The functions of cell organelles like the nucleus and mitochondria are critical to the processes on which life depends. But that does not mean they are easy to study. From a general biology course you probably learned facts like a nucleus controls the growth and reproduction of a cell and ribosomes are the sites of protein synthesis (Figure 13-1). But for a moment, take a step back and ask a more basic question, “How do we know these things?” Just seeing the microscopic structures inside a cell is hard enough. How can we know the way they actually work? For medical genetics, the next question is then obvious. What are the medical effects of a genetic change in the structure or activity of a cell organelle? Can that knowledge lead to appropriate treatment?
Figure 13-1. Electron microscopy opens cell ultrastructure to a world of detail that could not be imagined by early researchers. But seeing structure does not always explain function. (Reprinted with permission from Cheville, N., Ultrastructural Pathology, Iowa State University Press, Ames, IA. p 2, 1994.)
Asking how we learn things about a structure as tiny as a cell organelle leads us to an important insight about the way science, including medical science, progresses. The real champion is how human ingenuity can figure out a process, especially a small, elusive one hidden somewhere in the complexity of the body—or of a cell. Let’s step back in history to remind ourselves of both the limitations and the scientific revolutions that have been spurred by experimentation at the level of a cell. Then we will look at one specific example of discovering the role of a cell organelle.
Imagine yourself in the early 16th century. If you were a physician, not a common profession at the time, you would work with a limited and even misleading view of how the body functions. The prevailing explanation of life is a mystical one. But changes are underway. The publication of De humani corporis fabrica in 1543 by Andreas Vesalius helped introduce to science an emphasis on personal observation rather than a dependence on accepted authority. It replaced the authoritative position held by the studies that Galen published about 1300 years earlier. William Harvey’s De motu cordis (1628) traced the flow of blood to and from the heart and led to a mechanistic, rather than a mystical, explanation of nutrient and oxygen transport. The invention of optics for magnification, which began as a novel entertainment, opened biologists to the microscopic realm.
Robert Hooke described the cellular structure of bark in Micrographia (1665)–“… these pores, or cells, were not very deep, but consisted of a great many little Boxes, separated out of one continued long pore, by certain Diaphragms … .” This was the first use of the word “cells” to describe biological structure, because the images reminded him of the spartan cells in which monks slept. Thus, a previously unknown dimension was being opened to view. It was Matthias Schleiden and Theodor Schwann (1838 and 1839, respectively) who formalized the theory that all living material is composed of cells. The light microscope revealed basic elements of cell organization and tissue differences. But it was not until just after the Second World War that the electron microscope began to resolve ultrastructure sufficiently to see the internal organization of cells in detail.
Seeing cell organelles in sharp resolution is not the same as understanding their function. Indeed, a nondividing nucleus is not a very imposing image. Looking at it unveils little about how it works. Imagine for a moment you are a researcher interested in the function of an organelle like the nucleus. How can you correlate a microscopic cell particle with a defined function? Joachim Hämmerling explored this question experimentally, but not in the 16th century, or even the 18th. His experimental demonstration that the nucleus controls the growth and regeneration of eukaryotic cells was done as recently as the 1940s and early 1950s. This is an area of biological research that clearly illustrates in a powerful way the creative insight of good science. It also reminds us how important experimental organisms are in uncovering information that is valuable to medicine and other applied fields. We can learn things by taking advantage of the special characteristics of a model organism, where the same question would be difficult if not impossible to answer using human cells or tissue.
Hämmerling took advantage of the very large cell size of marine algae of the genus Acetabularia (Figure 13-2). An inch-long Acetabularia cell is composed of a cap and a stalk. A rhizoid or rootlet contains the nucleus. Such a large cell can be surgically dissected and its parts can be transferred or fused together. For example, when Hämmerling cut a cell into these three sections, the cap died and the rhizoid with its nucleus regenerated a new stalk and cap. Surprisingly, the stalk by itself initially regenerated a cap, but then when the new cap was removed, the stalk was not able to continue regenerating. It died. Something in the stalk allowed it to regenerate for a brief time, but the root containing the nucleus could do so repeatedly. We now understand that the temporary ability of the isolated stalk to regenerate is due to mRNA in the stalk. But mRNA cannot be replaced if the nucleus is missing. To confirm the nuclear role in a separate experiment, Hämmerling grafted the stalk from a fringed A. crenulata onto the rhizoid of the smooth-capped Acetabularia mediterranea. The regenerated cap took on characteristics associated with the nucleus, not the stem. With experiments like these, Hämmerling confirmed the functional role of this nuclear organelle.
Figure 13-2. Each Acetabularia is a single cell. (a) With a nucleus near the rootlet and with an umbrella that regenerates when foraged upon. The species Acetabularia mediterranea and A. crenulata differ in the shape of the umbrella at the top of each cell. (b) By removing and transplanting the cap, stalk, and nucleus containing rootlet, J. Hämmerling confirmed that genetic control of growth and regeneration resided in the nucleus.
(a: Reproduced with permission from Wolfgang Sterrer, originally appearing in Sterrer W. Marine Fauna and Flora of Bermuda. New York: John Wiley & Sons, 1986.)
The story of the growth of microscopy shows how advances in technology can open new vistas for biomedical discovery and for applications to benefit patients. We have seen this more recently in the case of microarrays and personalized medical treatment options. The questions may be old. What is always “new” is the creative way a researcher can devise to approach them. Being receptive to new tools and remaining open-minded to new explanations will always define the foundations for success at the frontiers of biomedical knowledge.
In this chapter, we focus on the microscopic basis of cell function and some of the medical consequences of abnormalities in selected key cell organelles. To illustrate these, we have chosen to put some microscopic images of normal and abnormal cells together for direct comparison. This means that references to figures may tend to jump back and forth a bit between sections. But we think the resulting image comparisons will be more meaningful this way.
Part 1: Background and Systems Integration
The cell has two domains, the nuclear and the cytoplasmic. The cytoplasm is composed of the fluid component (the cytosol) and the formed elements (organelles other than the nucleus) (Figure 13-3). The functions of these two domains are intricately interconnected. The nucleus and most of a cell’s other organelles, are bounded by membranes, although the composition and specific roles of the membrane will differ from one organelle to another. Transitory interconnection of membranes throughout the cell insures a high degree of molecular communication among cell components. Other non-membrane organelles, like ribosomes and the cytoskeleton, interact directly with molecules and formed elements in the cytoplasm. Not surprisingly, then, defects in organelle structure and function can have extensive consequences for all levels of biological activity.
Figure 13-3. An overview of the functions associated with primary organelles in an animal cell. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 70, 2011.)
All are critical for cell function, but because of their role in energy metabolism the mitochondria are among the most important for medical genetics. We will first review briefly some major types of microscopy, because this guides what can be visualized about cell and organelle organization. We will then explore both normal and defective organelle functions, with primary emphasis on the mitochondria.
Types of Microscopy
Cells are tiny. But it is a mistake to think that the more magnification, the better. One must choose a magnification that makes sense for the question being asked. Electron microscopy can provide fine resolution of the internal structures in a cell, but often the most useful diagnostic information will come from a less magnified view. To prepare material for different types of light or electron microscopy, the tissue must be stabilized and stained. Structures are fixed with chemicals like formaldehyde for light microscopy or glutaraldehyde for electron microscopy. Water is then removed so the tissue can be embedded in a non-aqueous medium like paraffin or plastic. From this block, thin sections are cut and stained. The choice of stain will control what aspects of the complex cell structure will be enhanced. Other techniques allow surfaces of large structures and even internal activities in living cells to be studied in detail.
