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InternalMed_Harrison. Eosinophils and neutrophils share similar morphology, many lysosomal constituents, phagocytic capacity, and oxidative metabolism. Eosinophils express a specific chemoattractant receptor and respond to a specific chemokine, eotaxin, but little is known about their required role. Eosinophils are much longer lived than neutrophils, and unlike neutrophils, tissue eosinophils can recirculate. During most infections, eosinophils appear unimportant. However, in invasive helminthic infections, such as hookworm, schistosomiasis, strongyloidiasis, toxocariasis, trichinosis, filariasis, echinococcosis, and cysticercosis, the eosinophil plays a central role in host defense. Eosinophils are associated with bronchial asthma, cutaneous allergic reactions, and other hypersensitivity states.


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InternalMed_Harrison. The distinctive feature of the red-staining (Wright’s stain) eosinophil granule is its crystalline core consisting of an arginine-rich protein (major basic protein) with histaminase activity, important in host defense against parasites. Eosinophil granules also contain a unique eosinophil peroxidase that catalyzes the oxidation of many substances by hydrogen peroxide and may facilitate killing of microorganisms.


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InternalMed_Harrison. Eosinophil peroxidase, in the presence of hydrogen peroxide and halide, initiates mast cell secretion in vitro and thereby promotes inflammation. Eosinophils contain cationic proteins, some of which bind to heparin and reduce its anticoagulant activity. Eosinophilderived neurotoxin and eosinophil cationic protein are ribonucleases that can kill respiratory syncytial virus. Eosinophil cytoplasm contains Charcot-Leyden crystal protein, a hexagonal bipyramidal crystal first observed in a patient with leukemia and then in sputum of patients with asthma; this protein is lysophospholipase and may function to detoxify certain lysophospholipids.


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InternalMed_Harrison. Several factors enhance the eosinophil’s function in host defense. T cell–derived factors enhance the ability of eosinophils to kill parasites. Mast cell–derived eosinophil chemotactic factor of anaphylaxis (ECFa) increases the number of eosinophil complement receptors and enhances eosinophil killing of parasites. Eosinophil CSFs (e.g., IL-5) produced by macrophages increase eosinophil production in the bone marrow and activate eosinophils to kill parasites.


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InternalMed_Harrison. Eosinophilia is the presence of >500 eosinophils per μL of blood and is common in many settings besides parasite infection. Significant tissue eosinophilia can occur without an elevated blood count. A common cause of eosinophilia is allergic reaction to drugs (iodides, aspirin, sulfonamides, nitrofurantoin, penicillins, and cephalosporins). Allergies such as hay fever, asthma, eczema, serum sickness, allergic vasculitis, and pemphigus are associated with eosinophilia. Eosinophilia also occurs in collagen vascular diseases (e.g., rheumatoid arthritis, eosinophilic fasciitis, allergic angiitis, and periarteritis nodosa) and malignancies (e.g., Hodgkin’s disease; mycosis fungoides; chronic myeloid leukemia; and cancer of the lung, stomach, pancreas, ovary, or uterus), as well as in Job’s syndrome, DOCK8 deficiency (see below), and CGD. Eosinophilia is commonly present in helminthic infections. IL-5 is the dominant eosinophil growth factor. Therapeutic administration of the cytokines IL-2


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InternalMed_Harrison. DOCK8 deficiency (see below), and CGD. Eosinophilia is commonly present in helminthic infections. IL-5 is the dominant eosinophil growth factor. Therapeutic administration of the cytokines IL-2 or GM-CSF frequently leads to transient eosinophilia. The most dramatic hypereosinophilic syndromes are Loeffler’s syndrome, tropical pulmonary eosinophilia, Loeffler’s endocarditis, eosinophilic leukemia, and idiopathic hypereosinophilic syndrome (50,000–100,000/μL). IL-5 is the dominant eosinophil growth factor and can be specifically inhibited with the monoclonal antibody mepolizumab.


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InternalMed_Harrison. The idiopathic hypereosinophilic syndrome represents a heterogeneous group of disorders with the common feature of prolonged eosinophilia of unknown cause and organ system dysfunction, including the heart, central nervous system, kidneys, lungs, gastrointestinal tract, and skin. The bone marrow is involved in all affected individuals, but the most severe complications involve the heart and central nervous system. Clinical manifestations and organ dysfunction are highly variable. Eosinophils are found in the involved tissues and likely cause tissue damage by local deposition of toxic eosinophil proteins such as eosinophil cationic protein and major basic protein. In the heart, the pathologic changes lead to thrombosis, endocardial fibrosis, and restrictive endomyocardiopathy. The damage to tissues in other organ systems is similar. Some cases are due to mutations involving the platelet-derived growth factor receptor, and these are extremely sensitive to the tyrosine kinase inhibitor


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InternalMed_Harrison. to tissues in other organ systems is similar. Some cases are due to mutations involving the platelet-derived growth factor receptor, and these are extremely sensitive to the tyrosine kinase inhibitor imatinib. Glucocorticoids, hydroxyurea, and IFN-α each have been used successfully, as have therapeutic antibodies against IL-5. Cardiovascular complications are managed aggressively.


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InternalMed_Harrison. The eosinophilia-myalgia syndrome is a multisystem disease, with prominent cutaneous, hematologic, and visceral manifestations, that frequently evolves into a chronic course and can occasionally be fatal. The syndrome is characterized by eosinophilia (eosinophil count >1000/μL) and generalized disabling myalgias without other recognized causes. Eosinophilic fasciitis, pneumonitis, and myocarditis; neuropathy culminating in respiratory failure; and encephalopathy may occur. The disease is caused by ingesting contaminants in L-tryptophan–containing products. Eosinophils, lymphocytes, macrophages, and fibroblasts accumulate in the affected tissues, but their role in pathogenesis is unclear. Activation of eosinophils and fibroblasts and the deposition of eosinophil-derived toxic proteins in affected tissues may contribute. IL-5 and transforming growth factor β have been implicated as potential mediators. Treatment is withdrawal of products containing L-tryptophan and the administration of


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InternalMed_Harrison. tissues may contribute. IL-5 and transforming growth factor β have been implicated as potential mediators. Treatment is withdrawal of products containing L-tryptophan and the administration of glucocorticoids. Most patients recover fully, remain stable, or show slow recovery, but the disease can be fatal in up to 5% of patients.


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InternalMed_Harrison. Eosinophilic neoplasms are discussed in Chapter 135e. Eosinopenia occurs with stress, such as acute bacterial infection, and after treatment with glucocorticoids. The mechanism of eosinopenia of acute bacterial infection is unknown but is independent of endogenous glucocorticoids, because it occurs in animals after total adrenalectomy. There is no known adverse effect of eosinopenia.


