The first four chapters have taken a long-range view of the way in which genes determine a phenotype through interconnected biochemical pathways and cell interactions. In this chapter we will focus on how the genetic information packaged in chromosomes is duplicated and distributed during cell division (Figure 5-1). Then in Chapter 6, we will explore how chromosome behavior during the formation and fusion of gametes determines the predictable genetic outcomes of a mating.
Figure 5-1. A chromosome spread (unsorted) with fluorescent probes for chromosome number 14. A nondividing cell is off to the side also showing the chromosome 14 probes. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
The process of nuclear division is very accurate. Yet, errors do occur and can lead to changes in chromosome number and structure with often severe or even fatal consequences. It is not easy to study the genetic control of nuclear division at more than a descriptive level. This is because some of the most powerful tools available to a genetic researcher are mutations. By seeing how a mutation alters a process, one can deduce the role of the normal gene. But mutations are difficult to isolate for the molecular and biochemical events governing mitosis, which is the complete duplication of the genome of a cell to produce two identical cells, and meiosis, which is the reduction division found in egg- and sperm-forming tissue. In order to collect and analyze the roles of mutations in a given trait, one must be able to breed and manipulate them. But mutations that prevent nuclear division naturally block that approach, forcing researchers to find new ways to explore the molecular control of genetic transmission. Most medical applications, however, simply depend upon understanding the inherent logic of mitosis and meiosis and the consequences of errors or other complications that affect them. That will be our focus in this chapter.
In addition to errors in nuclear division that lead to changes in chromosome number, various agents like radiation can alter chromosome structure. Changes in structure affect a block of genes and influence many otherwise independent biochemical processes. They can also cause complicated physical interactions between chromosomes that have serious secondary consequences. In this chapter we will first discuss the normal processes of chromosomal distribution in mitosis and meiosis, and then explore the kinds of errors that affect chromosome number and structure. There are many important medical examples of each.
Part 1: Background and Systems Integration
Overview of Nuclear Division as an Information Distribution System
Some of the basic terminology used to describe the genome was introduced in the first two chapters. We know, for example, that the nucleus of a diploid (2n) cell contains two copies of each gene. The subtleties of such a statement were discussed in Chapter 4 but do not affect our understanding of nuclear division. Each gene is located somewhere on one of the numerous strands of DNA we see in the microscope as chromosomes. Each chromosome is, therefore, a separate unit of information transmission, a copy of a linkage group. It might contain hundreds or even several thousand genes linked in a linear array on the same strand of DNA. There are as many different linkage groups in a species as there are genetically different types of chromosome, ignoring the minor differences that distinguish allelic forms of the same gene. A diploid individual carries two copies of each linkage group, i.e., a pair of homologous chromosomes. The goal of mitosis is to duplicate each chromosome and pass one copy of every chromosome into each of the two new nuclei of the diploid daughter cells.
The goal of meiosis, on the other hand, is more complex and in some ways more important. Meiosis is a reduction division involving two cycles. Instead of transmitting one copy of every chromosome, meiosis results in passing one copy of each kind of chromosome, i.e., one copy of each linkage group, to each haploid (1n) egg or sperm nucleus. The diploid (2n) nuclear composition is regenerated at fertilization, with one copy of each linkage group coming from each parent.
Although cancer, somatic mosaicism, and other outcomes involving chromosome-level changes can be significant, errors in mitosis will usually have only minor consequences, if they have any at all. The presence of one abnormal cell is hard to detect among so many normal ones in the body. Its abnormality and death go unnoticed. But an error in meiosis is far more serious, since it will affect the initial genome of the zygote produced at fertilization. Estimates range from 8% to 25% (with most authorities leaning toward the higher end of this range) of human fertilizations will result in spontaneous abortion, perinatal death, or severe developmental consequences due to changes in chromosome number from errors in meiosis or fertilization. After discussing the normal events of nuclear division, we will explore some of the consequences of errors in meiosis.
The Cell Cycle in Eukaryotes
Whether we are considering mitosis or meiosis, the phases of the cell cycle can be subdivided into two parts, interphase and the stages of nuclear division (Figure 5-2a, which shows the events for mitosis). Interphase is sometimes called the “resting phase,” but that is a misnomer. It may be a resting stage in the sense that it occurs between rounds of active nuclear division. But functionally, it is the most active time.
Figure 5-2a. The growth-duplication cycle of mitosis. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
During the G1 phase of interphase, active genes are being transcribed and are controlling the biochemical life of the cell. To be accessible to the enzymes of transcription, the chromosomes are in various degrees of uncoiling. That is why a stained microscopic preparation of interphase simply looks like a dark organelle with little internal structure other than one or more nucleoli. At this stage, each chromosome is a single DNA double-helix molecule complexed with nucleosomal proteins. In G1 the cell typically grows by duplicating cell contents, except for the nuclear material. Then, in response to a signal, such as cell age, reaching a critical cell size, or receiving a molecular trigger like a growth factor, a restriction point is reached. The cell becomes committed to make a transition into the S, or synthesis, phase. An example of activating this G1-S checkpoint by a growth factor is shown in Figure 5-2b.
Figure 5-2b. Activation of cell division by epidermal growth factor (EGF). EGF binds a pair of EGF receptors and causes them to form an active dimer and become phosphorylated. This attracts the GRB2 and other proteins intracellularly, ultimately activating the Ras protein by forming a Ras/GTP (i.e., guanosine triphosphate) complex. This complex activates the Raf-1 protein kinase that phosphorylates MEK, which then phosphorylates MAPK. This MAPK then activates transcription factors that initiate cell division. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Progress through the G1 (or G1-S) and the later G2 (or G2-M) checkpoints is regulated by the formation of complexes of specific cyclins and cyclin-dependent kinases (CDKs). CDKs regulate the activity of other proteins by phosphorylating them, thereby either activating or inactivating them, depending on the function of the target protein. The specific cyclins determine which target proteins are acted on. At the G1-S checkpoint, the cyclin-CDK complex activates proteins needed for DNA replication. At the later G2-M checkpoint, a different cyclin-CDK complex activates proteins responsible for condensation and other chromosomal changes. If damage such as a DNA break is detected, a checkpoint protein like p53 inhibits the formation of an active cyclin/CDK complex.
In some cell lineages, a division restriction point is delayed or stops. A nucleus can be temporarily inactive (Figure 5-3) so it is not preparing for a new cell division cycle, or it might be terminally differentiated and will never divide again. Such a cell is described as being in the G0 phase.
Figure 5-3. The G0 phase represents a cell that is no longer dividing, as in case of a terminally differentiated cell lineage. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
In the S (synthesis) phase, DNA replication occurs. To accomplish this complex task, the chromosomes must of course remain uncoiled, or uncondensed. But if we could visualize them in a microscope, we would see that the two single-stranded templates of the replicating parent DNA molecule separate and a pair of new complementary strands is constructed as described in Chapter 2. The two strands remain connected at the centromere, so when they condense during nuclear division, we are able to see the two copies for the first time (Figure 5-4) as sister chromatids. From here to the middle of nuclear division, each chromosome has twice the usual amount of DNA.
Figure 5-4. Chromosomes become highly coiled, or compacted, during early nuclear division (metaphase) and clearly show the position of the centromere attachment of the copies or the separation of the sister chromatids. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
The terminology concerning chromosome number and DNA content during this transition can be confusing, but it is simplified if one keeps a key definition in mind. The term “chromosome” literally means “colored body.” No matter how many chromosome arms it carries, anything connected together at the same centromere is one unit and, thus, one chromosome. To count chromosomes, you must simply count the number of centromeres. Chromosome number does not change between the beginning and the end of interphase. What changes is the amount of DNA in the nucleus. The C value is the amount of DNA in a haploid (1n) nucleus. The diploid cell at G1, therefore, has a DNA content of 2C. During S, this doubles to 4C. Nuclear division reduces it back to 2C in each of the two daughter cells at the end of mitosis or to 1C in each of the four nuclei that result from the meiotic reduction division to produce haploid eggs or sperm.
During G2, the cell makes final preparations for division of the nucleus and cytoplasm. A key event that continues from S into G2 is the error correction in DNA repair. The checkpoint between G2 and mitosis or meiosis (M) is not passed until repair activities have been completed. Timing of the substages of interphase will differ among species and as a function of how actively the tissue is dividing, with the period of interphase before S being the most variable. One estimate for dividing mouse fibroblasts is 9.1 hours for G1, 9.9 hours for S, 2.2 hours for G2, and 0.7 hours for mitosis (M).
Mitosis: Somatic Cell Division
The stages of mitosis (Figure 5-5) are defined for the convenience of talking about the details of the process. Keep in mind, however, that it is actually a continuous process. At the start, the DNA and chromosomal proteins have already duplicated in interphase, and the two sister chromatids of each replicated chromosome are still attached at the centromere (Figure 5-4). An additional cell organelle also comes into play. The centrosome containing a pair of centrioles is located in the cytoplasm near the nucleus. They produce the array of microtubules that move chromosomes during nuclear division.