Light microscopy can resolve tissue organization up to a little more than 1000 × with an oil immersion lens. There are three main types of light microscopy that allow different kinds of cell structure to be visualized. Bright field microscopy depends on staining cell components with dyes that are acidophilic (mostly proteins) or basophilic (primarily nucleic acids and certain sugars) (Figure 13-4). Osmium tetroxide covalently bonds to lipids. Some staining protocols involve double staining, such as defining the nucleus with one stain and counterstaining the cytoplasm with another. Other common stains, like Wright’s used for white blood cells, are a mixture of different stains (i.e., polychrome stains).
Figure 13-4. Stains help explain biochemical differences among cells and their organelles. Here hematoxylin, that behaves like a basic dye, colors the DNA of the nucleus and the RNA of the cytoplasmic ribosomes purple, but eosin, an acidic dye, colors the cytoplasmic proteins pink. These two types of staining are called basophilia and acidophilia, respectively. The neutral carbohydrates do not stain. (Courtesy of Paul Bell and Barbara Safiejko-Mroczka, Histology course web site, University of Oklahoma.)
A second type of light microscopy is phase contrast, a powerful tool to visualize living cells. This approach depends on the way light changes its speed when passing through structures that have different refractive indices. Phase contrast optics causes these structures to appear lighter or darker in the field of view. While imaging living cells, phase contrast can give detailed information about cell responses to stimuli (Figure 13-5). Nomarski differential interference can even produce detailed three-dimensional images. These are compared in Figure 13-6.
Figure 13-5. Phase contrast of living cells can allow observation of cell behavior and measurements of a response to chemical exposure. Here, the effect of neomycin on motility of human glioma cells in culture is compared: (a), before introduction of neomycin into the cell environment; (b), 2 minutes after exposure to neomycin. (Courtesy of Barbara Safiejko-Mroczka, University of Oklahoma.)
Figure 13-6. Phase contrast (left) and Nomarski (right) of the same field of fibroblasts in culture shows the three-dimensional capability of Nomarski. The cultured neural crest cells in these fields are alive. (Reprinted with permission from Junqueira, L., and J. Carneiro, Basic Histology. New York: McGraw-Hill, p 4, Fig. 1-3 b and c, 2003.)
A third approach to light microscopy uses fluorescent stains that have a high affinity to certain types of cellular molecules. This is a very powerful technique, because one can couple fluorescent materials to a large array of antibodies for almost any cellular molecule. Materials can be multiply-stained to visualize the physical relationships among molecules that are close together in the cell (Figure 13-7).
Figure 13-7. Immunofluorescence with multiple stains. The same human gingival fibroblast labeled using Bodipy conjugated phallacidin to reveal filamentous actin (left), and with anti-vinculin antibody and rhodamine conjugated secondary antibody to label vinculin present in focal contacts (center). The right-hand figure shows double-labeling of the F-actin and vinculin distributions in the same cell. (Courtesy of Barbara Safiejko-Mroczka, University of Oklahoma.)
There are two fundamental types of electron microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM requires very thin sections (on the order of 40-90 nm) prepared from tissue embedded in a resin block and stained with heavy metals like osmium tetroxide or uranyl acetate. Combining these with immuno-logical agents can target specific cell activities like the movement of a molecule through a membrane as seen in Figure 13-8. TEM magnification is related to the available energy for the electron beam, with powers up to 10,000,000 × now possible.
Figure 13-8. Molecule movement through the nuclear envelope, believed to be the passage of a ribosomal subunit through a nuclear pore (Cy, cytoplasm; N, nucleus). (Reprinted with permission from Stevens, B., and H. Swift. J. Cell Biol. 31:72, 1966.)
For SEM, a very thin coating of gold-palladium or other metal is applied via a plasma to the surface of a dry structure. A focused electron beam produces reflected or emitted electrons that are captured by a detector and projected as an image on a monitor. Although initially used to explore surface structures, internal organization can also be imaged after breaking or fracturing a section of tissue. In Figure 13-9, for example, freeze-fracturing of the double membrane surrounding the nucleus clearly shows the nuclear pores and inner membrane.
Figure 13-9. Freeze-fracturing of the nuclear membrane enables us to view the inner membrane and the nuclear pores that connect the cytosol with the nuclear region. (Reprinted with permission from Junqueira, L., and J. Carneiro, Basic Histology. New York: McGraw-Hill, p 56, Figure 3-7, 2003.)
An Overview of Cell Membranes
Membranes are composed of phospholipids and a range of protein and other organic molecule components (Figure 13-10). Artificial phospholipid membranes can form spontaneously, even in a test tube, because of the interaction between their hydrophilic region and the ubiquitous water environment surrounding them both inside and outside the cell. The hydrophobic fatty acid tails create a boundary between the two watery layers. While some cellular components can be anchored in a membrane, the lipid bilayer structure is somewhat fluid allowing molecules like protein receptors to move within it. Molecular interactions with water define many of the membrane’s cellular processes.
Figure 13-10. The fluid-mosaic model of membrane structure is based on the way membrane phospholipids, with both a hydrophobic and a hydrophilic region, will spontaneously form a bilayer in which proteins and other macromolecules can float. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 98, 2011.)
About half the molecular mass of a membrane is protein and half is lipid, although this will differ from one kind of membrane to another. A membrane protein is, however, much larger than a phospholipid, so the number of lipid molecules in a typical membrane outnumbers the proteins by 50 × or more. The diverse functions associated with different membranes are mainly a function of their protein component.
The plasma membrane that surrounds the cell serves as the communication interface with the environment, creating the intracellular domain and the surrounding extracellular environment. It is a very complex structure, with some specialized domains carrying out functions like regulating secretion or nutrient uptake. Lateral sides of a cell form a range of connections with adjacent cells that can influence intercellular interaction. Desmosomes and tight junctions anchor adjacent cells together, and gap junctions form molecular communication channels between adjacent cytosols. The extracellular matrix (ECM) serves animal cells in somewhat the same way as the cell wall in plants. Its primary components are glycoproteins, like collagen, which alone makes up about half the protein in a human body. Surface adaptations like cilia, flagella, and microvilli also play key roles in cell function. Microvilli are tiny fingerlike processes that increase the absorptive surface of an exposed membrane on cells like those lining the digestive tract. Cilia and flagella use dynein, one of the so-called “motor molecules” to move cells like sperm through the body or move materials like mucus past cells lining the respiratory tract to trap and eliminate dust and other particles.
A key characteristic of a biological membrane is its selective permeability. Because of its phospholipid component, lipids and lipid-soluble materials can generally pass through easily. Water and some small water-soluble molecules tend to diffuse beside the protein elements distributed in the membrane. In such cases, a specialized transport protein is not necessary. But many larger molecules and charged ions have very low permeability and require a membrane transport mechanism. In facilitated transport, a specialized protein creates a passage through which the targeted molecule can move passively. Active transport, on the other hand, requires energy from adenosine triphosphate (ATP) to generate protein shape changes that move a targeted molecule across the membrane, often against the passive movement of diffusion. Many of the variations seen in cell membrane activity are, therefore, due to differences in the presence of facilitated transport and active transport channels.
Because of its central role in life processes, heritable defects of the cell membrane are not common. Most defects associated with the plasma membrane are the result of toxins, trauma, or other disease influences. Among congenital disorders, however, we can identify examples of the inadequate number or even total absence of a specific membrane receptor, changes in cell adhesiveness, abnormalities in cell-to-cell communication, and the presence of exceptional proteins.
Mitochondria
As organelles, mitochondria are special. They have DNA and their replication is independent of the nucleus. They also have a key role in energy transformation—ATP synthesis—that makes them indispensible for life. These roles give mitochondria an especially important place in a survey of cell organelles.