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InternalMed_Harrison. The hyperimmunoglobulin E–recurrent infection syndrome, or Job’s syndrome, is a rare multisystem disease in which the immune and somatic systems are affected, including neutrophils, monocytes, T cells, B cells, and osteoclasts. Autosomal dominant mutations in signal transducer and activator of transcription 3 (STAT3) lead to inhibition of normal STAT signaling with broad and profound effects. Patients have characteristic facies with broad nose, kyphoscoliosis, and eczema. The primary teeth erupt normally but do not deciduate, often requiring extraction. Patients develop recurrent sinopulmonary and cutaneous infections that tend to be much less inflamed than appropriate for the degree of infection and have been referred to as “cold abscesses.” Characteristically, pneumonias cavitate, leading to pneumatoceles. Coronary artery aneurysms are common, as are cerebral demyelinated plaques that accumulate with age. Importantly, IL-17–producing T cells, which are thought responsible for


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InternalMed_Harrison. leading to pneumatoceles. Coronary artery aneurysms are common, as are cerebral demyelinated plaques that accumulate with age. Importantly, IL-17–producing T cells, which are thought responsible for protection against extracellular and mucosal infections, are profoundly reduced in Job’s syndrome. Despite very high IgE levels, these patients do not have elevated levels of allergy. An important syndrome with clinical overlap with STAT3 deficiency is due to autosomal recessive defects in dedicator of cytokinesis 8 (DOCK8). In DOCK8 deficiency, IgE elevation is joined to severe allergy, viral susceptibility, and increased rates of cancer.


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InternalMed_Harrison. Initial studies of WBC and differential and often a bone marrow examination may be followed by assessment of bone marrow reserves (steroid challenge test), marginated circulating pool of cells (epinephrine challenge test), and marginating ability (endotoxin challenge test) (Fig. 80-7). In vivo assessment of inflammation is possible with a Rebuck skin window test or an in vivo skin blister assay, which measures the 423 ability of leukocytes and inflammatory mediators to accumulate locally in the skin. In vitro tests of phagocyte aggregation, adherence, chemotaxis, phagocytosis, degranulation, and microbicidal activity (for


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InternalMed_Harrison. S. aureus) may help pinpoint cellular or humoral lesions. Deficiencies of oxidative metabolism are detected with either the nitroblue tetrazolium (NBT) dye test or the dihydrorhodamine (DHR) oxidation test. These tests are based on the ability of products of oxidative metabolism to alter the oxidation states of reporter molecules so that they can be detected microscopically (NBT) or by flow cytometry (DHR). Qualitative studies of superoxide and hydrogen peroxide production may further define neutrophil oxidative function.


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InternalMed_Harrison. Patients with leukopenias or leukocyte dysfunction often have delayed inflammatory responses. Therefore, clinical manifestations may be minimal despite overwhelming infection, and unusual infections must always be suspected. Early signs of infection demand prompt, aggressive culturing for microorganisms, use of antibiotics, and surgical drainage of abscesses. Prolonged courses of antibiotics are often required. In patients with CGD, prophylactic antibiotics (trimethoprim-sulfamethoxazole) and antifungals (itraconazole) markedly diminish the frequency of life-threatening infections. Glucocorticoids may relieve gastrointestinal or genitourinary tract obstruction by granulomas in patients with CGD. Although TNF-α-blocking agents may markedly relieve inflammatory bowel symptoms, extreme caution must be exercised in their use in CGD inflammatory bowel disease, because it profoundly increases these patients’ already heightened susceptibility to infection. Recombinant human IFN-γ, which


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InternalMed_Harrison. must be exercised in their use in CGD inflammatory bowel disease, because it profoundly increases these patients’ already heightened susceptibility to infection. Recombinant human IFN-γ, which nonspecifically stimulates phagocytic cell function, reduces the frequency of infections in patients with CGD by 70% and reduces the severity of infection. This effect of IFN-γ in CGD is additive to the effect of prophylactic antibiotics. The recommended dose is 50 μg/m2 subcutaneously three times weekly. IFN-γ has also been used successfully in the treatment of leprosy, nontuberculous mycobacteria, and visceral leishmaniasis.


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InternalMed_Harrison. Rigorous oral hygiene reduces but does not eliminate the discomfort of gingivitis, periodontal disease, and aphthous ulcers; chlorhexidine mouthwash and tooth brushing with a hydrogen peroxide–sodium bicarbonate paste help many patients. Oral antifungal agents (fluconazole, itraconazole, voriconazole, posaconazole) have reduced mucocutaneous candidiasis in patients with Job’s syndrome. Androgens, glucocorticoids, lithium, and immunosuppressive therapy have been used to restore myelopoiesis in patients with neutropenia due to impaired production. Recombinant G-CSF is useful in the management of certain forms of neutropenia due to depressed neutrophil production, including those related to cancer chemotherapy. Patients with chronic neutropenia with evidence of a good bone marrow reserve need not receive prophylactic antibiotics. Patients with chronic or cyclic neutrophil counts <500/μL may benefit from prophylactic antibiotics and G-CSF during periods of neutropenia. Oral


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InternalMed_Harrison. reserve need not receive prophylactic antibiotics. Patients with chronic or cyclic neutrophil counts <500/μL may benefit from prophylactic antibiotics and G-CSF during periods of neutropenia. Oral trimethoprim-sulfamethoxazole (160/800 mg) twice daily can prevent infection. Increased numbers of fungal infections are not seen in patients with CGD on this regimen. Oral quinolones such as levofloxacin and ciprofloxacin are alternatives.


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InternalMed_Harrison. In the setting of cytotoxic chemotherapy with severe, persistent lymphocyte dysfunction, trimethoprim-sulfamethoxazole prevents Pneumocystis jiroveci pneumonia. These patients, and patients with phagocytic cell dysfunction, should avoid heavy exposure to airborne soil, dust, or decaying matter (mulch, manure), which are often rich in Nocardia and the spores of Aspergillus and other fungi. Restriction of activities or social contact has no proven role in reducing risk of infection for phagocyte defects.


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InternalMed_Harrison. Although aggressive medical care for many patients with phagocytic disorders can allow them to go for years without a life-threatening infection, there may still be delayed effects of prolonged antimicrobials and other inflammatory complications. Cure of most congenital phagocyte defects is possible by bone marrow transplantation, and rates of success are improving (Chap. 139e). The identification of specific gene defects in patients with LAD 1, CGD, and other immunodeficiencies has led to gene therapy trials in a number of genetic white cell disorders. CHAPTER 80 Disorders of Granulocytes and Monocytes


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InternalMed_Harrison. CHAPTER 80 Disorders of Granulocytes and Monocytes Atlas of Hematology and Analysis of Peripheral Blood Smears Dan L. Longo Some of the relevant findings in peripheral blood, enlarged lymph nodes, and bone marrow are illustrated in this chapter. Systematic his-81e tologic examination of the bone marrow and lymph nodes is beyond the scope of a general medicine textbook. However, every internist should know how to examine a peripheral blood smear.