Figure 5-5. Stages of mitosis. (Photomicrographs © Dr. Conly L. Rieder, Wadsworth Center, Albany, New York 12201-0509. Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Prophase is a preparatory phase (pro = before). The chromosomes coil or condense into compact structures that can move easily within a cell and that can begin to be seen microscopically. The nuclear membrane breaks down, and the centrosomes divide and begin moving to opposite poles of the cell. As they separate, they generate an array of microtubules called the spindle, which is composed of tubulin.
In prometaphase, the sister chromatids become attached to the spindle by means of the kinetochore microtubules that extend from the kinetochore, a group of proteins that binds the centromeric DNA region (Figure 5-6). The microtubules from the spindle, called the polar microtubules, overlap near the equator of the cell and help keep the spindle poles separate. One reason this phase is important in medical genetics is that current cytogenetic standards use prometaphase chromosomes to establish the karyotype, or chromosome picture (e.g., Figure 5-23), for clinical evaluation. The chromosomes are less condensed then than they will be at later stages, so more detail can be seen with certain kinds of staining treatments.
Figure 5-6. The mitotic spindle is made up of kinetochore microtubules that bind to the chromosome’s kinetochore and polar microtubules that help keep the poles separate and the spindle in position. The kinetochore is made up of the centromeric DNA and two layers of kinetochore proteins that bind the kinetochore microtubules. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
During metaphase, the chromosomes line up at the equator between the centrosome poles. The chromosome still has two attached sister chromatids, with each attached to the opposite pole. Anaphase begins when the centromere divides and each chromatid is now a separate chromosome. Chromosome number has temporarily doubled, e.g., from 46 chromosomes (each with a pair of sister chromatids) to 92 in humans. The kinetochore microtubules shorten by dissociation of their tubulin subunits, so the chromosomes move toward the poles a bit like pieces in the classic Pac-Man computer game.
In telophase, generally the briefest phase of mitosis, events opposite to those of prophase occur. The chromosomes decondense; the spindle breaks down; and two new nuclear membranes form around the chromosomes at each pole. In addition, division of the cytoplasm, called cytokinesis, occurs when a contractile ring that includes actin and the motor protein myosin constricts the cell membrane to distribute the cytoplasm and its organelles into the two daughter cells (Figure 5-7).
Figure 5-7. Division of the cytoplasm in cytokinesis is seen in the formation of a cleavage furrow produced by a ring of actin filaments and myosin motor proteins. Cytokinesis divides the cytoplasm and its internal organelles into two daughter cells. (b: Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Meiosis: Producing Haploid Egg and Sperm Nuclei
In contrast to mitosis, which passes one duplicate copy of every chromosome to each of the daughter cells, meiosis reduces chromosome number by half. It does this in two cycles of division: prophase I, metaphase I, anaphase I, telophase I, followed by a second round with prophase II, and so forth (Figure 5-8). But it is not sufficient simply to cut chromosome number in half. Each haploid gamete must have one copy of each kind of chromosome, i.e., one copy of each linkage group. The process that allows this to occur centers on events in prophase I. In prophase I, as in the beginning of mitosis, the spindle forms, chromosomes condense, and the nuclear membrane breaks down. But instead of each chromosome attaching independently to the spindle microtubules, the homologous chromosomes pair together to form a bivalent. The process of pairing is called synapsis and involves the creation of a synaptonemal complex (Figure 5-9) that only forms between the chromatids of different homologous chromosomes. They do not form between sister chromatids. Consequently, there will be one bivalent for each type of chromosome, i.e., one bivalent for each linkage group. It is the bivalent that binds to the spindle so that at anaphase I, one of the chromosomes (still with the two sister chromatids attached at the centromere) moves to one pole while the other homologous chromosome moves to the opposite pole. Thus, chromosome number is reduced from the diploid set of chromosomes (2n) to two haploid (1n) nuclei by the end of the first meiotic division.
Figure 5-8. Meiosis involves two rounds of division and yields four haploid nuclei, each carrying one copy of each type of chromosome. Key events occur in prophase I of the first cycle including synapsis, or pairing, of homologous chromosome copies and recombination between them. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 5-9. In prophase I, the synaptonemal complex forms between homologous chromosomes. (a) An electron micrograph of a synaptonemal complex. (b) Diagram of the elements that make up the synaptonemal complex between chromatids. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Synapsis has at least two important functions in prophase I. First, it puts all of the copies of each linkage group into a separate cluster. This enables the cell to distribute one complete set of genetic information to each cell at the end of the first division. Synapsis keeps the homologous chromosomes together in a group. Second, there is an exchange between homologous chromosomes, called crossing over or recombination, which shuffles the alleles that are carried by the two homologues. Recombination is a powerful force in generating the enormous range of genetic variation that can be found among the offspring from each pair of parents. Since so many important events occur during prophase I, it is divided into subphases described in detail in Figure 5-10.
Figure 5-10. Events that occur during prophase I of meiosis include synapsis and recombination. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Following prophase I, the chromosomes move to the equator of the cell in metaphase I (Figure 5-11), and the homologous chromosomes are pulled to opposite poles in anaphase I. As we will see in Chapter 6, this separation, or segregation, of any genetic differences in the alleles carried by homologues is the basis of one of the fundamental Mendelian rules of genetic transmission. After a brief telophase I and cytokinesis, the second division proceeds as in mitosis, except that chromosome number is now haploid. The reduction division occurs during the first meiotic division. In humans, the result from each primary cell is four haploid sperm nuclei in spermatogenesis or one haploid egg nucleus plus three small haploid polar bodies in oogenesis (Figure 5-12).
Figure 5-11. The kinetochore microtubules from one pole are attached to only one of the chromatid pairs in a bivalent Thus, the chromosomes in a homologous pair are attached to different poles. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 5-12. Comparison of: (a) spermatogenesis, which can yield four haploid sperm, and (b) oogenesis, which yields one haploid egg cell and up to three haploid polar bodies, which degenerate. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
The Karyotype
A karyotype is a picture of chromosomal makeup (Figure 5-13). Chemicals like colchicine and its synthetic equivalent colcemid will bind and unassemble the microtubules of the spindle. Without a functioning spindle, cell division is halted at prometaphase and metaphase. Their condensed shapes clearly show relative chromosome sizes and centromere placements. These characteristics can then be used to arrange pairs of homologous chromosomes into a standardized pattern called a karyotype.
Figure 5-13. Karyotypes are ways of organizing and presenting the chromosomal makeup of an individual. (b: Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 4th ed. New York: McGraw-Hill, 2012.)
Additional information can come from modifying the basic staining protocols before the chromosomes are visualized microscopically. Giemsa is a polychromatic stain that darkens chromatin material uniformly. Chromosomes in dividing cells show up clearly, but modifications of technique can enhance structural detail. One example is G-banding, mentioned in Chapter 4 (Figure 4-19), which involves pretreating the chromosomes with the proteolytic enzyme trypsin before staining with Giemsa. The resulting dark bands are areas of heterochromatin, which is highly condensed chromosomal material. The intervening light bands are euchromatin. This produces a chromosome-specific pattern of dark and light bands that allows some degree of resolution for intrachromosomal changes in structure. Although small changes cannot be detected with these techniques, information about the karyotype of an individual can identify changes in chromosome number and large changes in chromosome structure that have clinical significance.
Overview of Changes in Chromosome Number and Structure
Species differ in the way their genomes are distributed among chromosomes. There is no correlation between the number of chromosomes and the developmental complexity of an organism. Likewise, genes of similar function are scattered among the chromosomes. There is no correlation between a particular chromosome and a particular body part or metabolic process. Chromosomes are simply the structures that link together and distribute genetic information from one cell generation to the next during mitosis and meiosis. Euploid is the normal chromosomal makeup of an individual (eu = true or normal; ploid = multiple). Deviations involving the loss or gain of one or more chromosomes are aneuploid, or “not true” multiples. A polyploid has “many” multiples of chromosomes, e.g., 3n triploids and 4n tetraploids. When they occur, changes in chromosome number are almost always much more serious than a single gene, or point, mutation, because so many different genes, and thus many biochemical processes, are involved. Chromosome number can also be altered by fusion or fission of the centromeric regions, although this phenomenon is typically of more importance when comparing chromosome arm homologies in related species.
Chromosome aberrations, or changes in structure, occur when the linkage of genes within and between chromosomes is altered (Figure 5-14). Changes in chromosome structure are most commonly due to breaks that are incorrectly repaired during replication. Chromosome breaks are very common. One estimate is that an average of 55,000 single-strand breaks and 9 double-strand breaks occur in DNA molecules in each nucleus each day. The vast majority of these are repaired, but if several affected strands are near each other, the broken ends can be reattached incorrectly.