Although a small amount of ATP synthesis occurs in the cytoplasm, most occurs in mitochondria (Figure 13-11). ATP carries a high energy phosphate bond (often symbolized ∼P). When that phosphate is removed leaving adenosine diphosphate (ADP) (ATP → ADP + ∼P), the high energy phosphate can transfer energy to other molecules and reactions. So it is not surprising that disorders of the mitochondria have their largest impact in tissues like the brain and muscles that have the greatest demand for energy through synthesis and use of ATP.
Figure 13-11. (a) Mitochondria in a cell, seen as dark elongated bodies. (b) An electron micrograph of a mitochondrion, showing the inner folded membrane (cristae). (Reprinted with permission from Karp, G., Cell and Molecular Biology: Concepts and Experiments, 3rd ed, Wiley, p 184, 2002.)
There are two functional domains in each mitochondrion, one in the fluid internal matrix and one embedded in the folded internal membrane, the cristae. Glucose digestion in the cell cytoplasm yields a small net gain of two ATP molecules plus two molecules of pyruvate, which enter the mitochondrion as acetyl-CoA (Figures 13-12 and 13-13). There, enzymatic breakdown by the Krebs (or citric acid) cycle (Figure 13-14) occurs in the mitochondrial matrix.
Figure 13-12. A summary of the metabolism of glucose in: (1) the cytoplasm, and in the mitochondria by (2) pyruvate breakdown, (3) the Krebs or citric acid cycle, and (4) oxidative phosphorylation (OXPHOS). (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 138, 2011.)
Figure 13-13. Glycolysis in the cytoplasm yields two molecules of pyruvate for each glucose molecule. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 139, 2011.)
Figure 13-14. One carbon is removed from pyruvate when it enters the mitochondrion as acetyl CoA. In the Krebs or citric acid, cycle the remaining two carbons are removed as CO2 and energy is passed to ATP and to carrier molecules that transfer it to the electron transport chain. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 143, 2011.)
Finally, sequential oxidation-reduction reactions by the cytochromes of oxidative phosphorylation (OXPHOS; also called the electron transport chain; Figure 13-15) produce most of the cell’s ATP yield. But the fact that mitochondria have their own DNA is what makes them stand out for medical geneticists as a special case of organelle biology. Key principles of mitochondrial inheritance are summarized in Table 13-1.
Figure 13-15. Oxidative phosphorylation, or OXPHOS, passes electrons along the chain of cytochromes in the mitochondrial inner membrane, the cristae. The final product is ATP synthesis with water as a byproduct. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 144, 2011.)
The human mitochondrial DNA (mtDNA) genome is present in about 5 to 10 copies per mitochondrion, with up to 1000 or so mitochondria per cell depending on the energy expenditure of each type of tissue. In fact in some cells like a frog egg the vast majority of the DNA in the cell is mtDNA, not nuclear DNA. In most mammalian tissues, however, about 1% of the cellular DNA is mtDNA. The human mitochondrial genome is a circular molecule of 16,569 bp and does not contain introns. Its use of the genetic code differs slightly from that of a nucleus. The UGA triplet codes for tryptophan instead of serving as a “stop codon,” and AGA and AGG serve as stop codons instead of coding for the amino acid arginine. AUA and AUU sometimes serve as start codons, in place of AUG.
Species differ widely in the number of genes encoded in their mtDNA genome. The human mitochondrial genome codes for only 13 of the 80 or so proteins needed to control the process of OXPHOS. The rest are coded in the nucleus and follow normal Mendelian genetic inheritance patterns. In contrast, mtDNA is passed from females to all their offspring. The mtDNA from the sperm is generally lost by the 2- to 4-cell stage. So, just because a disorder can be traced to a mitochondrial function does not mean it is going to follow maternal transmission rules. One needs to understand the specific genetic origin of each trait to determine its transmission pattern. In other words, it is important to distinguish clearly between a mitochondrial disorder and mitochondrial inheritance. Some examples of mitochondrial conditions are shown in Figure 13-16.
Figure 13-16. Abnormal mitochondria showing: (a) ragged red fibers, and (b) and crystalline inclusions in the mitochondrial matrix. (a: From Donald R. Johns in Karp, G., 2002, Cell and Molecular Biology: Concepts and Experiments, 3rd ed, Wiley, p 213, Figure 1; b: From Morgan-Hughes, J., and D. Landon, Engel, A., and C. Franzini-Armstrong, eds Mycology, 2nd ed, McGraw-Hill, 1994.)
One special phenomenon that affects cellular makeup is replicative segregation. This is a phenomenon that largely depends on chance (Figure 13-17). If there is just one type of mtDNA sequence in a cell, it is homoplasmic(“homo” = same; “plasmic” referring to the cytoplasm makeup). But mutations occur in mitochondria. If a cell has more than one mtDNA sequence, it is heteroplasmic. But the organelles are not regularly distributed at each cell division, so over time the daughter cells will change proportions, showing replicative drift, and can even return to homoplasmy.
Figure 13-17. The consequences of random sampling during replicative segregation of mitochondria is illustrated here by random samples of red and green chocolate candy pieces. Although drawn from the same pool of candy pieces, the random samples held different proportions each time.
Table 13-1. Basic Principles Associated With Mitochondrial Inheritance
The DNA outside the nucleus is under separate replication control. Mitochondria divide independently of the nucleus. It should, therefore, be no surprise that replication processes differ between the nucleus and this organelle. An important example is error correction. Mutation rate is much higher—perhaps even 10-fold or more higher—in mitochondria than in nuclear DNA. One possible explanation is that a mutation in a mitochondrion does not necessarily affect the function of the cell that contains it. It is functionally masked because there are so many nonmutant mitochondria to compensate for it. Selection against the new mutation may be zero. In addition, mtDNA does not have the histone associations that might mediate mutagenic effects. There is, therefore, a much lower selection pressure to favor efficient mtDNA repair enzymes.
Bottlenecks can affect mtDNA representation. A bottleneck is simply a reduction in number that leads to random sampling variation. In this case, the number of mtDNA molecules in oogonia is reduced to only about 1 to 30 copies. During oogenesis, the number increases again by about 100 ×. After fertilization, there is rapid nuclear DNA replication, but the number of mitochondria and mtDNA molecules does not change a lot. It is mainly affected by tissue specialization that reflects functional demands. In Chapter 15 we will return to this idea of bottlenecks and sampling error in a different context, the population.
In scientific applications, just as in our daily activity, a bottleneck means the same thing. It is a restriction. In biology, it is generally a restriction in numbers of individuals or component parts. Although a phenomenon like a genetic bottleneck may seem like a rare special case, it is not. It is just one of the many factors that commonly affect expression of inherited diseases. As a whole, we can view the influences on a biological trait as a threshold phenomenon. In earlier discussions, we explored variation in penetrance and expressivity. Below a threshold level, the trait is unexpressed, but above a threshold of contributing conditions it is exposed and changes the phenotype. Factors like the specific nature of the mutation, the proportion of the mtDNA that carries the mutation, and the relative dependence of the affected tissue on ATP and the processes of OXPHOS will determine a mutation’s phenotypic expression. We have seen that a change in a critical enzyme can change phenotypes a lot, but mutations in other steps might be masked. If that can be true of an enzyme, it can also be true for an organelle like a mitochondrion.