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InternalMed_Harrison. The examination of a peripheral blood smear is one of the most informative exercises a physician can perform. Although advances in automated technology have made the examination of a peripheral blood smear by a physician seem less important, the technology is not a completely satisfactory replacement for a blood smear interpretation by a trained medical professional who also knows the patient’s clinical history, family history, social history, and physical findings. It is useful to ask the laboratory to generate a Wright’s-stained peripheral blood smear and examine it. The best place to examine blood cell morphology is the feathered edge of the blood smear where red cells lie in a single layer, side by side, just barely touching one another but not overlapping. The author’s approach is to look at the smallest cellular elements, the platelets, first and work his way up in size to red cells and then white cells.


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InternalMed_Harrison. Using an oil immersion lens that magnifies the cells 100-fold, one counts the platelets in five to six fields, averages the number per field, and multiplies by 20,000 to get a rough estimate of the platelet count. The platelets are usually 1–2 μm in diameter and have a blue granulated appearance. There is usually 1 platelet for every 20 or so red cells. Of course, the automated counter is much more accurate, but gross disparities between the automated and manual counts should be assessed. Large platelets may be a sign of rapid platelet turnover, as young platelets are often larger than old ones; alternatively, certain rare inherited syndromes can produce large platelets. Platelet clumping visible on the smear can be associated with falsely low automated platelet counts. Similarly, neutrophil fragmentation can be a source of falsely elevated automated platelet counts.


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InternalMed_Harrison. Next one examines the red blood cells. One can gauge their size by comparing the red cell to the nucleus of a small lymphocyte. Both are normally about 8 μm wide. Red cells that are smaller than the small lymphocyte nucleus may be microcytic; those larger than the small lymphocyte nucleus may be macrocytic. Macrocytic cells also tend to be more oval than spherical in shape and are sometimes called macroovalocytes. The automated mean corpuscular volume (MCV) can assist in making a classification. However, some patients may have both iron and vitamin B12 deficiency, which will produce an MCV in the normal range but wide variation in red cell size. When the red cells vary greatly in size, anisocytosis is said to be present. When the red cells vary greatly in shape, poikilocytosis is said to be present. The electronic cell counter provides an independent assessment of variability in red cell size. It measures the range of red cell volumes and reports the results as “red cell distribution


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InternalMed_Harrison. be present. The electronic cell counter provides an independent assessment of variability in red cell size. It measures the range of red cell volumes and reports the results as “red cell distribution width” (RDW). This value is calculated from the MCV; thus, cell width is not being measured but cell volume is. The term is derived from the curve displaying the frequency of cells at each volume, also called the distribution. The width of the red cell volume distribution curve is what determines the RDW. The RDW is calculated as follows: RDW = (standard deviation of MCV ÷ mean MCV) × 100. In the presence of morphologic anisocytosis, RDW (normally 11–14%) increases to 15–18%. The RDW is useful in at least two clinical settings. In patients with microcytic anemia, the differential diagnosis is generally between iron deficiency and thalassemia. In thalassemia, the small red cells are generally of uniform size with a normal small RDW. In iron deficiency, the size variability and the RDW are


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InternalMed_Harrison. generally between iron deficiency and thalassemia. In thalassemia, the small red cells are generally of uniform size with a normal small RDW. In iron deficiency, the size variability and the RDW are large. In addition, a large RDW can suggest a dimorphic anemia when a chronic atrophic gastritis can produce both vitamin B12 malabsorption to produce macrocytic anemia and blood loss to produce iron deficiency. In such settings, RDW is also large. An elevated


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InternalMed_Harrison. RDW also has been reported as a risk factor for all-cause mortality in 81e-1 population-based studies (Patel KV et al: Arch Intern Med 169:515, 2009), a finding that is unexplained currently. After red cell size is assessed, one examines the hemoglobin content of the cells. They are either normal in color (normochromic) or pale in color (hypochromic). They are never “hyperchromic.” If more than the normal amount of hemoglobin is made, the cells get larger—they do not become darker. In addition to hemoglobin content, the red cells are examined for inclusions. Red cell inclusions are the following: 1. Basophilic stippling—diffuse fine or coarse blue dots in the red cell usually representing RNA residue—especially common in lead poisoning 2. 3. Nuclei—red cells may be released or pushed out of the marrow prematurely before nuclear extrusion—often implies a myelophthisic process or a vigorous narrow response to anemia, usually hemolytic anemia 4.


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InternalMed_Harrison. (Chap. 250e) 5. Polychromatophilia—the red cell cytoplasm has a bluish hue, reflecting the persistence of ribosomes still actively making hemoglobin in a young red cell Vital stains are necessary to see precipitated hemoglobin called Heinz bodies.


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InternalMed_Harrison. Red cells can take on a variety of different shapes. All abnormally shaped red cells are poikilocytes. Small red cells without the central pallor are spherocytes; they can be seen in hereditary spherocytosis, hemolytic anemias of other causes, and clostridial sepsis. Dacrocytes are teardrop-shaped cells that can be seen in hemolytic anemias, severe iron deficiency, thalassemias, myelofibrosis, and myelodysplastic syndromes. Schistocytes are helmet-shaped cells that reflect microangiopathic hemolytic anemia or fragmentation on an artificial heart valve. Echinocytes are spiculated red cells with the spikes evenly spaced; they can represent an artifact of abnormal drying of the blood smear or reflect changes in stored blood. They also can be seen in renal failure and malnutrition and are often reversible. Acanthocytes are spiculated red cells with the spikes irregularly distributed. This process tends to be irreversible and reflects underlying renal disease, abetalipoproteinemia, or


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InternalMed_Harrison. often reversible. Acanthocytes are spiculated red cells with the spikes irregularly distributed. This process tends to be irreversible and reflects underlying renal disease, abetalipoproteinemia, or splenectomy. Elliptocytes are elliptical-shaped red cells that can reflect an inherited defect in the red cell membrane, but they also are seen in iron deficiency, myelodysplastic syndromes, megaloblastic anemia, and thalassemias. Stomatocytes are red cells in which the area of central pallor takes on the morphology of a slit instead of the usual round shape. Stomatocytes can indicate an inherited red cell membrane defect and also can be seen in alcoholism. Target cells have an area of central pallor that contains a dense center, or bull’s-eye. These cells are seen classically in thalassemia, but they are also present in iron deficiency, cholestatic liver disease, and some hemoglobinopathies. They also can be generated artifactually by improper slide making.


