatomical region within which each one acts (Figure 3-19).
Figure 3-18. The organization of homeotic genes is highly conserved among taxonomically diverse animals. As a representative mammal, for example, mice have four homeobox (Hox) gene clusters that correspond with genes in Drosophila. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 3-19. The correspondence between Hox gene arrangement and the body segments in which they are expressed in mice. (Reprinted with permission from Hartwell LH, et al: Genetics: From Genes to Genomes. 3rd ed. New York: McGraw-Hill, 2008.)
Our discussion of developmental organization has focused on events defining body structure along the anterior-posterior and the dorsal-ventral axes. But there is another important dimension, laterality. We are essentially symmetrical, in organization if not in actual appearance. Yet, some internal organs are not. The heart, stomach, and liver do not develop symmetrically on the midline. Laterality in such structures is determined very early in development, and errors in signaling can lead to laterality defects like mirror image arrangement. In animals with less plasticity in developmental programming, early changes in cell lineage can yield distinct bilateral differences like the two-colored lobster in Figure 3-20. In Drosophila, bilateral mosaics called gynandromorphs, in which one half of the body is female and the other half is male, are used as experimental tools to study a variety of developmental mechanisms.
Figure 3-20. A rare laterality in color in a lobster caught by a Digby County, Nova Scotia, fisherman. (© Tina Comeau/Yarmouth Vanguard, Reprinted with permission from “Just how rare is a two-coloured lobster?” The Vanguard. Published on January 11, 2008. http://www.novanewsnow.com.)
Thus, a study of simple developmental systems in prokaryotes and invertebrates can give us an insight into the mechanisms that regulate the developmental events in more complex animals like ourselves. Genetic changes operating at each of the levels we have discussed here have parallels in medically important conditions.
Part 2: Medical Genetics
From a single fertilized egg, the development of the complex organism that will become a human being seems nothing less than miraculous. Understanding the process does not lessen the awe. The process of development is a series of steps that begins with the organization (set up) of the embryo, followed by progressive refinement of cells into specialized functions, leading to the organization of differentiated cells into tissues, and finally the formation of discrete anatomic structures.
As discussed earlier, this developmental cascade is under precise regulation by a series of programmed genetic “switches” that are intensely integrated. In the coordination of development, order and timing are crucial. At any given step in the process of development, something can go wrong. Structural congenital anomalies, or “birth defects,” are then the result of alterations in these normal developmental processes. Depending on the timing and the process involved, different types of anomalies may occur. Table 3-2 lists examples of disorders of embryonic organization with the corresponding levels of embryonic developmental control.
Table 3-2. Examples of Disorders of Embryonic Organization at Various Levels
General Principles of Embryology
Before we proceed with a discussion of congenital anomalies, a brief review of embryology is in order. It is beyond the scope of this text to review human embryology fully. However, a solid understanding of human embryology is critical to interpret congenital anomalies correctly.
Developmental periods
One of the best ways to conceptualize development is to organize it by developmental periods. As the organism develops, each period is characterized by the pattern of which critical structures and processes are emerging. The key point here is timing. Timing is critical both from the standpoint of which structure is developing when, and from knowledge about which processes are dependent on each other in a developmental sequence.
In a very real sense, the organization of a human embryo begins even before conception. Recent studies have demonstrated the central role of preconceptional influences on human development. In fact it probably begins during the middle part of the mother’s gestation! Beginning in the third fetal month, the oogonia begin to differentiate into oocytes. Oocyte formation in the female fetus requires organization of the cell that includes establishing polarity, intracellular sub-compartmentalization, and chemical gradients. Defects in these processes may have downstream effects on the development of the offspring from this individual. In the mature (adult) female, external (prepregnancy) influences can still affect fetal development presumably by their effects on the oocyte milieu. For example, abundant research has shown that the maternal use of relatively high doses of folic acid can significantly reduce the occurrence and recurrence of neural tube defects. The maximum effect can be achieved if the folate supplementation is started 2-3 months prior to conception, presumably due to changes in the oocyte prior to conception.