Molecular Traffic of the Endoplasmic Reticulum and Golgi Complex
Much of the molecular synthesis occurring in a cell is associated with an extensive membrane network called the endoplasmic reticulum (ER). Rough endoplasmic reticulum (RER; Figure 13-18) refers to the portion of this membrane that has attached ribosomes giving it a rough appearance in an electron micrograph. The proteins that are synthesized there can be transported along the RER to the Golgi complex (also called the Golgi apparatus or Golgi body), where they are further processed and then packaged into vesicles for transport within the cytoplasm or out of the cell. The smooth endoplasmic reticulum (SER), on the other hand, lacks attached ribosomes. It is primarily associated with detoxification and the synthesis of steroid hormones.
Figure 13-18. Endoplasmic reticulum with attached ribosomes is called rough endoplasmic reticulum (RER). (Reprinted with permission from Cheville, N., Ultrastructural Pathology, Iowa State University Press, Ames, IA, p 23, 1994.)
In addition to housing the cell’s synthetic biochemistry, the ER can engage in some kinds of quality control over protein secretion. To leave the ER, recently-synthesized proteins must be correctly folded or, if they are subunits of a more complex protein, they may need to be assembled. Abnormally-folded proteins and other defective molecules can be destroyed. Chaperone proteins help facilitate this process by anchoring abnormal proteins in the lumen of the ER from which they are transported back to the cytosol and digested. But this recycling also means that defects in these processes can lead to the abnormal retention of proteins, leading to serious ER storage diseases.
Endosomes, Lysosomes, and Other Membrane-Bounded Vesicles
Endosomes are membrane-bounded organelles that isolate materials that are newly-ingested into the cell (endocytosis). But there are many other similar vesicles carrying out a range of functions, such as sequestering various cell products or isolating chemical reactions in the cytoplasm. Lysosomes, for example, are the principal location of cellular digestion. Indeed, this is one way the cell can safely produce and use materials like proteolytic enzymes that would otherwise be fatal to a cell’s survival. A large number of lysosomal enzymes have been identified, and pathologies of the lysosomes involve releasing these enzymes into the cell or into extracellular spaces. Lysosomal storage diseases occur when they fail to digest properly (Figure 13-19).
Figure 13-19. (a) Gangliosidosis is a lysosomal storage disorder, Tay Sachs. (b) Zebra bodies, Hurler syndrome. (a: From Karp, G., 2002, Cell and Molecular Biology: Concepts and Experiments, 3rd ed, Wiley, p 315; b: From Cheville, N., Ultrastructural Pathology, Iowa State University Press, Ames, p 154, 1994.)
Peroxisomes typically contain one or more enzymes that remove hydrogen atoms from targeted molecules and produce hydrogen peroxide (H2O2) in an oxidation reaction with oxygen. An important aspect of this reaction is the breakdown of fatty acids, in which β-oxidation shortens the fatty acid chains to yield acetyl CoA used in various biosynthetic reactions. One key example is the synthesis of plasmalogens, the most abundant type of phospholipid in the myelin of nerve cells. Not surprisingly, peroxisomal abnormalities are often associated with nervous system disorders.
The Cytoskeleton
The cytoskeleton is a dynamic system of microtubules and filaments that are involved in cell shape, cell division, internal transport, and movement (Figure 13-20). There are three types of protein filaments in animal cells. Actin filaments (microfilaments) make the reversible transition from globular monomers to a filamentous two-strand polymer and thereby effect rapid changes in cell shape and movement. The microtubules, on the other hand, are more rigid hollow cylinders composed of the protein tubulin. They direct transport of materials, including organelles, within the cell. Finally, intermediate filaments are made up of a large, diverse group of proteins that can create a mesh-like network that gives mechanical strength to the cell. An example is the interconnected filaments that strengthen the elongated cellular structure of neurons.
Figure 13-20. Cell cytoskeleton shown as tubulin distribution in human fibroblasts labeled with anti-tubulin antibody and rhodamine conjugated secondary antibody. Left, untreated; right, treated with neomycin to induce formation of protrusions. (Courtesy of Barbara Safiejko-Mroczka, University of Oklahoma.)
Along this dynamic system of protein corridors, accessory proteins assist in assembly and disassembly of cytoskeletal components. Accessory proteins include the motor proteins, which use energy from ATP to change shape and move (or “walk”) along the filament pathways to transport organelles or other cell components to which they are attached. An important example of the cytoskeleton with specialized function is the mitotic spindle that moves chromosomes during cell division.
Cilia, Flagella, and Cell Surface Specializations
The cell surface is quite complex. Many functional proteins such as ion channels, gap junction proteins and receptors are distributed across the surface of cell membranes. Cilia and flagella are other specialized elements of the cell surface. Their structures are similar; both are composed of microtubules and the motor protein dynein (Figure 13-21). They also share many of the same genes that code for proteins common to both. In both, the core is composed of nine microtubules composed of a doublet fused together and with two single microtubules in the center. Pairs of dynein arms are attached to each microtubule doublet. When activated, these dynein arms “walk” up the adjacent microtubule causing the tubules to slide past each other in much the same way as actin and myosin filaments slide past each other in muscle contraction. But in the case of cilia and flagella, the tubules are connected at points along their length so the sliding actually causes the structure to bend. While they are quite similar, there are some distinct differences between cilia and flagella. Flagella are longer than cilia and typically beat with a whiplike motion (the Latin word for flagellum actually means ‘whip’). Cilia are shorter and more numerous on a cell surface. Their movement is described as being more of a coordinated, rhythmic wavelike movement. The movement of cilia is in two parts with power and recovery phases that have been likened to the breast stroke in swimming.
Figure 13-21. The structure of normal cilia and flagella. (Reprinted with permission from Brooker et al., Biology, 2nd ed, New York: McGraw-Hill, p 77, 2011.)
Flagella primarily function in cell motility. However, they may also function as sensory organs—being responsive to chemicals and temperature. In humans, the best known examples of flagella are the tails of sperm. Abnormalities of this structure (Figure 13-22) can lead to infertility from nonmotile sperm.
Figure 13-22. Defect of inner dynein arms. The loss of inner dynein arms is the ultrastuctural alteration that is most often seen (original magnification × 85,000).
(Reproduced, with permission, from Theegarten D, Ebsen M. Diagnostic Pathology, 6:115 doi:10.1186/1746-1596-6-115, 2011.)
There are two types of cilia: immotile (primary) and motile. The immotile cilia are primarily sensory in function and can be found on the surfaces of almost every cell. They serve as essentially cellular “antennae” to send and receive chemical signals. They are very important in cell-cell recognition and other critical interactions among cells. Motile cilia exist to move liquids across cell surfaces. They are typically found in the fallopian tubes and the respiratory epithelium.
The Nucleus and Centrioles
The focus of this chapter is on cell organelles that play a central role in certain heritable medical conditions. It may seem we have overlooked one of the most important of these, the nucleus. Along with the centrioles that generate the spindle for nuclear division, the nuclear region of a cell is clearly of critical importance for cell function. But for a moment, let’s look at the overall organelle, rather than the information coded within its chromosomes. Why do changes in the structure of this organelle not appear as a common source of medical problems? Although the answer may seem obvious, it is still worth highlighting. It is true that the nucleus controls the growth and reproduction of a cell. It, therefore, follows that, if the essential structure of the nucleus is abnormal, the cell will not be able to carry out its most fundamental role and will probably die.
This also illustrates a more general fact about genetic research. Genetics can identify the role of a gene by seeing the abnormalities that occur when the gene mutates. But a mutation affecting the ability of a cell to divide or to transcribe its encoded information cannot survive long. There is no phenotype to trace. The loss of such a valuable experimental approach helps us appreciate the ingenuity that researchers have brought to bear on questions about the function of the nucleus and other cell organelles. Extrapolating from mutations to understanding the normal function of a gene is what gives genetics its power as an experimental approach. But when the mutation causes the loss of the cell, that asset disappears. In spite of that limitation, the ingenuity of researchers is now drawing upon other approaches.