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InternalMed_Harrison. One last feature of the red cells to assess before moving to the white blood cells is the distribution of the red cells on the smear. In most individuals, the cells lie side by side in a single layer. Some patients have red cell clumping (called agglutination) in which the red cells pile upon one another; it is seen in certain paraproteinemias and autoimmune hemolytic anemias. Another abnormal distribution involves red cells lying in single cell rows on top of one another like stacks of coins. This is called rouleaux formation and reflects abnormal serum protein levels.


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InternalMed_Harrison. Finally, one examines the white blood cells. Three types of granulocytes are usually present: neutrophils, eosinophils, and basophils, in decreasing frequency. Neutrophils are generally the most abundant white cell. They are round, are 10–14 μm wide, and contain a lobulated nucleus with two to five lobes connected by a thin chromatin thread. Bands are immature neutrophils that have not completed nuclear condensation and have a U-shaped nucleus. Bands reflect a left shift in neutrophil maturation in an effort to make more cells more rapidly.


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InternalMed_Harrison. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears 81e-2 Neutrophils can provide clues to a variety of conditions. Vacuolated neutrophils may be a sign of bacterial sepsis. The presence of 1to 2-μm blue cytoplasmic inclusions, called Dle bodies, can reflect infections, burns, or other inflammatory states. If the neutrophil granules are larger than normal and stain a darker blue, “toxic granulations” are said to be present, and they also suggest a systemic inflammation. The presence of neutrophils with more than five nuclear lobes suggests megaloblastic anemia. Large misshapen granules may reflect the inherited Chédiak-Higashi syndrome. Eosinophils are slightly larger than neutrophils, have bilobed nuclei, and contain large red granules. Diseases of eosinophils are associated with too many of them rather than any morphologic or qualitative change. They normally total less than one-thirtieth the number of neutrophils. Basophils are even rarer than eosinophils in the


InternalMed_Harrison

InternalMed_Harrison. with too many of them rather than any morphologic or qualitative change. They normally total less than one-thirtieth the number of neutrophils. Basophils are even rarer than eosinophils in the blood. They have large dark blue granules and may be increased as part of chronic myeloid leukemia. Lymphocytes can be present in several morphologic forms. Most common in healthy individuals are small lymphocytes with a small dark nucleus and scarce cytoplasm. In the presence of viral infections, more of the lymphocytes are larger, about the size of neutrophils, with abundant cytoplasm and a less condensed nuclear chromatin. These cells are called reactive lymphocytes. About 1% of lymphocytes are larger and contain blue granules in a light blue cytoplasm; they are called large granular lymphocytes. In chronic lymphoid leukemia, the small lymphocytes are increased in number, and many of them are ruptured in making the blood smear, leaving a smudge of nuclear material without a surrounding


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InternalMed_Harrison. In chronic lymphoid leukemia, the small lymphocytes are increased in number, and many of them are ruptured in making the blood smear, leaving a smudge of nuclear material without a surrounding cytoplasm or cell membrane; they are called smudge cells and are rare in the absence of chronic lymphoid leukemia.


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InternalMed_Harrison. Monocytes are the largest white blood cells, ranging from 15 to 22 μm in diameter. The nucleus can take on a variety of shapes but usually appears to be folded; the cytoplasm is gray. Abnormal cells may appear in the blood. Most often the abnormal cells originate from neoplasms of bone marrow–derived cells, including lymphoid cells, myeloid cells, and occasionally red cells. More rarely, other types of tumors can get access to the bloodstream, and rare epithelial malignant cells may be identified. The chances of seeing such abnormal cells is increased by examining blood smears made from buffy coats, the layer of cells that is visible on top of sedimenting red cells when blood is left in the test tube for an hour. Smears made from finger sticks may include rare endothelial cells. PART 2 Cardinal Manifestations and Presentation of Diseases


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InternalMed_Harrison. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-1 Normal peripheral blood smear. Small lymphocyte in center of field. Note that the diameter of the red blood cell is similar to the diameter of the small lymphocyte nucleus. Figure 81e-3 Hypochromic microcytic anemia of iron deficiency. Small lymphocyte in field helps assess the red blood cell size. Figure 81e-2 Reticulocyte count preparation. This new methylene blue–stained blood smear shows large numbers of heavily stained reticulocytes (the cells containing the dark blue–staining RNA precipitates). Figure 81e-4 Iron deficiency anemia next to normal red blood cells. Microcytes (right panel) are smaller than normal red blood cells (cell diameter <7 μm) and may or may not be poorly hemoglobinized (hypochromic). Figure 81e-5 Polychromatophilia. Note large red cells with light purple coloring. Figure 81e-8 Spherocytosis. Note small hyperchromatic cells with-out the usual clear area in the center.


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InternalMed_Harrison. Figure 81e-5 Polychromatophilia. Note large red cells with light purple coloring. Figure 81e-8 Spherocytosis. Note small hyperchromatic cells with-out the usual clear area in the center. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-6 Macrocytosis. These cells are both larger than normal (mean corpuscular volume >100) and somewhat oval in shape. Some morphologists call these cells macroovalocytes. Figure 81e-9 Rouleaux formation. Small lymphocyte in center of field. These red cells align themselves in stacks and are related to increased serum protein levels. Figure 81e-7 Hypersegmented neutrophils. Hypersegmented neutrophils (multilobed polymorphonuclear leukocytes) are larger than normal neutrophils with five or more segmented nuclear lobes. They are commonly seen with folic acid or vitamin B12 deficiency.


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InternalMed_Harrison. Figure 81e-10 Red cell agglutination. Small lymphocyte and seg-mented neutrophil in upper left center. Note irregular collections of aggregated red cells. Figure 81e-11 Fragmented red cells. Heart valve hemolysis. Figure 81e-14 Elliptocytosis. Small lymphocyte in center of field. Elliptical shape of red cells is related to weakened membrane struc-ture, usually due to mutations in spectrin. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-12 Sickle cells. Homozygous sickle cell disease. A nucle-ated red cell and neutrophil are also in the field. Figure 81e-15 Stomatocytosis. Red cells characterized by a wide transverse slit or stoma. This often is seen as an artifact in a dehydrat-ed blood smear. These cells can be seen in hemolytic anemias and in conditions in which the red cell is overhydrated or dehydrated.