The process of in utero development can be divided into major time periods typically marked from the time of conception. It is important to note that an alternative designation can be used that defines the periods of development in relationship to the last menstrual period (LMP). By this designation, pregnancies are dated in weeks beginning from the first day of the woman’s LMP. On average, ovulation occurs on day 14 of the menstrual cycle, and conception occurs about 2 weeks after her LMP. Thus a woman’s “obstetrical date” of 6 weeks would be 2 weeks after her first missed period. This definition is preferentially used in perinatal assessments such as prenatal ultrasounds. This is in contrast to the embryologic date (the age of the embryo). The obstetric date is about 2 weeks longer than the embryologic date. For the purposes of identifying critical periods for the occurrence of congenital anomalies, the embryologic date is used. The clinically important time periods of in utero development can be defined as:
• Early pregnancy (first 3 weeks)
• First trimester (3-11 weeks)
• Second trimester (12-25 weeks)
• Third trimester (26-40 weeks)
Embryonic development essentially refers to the time of organ formation. For practical purposes this corresponds to the first trimester. All major structural features of the individual are in place by the end of the 11th week, with one major exception. Macroscopic structural changes can still be seen in the central nervous system until about the middle of pregnancy. The last major structural development that can be seen in developing humans is the corpus callosum of the brain. The completion of its development is seen at about 21 weeks.
The response of the embryo or fetus to various deleterious factors differs by the embryologic period. In brief, major insults in the first 3 weeks of pregnancy result in pregnancy loss. Problems during the first trimester result in malformations (discussed further later). Insults in the second and trimester tend to disrupt growth and organization more at the cellular level.
Key factors in normal morphogenesis
Conceptually, most congenital anomalies can be understood as the disruption of specific normal processes in development. Some of the key processes to be considered are:
1. Cell growth rateas a “force” in human development. Many observable physical features can be explained by differences in cell growth rate. For instance, things like fingerprint patterns are the results of relative growth rates of the cells of the finger tip pads. Higher elevated pads (associated with increased cell growth) will result in whorl patterns, whereas lower elevated pads tend to produce arch patterns. The so-called “embryopathic face” is a set of common facial features, such as underdevelopment of the mid-face, flattened nasal bridge, thin upper lip, and smoothened philtum. These features can be caused by many factors (including teratogens) that work by the common mechanism of reducing the overall rate of cellular growth.
2. Cell migration. In the normal process of embryonic development, many specialized cells need to migrate from their place of origin to their definitive position in the body. Migrational defects occur when the movement of cells from one place to the next is interrupted.
3. Cell-cell interactions. Throughout development, specialized cells are being defined. Among cell types, there is a need for interactions between these groups. In the normal process of development and organization, interactions among cells, such as induction, adhesion, and destruction, are critical. Interference with these interactions may produce areas of dysplasia. It is also important to note that most of these processes are normal events during embryogenesis. At the conclusion of embryonic development, they should be rendered inactive. Reactivation of some of these processes plays a central role in the ontogeny of cancer.
4. Selected cell death. Certain cells are developmentally preprogrammed for apoptosis (cessation of function). Eliminating these cells is just as crucial for normal development as the generation of cells in the first place. For example, failure of the (normal) programmed cell death for those cells between developing fingers will result in syndactyly (fusion of the digits).
5. Growth factors/hormones. Cell growth and migration are directly influenced by a variety of hormones and growth factors. These substances are typically produced elsewhere and are then transported to the target site to direct those cells in further development. Many of these factors exert their influence under a narrowly-defined set of chemical concentrations established by interactive gradients.
Congenital Anomalies
The term congenital anomaly literally means “something not right at birth.” In the broadest sense, any abnormality present at birth and its resulting phenotype could be classified as a “birth defect.” This could be an abnormality of structure, function, or body metabolism (i.e., an inborn error of body chemistry) present at birth that results in physical or mental disability, or is fatal. Taken to the extreme any mutation present at conception and its resulting phenotype could be classified as a congenital anomaly. For instance, a seminar was held several years ago entitled “Breast Cancer and Other Birth Defects.” Their reference was to heritable abnormalities of certain “cancer genes” that give a genetic predisposition to breast cancer. For the purposes of this chapter, however, we will limit our discussions to structural congenital anomalies.
Epidemiology
Overall, 4 million babies are born each year in the United States. Approximately 150,000 of these are born with a congenital anomaly. Roughly half of these are detectable as part of the child’s first physical examination shortly after birth. The other half may be hidden from view and not readily detected initially.