Nuclear size and shape give hints about its level of activity. A nucleus that is large, lightly staining, and with an enlarged nucleolus is probably transcriptionally active. A large basophilic nucleus may have an elevated DNA level, but a small one may be less active with transcription down-regulated. Certain cells like lymphocytes may have irregular nuclei normally. However, unusual nuclear shapes are often found in cells of malignant tumors.
In this overview of cell ultrastructure, we have limited our attention to some of the main organelles that are involved in human genetic disease. In the next section, we will discuss examples of how abnormalities in organelle structure and function can explain some heritable conditions.
Part 2: Medical Genetics
Introduction
The function and importance of subcellular organelles has been detailed in the first section of this chapter. As with most other chapters in this book, the Medical Genetics correlate is what happens when things go wrong. Each organelle has a specific set of functions. When those functions are impaired, there are usually clinical consequences. One of the most fascinating aspects of these correlations has been the discovery of what is seen clinically in these situations. Often, the outcomes are quite unexpected. If one were to try and predict the outcomes based on knowledge of structure and function, you would often be wrong. For instance, as discussed below, no one would ever have predicted that mutations in the laminin proteins of the nuclear membrane would be associated with progeria (premature aging syndromes). Conversely, in this era of genomics, the typical scenario is to start with the disorder and then search the entire genome rather than think about the condition and go check out logical candidates.
In this section we will discuss mitochondrial disorders in detail, as these represent an important group, both numerically and in treatment potential. For the rest of the organelles to be discussed, we will focus mainly on the known disease entities associated with dysfunction of each type.
Mitochondria
It is extremely important to discuss mitochondrial disorders. Collectively they are actually quite common. The range of symptoms associated with these conditions encompasses just about every symptom group described, i.e., they are in the differential diagnosis of almost any human malady. Still, they are commonly underdiagnosed. If the clinician does not consider them, they will simply be overlooked. Besides the relatively common occurrence of these conditions, there is also treatment potential for many.
The key to understanding mitochondrial disorders is to consider their primary role as generators of biologic energy. Mitochondria exist to facilitate aerobic energy generation through OXPHOS. The reliance on OXPHOS for energy varies during developmental stages and among tissues. During embryonic development, the blastocyst stage is a time of increased oxygen consumption associated with increased numbers of mitochondria and mtDNA. For the rest of the first half of gestation there is low oxygen tension with a major reliance on glycolysis: i.e., OXPHOS is not heavily utilized. From the last half of gestation through the first 10 years post-natally, OXPHOS activity rises with a gradual decrease to adult levels by about 20 years of age.
OXPHOS gene expression in different tissues varies with changes in mtDNA levels, nuclear OXPHOS genetic activity, nuclear-cytoplasmic interactions, and environmental influences. Mitochondrial density is related to the overall number of mitochondria per cell as well as the number of mtDNA copies per mitochondrion. These factors vary greatly among tissues. Those tissues that have functions that have higher energy needs tend to have more mitochondria. In humans, the mtDNA content is highest in the brain, eyes, muscle, liver, kidney, and heart (in decreasing order) with the overall involvement of these organs reflecting this fact (i.e., CNS dysfunction is the most common presenting problem in mitochondrial disorders).
The pathophysiology of mitochondrial dysfunction is complex and involves interactions with multiple other physiological systems. Mitochondrial disorders include multiple conditions that share in common structural and functional differences in the mitochondria. This includes defects in aerobic cell metabolism, the electron transport chain, the Krebs’s cycle or any of several combinations of these. In general, mitochondrial damage results in decreased mitochondrial activity (including superoxide dismutase) plus the inability to regenerate proteins. This results in the generation of metabolic by-products including free radicals that in turn will damage proteins (structural and enzymatic) and the mtDNA itself. This leads to further elevation of anaerobic by-products with further decline in mitochondrial functions.
Mitochondrial inheritance has been discussed in detail in the first section of this chapter. If you are not quite clear on the details, you are encouraged to go back and review this. Of paramount importance is that clear distinction needs to be made between mitochondrial disorders and mitochondrial inheritance. The term “mitochondrial disorder” refers to recognizable clinical disorders that are due to dysfunction of the mitochondria. It is important to remember that the mtDNA only codes for 13 of the large number of proteins needed for normal mitochondrial metabolic functioning. Thus the vast majority of mitochondrial proteins are encoded in the nuclear genome. It then follows that many mitochondrial disorders are inherited as Mendelian traits (see Clinical Correlation section). Mitochondrial inheritance, then, refers to pattern of transmission seen in those mitochondrial disorders in which the causative mutation is located in the mtDNA. For these conditions, the unique pattern of mitochondrial inheritance as described earlier (maternal transmission) holds. As of the time of this writing, mutations in 13 mitochondrial genes and 228 nuclear genes have been reported in association with human medical disorders.
The complex requisite interaction between the mitochondrial genome and the nuclear genome cannot be overstated. For the mitochondria to work appropriately, both genomes have to be working correctly and be carefully synchronized. Because both genomes may code for proteins involved in the same pathway(s), certain disorders of mitochondrial function can be inherited as either classic Mendelian or mitochondrial inheritance. For instance, diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) is a well-described entity. It has long been recognized that the primary pathophysiology of this condition involves mitochondrial physiology. It is now known that DIDMOAD can be caused by mutations in the mitochondrial genome or by mutations in an autosomal gene at chromosome locus 4p16. Obviously the familial implications of these two are markedly different. Thus, when a patient is diagnosed with DIDMOAD, careful identification of the underlying genetic etiology is critical. One cannot simply infer an inheritance mode based solely on the clinical impressions.
The clinical characteristics of mitochondrial disorders are tied to their roles in energy metabolism. Typically they are progressive in nature. They exhibit high intra- and inter-familial variability. The overall phenotype depends on multiple factors such as the level of heteroplasmy, the distribution of heteroplasmy (i.e., among which cells, tissues and organs), other modifier genes (including those in the nuclear genome), the timing of the life cycle (the level of heteroplasmy can change over the life of an individual), and the specific biologic threshold of a particular function.
As noted earlier, mitochondrial disorders have a myriad of presentations. They can present as almost anything and at any time in a person’s life. Simply, if you do not think about them you will miss them. Mitochondrial disorders often present in childhood. Table 13-2 lists the most common presenting symptoms and the time in childhood when they usually present. In pediatric intensive care units, children are often seen with “multisystem failure.” The commonly considered causes of this are infectious and toxic etiologies. However, many of these children actually have mitochondrial disorders–often undiagnosed. The presentations of mitochondrial disorders are somewhat different in adults than in children. The most common presenting symptoms in adulthood are listed in Table 13-3. Another important consideration of mitochondria in adult health is their role as contributing factors to other primary pathologic conditions. Changes in mtDNA have been shown to contribute to a number of late onset processes such as Alzheimer disease, Parkinsonism, and multiple sclerosis. The accumulation of mitochondrial mutations undoubtedly plays a large part in the “normal” aging process. Presbycusis (age associated hearing loss) is particularly tied to mitochondrial dysfunction. It is also important to note that there are a few presenting findings that are strongly associated with mitochondria disorders. The finding of “ragged red fibers” on a muscle biopsy (Figure 13-16) is essentially pathognomonic for mitochondrial disorders. Likewise, in any patient with chronic progressive external ophthalmoplegia (CPEO) mitochondrial disease should be considered.