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InternalMed_Harrison. Figure 81e-13 Target cells. Target cells are recognized by the bull’s-eye appearance of the cell. Small numbers of target cells are seen with liver disease and thalassemia. Larger numbers are typical of hemoglobin C disease. Figure 81e-16 Acanthocytosis. Spiculated red cells are of two types: acanthocytes are contracted dense cells with irregular membrane projections that vary in length and width; echinocytes have small, uniform, and evenly spaced membrane projections. Acanthocytes are present in severe liver disease, in patients with abetalipoproteinemia, and in rare patients with McLeod blood group. Echinocytes are found in patients with severe uremia, in glycolytic red cell enzyme defects, and in microangiopathic hemolytic anemia. Figure 81e-17 Howell-Jolly bodies. Howell-Jolly bodies are tiny nuclear remnants that normally are removed by the spleen. They appear in the blood after splenectomy (defect in removal) and with maturation/dysplastic disorders (excess production).


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InternalMed_Harrison. Figure 81e-20 Reticulin stain of marrow myelofibrosis. Silver stain of a myelofibrotic marrow showing an increase in reticulin fibers (black-staining threads). Figure 81e-18 Teardrop cells and nucleated red blood cells char-acteristic of myelofibrosis. A teardrop-shaped red blood cell (left panel) and a nucleated red blood cell (right panel) as typically seen with myelofibrosis and extramedullary hematopoiesis. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-21 Stippled red cell in lead poisoning. Mild hypochro-mia. Coarsely stippled red cell. Figure 81e-19 Myelofibrosis of the bone marrow. Total replace-ment of marrow precursors and fat cells by a dense infiltrate of reticu-lin fibers and collagen (H&E stain). Figure 81e-22 Heinz bodies. Blood mixed with hypotonic solution of crystal violet. The stained material is precipitates of denatured hemoglobin within cells.


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InternalMed_Harrison. Figure 81e-22 Heinz bodies. Blood mixed with hypotonic solution of crystal violet. The stained material is precipitates of denatured hemoglobin within cells. Figure 81e-23 Giant platelets. Giant platelets, together with a marked increase in the platelet count, are seen in myeloproliferative disorders, especially primary thrombocythemia. Figure 81e-26 Normal eosinophils. The film was prepared from the buffy coat of the blood from a normal donor. E, eosinophil; L, lymphocyte; N, neutrophil. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-27 Normal basophil. The film was prepared from the buffy coat of the blood from a normal donor. B, basophil; L, lymphocyte. Figure 81e-24 Normal granulocytes. The normal granulocyte has a segmented nucleus with heavy, clumped chromatin; fine neutrophilic granules are dispersed throughout the cytoplasm.


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InternalMed_Harrison. Figure 81e-24 Normal granulocytes. The normal granulocyte has a segmented nucleus with heavy, clumped chromatin; fine neutrophilic granules are dispersed throughout the cytoplasm. Figure 81e-25 Normal monocytes. The film was prepared from the buffy coat of the blood from a normal donor. L, lymphocyte; M, monocyte; N, neutrophil. Figure 81e-28 Pelger-Ht anomaly. In this benign disorder, the majority of granulocytes are bilobed. The nucleus frequently has a spectacle-like, or “pince-nez,” configuration. Figure 81e-29 Dle body. Neutrophil band with Döhle body. The neutrophil with a sausage-shaped nucleus in the center of the field is a band form. Döhle bodies are discrete, blue-staining nongranular areas found in the periphery of the cytoplasm of the neutrophil in infections and other toxic states. They represent aggregates of rough endoplasmic reticulum.


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InternalMed_Harrison. Figure 81e-32 Aplastic anemia bone marrow. Normal hematopoi-etic precursor cells are virtually absent, leaving behind fat cells, reticu-loendothelial cells, and the underlying sinusoidal structure. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-30 Chédiak-Higashi disease. Note giant granules in neutrophil. Figure 81e-33 Metastatic cancer in the bone marrow. Marrow biopsy specimen infiltrated with metastatic breast cancer and reactive fibrosis (H&E stain).


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InternalMed_Harrison. Figure 81e-33 Metastatic cancer in the bone marrow. Marrow biopsy specimen infiltrated with metastatic breast cancer and reactive fibrosis (H&E stain). Figure 81e-31 Normal bone marrow. Low-power view of normal adult marrow (hematoxylin and eosin [H&E] stain), showing a mix of fat cells (clear areas) and hematopoietic cells. The percentage of the space that consists of hematopoietic cells is referred to as marrow cellularity. In adults, normal marrow cellularity is 35–40%. If demands for increased marrow production occur, cellularity may increase to meet the demand. As people age, the marrow cellularity decreases and the marrow fat increases. Patients >70 years old may have a 20–30% marrow cellularity. Figure 81e-34 Lymphoma in the bone marrow. Nodular (follicular) lymphoma infiltrate in a marrow biopsy specimen. Note the character-istic paratrabecular location of the lymphoma cells. PART 2 Cardinal Manifestations and Presentation of Diseases


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InternalMed_Harrison. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-35 Erythroid hyperplasia of the marrow. Marrow aspirate specimen with a myeloid/erythroid ratio (M/E ratio) of 1:1–2, typical for a patient with a hemolytic anemia or one recovering from blood loss. Figure 81e-36 Myeloid hyperplasia of the marrow. Marrow aspi-rate specimen showing a myeloid/erythroid ratio of ≥3:1, suggesting either a loss of red blood cell precursors or an expansion of myeloid elements. Figure 81e-37 Megaloblastic erythropoiesis. High-power view of megaloblastic red blood cell precursors from a patient with a macrocytic anemia. Maturation is delayed, with late normoblasts showing a more immature-appearing nucleus with a lattice-like pattern with normal cytoplasmic maturation.


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InternalMed_Harrison. Figure 81e-38 Prussian blue staining of marrow iron stores. Iron stores can be graded on a scale of 0 to 4+. A. A marrow with excess iron stores (>4+); B. normal stores (2–3+); C. minimal stores (1+); and D. absent iron stores (0). Figure 81e-39 Ringed sideroblast. An orthochromatic normoblast with a collar of blue granules (mitochondria encrusted with iron) sur-rounding the nucleus. Figure 81e-42 Acute erythroleukemia. Note giant dysmorphic erythroblasts; two are binucleate, and one is multinucleate. Figure 81e-40 Acute myeloid leukemia. Leukemic myeloblast with an Auer rod. Note two to four large, prominent nucleoli in each cell. Figure 81e-43 Acute lymphoblastic leukemia. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-41 Acute promyelocytic leukemia. Note prominent cytoplasmic granules in the leukemia cells. Figure 81e-44 Burkitt’s leukemia, acute lymphoblastic leukemia. Figure 81e-45 Chronic myeloid leukemia in the peripheral blood.