The overall incidence of a major congenital anomaly is around 4% of all live born infants. This represents a baseline or “population” incidence. It represents the lowest relative risk that exists for a given pregnancy—a number from which no couple is exempt. Few couples approach a pregnancy with the expectation of a child with a birth defect. Yet, this baseline number means that 1 in 25 couples are expected to experience the birth of a child with a medically significant problem. Obviously, other factors such as family history, medical conditions, environmental factors, ethnicity, and genetic factors can modify, i.e., increase, these risks. Interestingly, there are no well-identified factors that can actually lower this 4% risk.
If the actual number of congenital anomalies per live birth is calculated, it is more than double (9%) the rate of newborns with an anomaly. The reason for this, of course, is that some children have more than one anomaly. In fact the presence of one congenital anomaly carries a 50% chance that there is a second significant anomaly present. This is critically important in the evaluation of a child with a congenital anomaly. Once a structural congenital anomaly is identified, it is imperative that a search for potentially associated anomalies be implemented. Table 3-3 lists the most commonly found congenital anomalies and the incidence of these at birth.
Table 3-3. Most Common Structural Congenital Anomalies
Trends
Because of the enormous impact of these conditions, most state health departments have some sort of birth defects registry in place. These registries vary from state to state in their scope and policies. The common link is the goal to track the number and types of congenital anomalies occurring in that state for any given year. These projects have been ongoing for decades now. In addition, several federal initiatives have been implemented to coordinate and compare data collection efforts across states in the United States and worldwide. As might be expected, there are distinct regional variances. But, in general, the information obtained is fairly consistent and identifies several common themes and trends.
The overall birth defect rates have been surprisingly consistent over the past several decades. A few changing trends have been identified. Neural tube defects (excluding anencephaly) have steadily decreased. Notably, this decline seems to have started before the advent of folate supplementation or fortification. The occurrence of congenital heart malformations has been steadily increasing over the past several years for reasons still unknown. Other anomalies showing an increasing incidence include obstructive uropathies and certain neurodevelopmental disorders.
Consistently higher birth defect rates are reported in the southern United States and parts of the Midwest as compared to other parts of the United States. It is likely that these differences are not related to an actual increase in congenital anomalies in these regions, but rather are a function of the tracking systems in place. There is a direct correlation in which the states that have a “better” tracking system have a higher reported incidence of birth defects; i.e., a correlation of higher numbers with better reporting.
Congenital anomalies affect people of all racial and ethnic groups. While certain specific genetic conditions clearly do occur with a higher frequency in certain ethnic groups, in general these differences are relatively small for congenital anomalies. The birth defect rates are mildly increased in African Americans and Hispanics in the United States, as compared to Caucasians. Potentially this may be related to socioeconomic factors, although there is some suggestion that this may not be the sole answer.
Impact
The medical and fiscal impact of congenital anomalies is staggering. This is due in part to the sheer number of these conditions as well as the magnitude of their effects. Persons with congenital anomalies are affected in their medical care and can suffer serious health, emotional, and social burdens. The burdens affect not only the child, but also that child’s family and society as a whole. In one study, the estimated lifetime expense associated with 12 selected, isolated birth defects was calculated to be more than $8 billion, ranging from $140,000 to $700,000 per child.
One of the most notable impacts of birth defects is their effect on longevity. Consistently, birth defects and prematurity are fairly well tied as the two leading causes of death among infants in the United States. Birth defects account for an estimated 20% of infant deaths per year, which translates to 6,500 deaths annually. Infants with major congenital anomalies have a six-fold higher incidence rate of infant deaths when compared to those without congenital anomalies. Forty-five percent of all deaths in the neonatal intensive care units are due to congenital anomalies. For African Americans, they represent the second most common cause, with preterm labor or low- birth-weight infants being the most common cause of infant mortality. They are also the second leading cause in Native Americans and Alaskan natives.
Birth defects account for a similar proportion of total deaths for children aged 1 to 14. They account for approximately 15.5% of deaths among children 1 to 4 years old; 8% in the 5- to 9-year-old age group; and 6% in the 10- to 14-year-old category. In fact, among children aged 1 to 14, one study estimated that birth defects could account for 21.5% of total deaths.
Types of Congenital Anomalies
Individual congenital anomalies can be classified according to the pathogenetic mechanism responsible for their occurrence. In general four distinct mechanisms have been purported: malformations, deformations, disruptions, and dysplasias. Clinical geneticists are meticulous about the use of these terms in the classification of congenital anomalies. The student should be cautious when reviewing the medical literature in this regards. Outside of the discipline of clinical genetics the terms may be applied more loosely.