Table 13-2. Childhood Presentations of Mitochondrial Disorders
Neuromuscular symptoms (44%)
Non-neuromuscular symptoms (56%)
Liver, heart, kidney, GI, endocrine, hematologic, dermatologic
Age of Onset:
Neonatal (<1 month): 36%
Infantile (1-24 months): 44%
Childhood (>24 months): 20%
Table 13-3. Adult Presentations of Mitochondrial Disorders
Ataxia
Deafness
Diabetes
Myopathies
Neuropathy
Vision loss
Monoallelic
Triallelic
Uniparental disomy
Several mitochondrial “syndromes” have been described. These are a small number of specific disorders that have been described where mitochondrial mutations are either inherited or occur early enough in development to dominate most cells. These disorders characteristically affect muscle and nervous tissue, particularly the optic tracks. One such example is Leber hereditary optic neuropathy (LHON) shown in Figure 13-23. Patients with LHON exhibit a progressive optic nerve atrophy beginning early in adulthood. The condition has been reported with at least 18 different mutations in the mitochondrial genome. Table 13-4 lists several more examples of mitochondrial syndromes. In reality, the description of mitochondrial syndromes represents an artificial distinction clinically. Such descriptions reflected the functional limitation of knowledge and technology (for testing). It is now known that there are multiple phenotypes associated with same mutation and multiple mutations associated with the same phenotype. Thus the best practice these days is to describe the phenotype carefully and then pair it with the specific genetic abnormality identified (genotype) in a particular patient or family.
Figure 13-23. Retinal photograph of Leber hereditary optic neuropathy (LHON). The retina in this patient demonstrates central retinal vessel vascular tortuosity and optic atrophy.
Table 13-4. Examples of Mitochondrial “Syndromes”
At the present time, advances in genetic testing technology (see Chapter 11) have greatly increased the ability to diagnose mitochondrial diseases. It is relatively easy and not extremely costly these days to sequence the entire mitochondrial genome. Testing for nuclear gene abnormalities is significantly more expensive and time consuming. Undoubtedly, this will get much better as NextGen sequencing is developed as a clinical testing modality. For now, the diagnostic approach to mitochondrial disorders is best accomplished as an algorithmic approach using the family history, clinical symptoms, and the medical history to direct the type and order of tests.
Molecular Traffic of the Endoplasmic Reticulum and Golgi Complex
The ER has a variety of different functions. Its functions can differ depending on the individual type, function, and needs of a given cell. The ER can even adapt with time in response to cell needs. One of its primary functions is in facilitating protein folding and transport. It plays a major role in post-translational protein modification including glycosylation and disulfide bond formation. The Golgi complex processes and packages proteins from the ER.
Several conditions have been found to be due to disorders of the ER/Golgi complex functions. In the Clinical Correlation section of Chapter 2, congenital disorders of glycosylation are discussed in detail. As noted earlier, the process of glycosylation occurs in the ER. Some disorders of myelination (Charcot-Marie-Tooth disease, Pelizaeus-Merzbacher disease, the vanishing white matter syndrome, and spastic paraplegia type 17) are due to ER dysfunction. Other ER problems include the skeletal dysplasia pseudoachondroplasia which is an ER storage disorder, some endocrinopathies (congenital hypothyroid goiter, diabetes insipidus), familial hypercholesterolemia, and congenital lipo-dystrophy 2. One disorder attributed to problems of the Golgi complex is the I-cell storage disorder (the abnormal enzyme phosphotransferase is a Golgi enzyme). Also Golgi dysfunction has been implicated in Alzheimer disease pathophysiology.
Endosomes, Lysosomes, and Other Membrane-Bounded Vesicles
Membrane bound vesicles serve multiple functions. They are involved in endocytosis and other protein transport, storage of multiple compounds, and complex enzymatic processes. Endosomes are endocytic vacuoles that transport molecules to the lysosomes. Niemann Pick C is caused by abnormal endo-cytic transport of lipids, particularly cholesterol.
Lysosomes are vesicles that contain enzymes that are acid hydrolases. They function to clear compounds from the cell. In general the enzyme functions of the lysozymes are much less specific than those involved in the synthesis of biochemicals. They essentially function in the intracellular “digestion” of macromolecules. In addition, in many organisms, lysosomes can be involved in programmed cell death. Disorders in lysosomal function lead to lysosomal storage disorders (LSDs). Because lysozymes work in clearing compounds from the cells, the end result of lysozyme dysfunction is the progressive accumulation of these compounds within the lysozymes. Over time the continued accumulation of compounds that should be cleared leads to engulfed lysozymes. As the lysozymes continue to enlarge they disrupt all aspects of cell function.
Pompe disease is a lysosomal storage due to deficiency of the enzyme acid maltase (acid alpha-glucosidase). This enzyme nonspecifically degrades glycogen in the lysosomes to clear this compound from the cells. Because glycogen is found abundantly in the muscles, glycogen accumulates rapidly in these cells (Figure 13-24). Progressive accumulation of glycogen in the muscles leads to muscle deterioration and weakness and a hypertrophic cardiomyopathy (Figure 13-25). Without treatment, infants with classic (complete) Pompe disease usually die in the first year of life. There are many types of lysosomal storage disorders which share in common the mechanism of lysosomal congestion, but differ by the accumulated chemicals and the clinical features of the condition. Table 13-5 provides a list of some of the known lysosomal storage disorders.
Figure 13-25. (a) Severely hypotonic infant with Pompe disease (also known as glycogen storage disease type II). (b) Postmortem picture of a heart in a patient with Pompe disease showing a severe cardiomyopathy secondary to massive stores of glycogen.
Table 13-5. Examples of Lysosomal Disorders
Hydrolase deficiencies
Glycogen storage disease type II–Pompe disease
Glycoproteinoses—Mannosidosis
Mucopolysaccharidosis–Hurler syndrome, Hunter syndrome (Total of 7 types with multiple subtypes)
Neutral lipid storage–Wolman disease (cholesterol ester storage disease, CESD)
Pycnodysostosis—Cathepsin K deficiency
Sphingolipidoses—Niemann-Pick types A and B
Lysosomal transport disorders
Cystinosis
Sialic acid storage
Figure 13-24. (a) Normal muscles fibers in microscopic section. (b) Muscle biopsy of a child with Pompe disease. Note the massively congested lysosomes, which are full of glycogen, and the distorted normal architecture of the muscle fibers. (Reproduced, with permission, from Amalfitano A, Bengur AR, Morse RP, Majure JM, Case LE, Veerling DL, Mackey J, Kishnani P, Smith W, McVie-Wylie A, Sullivan JA, Hoganson GE, Phillips JA 3rd, Schaefer GB, Charrow J, Ware RE, Bossen EH, Chen YT. Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial. Genet Med. 2001 Mar-Apr;3(2):132-138.)
Significant interest in the lysosomal storage disorders has emerged over the past several years due to their therapeutic potential. As mentioned before, these are progressive disorders that have the potential for reversal of symptoms. Over the years many strategies have been tried that involve multiple ways to replace/insert normal enzyme into the patient to reverse the lysosomal storage. Currently two treatment modalities (Figure 13-26) are being used to treat these conditions: tissue transplantation or direct infusion of bioengineered enzyme (enzyme replacement therapy or ERT). Because treatment is most effective the earlier it is started, it has been suggested that newborn screening for LSDs should be strongly considered. This will be discussed in more detail in Chapter 14.
Figure 13-26. Adolescent male with mucopolysaccharide storage disorder type II (Hunter syndrome). Hunter syndrome is X-linked. He is also pictured with his affected brother in Fig. 8-10.