Figure 5-14. Chromosome aberrations are changes in chromosome structure. (a) Deletion is the loss of a segment of chromosome. (b) Duplication is the insertion of a section of chromosome so that two copies of each affected gene are present. (c) Inversions can occur when two breakpoints are reattached at alternate ends. (d) Simple translocation is the movement of a section of one chromosome to a different linkage group. (e) Reciprocal translocation involves the exchange of sections between nonhomologous chromosomes. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Three types of aberrations can affect the genetic content of an individual chromosome. If two breaks are repaired so that the intervening segment is left out, a portion of the chromosome is no longer attached to a centromere and is lost from the nucleus the next time it divides. This yields a deletion or deficiency. Various mechanisms can cause a portion of the chromosome to be present twice, duplication.The order of genes along a chromosome can also change. For example, if two breaks in the chromosome are repaired so that alternate ends are attached, the intervening segment is now reversed, creating an inversion. In addition to the obvious changes in genetic content caused by these aberrations, especially in duplications and deficiencies, point mutations occur if the DNA breakpoints happen to be within the coding region of a gene. Furthermore, topological relationships among synapsed chromosomes in prophase I of meiosis can cause secondary consequences for the genetic composition of a fertilized egg. These are described in more detail later.
Finally, aberrations can affect more than one chromosome at a time. When a portion of one chromosome is reattached to a chromosome from a different linkage group, the result is called a translocation.Simple translocations involve the movement of a piece of one chromosome to another. When this translocated chromosome is passed to an offspring, there are extra copies of the genes carried in the translocated region. Reciprocal translocationsinvolve the complementary exchange of segments between two nonhomologous chromosomes. If both translocated chromosomes are passed to the offspring, there is no overall change in genome content. But if only one of them is passed on, the offspring will carry an unbalanced genome content. Thus, translocations change how genes are arranged in linkage groups and can have secondary consequences due to the way the altered chromosomes segregate in meiosis. We will look at the consequences of these abnormalities in more detail next.
Aneuploidy: Errors in Segregation
Aneuploidy is a deviation from the normal chromosome complement involving less than a full haploid set of chromosomes. The breakdown of a spindle microtubule, delayed division of the centromere connecting two sister chromatids, and other events can lead to the failure of chromosomes to segregate properly to opposite poles during division. Although this can occur in both mitosis and meiosis, our focus here will be on meiotic errors.
Failure to separate, or “segregate,” normally can occur at either the first or the second meiotic division (Figure 5-15). The term used to describe this kind of error, nondisjunction, is actually a double negative. “Junction” (to join) means to go together, so disjunction is not to go together, i.e., to separate. Nondisjunction is, therefore, not to separate, thus go together. The result is a gamete with either two copies or no copy of one or more chromosomes. When such a gamete combines with a normal gamete at fertilization, the resulting genome will be unbalanced by having abnormal numbers of active genes coding for their protein products. This will affect a large number of independent developmental and physiological processes. Most such affected embryos will die early. If an extra copy of a chromosome is present, there will then be three copies of that linkage group, a trisomic (“tri” is three, “som” is body). Alternatively, if the abnormal gamete has lost its copy of that chromosome, fertilization will result in only one copy of the linkage group, a monosomic (“mono” is one), coming from the normal gamete fertilizing the gamete that is deficient. In humans, most trisomics and all but one type of monosomic typically die early in development. Most special cases involve aneuploidy of the X or Y chromosomes. Since females carry two X chromosomes but males have only one, it is normal to have a difference in the copy number, or dosage, of all X-linked genes when comparing the two genders. One mechanism that compensates for this difference in copy number, X-chromosome inactivation, will be discussed next. Here we will simply point out that the mechanism that allows males and females to develop normally with different numbers of X chromosomes can also allow development to proceed fairly normally if aneuploidy of a sex chromosome occurs.
Figure 5-15. Nondisjunction is a failure of the separation of homologues at the first or second meiotic division to yield cells that are missing or have extra copies of a chromosome. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
X-chromosome Inactivation in Mammals
In mammals, the number of copies of X-linked genes will differ between males (with one X chromosome) and females (with two). To balance this difference, i.e., to accomplish “dosage compensation,” almost all genes on one X chromosome in a cell are inactivated (Xi) by a process called Lyonization after its discoverer, Mary Lyon. Important exceptions will be discussed later. Lyonization involves the tight coiling of all X chromosomes except for one that is left genetically active (Xa). X inactivation is permanent in the somatic cells but has to be reversible in the development of the germ cells. In summary, X inactivation occurs early in embryogenesis, it is random, and it is clonal in that, once inactivated, that same X chromosome remains inactive in somatic daughter cells.
In interphase nuclei, the inactivated X can be seen as a dark spot or Barr body, named for Murray Barr, who first described them in cells from female cats. If additional X chromosomes are present due to errors in segregation, they are also Lyonized to produce additional Barr bodies. The nucleus shown in Figure 5-16, for example, has three Barr bodies in an abnormal cell containing a total of four X chromosomes in addition to the normal 22 pairs of autosomes (2n = 48).
Figure 5-16. Three Barr bodies in an abnormal cell containing a total of four X chromosomes. Barr bodies are formed by the tight coiling, or Lyonization, of all except one X chromosome, so normal female nuclei have one Barr body.
X-chromosome inactivation is an example of epigenetic modification. In this phenomenon, a gene or in this case a chromosome becomes inactivated during an individual’s lifetime. X inactivation is passed on to daughter cells during cell division, yielding patterns like the black and orange patch pattern seen in calico cats (Figure 5-17). Inactivation occurs randomly, so a female is really a patchwork of genetic expressions, a “functional mosaic,” for any genes that differ between her two X chromosome copies. But in a broader context, one should be careful when using the term “mosaic,” because in medical genetics it is usually reserved to describe differences in genetic makeup, not simply expression.
Figure 5-17. Calico cats are females that are heterozygous for black and orange alleles carried on randomly inactivated X chromosomes to yield patches of orange and black fur, respectively. (Courtesy of Sarah M. Granlund.)
The mechanism of X inactivation involves a limited amount of blocking factor protein that binds to an X chromosome and blocks its inactivation. All other X chromosomes are left unprotected and are inactivated. The X inactivation center (XIC) is thought to control this chromosomal silencing process by binding the blocking factor protein. Indeed, if the XIC is translocated to an autosome, that autosome will become inactivated.
Polyploidy
Polyploidy is a change in chromosome number that involves multiples of a full haploid set. It is commonly found in plants, where it is an important mechanism for speciation. Many crop plants are polyploids derived from wild ancestors. Examples include coffee (4×, 6×, 8×), bananas (3×), bread wheat (6×), and common tobacco (4×). Agricultural geneticists can artificially induce polyploidy to combine genomes from different plant species. For some reason, polyploidy is tolerated much less well in animal development. In humans polyploidy is usually fatal at an early stage of development. Among the possible causes, polyploidy will result if more than one sperm enters the egg simultaneously or if there is a failure of separation of haploid nuclei during meiosis in the developing egg cell.
Changes in Chromosome Content: Deletions and Duplications
The terms “deletion” and “deficiency” are interchangeable and refer to the loss of a section of DNA that can range in size from simply affecting a region of one gene to encompassing tens or even hundreds of linked genes. If it is limited to a single gene, it may be difficult to tell a deletion from a nucleotide substitution or other point mutation. One way to identify them is by DNA sequencing or measuring the size of fragments amplified by the polymerase chain reaction, PCR (see Chapter 2). A small deletion will yield an amplified DNA fragment that is smaller than one from a simple base substitution mutation in which all nucleotides are present.
Figures 5-18 and 5-19 show some of the ways a change can be made in chromosome content. Typically, homozygosity for a deletion is lethal (it is equivalent to being homozygous for a large number of damaging point mutations), and heterozygosity can have severe effects on development. A deletion can also affect the phenotype if it happens to be heterozygous with a recessive mutant allele on the “normal” homologue. Since the dominant is missing in the deleted chromosome, the sole recessive allele is expressed phenotypically. This phenomenon is sometimes called pseudodominance. In experimental organisms like Drosophila, deletion mapping is a powerful tool for analyzing linkage relationships and for manipulating developmental processes.
Figure 5-18. Loss of genetic material can occur from (a) terminal deletions in which the broken end of a chromosome is lost, or (b) interstitial deletions involving two breaks and the loss of the intervening section of the chromosome. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 5-19. Crossing over between two improperly aligned homologues can generate deletions and duplications by recombination. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Duplications are regions of a chromosome that occur twice, so that the diploid has a total of three copies of each gene in the duplication. The duplicated copies can be directly adjacent, called tandem duplications, or can be located at a distance from each other as dispersed duplications. On balance, a duplication generally has a smaller effect on development than does a deletion of the same size. But in both cases, deletions and duplications essentially alter the gene dosage, which changes the amount of protein produced when the genes are active. This can cause an imbalance in biochemical processes throughout the body.
Changes in Chromosome Organization: Inversions
As is true for all aberrations, there are several mechanisms that will cause a change in the order of genes on a chromosome. One way is for two simultaneous breaks to occur on portions of a chromosome that happen to be coiled next to each other in the interphase nucleus. A change in order will occur if the broken ends are misattached (Figure 5-20). If the two breaks are in different arms so that the centromere is included within the inverted region, it is called a pericentric inversion (peri = around, as in perimeter). If both endpoints of the inversion are in the same arm, it is a paracentric inversion (para= beside, as in paramedic). A pericentric inversion will change chromosome appearance if the breaks are not symmetrical around the centromere. Other than possible point mutations at the breakpoints, the genetic content of the chromosome is not altered in either type. But that does not mean that inversions are without consequences for the carrier. The consequences are expressed in a different way, specifically as a reduction in genetically normal gametes. This is due to the physical looping of one chromosome to allow its genes to pair with their homologous copies on the other chromosome in an inversion heterozygote during synapsis. This will result in chromosome anomalies if crossing over occurs within the synapsed inverted region in prophase I of meiosis (Figure 5-21).