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InternalMed_Harrison. Figure 81e-44 Burkitt’s leukemia, acute lymphoblastic leukemia. Figure 81e-45 Chronic myeloid leukemia in the peripheral blood. Figure 81e-48 Adult T cell leukemia. Peripheral blood smear show-ing leukemia cells with typical “flower-shaped” nucleus. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-46 Chronic lymphoid leukemia in the peripheral blood. Figure 81e-49 Follicular lymphoma in a lymph node. The normal nodal architecture is effaced by nodular expansions of tumor cells. Nodules vary in size and contain predominantly small lymphocytes with cleaved nuclei along with variable numbers of larger cells with vesicular chromatin and prominent nucleoli. Figure 81e-47 Sézary’s syndrome. Lymphocytes with frequently convoluted nuclei (Sézary cells) in a patient with advanced mycosis fungoides. Figure 81e-50 Diffuse large B cell lymphoma in a lymph node.


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InternalMed_Harrison. Figure 81e-50 Diffuse large B cell lymphoma in a lymph node. The neoplastic cells are heterogeneous but predominantly large cells with vesicular chromatin and prominent nucleoli. Figure 81e-51 Burkitt’s lymphoma in a lymph node. Burkitt’s lym-phoma with starry-sky appearance. The lighter areas are macrophages attempting to clear dead cells. Figure 81e-54 Lacunar cell; Reed-Sternberg cell variant in nodular sclerosing Hodgkin’s disease. High-power view of single mononuclear lacunar cell with retracted cytoplasm in a patient with nodular scleros-ing Hodgkin’s disease. CHAPTER 81e Atlas of Hematology and Analysis of Peripheral Blood Smears Figure 81e-52 Erythrophagocytosis accompanying aggressive lymphoma. The central macrophage is ingesting red cells, neutro-phils, and platelets. (Courtesy of Dr. Kiyomi Tsukimori, Kyushu University, Fukuoka, Japan.) Figure 81e-55 Normal plasma cell.


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InternalMed_Harrison. Figure 81e-55 Normal plasma cell. Figure 81e-53 Hodgkin’s disease. A Reed-Sternberg cell is present near the center of the field; a large cell with a bilobed nucleus and prominent nucleoli giving an “owl’s eyes” appearance. The majority of the cells are normal lymphocytes, neutrophils, and eosinophils that form a pleiomorphic cellular infiltrate. Figure 81e-56 Multiple myeloma. PART 2 Cardinal Manifestations and Presentation of Diseases Figure 81e-57 Serum color in hemoglobinemia. The distinctive red coloration of plasma (hemoglobinemia) in a spun blood sample in a patient with intravascular hemolysis. AcknowledgmentFigures in this e-chapter were borrowed from Williams Hematology, 7th edition, M Lichtman et al (eds). New York, McGraw-Hill, 2005; Hematology in General Practice, 4th edition, RS Hillman, KA Ault, New York, McGraw-Hill, 2005.


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InternalMed_Harrison. 425part 3: Genes, the Environment, and Disease of the genome (and epigenome) in various malignancies has led toprinciples of human Genetics fundamental new insights into cancer biology and reveals that the genomic profile of mutations is in many cases more important in J. Larry Jameson, Peter Kopp determining the appropriate chemotherapy than the organ in which


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InternalMed_Harrison. The prevalence of genetic diseases, combined with their potential severity and chronic nature, imposes great human, social, and financial burdens on society. Human genetics refers to the study of individual genes, their role and function in disease, and their mode of inheritance. Genomics refers to an organism’s entire genetic information, the genome, and the function and interaction of DNA within the genome, as well as with environmental or nongenetic factors, such as a person’s lifestyle. With the characterization of the human genome, genomics complements traditional genetics in our efforts to elucidate the etiology and pathogenesis of disease and to improve therapeutic interventions and outcomes. Following impressive advances in genetics, genomics, and health care information technology, the consequences of this wealth of knowledge for the practice of medicine are profound and play an increasingly prominent role in the diagnosis, prevention, and treatment of disease (Chap. 84).


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InternalMed_Harrison. Personalized medicine, the customization of medical decisions to an individual patient, relies heavily on genetic information. For example, a patient’s genetic characteristics (genotype) can be used to optimize drug therapy and predict efficacy, adverse events, and drug dosing of selected medications (pharmacogenetics) (Chap. 5). The mutational profile of a malignancy allows the selection of therapies that target mutated or overexpressed signaling molecules. Although still investigational, genomic risk prediction models for common diseases are beginning to emerge.


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InternalMed_Harrison. Genetics has traditionally been viewed through the window of relatively rare single-gene diseases. These disorders account for ~10% of pediatric admissions and childhood mortality. Historically, genetics has focused predominantly on chromosomal and metabolic disorders, reflecting the long-standing availability of techniques to diagnose these conditions. For example, conditions such as trisomy 21 (Down’s syndrome) or monosomy X (Turner’s syndrome) can be diagnosed using cytogenetics (Chap. 83e). Likewise, many metabolic disorders (e.g., phenylketonuria, familial hypercholesterolemia) are diagnosed using biochemical analyses. The advances in DNA diagnostics have extended the field of genetics to include virtually all medical specialties and have led to the elucidation of the pathogenesis of numerous monogenic disorders. In addition, it is apparent that virtually every medical condition has a genetic component. As is often evident from a patient’s family history, many common disorders


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InternalMed_Harrison. of numerous monogenic disorders. In addition, it is apparent that virtually every medical condition has a genetic component. As is often evident from a patient’s family history, many common disorders such as hypertension, heart disease, asthma, diabetes mellitus, and mental illnesses are significantly influenced by the genetic background. These polygenic or multifactorial (complex) disorders involve the contributions of many different genes, as well as environmental factors that can modify disease risk (Chap. 84). Genome-wide association studies (GWAS) have elucidated numerous disease-associated loci and are providing novel insights into the allelic architecture of complex traits. These studies have been facilitated by the availability of comprehensive catalogues of human single-nucleotide polymorphism (SNP) haplotypes generated through the HapMap Project. The sequencing of whole genomes or exomes (the exons within the genome) is increasingly used in the clinical realm in order to


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InternalMed_Harrison. polymorphism (SNP) haplotypes generated through the HapMap Project. The sequencing of whole genomes or exomes (the exons within the genome) is increasingly used in the clinical realm in order to characterize individuals with complex undiagnosed conditions or to characterize the mutational profile of advanced malignancies in order to select better targeted therapies.


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InternalMed_Harrison. Cancer has a genetic basis because it results from acquired somatic mutations in genes controlling growth, apoptosis, and cellular differentiation (Chap. 101e). In addition, the development of many cancers is associated with a hereditary predisposition. Characterization the tumor originates. Hence, comprehensive mutational profiling of malignancies has increasing impact on cancer taxonomy, the choice of targeted therapies, and improved outcomes.