Peroxisomes are amazingly complex organelles. To date over 70 enzymatic functions have been identified in the peroxisomes. Some of their known major functions include β-oxidation of very long and long chain fatty acids, peroxide-based respiration, plasmalogen and bile acid synthesis, and glyoxylate transamination. There are two categories of peroxisomal disorders. Type 1 disorders involve multiple enzymes. These are typically disorders of the formation and assembly of the organelles themselves. Type 1 peroxisomal disorders include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and rhizomelic chondrodysplasia punctata. Type 1 peroxisomal disorders are typically severe conditions that affect overall brain function. Type 2 disorders are those in which a single enzymatic function of the peroxisome is affected. Examples of these conditions include X-linked adrenoleukodystrophy (ABCD1 gene), Pseudo-Zellweger syndrome (PMP70 gene), Adult Refsum disease (phytanic acid oxidase), and hyperoxaluria (alanine-glyoxylate aminotransferase).
The Cytoskeleton
Early thoughts on cellular organization were that the cytoplasm was an amorphous collection of fluid bound by the cell membrane and separated from the nucleus by its own membrane. It is now known that the cytoplasm is not merely a buffered solution of free floating enzymes and other biochemicals, but rather it contains a series of complex structural (filamentous) proteins that exist for cellular architectural support and organization. The collective term given to these filaments is the “cytoskeleton.” The proteins of the cytoskeleton come in several varieties, which include actin microfilaments (6 nm), intermediate filaments (10 nm), and the microtubules (23 nm). The intermediate filaments are composed of several important subunits including vimentin, keratins, and lamins.
Actins are microfilament proteins of the cytoskeleton. They are highly conserved across species and are one of the most prevalent proteins in cells. They have several functions including cell support, cell mobility, and molecular trafficking. Mutations in actin have been shown to cause a congenital myopathy known as nemaline myopathy. Filamins are cytoskeletal proteins that interact with actin as a basic anchoring protein. Several filamin A disorders have been described including frontometaphyseal dysplasia, Melnick Needles syndrome, otopalataldigital syndromes 1 and 2, and periventricular heterotopia.
The protein dystrophin is a component of the sub-sarcolemmal cytoskeleton. It is coded for by one of the largest known human genes (almost 2 Mb). The dystrophin gene is located at Xp21. The dystrophin protein acts as a biological “shock absorber” during muscle cell contraction. Abnormalities of this protein result in unrestrained contraction, which ultimately leads to disruption of the cellular membrane and muscle cell loss (Figure 13-27). The dystrophinopathies are a group of related disorders due to abnormalities of dystrophin. The phenotype of Duchenne muscular dystrophy (DMD) is most often associated with complete dystrophin dysfunction. DMD is an X-linked recessive disorder that, therefore, primarily affects boys. The typical presentation is that of a normal young boy who begins to develop difficulties walking at around 4 to 5 years old. Around this same time, hypertrophy of the calves is noted. Thereafter, progressive muscle wasting/weakness are noted. Most commonly young men with DMD die in young adulthood (Figure 13-28).
Figure 13-27. (a) Histopathologic picture of normal muscle. (b) Slide of dystrophic muscle. Note irregular fiber size and staining and the multiple central nuclei.
Figure 13-28. (a) Young man with Duchenne muscular dystrophy demonstrating a positive “Gower sign.” When asked to pick up a reflex hammer off of the floor, he cannot straighten back up without pushing off of his legs. This is a sign of proximal muscle weakness. (b) Adolescent male with more advanced Duchenne muscular dystrophy. He is now non-ambulatory due to advanced muscle deterioration.
Epidermolysis bullosa refers to a group of skin disorders characterized by blistering of the skin associated with minimal pressure, friction or trauma. The disorder is genetically heterogeneous. Most commonly, it shows autosomal dominant inheritance; a few types show autosomal recessive inheritance. The common pathophysiology of these conditions is shearing within the epidermal cells. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder that has a blister pattern affecting mainly the hands and feet (Figure 13-29). EBS is caused by mutations in the cytoskeletal keratins, keratin-5 or keratin-14 gene.
Figure 13-29. Lower extremities of an infant with epidermolysis bullosa. These patients have recurrent generalized blistering especially in pressure prone areas. The blistering typically does not scar. Dermatopathology shows cleavage within basal keratinocytes.
Other disorders reported in association with mutations of cytoskeletal proteins include hepatic cirrhosis, chronic pancreatitis, pulmonary fibrosis, red blood cell membrane disorders (elliptocytosis and spherocytosis), and hearing loss. Disorders of the cytoskeleton have also been implicated in the pathogenesis of neurodegeneration, heart failure, and cancer (invasion).
The Nucleus and Centrioles
As with the cytoskeleton, mutations that lead to abnormal proteins involved in the nuclear membrane and centri-ole have been associated with recognized human disorders. The nuclear lamina is a dense collection of intermediate filaments and membrane associated proteins on the inner portion of the nuclear membrane. It has many important roles including providing mechanical support to the membrane, regulating DNA replication and cell division, chromatin organization, and anchoring the nuclear pore complexes embedded in the nuclear envelope. Lamins are filamentous proteins of the nuclear lamina. Currently three lamin protein genes (LMNA, LMNB2, LMNB1) have been associated with 13 known disorders, including 11 discrete phenotypes caused by LMNA mutations such as Hutchinson-Gilford progeria (Figure 13-30), Emery-Dreifuss muscular dystrophy, mandibuloacral dysplasia, generalized lipodystrophy, and restrictive dermopathy.
Figure 13-30. Young child with Hutchinson-Gilford progeria. Note the appearance of premature aging.
The centriole is a relatively small, tubular organelle located in the cytoplasm close to the nucleus. Its primary role appears to be involved in the nuclear division. The centriole is a self-replicating organelle. The hydrolethalus syndrome is a severe multiple anomaly syndrome. As the name implies, the outcome is very poor. Seventy percent of cases are still born; the remaining infants die shortly after birth. The condition is associated with a long list of congenital anomalies. The most common include complex central nervous system malformations, atrioventricular canals, polydactyly, stenosis of the airway, and abnormal pulmonary lobulations. Hydrolethalus syndrome is known to be caused by mutations in a gene designated HYLS-1. The protein product of this gene is a core centriolar protein that links the core centriole architecture to cilia.
Cilia, Flagella, and Cell Surface Specializations
There are two major types of cilia found on the surfaces of human cells. Immotile (primary) cilia have a primarily sensory function and occur on almost every cell type. Motile cilia are found on specialized cells that have specific needs for motility. They are found in high concentrations on the cells of the lining of respiratory tract, middle ear, and fallopian tubes.
Immotile (primary) cilia
Since primary cilia are found on most cell types, abnormalities of these structures have been associated with a large variety of clinical problems. Reported disorders associated with disrupted primary ciliary function include hepatic cystic disease, polycystic kidney disease (Figure 13-31), retinal dystrophies, ocular colobomas, infertility, polydactyly, and brain malformations (Figure 13-32). In the past few years, several well-described conditions have been shown to be disorders of the cilia. Collectively they have been called “ciliopathy syndromes.” Table 13-6 lists some of these disorders. These conditions have in common abnormalities of the primary cilia and multiple congenital anomalies that include varying combinations of the anomalies noted above, plus other unique findings. Bardet-Biedel syndrome described in Chapter 12 (Figure 12-14) is a well-known disorder of the primary cilia.
Figure 13-31. Renal ultrasounds showing multiple cysts in both kidneys. This child has autosomal recessive polycystic kidney disease.