Figure 5-20. Inversions occur when the ends of chromosome segments are reattached incorrectly. The products can be classified as (a) pericentric or (b) paracentric, depending on whether the centromere is included or not included in the inversion. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
As we saw earlier, at synapsis in prophase I, the synaptonemal complex forms between the identical regions of the non-sister chromatids all along the length of the paired chromosomes. The only way for that to happen in an inversion heterozygote is for one of the strands to form a loop as shown in Figure 5-21. Recombination occurs during synapsis. So, if a recombination event occurs within the inversion loop, the strands become attached so that deleted and duplicated chromosomes are produced. You can demonstrate this yourself by tracing one of the chromosome strands beginning at the top arrow of Figure 5-21a; at the recombination point, the path crosses to the other chromosome and ends at the second arrow. The result is a chromosome that is duplicated for the normal sequence to the left of the first breakpoint and deleted for the region to the right of the second breakpoint. If the inversion is paracentric (Figure 5-21b), the centromere will be duplicated in one of the crossover products yielding a dicentric chromosome that forms a bridge between the separating nuclei when the centromeres move to opposite poles in anaphase I. The other crossover product has no centromere and is considered an acentric fragment, which is left behind when the nucleus divides. Neither type of gamete will yield a viable zygote, so fertility is reduced in inversion heterozygotes.
Figure 5-21. Crossing over in an inversion heterozygote yields deletions and duplications in half the haploid products of meiosis. In a paracentric inversion heterozygote, the centromere will be included in the segment that is either duplicated or deleted. This will result in dicentric bridges or acentric fragments, respectively. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Translocations
Translocations are the main type of chromosome aberration affecting two different chromosomes at the same time. If the exchange is reciprocal, so that a fragment from the first chromosome becomes attached to the centromere-bearing second chromosome, and vice versa, the genetic content of the cell is not affected except for possible point mutations at the breakpoints as in an inversion. But, as in inversion heterozygotes, reciprocal translocation heterozygotes can suffer severe reductions in fertility due to segregation in meiosis. The mechanism behind this is illustrated in Figure 5-22 and is not as complicated as it may initially appear. It essentially comes down to how the centromeres happen to attach to the meiotic spindle.
Figure 5-22. Translocation products depend on how the homologous centromeres attach to the spindle during meiosis. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
In our earlier discussion of meiosis, we pointed out that independent assortment is the result of the randomness with which different pairs of synapsed chromosomes attach to the spindle. If one bivalent is heterozygous Aa and a second bivalent is heterozygous Bb, the two sets of centromeres could attach so that the two dominant alleles are toward the same pole. In that case one gamete gets both dominant alleles, AB, and the other gets both recessive alleles. Or they could attach with the dominant-carrying strands facing opposite poles, so each gamete gets one dominant allele and one recessive, that is, they will be either Ab or aB. Think of segregation of centromeres in a reciprocal translocation heterozygote the same way.
To interpret Figure 5.22, you must imagine that the centromeres are attached to the spindle and that the poles are at the top and bottom of the figure. The left-hand portion of the figure is equivalent to one bivalent, and the right-hand side is a second bivalent. They interact because parts of their structures are synapsed to different homologues, but the key is how the centromeres attach to the spindle and segregate. Let us consider the case of adjacent segregation first (central panel of Figure 5-22). That is what will result if the centromeres are laid out as in the top part of the figure. The upper centromere of each bivalent will move to the top pole and the lower centromere of each bivalent will move the opposite direction. At the end of that first meiosis, each haploid nucleus has one normal and one translocated chromosome, thus being duplicated for some regions and deficient for others. These will not yield viable zygotes. But now imagine that the centromeres of one of the bivalents are flipped, so that the upper left centromere segregates along with the lower right centromere, and the lower left goes with the upper right. This is alternate segregation and results in one haploid having the two normal chromosomes and the other haploid getting both of the translocated chromosomes. The genetic content is balanced and the zygotes they create will each have a complete diploid genome.
An important example of this type of aberration is the Robertsonian translocation, in which two nonhomologous acrocentric or telocentric chromosomes become fused at their centromeres to produce one linkage group. This results in a reduced chromosome number that may or may not have a phenotypic effect, depending on whether any coding DNA is lost in the fusion.
Somatic Mosaics
The changes in chromosome makeup described so far in meiosis affect every cell in the offspring’s body. But changes in chromosome number and structure can also occur during mitosis. The effect of such somatic changes will be limited to the lineage of cells that derive from the original error. They lead to an individual that is a cellular mosaic of different genotypes. Indeed, at some level, we are all probably mosaics of slight genetic differences that have occurred during our development. Most will involve point mutations of genes that may not even be transcribed in the specialized cell type in which they are found. A few will involve changes in chromosome number or structure that can cause serious medical conditions like certain cancers.
Another related phenomenon should be mentioned here. It is autonomous gene expression.
“Autonomous” means self-contained. In the context of gene expression it refers to a gene that affects only the biochemical activities of the cell in which it acts. Its gene product is not diffusible, so mutant cells in such a mosaic patch cannot be helped by normal tissue surrounding them. The phenotype for a visible trait would, therefore, be a mosaic spot or patch.
The Unique Nature of the Y Chromosome
Comparatively few genes are located on the human Y chromosome. Those that are unique to the Y, so-called holandric genes, include the Sry (sex-determining region Y) gene that is necessary for normal male-specific development. It specifies testis determination and promotes the synthesis of testosterone. In addition, a few genes are found in small areas of homology between the X and Y chromosomes called pseudoautosomal regions. These promote pairing of the X and Y to assist in proper segregation during meiosis. Like genes on the human X chromosome, those in the pseudoautosomal region of the Y can show recombination. But they do not undergo Lyonization, or X chromosome inactivation, so this small region of X and Y homology behaves like an autosomal region.
Part 2:Medical Genetics
Introduction
Mitosis and meiosis are intricate and coordinated. Like most biological systems, however, things may not happen perfectly. The discipline of cytogenetics is the study of what happens when the processes of mitosis or meiosis do not go according to design or plan.
Cytogenetic abnormalities are important conditions to know about, because of their historic standing as well as the relative frequency of these conditions in general. The determination of the human chromosome count at a modal number of 46 was only correctly assigned in 1956. Shortly thereafter in 1959, trisomy 21 (an extra, i.e., third, copy of chromosome 21) was associated with Down syndrome as the first identified genetic marker of a medical condition. Thus, cytogenetic anomalies became the first genetic abnormalities that were understood at an etiologic level. Advances in cytogenetic technology have occurred at a rapid pace (Refer to Tables 1-3 and 1-4 for an overview). For the medical student, cytogenetic abnormalities are also important as frequently asked questions on board examinations. A student should be aware of the (historically) major chromosomal syndromes. This chapter will provide brief descriptions and highlights of these conditions; however, these descriptions are not meant to be comprehensive. Specifically, this is not a dysmorphology textbook. For more details on any given condition, you are referred to the most recent edition of any of several good references at the end of this chapter.
Chromosome Abnormalities
Chromosomal abnormalities are relatively common (Table 5-1). Prior to conception 5% of sperm and 50% of oocytes have abnormal chromosome complements. Not surprisingly, then, 50% of conceptions are expected to have an abnormal chromosome makeup. At the time of live term births, however, only 0.8% of infants will have a chromosome abnormality. As these numbers clearly suggest, chromosome abnormalities have a strong association with early pregnancy loss (Tables 5-1 and 5-2). In fact, 95% of all chromosomal abnormalities present at conception are not live-born.
Table 5.1 Estimated Frequencies of Chromosome Abnormalities (Perinatal)
Table 5.2 Estimated Frequencies of Chromosomal Anomalies
By way of definition, pregnancy loss at less than 6 weeks post conception would be classified as an “early loss.” Losses between 6 and 22 weeks are termed “miscarriage” or “spontaneous abortion.” Losses later than or equal to 23 weeks are best classified as “stillborn” infants. Other terms would include intrauterine fetal death and/or products of conception in reference to the tissue that is lost with a spontaneous miscarriage.
Cytogenetic analysis of first trimester products of conception show 65% will have an abnormal chromosome count. In contrast, only 1.5% are abnormal by mid gestation (approximately 20 weeks), and, as mentioned above, only 0.8% of live-born children will have a chromosome imbalance.
Recurrent pregnancy losses may sometimes be perceived as infertility, because the conceptions are actually not recognized and the pregnancy is lost early. A chromosome analysis of the couple is part of the workup for infertility. Likewise, in the case of recognized miscarriages, any couple who has experienced three or more spontaneous miscarriages should be offered chromosomal analysis. In this setting, 10% of the time, one of the partners carries a balanced chromosome rearrangement.