A malformation, as the term would imply, is a congenital anomaly in which the tissue is malformed, i.e., it did not form correctly from the start. As noted in our earlier discussions, the vast majority of malformations occur in the first trimester of pregnancy given the timing of normal embryogenesis. A distinction is made between major and minor malformations. Major malformations are defined as those that have significant clinical implications and are not found in the general (normal) population (Figure 3-21). In contrast, minor malformations do not produce clinically significant problems and may occur in a small number of “normal” individuals (Figure 3-22). Current estimates suggest that 15% of newborns will have a single minor anomaly (not including dermatoglyphic changes) when carefully assessed by a trained dysmorphologist. In addition about 1% of all newborns will have two or more minor anomalies. Although minor malformations are not of major clinical import, they are very important in the assessment of persons with congenital anomalies as clues to more serious problems and the recognition of malformation syndromes. As the number of detected minor malformations goes up, the chance of having a major malformation likewise increases. As such, it is recommended that any individual with three or more minor malformations have a formal assessment looking for major malformations. It is also important to note that minor malformations may present as a familial trait. In this context, it is important to define whether this represents a normal familial variant or is an indicator of a heritable disorder. From a mechanistic standpoint, malformations can result from the lack of development (agenesis) or underdevelopment (hypogenesis) of a given structure. There may also be abnormal migration of cells (heterotopia) or whole organs (ectopia.) Finally there may be incomplete closure or separation.
Figure 3-21. Two examples of major malformations. (a) Bilateral cleft lip and palate. (b) Agenesis (absence) of the radius.
Figure 3-22. Two examples of minor malformations. (a) A single transverse palmar crease. Most individuals have two creases in the palms. About 2% of the general population has a single crease. In contrast, over 90% of patients with Down syndrome have a single crease. (b) Widely spaced nipples. Note that the nipple is laterally placed almost to the axilla.
Deformations represent another mechanism that causes congenital anomalies (Figure 3-23). Deformational changes are the results of mechanical forces applied to otherwise normally developing structures. In the case of a deformation, the magnitude and the direction of the applied force can often be deduced from careful inspection of the changes coupled with knowledge of normal perinatal anatomy and physiology (Figure 3-24). The mechanical forces that lead to deformations can arise from a variety of sources. In general they are factors that somehow constrain or apply a force to the fetus. Deformations can be of maternal or fetal origin. Maternal factors that may cause deformational anomalies include a small mother, a small uterus, uterine malformations (bicornuate uterus, septate uterus, and so forth), primigravid pregnancy, or oligohydramnios. Fetal factors would include multiple gestation, a large fetus, other fetal anomalies, fetal hypo-mobility, or oligohydramnios. (Note that oligohydramnios appears on both lists as it may occur due to maternal factors such as amniotic leakage or fetal factors such as oliguria.) Table 3-4 lists some of the commonly found deformations.
Figure 3-23. Deformational plagiocephaly. (a) Note the asymmetry of the cranium with a deformational change in the shape to a rhomboid configuration. (b) Corresponding CT scan of the head. Note the asymmetry of the position of the ears.
Figure 3-24. Schematic demonstrating how deformational plagiocephaly may be induced by early descent into the pelvis. (a) Infant engaged in pelvis. (b) Contralateral pressure on the skull from opposite sides of the pelvic rim. (Redrawn from Bruneteau RJ, Mulliken JB: Frontal plagiocephaly: synostotic, compensational, or deformational. Plast Reconstr Surg. 1992; 89(1):21-31.)
Table 3-4. Commonly Noted Deformations
Normally developing tissue may be subjected to insults that result in actual loss of cells and/or tissue. The resultant anomalies are due to the effects of the missing cells. This type of anomaly is referred to as a disruption(Figure 3-25). A variety of insults may occur during pregnancies that result in a disruption. The most common mechanism is some sort of vascular accident (hemorrhage, occlusion, ischemia, or constriction). Other mechanisms include radiation or infection. Table 3-5 lists some of the commonly found disruptions.
Figure 3-25. Two examples of disruptions. (a) Terminal reduction of the digits due to constriction from amniotic bands. (b and c) Hemifacial microsomia. Photo and CT scan demonstrating underdevelopment of the left side of the jaw. This anomaly is most commonly due to occlusion of the stapedial artery in the developing fetus.