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InternalMed_Harrison. Genetic and genomic approaches have proven invaluable for the detection of infectious pathogens and are used clinically to identify agents that are difficult to culture such as mycobacteria, viruses, and parasites, or to track infectious agents locally or globally. In many cases, molecular genetics has improved the feasibility and accuracy of diagnostic testing and is beginning to open new avenues for therapy, including gene and cellular therapy (Chaps. 90e and 91e). Molecular genetics has also provided the opportunity to characterize the microbiome, a new field that characterizes the population dynamics of bacteria, viruses, and parasites that coexist with humans and other animals (Chap. 86e). Emerging data indicate that the microbiome has significant effects on normal physiology as well as various disease states.


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InternalMed_Harrison. Molecular biology has significantly changed the treatment of human disease. Peptide hormones, growth factors, cytokines, and vaccines can now be produced in large amounts using recombinant DNA technology. Targeted modifications of these peptides provide the practitioner with improved therapeutic tools, as illustrated by genetically modified insulin analogues with more favorable kinetics. Lastly, there is reason to believe that a better understanding of the genetic basis of human disease will also have an increasing impact on disease prevention.


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InternalMed_Harrison. The astounding rate at which new genetic information is being generated creates a major challenge for physicians, health care providers, and basic investigators. Although many functional aspects of the genome remain unknown, there are many clinical situations where sufficient evidence exits for the use of genetic and genomic information to optimize patient care and treatment. Much genetic information resides in databases or is being published in basic science journals. Databases provide easy access to the expanding information about the human genome, genetic disease, and genetic testing (Table 82-1). For example, several thousand monogenic disorders are summarized in a large, continuously evolving compendium, referred to as the Online Mendelian Inheritance in Man (OMIM) catalogue (Table 82-1). The ongoing refinement of bioinformatics is simplifying the analysis and access to this daunting amount of new information.


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InternalMed_Harrison. THE HUMAN GENOME Structure of the Human Genome • Human Genome Project The Human Genome Project was initiated in the mid-1980s as an ambitious effort to characterize the entire human genome. Although the prospect of determining the complete sequence of the human genome seemed daunting several years ago, technical advances in DNA sequencing and bioinformatics led to the completion of a draft human sequence in 2000 and the completion of the DNA sequence for the last of the human chromosomes in May 2006. Currently, facilitated by rapidly decreasing costs for comprehensive sequence analyses and improvement of bioinformatics pipelines for data analysis, the sequencing of whole genomes and exomes is used with increasing frequency in the clinical setting. The scope of a whole genome sequence analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli


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InternalMed_Harrison. analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli genome. If the human DNA sequence were printed out, it would correspond to about 120 volumes of Harrison’s Principles of Internal Medicine.


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InternalMed_Harrison. In addition to the human genome, the genomes of numerous organisms have been sequenced completely (~4000) or partially (~10,000) (Genomes Online Database [GOLD]; Table 82-1). They include, among others, eukaryotes such as the mouse (Mus musculus), Chapter 82 Principles of Human Genetics Catalog of Published Genome-Wide Association Studies Office of Biotechnology Activities, National Institutes of Health American College of Medical Genetics and Genomics American Society of Human Genetics MITOMAP, a human mitochondrial genome database Dolan DNA Learning Center, Cold Spring Harbor Laboratories The Online Metabolic and Molecular Bases of Inherited Disease (OMMBID)


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InternalMed_Harrison. MITOMAP, a human mitochondrial genome database Dolan DNA Learning Center, Cold Spring Harbor Laboratories The Online Metabolic and Molecular Bases of Inherited Disease (OMMBID) The Jackson Laboratory http://www.ncbi.nlm.nih.gov/ http://www.genome.gov/ http://www.genome.gov/ GWAStudies/ http://www.ensembl.org http://www.ncbi.nlm.nih.gov/ omim http://oba.od.nih.gov/oba http://www.acmg.net/ http://www.ashg.org http://cgap.nci.nih.gov/ http://www.genetests.org/ http://www.genomesonline.org/ http://www.genenames.org/ http://www.mitomap.org/ http://www.hapmap.org/ http://www.genome.gov/10005107 http://www.dnalc.org/ http://www.ommbid.com/ http://omia.angis.org.au/ http://www.jax.org/ http://www.informatics.jax.org Broad access to biomedical and genomic information, literature (PubMed), sequence databases, software for analyses of nucleotides and proteins Extensive links to other databases, genome resources, and tutorials


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InternalMed_Harrison. Extensive links to other databases, genome resources, and tutorials An institute of the National Institutes of Health focused on genomic and genetic research; links providing information about the human genome sequence, genomes of other organisms, and genomic research Maps and sequence information of eukaryotic genomes Online compendium of Mendelian disorders and human genes causing genetic disorders Information about recombinant DNA and gene transfer; medical, ethical, legal, and social issues raised by genetic testing; medical, ethical, legal, and social issues raised by xenotransplantation Extensive links to other databases relevant for the diagnosis, treatment, and prevention of genetic disease Information about advances in genetic research, professional and public education, social and scientific policies Information about gene expression profiles of normal, precancer, and cancer cells


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InternalMed_Harrison. Information about advances in genetic research, professional and public education, social and scientific policies Information about gene expression profiles of normal, precancer, and cancer cells International directory of genetic testing laboratories and prenatal diagnosis clinics; reviews and educational materials Gene names and symbols A compendium of polymorphisms and mutations of the human mitochondrial DNA Catalogue of haplotypes in different ethnic groups relevant for association studies and pharmacogenomics Encyclopedia of DNA Elements; catalogue of all functional elements in the human genome Educational material about selected genetic disorders, DNA, eugenics, and genetic origin Online version of the comprehensive text on the metabolic and molecular bases of inherited disease Online compendium of Mendelian disorders in animals Information about murine models and the mouse genome Mouse genome informatics


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InternalMed_Harrison. Online compendium of Mendelian disorders in animals Information about murine models and the mouse genome Mouse genome informatics Note: Databases are evolving constantly. Pertinent information may be found by using links listed in the few selected databases.


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InternalMed_Harrison. Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster; bacteria (e.g., E. coli); and Archaea, viruses, organelles (mitochondria, chloroplasts), and plants (e.g., Arabidopsis thaliana). Genomic information of infectious agents has significant impact for the characterization of infectious outbreaks and epidemics. Other ramifications arising from the availability of genomic data include, among others, (1) the comparison of entire genomes (comparative genomics), (2) the study of large-scale expression of RNAs (functional genomics) and proteins (proteomics) to detect differences between various tissues in health and disease, (3) the characterization of the variation among individuals by establishing catalogues of sequence variations and SNPs (HapMap Project), and (4) the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.