Figure 13-32. Axial T1 brain image showing the “molar tooth sign,” a very characteristic brain malformation seen with Joubert syndrome. Joubert syndrome is a known “ciliopathy” disorder. (Reprinted with permission from Macferran KM, Buchmann RF, Ramakrishnaiah R, Griebel ML, Sanger WG, Saronwala A, Schaefer GB. Pontine tegmental cap dysplasia with a 2q13 microdeletion involving the NPHP1 gene: insights into malformations of the mid-hindbrain. Semin Pediatr Neurol. 2010 Mar; 17(1):69-74.)
Table 13-6. Examples of Ciliopathies: Disorders of Primary (Immotile) Cilia
Biedel-Bardet syndrome
Cerebellar vermis hypoplasia or aplasia, oligophrenia, congenital ataxia, ocular coloboma, and hepatic fibrosis (COACH) syndrome
Joubert syndrome
Meckel-Gruber syndrome
Nephronopthisis
Oral-facial-digital syndromes I, VI
Polycystic kidney disorders
Senior-Lokien syndrome
Motile cilia and flagella
Motile cilia and flagella are structurally very similar. In fact, flagella are essentially very long motile cilia. The majority of genes that code for both types of these structures are the same. Motile cilia are important for regional cell motility and for movement of fluids across the cell surface. Flagella serve a primary function in locomotion. Disorders of motile cilia and flagella, reflect their known distribution in cells (epithelium of the respiratory tract, middle ear, and the fallopian tubes as well as the tails of sperm).
A primary disorder of flagella has been described. In cases of male infertility, spermatic analysis is a first round assessment. Morphologically abnormal sperm termed “stump tail” or “short tail” sperm have been described. These sperm are described as having short, thick, and irregular flagella. On electron microscopic examination they are noted to have findings of dysplasia of the fibrous sheath. The primary problem ultimately appears to be dysplastic development of the axonemal and periaxonemal cytoskeleton of the spermatic flagella. Genetic studies in individuals with this anomaly have identified mutations in several related genes. The A-kinase anchor proteins (AKAPs) are found in the fibrous sheaths of sperm. Their function is to direct protein kinase A activity by anchoring the enzyme close to its substrates. Several patients with short tail spermatic abnormalities have been reported with abnormalities of these genes. Reported mutations include partial deletions in the Akap3 (12p13) and Akap4 (Xp11.22) genes and complete deletions of Akap4.
Because of the shared structural identity of motile cilia and flagella, mutations in the genes that code for any of their common structural proteins may affect both of these structures.
Kartagener syndrome (also called primary ciliary dyskinesia or PCD) is a well-described entity. The primary manifestations of PCD are situs abnormalities, chronic respiratory problems, and infertility. It is easy to understand these symptoms in light of ciliary and flagellar dysfunction. An interesting observation is that only 50% of patients with Kartagener syndrome will have situs abnormalities. The explanation for this appears to be that the correct placement of organs depends in part on the directional beating of cilia. If ciliary motion is impaired, the organs can essential “fall” randomly in either direction. Thus by chance, they will fall into the normal position about half the time. The other half of the time they will fall in the opposite (abnormal) direction. The condition shows marked genetic heterogeneity. To date at least 16 loci have been associated with the disorder. All of the associated genes share in common a primary function of the ciliary/flagellar mechanism. These include abnormalities of some of the key structural proteins (dyneins), components of the radial spokes, assembly of the units, and stabilizers of the assembled structures.
Part 3: Clinical Correlation
As noted earlier, certain clinical signs and symptoms are strongly associated with mitochondrial dysfunction. The presence of such findings provides the clinician with important clues as to the possible origin of a patient’s disease. In the early 1990s several families were reported with problems that clearly appeared to be mitochondrial in origin. Affected individuals had progressive external ophthalmople-gia (inability to move the globe of the eye), myopathy with ragged red fibers seen on biopsy (Figure 13-16), and neurosensory hearing loss: there is a failure in the tissues of high energy requirement causing a combination of muscle, sight, and hearing problems. For medical geneticists this pattern of problems screams of mitochondrial dysfunction. Review of the pedigrees, however, showed vertical transmission with several instances of male-to-male transmission suggestive of autosomal dominant inheritance. However, additional investigation into these families revealed mutations in the mitochondrial genome (usually large scale and often multiple deletions) in the affected individuals. The other fascinating observation in these individuals was that each of them had different mitochondrial mutations! Ultimately it has been shown that all of this information can be explained by a novel genetic mechanism. These disorders are now known to be due to mutations of nuclear-encoded proteins that disrupt the integrity of the mitochondrial genome. To phrase this another way, the mutations in the nuclear genes affect the normal replication of the mitochondrial DNA. What is observed in the families then is a Mendelian pattern of transmission of randomly generated mitochondrial mutations.
These families highlight a very important biological fact. Having the different proteins in a single organelle encoded by two or more independent genomes requires a high level of coordination. In this light, a new class of genes has been described that are involved in this process. Regulators of organelle gene expression (ROGEs) are nuclear genes that—as the name implies–regulate the mitochondrial genes. This regulation of mitochondrial gene expression usually occurs via posttranscriptional mechanisms (transcript maturation and translation). ROGEs have been shown to have multiple mechanisms of influence on the mitochondrial genome. Examples of some of these include influences on mitochondrial formation, level of OXPHOS activity, balance between aerobic and anaerobic metabolism, and the removal of dysfunctional mitochondria.
Board-Format Practice Questions
1. Which of the following is a characteristic of mitochondrial inheritance?
A. Male-to-male transmission.
B. Replicative segregation.
C. Expression in homozygotes.
D. A spontaneous mutation rate lower than that of nuclear genes.
E. Bottlenose phenomenon.
2. A patient (20-year-old woman) presents to your clinic wanting to be tested for a mitochondrial disorder. She tells you that her bother has been diagnosed with a mitochondrial disorder. You would tell her which of the following?
A. Because her brother is affected, and the condition is mitochondrial, she could not be affected.
B. Since she does not have any symptoms now as a young adult, she could not be affected.
C. If she has the same condition as her brother, she should be affected to the same degree as he is.
D. The most helpful information would be to know the particular mutation that is present in her brother.
E. She should not have any children and should have a sterilization procedure soon.
3. You are evaluating a child with multisystem problems. As you ponder ordering some diagnostic tests, you could appropriately decide which of the following?
A. Because the child has chronic respiratory problems you should test for peroxisomal disorders.
B. Perform a renal biopsy.
C. Because the child is showing severe premature aging, you should do testing for genes coding for the nuclear membrane proteins.
D. You can safely assume the condition is autosomal dominant and counsel as such—avoiding doing any tests.
E. Because multiple organ systems are involved, testing won’t be able to give you an answer, so you should not do any tests.
4. You see a patient in the newborn nursery. The child has multiple congenital anomalies. As part of a thorough evaluation you discover that the child has polydactyly, polycystic kidneys, retinitis pigmentosa, and a “molar tooth sign” on the brain MRI. Given this pattern of anomalies, you would conclude that the primary pathogenesis of this child’s problems is likely due to abnormalities of:
A. Primary cilia.
B. Mitochondria.
C. Peroxisomes.
D. Lysosomes.
E. Endosomes.
5. You see a new patient who is a 2-month-old boy. He is seeing you because his brother has Hunter syndrome (an X-linked lysosomal storage disorder). On your exam he has no dysmorphic features and is exhibiting normal growth and development. His parents have multiple questions. Which of the following would be a correct statement to share with them?
A. Since Hunter syndrome is X-linked and he is not a female, he will not have it.
B. Since he looks normal at this point, he probably does not have Hunter syndrome.
C. Since he had newborn screening and that was normal, he could not have Hunter syndrome.
D. Enzyme replacement therapy is now available for Hunter syndrome. Early confirmation of his status would be important.
E. Lysosomal transplantation should be performed soon on this child.