Table 3-5. Commonly Noted Disruptions
Dysplasia (Figure 3-26) literally means “bad form.” A dysplasia represents aberrant formation specifically at the level of the organization of cells into tissues (dyshistogenesis). As such they tend to occur later in development and somewhat independently of morphogenesis. Morphogenesis is exclusively prenatal in origin, whereas histogenesis continues postnatally in all tissues that have not undergone end differentiation. A unique aspect of congenital anomalies that are dysplastic in nature is that they may predispose to cancer later in life.
Figure 3-26. Imaging studies of a patient with polyostotic fibrous dysplasia. (a and b) MRI slices of the cranium. Note the markedly abnormal configuration (dysplasia) in the cranial bones. (c) X-rays of the forearms also demonstrating dysplastic changes.
Patterns of Congenital Anomalies
As mentioned earlier, congenital anomalies may occur as isolated anomalies, but frequently multiple anomalies occur in the same individual. In fact, if one structural congenital anomaly is present in a patient, there is a 50% chance that that person has one or more additional anomalies (i.e., multiple congenital anomalies). If multiple anomalies are present, then the next step in the evaluation process is to try and identify a specific pattern that ties the multiple findings together.
Three major categories of patterns of multiple anomalies have been defined. Syndromes are patterns of congenital anomalies of more than one organ system with a common etiology. Sotos syndrome (Figure 3-27) is characterized by overgrowth, macrocephaly, an advanced bone age, a characteristic facial appearance, and neurodevelopmental and neurobehavioral changes. Over 90% of patients with Sotos syndrome have a mutation in a gene called NSD1. There are hundreds of other well-described syndromes.
Figure 3-27. Young girl with Sotos syndrome.
Associations refer to the known occurrence of certain anomalies that happen too often to be by chance, but without a defined etiology. Associations often are designated by acronyms that detail the most common features. VACTERL association (Figure 3-28) defines an association of Vertebral anomalies, Anal atresia (imperforate anus), Cardiac malformations, Tracheal-Esophageal fistula, Renal anomalies, and Limb anomalies. (Earlier reports defined the condition as the VATER association.) Not every patient has every one of the listed anomalies. Again, what is found is the association—these anomalies are found together at a frequency too often to be likely by chance. The diagnostic criteria for this condition require that at least three of the highlighted features be present. The key distinction from syndromes is the lack of an identified common etiology.
Figure 3-28. Infant with VACTERL (VATER) association. (a) Whole body of infant. (b) Note forearm anomalies due to radial hypogenesis. (c) Multiple anomalies of cervical vertebrae.
Because development is so interconnected, an early change can have a “snowball effect” on other components of development. The cascading effect can lead to a reproducible pattern of anomalies. This type of multiple anomaly pattern is referred to as a sequence. The Robin sequence (sometimes called the Pierre Robin sequence) is an example of such a pattern (Figure 3-29). It is often errantly referred to as the Robin syndrome. By strict definition, it is not a syndrome, rather it is best designated a sequence. In this condition, there is only one primary anomaly, micrognathia (small jaw). A smaller jaw tends to displace the tongue posteriorally. If this occurs before 9 weeks of gestation (prior to the closure of the lateral palatine ridges), the abnormally displaced tongue physically interferes with the closure of the palate. This results in a cleft palate that is somewhat different in its configuration than the typical cleft palate. While the typical cleft is a linear defect, the cleft palate seen in the Robin sequence will have a ‘U’ or wedge- shaped configuration. Secondary changes are often seen because of malnutrition, the combination of a cleft and a small jaw making feeding quite difficult. The end result is a child with what appears to be a syndrome, but in actuality it has a series of problems cascading from a single early anomaly.
Figure 3-29. Infant with Robin sequence. (a) Small recessed chin (retromicrognathia). (b) ‘U-shaped’ cleft palate.
Etiology of Congenital Anomalies
When a person is identified as having a congenital anomaly, the first question usually raised is “Why?” For most people there is a strong desire to know the cause (etiology) of the abnormality. In addition, there are often medically important pieces of information that are tied to this answer. Besides simply knowing the cause, identifying an etiology is helpful in defining recurrence risks, prognosis, associated co-morbidities, and even potential targeted therapies. Thus, when a child is born with a congenital anomaly, a consultation with a clinical geneticist is typically requested. The role of the geneticist is to identify the pattern of anomalies and determine the etiology, if at all possible. If an etiology can be determined, then detailed counseling can be provided to the family along the lines noted. The discipline of dysmorphology is the art and science of discerning recognizable patterns of congenital anomalies. Some would consider this a separate discipline and request themselves to be called dysmorphologists. We would tend to characterize it as a subdiscipline within clinical genetics. The seminal publication in this arena is Smith’s Recognizable Patterns of Human Malformations which was originally penned by Dr. David Smith, the man regarded as the father of the discipline of dysmorphology. This book is currently in its 6th edition, ever expanding to include additional syndromes and updates on the molecular genetics of each condition. Several other excellent published collections of syndromes and their descriptions are available.