The occurrence of chromosome imbalances has a strong association with advanced maternal age (Table 5-3). As mothers age, there is an increase in abnormalities due to chromosomal nondisjunction. These meiotic errors are presumably related to the normal status of human oocytes. Human female fetuses typically have several million oocytes around mid-gestation. By term birth, this number has been culled down to a few hundred thousand functional cells, although only about 400 will actually be released at ovulations through the reproductive life of the female. These oocytes have started meiosis, but are arrested mid-process at the specialized stage called dictyotene in prophase I. Meiosis I is not completed until ovulation, and meiosis II is not accomplished until fertilization. Presumably, the longer period of time that these cells stay in this suspended state predisposes to a greater chance of nondisjunction when the process is finally allowed to proceed. The rise in nondisjunction is first noticed with maternal ages in the mid thirties, and rises asymptotically after 40.
Table 5.3 Frequency of Down Syndrome in Association With Maternal Age
Although the association of an advanced maternal age with the occurrence of Down syndrome is well known, it should be pointed out that this association holds true for all types of nondisjunction. It is also important to point out that this increase occurs on a per pregnancy basis. Thus, nondisjunction occurs more often in older mothers per pregnancy. As there are many more pregnancies in younger mothers, there are actually more children born with Down syndrome and other chromosome abnormalities to younger mothers.
Laboratory Diagnosis of Chromosome Abnormalities
As described earlier, a karyotype is a conventional representation of chromosome structure and number (Figure 5-23). The method used in most clinical diagnostic settings is G banded chromosomes. The current standard is what is referred to as high resolution or prometaphase chromosomes. This typically would represent 700 to 800 recognizable bands on the karyotype.
Figure 5-23. Normal karyotypes. G banded. High resolution (prometaphase). (a) Female (b) Male. (Courtesy of Dr. Warren G. Sanger, University of Nebraska Medical Center.)
Recent advances in cytogenetics have sprung from the advancement of a technology known as fluorescent in situhybridization (FISH). Basically FISH utilizes a synthesized probe that is composed of the complementary sequence to a known segment of DNA. The probe is attached to a fluorescent marker. The probe strand is then applied to the patient’s DNA. If the complementary strand is present, the two will hybridize. The presence of the hybridization can then be detected by the presence of fluorescence as seen under the microscope (Figure 5-24).
Figure 5-24. Flourescent in situ hybridization (FISH). (a) Schematic of process. (b) Idiogram showing a deletion on chromosome 22.
Fluorescent in situ hybridization technology has truly revolutionized the practice of clinical genetics. The first readily available clinical uses of this technique were single locus FISH. These studies led to the description and definition of the etiology of specific recognizable conditions due to duplications or deletions too small to be detected by even high resolution chromosome studies (Figure 5-25). Collectively, these disorders may be called contiguous gene syndromes. These conditions are characterized by recognizable patterns of multiple malformations and anomalies due to the duplication or deletion of several genes that are situated together at a particular chromosomal locus. These are discussed in more detail later.
Figure 5-25. FISH study demonstrating a 15q deletion in the Prader-Willi/Angelman syndrome region. (Courtesy of Dr. Warren G. Sanger, University of Nebraska Medical Center.)
Fluorescent in situ hybridization technology has a myriad of other applications in the clinical setting. An assembly of FISH probes that provide coverage of entire chromosomes may be used for chromosome painting. This may be quite helpful in sorting out complex rearrangements, identifying the origin of marker chromosomes, and so forth. A significant advantage of FISH technology over standard karyotype analysis is that FISH does not require actively dividing cells. In order to create a traditional karyotype, living cells must be allowed to divide. Then cell division must be stopped by biochemical methods to halt further progression. In order to visualize a “chromosome,” the cell cycle must be stopped somewhere between metaphase and prophase. But FISH studies can be performed on any target nucleic acid segment regardless of what point of the cell cycle it is in. It can be used for any segment of nucleic acid, even for those outside a cell. This offers a great advantage in the clinical realm allowing for its application in many different settings and providing more rapid diagnostics (Figure 5-26).
Figure 5-26. Different applications of FISH technology. (a) Whole chromosome painting. (b) Interphase (amniocentesis) FISH showing Trisomy 13. (c) Interphase (amniocentesis) FISH showing XX/XY mosaicism. (Courtesy of Dr. Warren G. Sanger, University of Nebraska Medical Center.)
The areas underneath the telomeres of the chromosomes are regions prone to rearrangements and mismatches. Further expansion of the applications of FISH studies led to the expansion from single locus FISH to what was known as sub-telomeric FISH panels (Figure 5-27). This panel, developed around 1998 to 1999, included approximately 40 probes that corresponded to the sub-telomeric regions of the chromosomes (note, there are not 46 probes since the acrocentric chromosomes do not have telomeres for a short arm). The advent of a sub-telomeric FISH is now only of historic interest as it has already been supplanted by further refined techniques. But at the time it was introduced, it represented an important advancement in cytogenetic diagnostics. Published studies around 1999 to 2000 or so, showed the diagnostic yield of sub-telomeric FISH (chances of finding a positive result) in mental retardation was 7.4% for moderate levels of mental retardation and 0.5% for mild mental retardation, with an average around 3%. This made sub-telomeric rearrangements the most common identifiable cause of moderate mental retardation in humans (i.e., a calculated incidence in the general population of 0.22% as compared to the incidence of Trisomy 21 at 1 in 800 [0.13%]).
Figure 5-27. Sub-telomeric FISH study with chromosome 3 example. (Reprinted with permission from Clarkson B, Pavenski K, Dupuis L, et al. Detecting Rearrangements in Children Using Subtelomeric FISH and SKY. American Journal of Medical Genetics 107:267-274, 2002.)
Further expansion of FISH technology beyond the sub-telomeric FISH panel included the development of a technology called array comparative genomic hybridization (aCGH). Array comparative genomic hybridization refers to the process of comparing sample DNA to known reference DNA and looking for changes in copy numbers (either duplications or deletions). Application of aCGH technology on small microscopic slides with multiple imbedded wells is referred to as a microarray (Figure 5-28). Hence array comparative genomic hybridization (aCGH) refers to the use of aCGH on a microarray platform. This technology is an ever-evolving one. The original aCGH platforms had approximately 400 probes but were rapidly replaced by platforms that included 2000, then 40,000, then 105,000. Now a few laboratories offer aCGH panels of 180,000 probes. These platforms offer coverage of less than 1 megabase (Mb) intervals across the entire genome, although the intervals are not actually distributed evenly. To put this in perspective, the size of the single dystrophin gene is approximately 1.8 Mb. Still, it would not surprise anyone if, as you read this, these numbers are already outdated. In fact on the horizon aCGH technology may even be replaced by emerging technology such as whole exonic sequencing.
Figure 5-28. Microarray-based comparative genomic hybridization. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
With these advances in technology, the boundaries between molecular genetics and cytogenetics have blurred. Currently this hybrid discipline is referred to by many as molecular cytogenetics. Array CGH and related modern whole genome screening techniques are discussed in additional detail in Chapter 11.
Chromosome Aneuploidy
Aneuploid literally means “not the correct multiple or number.” Aneuploidy then is the state of being aneuploid. Thus, chromosomal aneuploidy refers to the situation in which an individual possesses an abnormal chromosome number. Table 5-4 lists some of the more important or common human chromosomal aneuploidy syndromes. These conditions are important to recognize for several reasons. As mentioned earlier, historically these were the first described conditions with an identifiable genetic etiology. The general public is well aware of some of these conditions—and often has significant misconceptions. And, not least important, they appear frequently in questions on standardized examinations.
Table 5.4 Conventional Chromosomal Syndromes
One variant of chromosome number is referred to as polyploidy. Triploidy means 69 chromosomes with a full three copies of every chromosome (Figure 5-29). It is a very common occurrence in conceptions. But, as with most chromosome imbalances, the vast majority of such conceptions result in a spontaneous miscarriage (Table 5-2). In fact, it is estimated that approximately 11% of all spontaneous miscarriages have a triploid karyotype. Occasionally there is a live birth of an individual with a triploid chromosome count. This is much more likely if the individual is mosaic for this change (Figure 5-30). Overall it is estimated that approximately 1 in 10,000 live births may have this abnormality. The karyotype in triploid individuals can be 69, XXX, 69, XXY or 69, XYY. Triploidy most often results from a single egg being fertilized by two sperm (dispermy), resulting in an extra set of chromosomes from the father (diandry). It can also occur from digyny, in which the full extra set is from the mother.
Figure 5-29. Karyotype with Triploid count (69 XYY). Likely due to dispermy. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
Figure 5-30. Patient with diploid/triploid mosaicism. (a) Infant. (b) Young adult.
Fetuses with dygyny tend to have a relatively small placenta with a better developed fetus. Conversely, fetuses with dyandry are less well developed, and there is a large abnormal placenta. These variations in clinical expression can be explained by differences in what is known as imprinting. For further information on imprinting, please refer to Chapter 12, which addresses Atypical Inheritance.