As one might expect, there are a myriad of causes of congenital anomalies. All known categories of etiologies have been identified in congenital anomalies. Depending upon the particular anomaly, the etiology may be chromosomal, single gene, teratogenic, or multifactorial. More complex genetic changes may also be involved. Traditionally, it has been noted that only about 20% of all congenital anomalies have an identifiable cause. Recent advances in genetic testing are clearly improving this yield. Although we are not aware of any recent study that has been published to quantify and substantiate this, our clinical experience would suggest that currently this diagnostic yield has at least doubled and is on the order of 40% or greater. Clearly further advances will continue to improve the diagnostic yield for congenital anomalies.
In general it is easier to identify the etiology if multiple anomalies are present. Identifying the etiology for single anomalies has historically been quite difficult. But recent advances in diagnostic testing (see Chapter 11) have provided powerful tools that are allowing the identification of the etiology even for isolated (single) anomalies. Some of this can be attributed to the better recognition and understanding of the range of expression of certain conditions. For example, submicroscopic deletions (Chapter 5) on a specific region of chromosome 22, designated as a “22q11.2 deletion” are associated with several patterns of multiple anomalies involving the face/palate, heart, parathyroid glands, thymus, and other organs. When multiple anomalies are seen in conjunction with this deletion, it is often possible to identify an associated recognizable pattern (syndrome) including DiGeorge syndrome (Figure 3-30) and Shprintzen (velo-cardio-facial) syndrome among others. The spectrum of abnormalities seen in conjunction with this deletion is staggering. In fact, the total number of anomalies reported with this deletion is now over 180! The phenotypic range seen with this deletion goes from easily recognizable multiple anomaly syndromes to apparently unaffected individuals. Within this range it is known that some individuals may have only single organ involvement. It is now known that 17% of persons with isolated congenital heart malformations will have a 22q11.2 deletion and 30% of persons with a specific subcategory of heart malformations (conotruncal heart malformations) will have this deletion. Thus, even though the diagnostic yield is nowhere near 100%, these sort of advances have ushered in the era of identifying some causes of congenital anomalies—even at the single organ level.
Figure 3-30. Young girl with a known 22q11.2 deletion. She has learning disabilities, cleft palate, congenital heart disease, immune deficiency, and minor facial dysmorphisms. Her phenotype is best characterized as DiGeorge syndrome.
For much of the rest of this book, we will be discussing two major parameters of human diseases: etiology (the cause) and pathogenesis (the mechanism). In future chapters we will be reviewing in detail etiologies such as single gene disorders, chromosomal abnormalities, gene-environment interactions, and even more complex etiologies such as epigenetic causes. In the context of the discussions in this chapter about congenital anomalies, one specific category of etiology, teratogens, warrants a more detailed discussion here.
Teratogens are environmental agents that can cause birth defects if the mother is exposed to the agent during pregnancy. The maternal exposure is transmitted to the fetus and can invoke congenital anomalies. Early medical thinking had envisioned the “womb” as a highly protective environment that would shield the developing baby from any and all outside influences. Modern understanding recognizes that the maternal-fetal interface is by no means impervious to outside agents. In fact hundreds if not thousands of environmental factors are now known to affect fetal development adversely in the right setting. Some of the major categories of known teratogens include legal and illegal drugs, prescribed medications, herbal and other homeopathic compounds, maternal medical conditions, and environmental/occupational exposures. Table 3-6 lists many of the more common/important human teratogens. The teratogenic potential of any given agent (the likelihood that it could induce a birth defect) is dependent upon a complicated series of interacting characteristics of the agent and the particular exposures. Table 3-7lists such factors.
Table 3-6. Important Human Teratogens
Table 3-7. Factors That Influence the Potential Teratogenicity of a Particular Agent/Exposure
With a better understanding of the importance of teratogenic agents, several major efforts have emerged to identify such agents, report on their characteristics, measure their impact, and establish prevention measures. Large databases available to clinicians and researchers such as TERIS and Reprotox have been established and are constantly being updated. Groups like the Organization of Teratogen Information Specialists (OTIS), the Centers for Disease Control (CDC), the National Institutes for Health (NIH), and the March of Dimes (MOD) have all had major programmatic efforts in understanding and preventing teratogenic birth defects. Herein is by far the most important feature of teratogen-induced birth defects—they are preventable! (For many of the other known causes of congenital anomalies, prevention is simply not possible at this time.) The key to prevention of teratogen-induced congenital anomalies is simple—avoid exposures. This of course is much easier said than done. Some exposures are simply unavoidable. Sometimes the mother’s health requires that she take a particular medication. While the drug may have teratogenic potential, it would be more harmful to both mother and child if the mother were not treated.
Likewise, the science may be simple but the implementation extremely difficult. Fetal alcohol syndrome is a recognizable pattern of malformations seen with in utero exposure of the fetus to alcohol (ethanol). Fetal alcohol syndrome (FAS) is identified by characteristic facial changes, abnormalities of the growth of the head and body, and neurologic deficits (Figure 3-31). Not all children exposed to alcohol in the womb will have FAS. Of those children exposed to “significant” amounts of alcohol in utero, 1/3 will have the full expression of FAS, another 1/3 will have neurodevelopmental and neurobehavioral problems without any physical features, and the final 1/3 will have no apparent effects. The reasons for this wide range expression can be attributed to genetic differences in the maternal and the fetal genome as well as environmental modifiers. Collectively, then the full range of problems seen with in utero alcohol exposure are best termed Fetal Alcohol Spectrum Disorders (FASD). The magnitude of problems produced by alcohol teratogenesis cannot be understated. FASDs represent the most common preventable cause of mental retardation. As a group they may account for up to 30% of all neurodevelopmental disabilities. The total costs of habilitation for individuals with Fetal Alcohol Syndrome (FAS) have been estimated at over $1 million for a lifetime. Thus it is imperative that all health care providers recognize one simple health care recommendation. The only appropriate advice to give is that there should be zero alcohol use at any time for any pregnancy! As already stated, this is easier said than done. In order to avoid any exposure, a woman must not drink at any point in pregnancy. Since the average gestational age that a woman identifies pregnancy is around 8-9 weeks, it is not enough simply to quit drinking when she recognizes that she is pregnant. Most importantly, health care providers should be aware of this risk and appropriately counsel their patients. As unbelievable as it might seem, there are still practitioners who will advise their pregnant patients to drink a small amount of alcohol during pregnancy to “calm their nerves”! We sincerely hope that you as a conscientious health care provider will instead make the only wise prescription for your patients. No alcohol during pregnancy. None; never.
Figure 3-31. Adolescent male with FAS. This child originally presented with short stature of unexplained etiology.
Evaluation of the Patient With Congenital Anomalies
The approach to the person with a congenital anomaly is largely similar to most other medical assessments. The evaluation revolves around the typical “history and physical” approach. The difference in the evaluation of a person with a congenital disorder is largely a greater emphasis on certain components of the exam and history. Clearly much more attention is paid to the family and prenatal histories. The examination goes beyond a standard physical exam to include quantifying morphologic alterations. Growth patterns—both pre- and postnatal—are emphasized. All of the collected data are then interpreted in light of known embryologic principles. Finally testing is performed if indicated, and counseling is given regarding etiology, recurrence risk, and expected outcomes (Figure 3-32).
Figure 3-32. A schematic outlining the diagnostic approach to the child with congenital anomalies.
Part 3:Clinical Correlation
The definitions provided above are precise in their meaning and intention. They convey critical information about the nature and feature of a given congenital anomaly. As such, clinical geneticists are meticulous in their use of these terms. Two clinical examples are provided below that highlight the types of information contained in these terms.
1. Potter syndrome as originally described in 1946 was the set of features seen in newborns with renal abnormalities and characteristic facies (hypertelorism, flattened nasal structures, retrognathia, and large, low-set ears lacking in cartilage) (Figure 3-33). Other commonly associated changes include pulmonary hypoplasia and bone and joint abnormalities.
Figure 3-33. A stillborn infant with Potter anomalad. On autopsy, complete absence of the kidneys was noted. In this photograph note the multiple deformational changes of the face and joints.