Tetraploidy (92 chromosomes) is common in spontaneous miscarriages but is typically not found in live born infants.
Sex chromosome aneuploidy
Because of the unique nature of the sex chromosomes, aneuploid situations are better tolerated. Specifically, sex chromosome aneuploidy is seen more commonly in live born infants than is autosome aneuploidy. At conception, the occurrence is likely similar, but autosome aneuploidy is more likely to be lost as a spontaneous miscarriage.
1. Turner syndrome. A missing X chromosome (45, X) is the only whole chromosome monosomy that is compatible with postnatal life in humans. The phenotype was described by Henry Turner in 1938 with the condition now having the eponym of Turner syndrome. The initial description was that of girls with short stature, pubertal failure, cubitus valgus and webbed neck (Figure 5-31). Other features may include a broad chest with wide spaced nipples and angulated nails. A variety of structural anomalies may occur. The two most important ones to note are coarctation of the aorta and a “horseshoe” kidney. Many of the clinical features in Turner syndrome can be assigned as being secondary to congenital lymphedema (Figure 5-32). The discovery of a missing X chromosome as the cause of Turner syndrome was made by Charles Ford in 1959.
Figure 5-31. Turner syndrome. These photographs were taken from the original pictures used by Dr. Henry Turner in his 1938 publication. Black and white photographs were cut and pasted on black cardboard. Hand lettering was with a white ink pen. (Courtesy G.B. Schaefer; originals donated to the archives of the Endocrine Society of America.)
Figure 5-32. Spontaneous miscarriage of fetus with Turner syndrome. Note striking lymphedema, which can explain most of the somatic features associated with Turner syndrome.
Depending on the presentation of a particular girl, the diagnosis of Turner syndrome may be made at various points in her life. On occasion, infants with Turner syndrome can be recognized due to the presence of suspicious somatic features. The congenital lymphedema may persist after birth and show up as edema of the dorsum of the hands and feet (Figure 5-33). Differential pulses may indicate a coarctation of the aorta, which, if present in a newborn female, may warrant further investigation into possible Turner syndrome. But many girls with Turner syndrome will not have any identifiable features at birth. The most consistent clinical features seen in individuals with a 45, X karyotype are short stature and primary ovarian failure. Both of these would obviously present later in life.
Figure 5-33. Dorsal edema of the (a) feet and (b) hands in two infants with Turner syndrome. (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
This offers a situation in which early proactive awareness by a physician can have significant benefits for the patient. It is important to recognize Turner syndrome early, because of the advantages of using growth hormone to increase final adult height. Therefore, even in the absence of any physical features, girls with an otherwise unexplained short stature should have a karyotype performed (Figure 5-34).
Figure 5-34. Young girl with Turner syndrome; no appreciable dysmorphic features of Turner syndrome.
In general, and contrary to what is reported in the older literature, Turner syndrome is associated with normal intelligence. These women have distinct neuropsychological changes, including consistent problems with visual–spatial integration and often difficulties in mathematics.
The Turner syndrome phenotype may be associated with several chromosomal imbalances. Approximately half of the girls with Turner syndrome have a straight 45, X karyotype (Figure 5-35). Another 25% have some form of sex chromosome mosaicism, with the remainder having a variety of other chromosomal rearrangements including isochromosome Xq, deletion Xq, or deletion Xp. Turner syndrome is estimated to occur in 1 in 2500 live births.
Figure 5-35. Karyotype 45, X. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
Klinefelter syndrome. Klinefelter syndrome (Figure 5-36) is a disorder found in males associated with a 47, XXY karyotype (Figure 5-37a). The major clinical features of Klinefelter syndrome are attributable to hypogonadism (primary testicular failure). Because of the primary testicular failure, these individuals often present with gynecomastia, infertility, and a “eunichoid habitus.” Hypo-mentation (decreased IQ) is relatively common in this condition; however, many individuals with this condition have normal intellect. The overall mean IQ with Klinefelter syndrome is around 90. This condition occurs in approximately 1 in 600 newborn males.
Figure 5-36. Young man with Klinefelter syndrome. (a) Tall stature with “eunichoid habitus”; (b) Small testes.
It is important to note that 40%-50% of males with Klinefelter syndrome have no discernable physical features. They only present as male infertility due to azoospermia (no production of sperm). For that reason, a semen analysis with a follow up karyotype is indicated as part of the infertility workup.
Variants of Klinefelter syndrome include 48, XXXY (Figure 5-37b) and 49, XXXXY. In general, these individuals have a Klinefelter-like phenotype. The primary differences are that with an increasing number of X’s, there is an increase in growth restriction and decrease in IQ. Many individuals with these variants may have some mild craniofacial and skeletal changes not otherwise seen in 47, XXY Klinefelter syndrome.
Figure 5-37. (a) Karyotype 47, XXY. (b) Karyotype 48, XXXY. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
3. XXX Syndrome. Women with a 47, XXX karyotype (Figure 5-38) are typically reported as having “no pattern of malformations.” Pubarche and fertility are felt to be typically normal. As compared to the general population, females with 47, XXX tend to be a little taller and have a slightly decreased average head circumference. As a group they tend to have a slightly higher incidence of learning disabilities, developmental disabilities, discoordination, and an increase in behavioral problems (Figure 5-39).
Variants of the 47, XXX syndrome include 48, XXXX and 49, XXXXX. Again, as the number X’s increases, the overall IQ decreases. Some individuals who have XXXX and XXXXX have been reported to have facies not unlike that seen in Down syndrome.
Figure 5-38. Karyotype 47, XXX. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
4. XYY. Individuals with a 47, XYY karyotype (Figure 5-39) have been reported to have subtle features of slightly taller than expected stature, discoordination, speech delays, behavior differences, and learning disabilities. These men have normal fertility. Occasionally reported features include large teeth, prominent glabella, long arms and legs, and difficult to control acne. The overall incidence is felt to be 1 in 700 to 1 in 1000 males. Early reports in the 1970s showed an increased incidence of XYY karyotype in men that were incarcerated. Sociobiological interpretation raised the question of an XYY karyotype being associated with a “criminal phenotype.” It has been shown that this condition is not associated with a criminal phenotype, but in fact the increased incidence noted in the prison population represents a bias of ascertainment—presumably not due to increased chances of committing crime, but, of being caught. They are tall, clumsy, and mentally slow.
Figure 5-39. Karyotype 47, XYY. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
Trisomies
Trisomy refers to the presence of a single extra full chromosome. Trisomies of all autosomes occur with a relatively high frequency in conceptions, but most are lost as miscarriages. Only three whole chromosome (non-mosaic) aneuploidies are compatible with postnatal life in humans. These are Trisomy 13, 18, and 21.
1. Trisomy 21 (Down syndrome). Down syndrome was described by Langdon Down in 1866. Individuals with Down syndrome have a readily recognizable facial appearance described as flattening of the facial profile, small nose, epicanthal folds, and Brushfield spots (focal areas of dysplasia on the iris). They may have notably short fifth fingers, a wide gap between the first and second toes, and single transverse palmar creases (Figure 5-40). In 1959, the karyotypic association of Trisomy 21 in association with the phenotype of Down syndrome was described (Figure 5-41). Down syndrome is estimated to occur at 1 in 800 live births.
Figure 5-40. Down syndrome. (a) Typical facial features. (b) Eye showing Brushfield spots (small light colored spots within the iris due to focal dysplasia of the connective tissue). (c) Single transverse palmar (simian) crease. (d) Wide gap between first and second toes.
Figure 5-41. Karyotype 47, XX, +21. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
The vast majority (95%) of individuals with Down syndrome have whole chromosome aneuploidy (trisomy 21) as the etiology. Another 4% have a variety of translocations, most importantly a 14:21 Robertsonian translocation. Approximately 1% of individuals with Down syndrome have mosaicism.
Individuals with Down syndrome have a variety of health issues that are important to be aware of. These issues need to be addressed as a condition-specific series of recommended additions to the “typical” health care maintenance regimen as these patients are followed in their medical home.
• Individuals with Down syndrome have a variety of neurologic problems. These can include lower than average IQ, Alzheimer disease, low muscle tone (hypotonia), and vision and hearing problems.
• Congenital heart malformations occur in about 50% of the cases. The most common heart anomaly is an atrial-ventricular canal which, if large enough, may have no associated cardiac murmur. Thus, an echocardiogram is recommended for all people with Down syndrome at the time of diagnosis, regardless of the presence or absence of a murmur.
• Other structural changes in Down syndrome include atlanto-occipital instability of the spine and a variety of different GI obstructions. Acquired hypothyroidism occurs much more often in these individuals.
• The overall incidence of leukemia is approximately 11-fold greater than in the general population.
2. Trisomy 13 (Patau syndrome). Trisomy 13 is associated with multiple congenital abnormalities and severe cognitive impairments. Structural changes include cleft lip and cleft palate, microphthalmia, polydactyly, microcephaly, and congenital heart disease (Figure 5-42). The etiology is either nondisjunction or an inherited translocation of chromosome 13 (Figure 5-43). The incidence is estimated at 1 in 5000 births.