Subsequently other newborns were reported with identical physical features, but with normal kidneys. The physical features in these infants could be attributed to low levels of amniotic fluid (oligohydramnios). The link in this situation is that 80% of amniotic fluid is derived from fetal urine. Thus fetuses with poorly functioning kidneys will have oligohydramnios. In the other group—the newborns with Potter-like features and normal kidneys—there were other causes of the oligohydramnios such as amniotic fluid leakage.
Thus, to apply the terms above accurately, one would say:
• The renal anomalies are malformations.
• The facial changes and the bone and joint changes are deformations.
• The facial changes and the bone and joint deformations are secondary to oligohydramnios with this pattern, then, best termed the oligohydramnios sequence.
• The designation Potter syndrome, then, is actually a misnomer. But, given the historical significance of Dr. Potter’s description, many choose to keep this designation and reserve its use for the specific situation of the oligohydramnios sequence when it is caused by bilateral renal agenesis.
2. CHARGE association was originally described in 1979 as a constellation of Coloboma of the eyes, Heart anomalies, choanal Atresia, Retarded growth and development, Genital anomalies, and Ear abnormalities α hearing loss with the acronym designating the key features (Figure 3-34). Most cases identified were sporadic (negative family history). At the time of its description, CHARGE had no known etiology. Much of the literature about CHARGE noted that the features often overlapped with other developmental field defects, especially those known to be due to vascular disruptions. Thus, the designation CHARGE association was appropriately given. As molecular diagnostic testing advanced, a small number of patients with CHARGE were found to have 22q11.2 deletions. Scattered patients were identified with various other chromosome imbalances. Then, in 2006, patients were identified with CHARGE association who had identifiable mutations in a gene called CHD7. Further studies have shown that over 2/3 of patients with CHARGE harbor a CHD7 mutation. With this knowledge of the etiology of CHARGE (albeit heterogeneous), the designation has changed. Officially, it is now the CHARGE syndrome. This change in terms accurately reflects the change in the knowledge of the basis of this condition. Still, those reviewing the literature may find this shift in terminology confusing.
Figure 3-34. (a, b, and c) A child with CHARGE syndrome. This young man has a confirmed CHD7 gene mutation.
Board-Format Practice Questions
1. Most people have two major linear creases across the palm of their hands (take a look). Two percent of people in the general population have a single transverse palmar crease (a so-called “simian crease”). This finding is very frequent in patients with Down syndrome. The single transverse palmar crease would be an example of
A. an association.
B. a sequence.
C. a syndrome.
D. a malformation.
E. a disruption.
2. Porencephalic cysts are fluid-filled spaces in the brain that are left after the death of brain cells. A porencephalic cyst identified in an infant at 2 hours of age is the result of a
A. malformation, and must have occurred before 11 weeks of pregnancy.
B. malformation, but since the organ affected is the brain, must have occurred before 22 weeks of pregnancy.
C. deformation.
D. disruption.
E. transfected DNA sequence.
3. Dysmorphology (the area of clinical genetics concerned with diagnosis and etiology) classifies congenital anatomic malformations according to pathophysiology. This classification includes malformations, deformations, and disruptions. A fourth type exists in which there is abnormal organization of cells into tissues with its morphologic consequences. This would be defined as
A. dysautonomia.
B. dysdiadokinesis.
C. distichiasis.
D. dysergia.
E. dysplasia.
4. Birth defects
A. are uncommon disorders that most practitioners will rarely see.
B. in general are decreasing in frequency.
C. are often associated with infant mortality.
D. are less common in the South as compared to the rest of the United States.
E. rarely are seen with more than one anomaly per person.
5. Occasionally, an infant is born in which both kidneys are missing because they simply never developed. Since over 75% of amniotic fluid is formed by fetal urine, fetuses with no kidneys produce very little amniotic fluid, a condition called oligohydramnios. If oligohydramnios is present, then there are a variety of changes such as flattening of the face and misshaped joints that occur because the fetus cannot move about freely in the womb. The best description of these anomalies would be
A. the kidney anomalies are deformations; the face and joint changes are malformations.
B. the kidney anomalies are malformations; the face and joint changes are deformations.
C. the kidney anomalies are disruptions; the face and joint changes are malformations.
D. the kidney anomalies are malformations; the face and joint changes are disruptions.
E. the kidney anomalies are disruptions; the face and joint changes are deformations.