Figure 5-42. Two day old girl with Trisomy 13 (Patau syndrome). (a) Craniofacial features include microcephaly with sloping forehead, supraorbital creases, and broad triangular nose. (b) Low set pinnae with abnormal helices. (c) Postaxial polydactyly. (d) “Rocker bottom feet” (prominent calcanei). (e) Another child Trisomy 13 with cutis aplasia of the scalp.
Figure 5-43. Karyotype 47, XY, +13. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
3. Trisomy 18 Edwards syndrome. A similar phenotype is seen in Trisomy 18 (Edwards syndrome). Clinical features include severe growth deficiency, mental retardation, clinched fist with overlapping fingers, “rocker bottom” feet, and congenital heart disease (Figure 5-44). The etiology is also nondisjunction or inherited translocation, but of the 18th chromosome (Figure 5-45). The estimated incidence is 1 in 3000.
Figure 5-44. Patient with Trisomy 18 (Edward syndrome).
Figure 5-45. Karyotype 47, XY, +18. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
From a management standpoint, the most critical issue for Trisomy 13 and 18 is a decrease in the overall life expectancy. Table 5-5 summarizes the reported data on the longevity expectations of these two conditions. It is important to highlight that the last column shows that death by year one is estimated to be about 90% for both conditions. It is critical that the clinicians working with families who have newborns with these severe conditions be aware of the fact that 90% is not 100%. Long-term survival with Trisomy 13 and 18 have been reported on occasion in almost every genetic practice. It is, therefore, important to give the family the most accurate information. One should not downplay the ominous statistics, but yet be aware of the correct statistics.
Table 5.5 Published Longevity Statistics for Trisomy 13 and Trisomy 18
Chromosome Mosaicism
Chromosomal changes due to mitotic errors that occur after conception can result in chromosomal mosaicism i.e., not every cell has the same chromosome makeup. (The concept of mosaicism is discussed in additional detail in Chapter 12 Atypical Inheritance). The number and distribution of the chromosomally abnormal cells in an individual will vary depending upon the timing and on which progenitor cells the abnormality begins with. It follows that the phenotype can be strikingly variable from being almost the same as “the full blown” condition to a non-expressed one. Figure 5-46 shows a young girl who was evaluated for mild developmental delays. Her cytogenetic testing revealed mosaicism for Down syndrome (7 out of 30 cells tested). As can be seen from the picture, her craniofacial features truly do not bear a resemblance to the phenotype of Down syndrome. In fact, the only appreciably abnormal physical feature seen was a wide gap between her first and second toes. Undoubtedly, however, this mild mosaicism is the cause of her developmental delays.
Figure 5-46. (a) Young girl with mosaicism for Trisomy 21 (7 of 30 cells in peripheral blood with trisomy). She presented with mild developmental delays. (b) The only physical feature of Down syndrome was a wide gap between the first and second toes.
Another important feature to highlight is the association between chromosomal mosaicism and skin pigmentary changes. This arises from the embryology of the neural crest cells. After separating from the neural tube, some of the neural crest cells migrate extensively and give rise to many different types of differentiated cells. These cell types include neurons and glial cells of the nervous systems, medulla cells of the adrenal gland, the melatonin producing cells of the epidermis, and skeletal and connective tissue components of the craniofacial complex. This known association may provide the critical clue in defining an etiology of a particular problem. Specifically, skin pigmentary changes that follow a pattern known as Blaschko lines may indicate an underlying chromosomal mosaicism. Hence, part of the diagnostic work-up of individuals with neurologic problems (seizures, mental retardation, and so forth) and skin pigmentary changes includes a skin biopsy to obtain cultured fibroblasts for karyotypic analyses on another tissue (Figure 5-47).
Figure 5-47. Hyperpigmented skin patches associated with chromosomal mosaicism. (a) large clonal patches of pigment. This patient has diploid/triploid mosaicism. (b) Hyperpigmented swirls on back. This patient has mosaicism for tetrasomy 12p. Both patients had normal blood chromosome studies.
Changes in Chromosome Structure
Not all chromosome imbalances are changes in the number of entire chromosomes. Partial changes in chromosome structure include duplications, deletions, and translocations. The phenotype associated with partial structural chromosomal changes (duplications/deletions) depends on many factors, including the size of the change, which region of which chromosome is involved, and the particular genes present in the affected region. In general, duplications may not be as problematic as deletions. While these factors intuitively make sense, there is much variation that cannot be explained by these factors alone. Many other modifiers are also likely at work.
Translocations may be either balanced or unbalanced. Balanced translocations are defined as those rearrangements in which there is a change in the position but not the actual amount of genetic material. Conversely, unbalanced translocations occur when there are changes in both the amount and location. In theory, persons carrying balanced translocations should have no clinical effects from the rearrangement. The major implication of carrying a balanced translocation is the possibility of passing on an unbalanced rearrangement to the next generation. As explained in the first section, the possible outcomes in the offspring of a carrier of a balanced translocation would be:
1. normal chromosomes
2. unbalanced rearrangement, or
3. a balanced rearrangement like the parent.
The clinical sequelae of an unbalanced translocation are that of producing a deletion or duplication—with the same implications for these imbalances as noted earlier.
When individuals are identified with chromosome rearrangements (either balanced or unbalanced) as part of a diagnostic evaluation, it is absolutely indicated to do a karyotype on the parents. In the situations of chromosome structural changes, there is a possibility that one of the parents is a carrier of a balanced translocation. Up to 10% of the time, one of the parents of a child with an unbalanced translocation may themselves have a balanced translocation. Particularly important are certain rare abnormalities such as tandem duplication of chromosomes. Figure 5-48a shows a 13:13 translocation present in a newborn male with features of Trisomy 13. Even though this individual has 46 chromosomes, he is functionally trisomic for 13 and had the full array of clinical features. Karyotypic analysis of this individual’s parents showed that the mother had 45 chromosomes with one of the chromosomes being the 13:13 hybrid chromosome. Thus, the only possible reproductive outcomes for this individual were either to have a child that was monosomic for 13 (and invariably would miscarry) or have a child with Trisomy 13 (Figure 5-48b).
Figure 5-48. Two karyotypes with Trisomy 13 due to a 13:13 translocation (both from amniocentesis). (Courtesy Dr. Warren G. Sanger, University of Nebraska Medical Center.)
An important observation has been noted by clinicians in this regards for decades. A child might have been seen for an evaluation because of multiple congenital anomalies, dysmorphic features, and/or cognitive deficits. As part of that evaluation, cytogenetic studies were performed, and the child was noted to have a balanced translocation. In order to clarify this abnormality, the next step was to perform parental studies. Not infrequently, one of the parents (with no clinical abnormalities) would be found to have the same balanced rearrangement. The logical interpretation would be that this was a coincidental finding unrelated to the child’s problems, since an unaffected parent had the same chromosome change. However, this situation was seen all too commonly in these types of evaluations, leading many clinicians to suspect that the situation was not as straightforward as it seemed. With advances in molecular techniques, it has now become apparent the transmission of an apparently balanced rearrangement from a parent to the child may involve further changes, such as small duplications or deletions around the breakpoints (or elsewhere), that were too small to detect at the resolution of a karyotype.
Structural rearrangements involving the X chromosome can influence the process of Lyonization. As such, different patterns of expression may be seen. In the event of a structurally abnormal X chromosome (i.e., one with a deletion), the abnormal X is preferentially inactivated, leaving the normal X active. But with balanced X-autosome translocations, the normal X chromosome is the one that is usually inactivated. It has been hypothesized that this is because inactivation of the X-autosome translocated chromosome is probably lethal. These differing patterns are observed phenomena, not events that could be predicted.
Chromosome Structural Changes Associated with Well-Described Syndromes
Prior to FISH, a handful of conditions were identified with specific structural chromosomal imbalances big enough to see in karyotypes. These conditions had a recognizable phenotype that allowed the identification of a described syndrome in association with the specific chromosome imbalance. Historically, two are worth mentioning.
1. Wolf-Hirschhorn syndrome (4p-). Deletion of the terminal end of the short arm of chromosome 4 has been associated with a condition known as Wolf-Hirschhorn syndrome (Figures 5-49 and 5-50). Patients with Wolf-Hirschhorn syndrome have microcephaly, a “characteristic” facial appearance due to a very prominent nasal bridge, mental deficiency, and cleft lip with or without cleft palate. This can either be de novo or inherited.
Figure 5-49. Young girl with Wolf-Hirschhorn syndrome (4p-). (a) 9-months old. (b) 6-years old. (c) 9-years old. (d) 18-years old.
Figure 5-50 (a) Karyotype 46, XX, deletion 4p. (b) High resolution chromosome 4 pair showing deletion 4p (arrow). (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
2. Cri-du-chat (5p-) syndrome. Figure 5-51 shows an individual with a deletion of the terminal portion of the short arm of chromosome 5 (Figure 5-52). This particular deletion has been associated with a unique phenotype (syndrome) known as Cri-du-chat, which is the French term for “cry of the cat.” This cry is distinctive and truly reflective of its name. This condition can sometimes be suspected upon hearing the characteristic cry. Other physical features include low-birth weight, microcephaly, hypertelorism, rounded facies, micrognathia, and severe cognitive deficits. This may be a de novo or inherited abnormality.
Figure 5-51. Young girl with Cri-du-chat syndrome (5p-). (Reprinted with permission from Brooker RJ: Genetics: Analysis & Principles, 3rd ed. New York: McGraw-Hill, 2008.)
Figure 5-52. Karyotype 46, XX, del 5p. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska)
Contiguous Gene Syndromes
Wolf Hirschhorn syndrome and Cri-du-chat syndrome represent well-described “contiguous gene syndromes” (Table 5-6). Such disorders are conditions that have a recognizable phenotype with an associated structural chromosome change. The changes may be large enough to be seen on a karyotype or small enough that a molecular cytogenetic testing modality (such as FISH) is needed to identify it. The multiple genes involved sometimes can actually predict phenotypes. The clinical manifestations can be attributed to multiple genes that are located in tandem on the chromosomes which are either deleted or duplicated. Sometimes knowledge of the genes involved may provide direct insight into the phenotype. More often, however, it is not possible to predict the phenotype based upon the genes involved.
Table 5.6 Important Contiguous Gene Syndromes
The characteristics of contiguous gene syndromes are:
1. They have a recognizable phenotype.
2. Typically the individual is heterozygous for the genetic change.
3. The condition shows familial transmission that looks Mendelian in nature (dominant transmission).
4. They typically show markedly variable expression.
A classic example of a contiguous gene syndrome is Williams syndrome. Williams syndrome has a recognizable phenotype associated with short stature, pixie or “elfin like” facial features, a stellate pattern to the iris, hypercalcemia, and a variety of vascular changes (Figure 5-53). The facial features are somewhat subtle. In the pre-FISH era, the clinical diagnosis could often be difficult. Ultimately, Williams syndrome has been shown to be associated with a deletion at 7q11.23 (Figure 5-54). One gene in this region is the elastin gene which codes for a protein that, as the name would imply, gives elasticity to connective tissues. Many of the somatic changes seen in Williams syndrome can be attributed to the deletion of the elastin gene. Other deleted genes in this region are related to the other changes, such as cognitive and behavioral abnormalities, seen in these patients.
Figure 5-53. A young girl with Williams syndrome. (a) Note classic facial changes. (b) “Stellate” pattern of the irises.
Figure 5-54. FISH study showing 7q deletion seen with Williams syndrome. (Reproduced with permission of Warren G. Sanger, PhD, University of Nebraska Medical Center, Omaha, Nebraska.)
The advent of microarray comparative genomic hybridization has greatly expanded the list of identifiable contiguous gene disorders. An interesting phenomenon is now frequently encountered that is an important issue for families. Microarray studies are now identifying conditions that bear no eponymic designation, i.e., there is no name to the syndrome. For these conditions, the actual “name” is the cytogenetic description. Surprisingly, this has turned out to be concerning to some families. One such example is the 1q21.1 deletion. This particular micro-deletion was first described in 2008. With microarray technology, it has been found to be a relatively common deletion in patients with mild neurodevelopmental disorders or congenital heart malformations. The phenotypic range is broad and includes speech and language delays, behavioral changes, mild dysmorphic facies, and congenital heart malformations in some (Figure 5-55a). The gene for thrombocytopenia-aplasia of the radius (TAR) is in this region. Depending on the size of the abnormality, it may be deleted as part of this change. If the TAR gene is deleted, patients have been noted to have variable expression of radial anomalies and/or thrombocytopenia (Figure 5-55b). There is no eponym associated with this condition. The actual designation is “1q21.1 deletion.”
Figure 5-55. Two patients with a 1q21.1 deletion discovered on microarray studies. (a) Middle-aged girl who presented with mild developmental delays and behavioral problems. (b) Young boy with 1q21.1 deletion with developmental delays, hypotonia, and radial hypoplasia.
Part 3:Clinical Correlation
DiGeorge syndrome (DGS) was described by the pediatric endocrinologist Angelo DiGeorge in 1968. The condition represents a field defect of the developing third and fourth branchial arches of the human embryo. The pattern of anomalies seen in DiGeorge syndrome reflects the embryological derivatives of this region. Patients with DGS have hypoplasia or aplasia of the parathyroid glands with subsequent hypocalcemia. Absence of the thymus produces a T-cell deficiency with immune dysfunction. The third and fourth branchial arches contribute to the “upper heart”, with disruption of this part of development leading to congenital heart malformations classified as conotruncal heart anomalies. Minor changes of the lower face and ears may also occur (Figure 5-56a).
Figure 5-56. Two patients with 22q11.2 deletions.
(a) Phenotype = partial DiGeorge syndrome. (b) Phenotype is that of velo-cardio-facial (Shprintzen) syndrome. She has typical facial changes, palatal cleft and conotruncal heart defect. (b: Courtesy of Dr. Nancy Mendelsohn, Children’s Hospitals and Clinics of Minnesota)
Robert Shprintzen, a speech-language pathologist, and colleagues described the syndrome that bears his name in 1978. Shprintzen syndrome is also called velo-cardio-facial syndrome, describing the major features of the condition. Changes in the development of the palate are common. This includes structural malformations such as cleft palate and functional defects such as velo-palatal insufficiency. Other features include conotruncal heart malformations and characteristic facial features including a distinctive nasal appearance. Patients with Shprintzen syndrome (Figure 5-56b) have a high incidence of learning disabilities and neuropsychiatric problems as well.
With advances in cytogenetic techniques in the early 1980s, several reports began to appear of patients with either DiGeorge or Shprintzen syndrome with deletions in the 22q1 region. With the introduction of FISH studies, 90% of patients with either condition were found to have deletions in the region 22q11.2. Subsequent investigations found 22q11.2 deletions in a smaller number of patients with a variety of other described syndromes such as Opitz BBB syndrome, CHARGE syndrome, and Cayler cardiofacial syndrome (also known as asymmetric crying facies). Even more fascinating, it was discovered that almost 30% of patients with isolated (non-syndromic) conotruncal malformations have a 22q11.2 deletion. To date over 180 different malformations have been reported in association with this contiguous gene disorder. Given the association with so many different syndromes and anomalies, it is best at this point in time to refer to the spectrum of 22q11.2 deletions as the overriding designation. It is estimated that 1 in 4000 individuals may have a 22q11.2 deletion.
As this deletion is transmitted through the generations, there is marked inter-familial and intra-familial variability. The inheritance pattern is best described as looking like autosomal dominant inheritance with variable expression and incomplete penetrance. A discussion of these terms in the next chapter, Chapter 6 on Mendelian Inheritance, will clarify this relationship and build on this introduction.
Board-Format Practice Questions
1. In regards to chromosome aneuploidies in humans:
A. They are rare at the time of conception.
B. They are seen more often as pregnancy progresses.
C. If present at conception, they most often end in a miscarriage.
D. All whole chromosome trisomies are incompatible with postnatal life.
E. All whole chromosome monosomies are incompatible with postnatal life.
2. The incidence of chromosome abnormalities at the time of conception is:
A. 1/120
B. 1/75
C. 1/65
D. 1/10
1/2
3. Turner Syndrome:
A. is the only whole chromosome monosomy seen in live births in humans.
B. the most common congenital heart disease is a ventricular septal defect.
C. is milder in boys.
D. girls with this condition are usually tall for age.
E. is more accurately described as Turner’s syndrome.
4. In regards to chromosome disorders in humans:
A. they occur rarely and are not of major clinical importance.
B. they may show a paternal age effect.
C. aneuploidies for the sex chromosomes occur more commonly than for the autosomes in live births.
D. whole chromosome aneuploidies can be seen in patients for any of the 23 pairs.
E. the most common clinical outcome in a chromosome abnormality is a newborn with a birth defect.
5. In regards to contiguous gene syndromes:
A. Clinical syndromes are usually associated with homozygosity of the deletion.
B. They usually track through a family like an autosomal recessive trait.
C. They usually occur sporadically, but may be familial.
D. Because they occur on the same part of the chromosome, there is very little variability in the phenotype.
E. Diagnosis by chromosome analysis is more sensitive and practical than FISH testing.
Supplementary Readings
Cassidy, S., and J. Allanson. Management of Genetic Syndromes, 2nd ed. Hoboken, NJ: Wiley-Liss Publishing; 2004.
Gardener, R. and G. Sullivan. Chromosome Abnormalities and Genetic Counseling, 3rd ed. Oxford: Oxford University Press; 2004.
Hennekam, R., J. Allanson, and I. Krantz. Gorlin’s Syndromes of the Head and Neck. Oxford: Oxford University Press, 2010.
Jones, K. Smith’s Recognizable Patterns of Human Malformations, 6th ed. Philadelphia: Elsevier, Saunders Publishing, 2005.
Shprintzen, R. Genetics, Syndromes and Communication Disorders. San Diego: Singular Publishing Group, 1997.