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InternalMed_Harrison. cHromosomes The human genome is divided into 23 different chromosomes, including 22 autosomes (numbered 1–22) and the X and Y sex chromosomes (Fig. 82-1). Adult cells are diploid, meaning they contain two homologous sets of 22 autosomes and a pair of sex chromosomes. Females have two X chromosomes (XX), whereas males have one X and one Y chromosome (XY). As a consequence of meiosis, germ cells (sperm or oocytes) are haploid and contain one set of 22 autosomes and one of the sex chromosomes. At the time of fertilization, the diploid genome is reconstituted by pairing of the homologous chromosomes from the mother and father. With each cell division (mitosis), chromosomes are replicated, paired, segregated, and divided into two daughter cells.


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InternalMed_Harrison. structure of Dna DNA is a double-stranded helix composed of four different bases: adenine (A), thymidine (T), guanine (G), and cytosine (C). Adenine is paired to thymidine, and guanine is paired to cytosine, by hydrogen bond interactions that span the double helix (Fig. 82-1). DNA has several remarkable features that make it ideal for the transmission of genetic information. It is relatively stable, and the double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division. Complementarity also allows the transmission of genetic information from DNA → RNA → protein (Fig. 82-2). mRNA is encoded by the so-called sense or coding strand of the DNA double helix and is translated into proteins by ribosomes.


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InternalMed_Harrison. The presence of four different bases provides surprising genetic diversity. In the protein-coding regions of genes, the DNA bases are arranged into codons, a triplet of bases that specifies a particular amino acid. It is possible to arrange the four bases into 64 different triplet codons (43). Each codon specifies 1 of the 20 different amino acids, or a regulatory signal such as initiation and stop of translation. Because there are more codons than amino acids, the genetic code is degenerate; that is, most amino acids can be specified by several different codons. By arranging the codons in different combinations and in Nucleosome core Histone H2A, H2B, H4 p, short arm Centromere Solenoid q, long arm


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InternalMed_Harrison. Nucleosome core Histone H2A, H2B, H4 p, short arm Centromere Solenoid q, long arm FIGURE 82-1 Structure of chromatin and chromosomes. Chromatin is composed of double-strand DNA that is wrapped around histone and nonhistone proteins forming nucleosomes. The nucleosomes are further organized into solenoid structures. Chromosomes assume their characteristic structure, with short (p) and long (q) arms at the metaphase stage of the cell cycle. various lengths, it is possible to generate the tremendous diversity of primary protein structure.


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InternalMed_Harrison. various lengths, it is possible to generate the tremendous diversity of primary protein structure. DNA length is normally measured in units of 1000 bp (kilobases, kb) or 1,000,000 bp (megabases, Mb). Not all DNA encodes genes. In fact, genes account for only ~10–15% of DNA. Much of the remaining DNA consists of sequences, often of highly repetitive nature, the function of which is poorly understood. These repetitive DNA regions, along with nonrepetitive sequences that do not encode genes, serve, in part, a structural role in the packaging of DNA into chromatin (i.e., DNA bound to histone proteins, and chromosomes) and exert regulatory functions (Fig. 82-1).


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InternalMed_Harrison. Genes A gene is a functional unit that is regulated by transcription (see below) and encodes an RNA product, which is most commonly, but not always, translated into a protein that exerts activity within or outside the cell (Fig. 82-3). Historically, genes were identified because they conferred specific traits that are transmitted from one generation to the next. Increasingly, they are characterized based on expression in various tissues (transcriptome). The size of genes is quite broad; some genes are only a few hundred base pairs, whereas others are extraordinarily large (2 Mb). The number of genes greatly underestimates the complexity of genetic expression, because single genes can generate multiple spliced messenger RNA (mRNA) products (isoforms), which are translated into proteins that are subject to complex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing


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InternalMed_Harrison. that are subject to complex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing regions between the exons that are spliced out of precursor RNAs during RNA processing. The gene locus also includes regions that are necessary to control its expression (Fig. 82-2). Current 427 estimates predict 20,687 protein-coding genes in the human genome with an average of about four different coding transcripts per gene. Remarkably, the exome only constitutes 1.14% of the genome. In addition, thousands of noncoding transcripts (RNAs of various length such as microRNAs and long noncoding RNAs), which function, at least in part, as transcriptional and posttranscriptional regulators of gene expression, have been identified. Aberrant expression of microRNAs has been found to play a pathogenic role in numerous diseases.


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InternalMed_Harrison. sinGle-nucleotiDe PolymorPHisms An SNP is a variation of a single base pair in the DNA. The identification of the ~10 million SNPs estimated to occur in the human genome has generated a catalogue of common genetic variants that occur in human beings from distinct ethnic backgrounds (Fig. 82-3). SNPs are the most common type of sequence variation and account for ~90% of all sequence variation. They occur on average every 100 to 300 bases and are the major source of genetic heterogeneity. Remarkably, however, the primary DNA sequence of humans has ~99.9% similarity compared to that of any other human. SNPs that are in close proximity are inherited together (e.g., they are linked) and are referred to as haplotypes(Fig. 82-4). The HapMap describes the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations. The haplotype map information, referred to as HapMap, is greatly facilitating GWAS designed to elucidate the complex


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InternalMed_Harrison. and how they are distributed among individuals within and among populations. The haplotype map information, referred to as HapMap, is greatly facilitating GWAS designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders (see below). Moreover, haplotype analyses are useful to assess variations in responses to medications (pharmacogenomics) and environmental factors, as well as the prediction of disease predisposition.


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InternalMed_Harrison. coPy number variations Copy number variations (CNVs) are relatively large genomic regions (1 kb to several Mb) that have been duplicated or deleted on certain chromosomes (Fig. 82-5). It has been estimated that as many as 1500 CNVs, scattered throughout the genome, are present in an individual. When comparing the genomes of two individuals, approximately 0.4–0.8% of their genomes differ in terms of CNVs. Of note, de novo CNVs have been observed between monozygotic twins, who otherwise have identical genomes. Some CNVs have been associated with susceptibility or resistance to disease, and CNVs can be elevated in cancer cells.


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InternalMed_Harrison. Replication of DNA and Mitosis Genetic information in DNA is transmitted to daughter cells under two different circumstances: (1) somatic cells divide by mitosis, allowing the diploid (2n) genome to replicate itself completely in conjunction with cell division; and (2) germ cells (sperm and ova) undergo meiosis, a process that enables the reduction of the diploid (2n) set of chromosomes to the haploid state (1n).


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InternalMed_Harrison. Prior to mitosis, cells exit the resting, or G0 state, and enter the cell cycle (Chap. 101e). After traversing a critical checkpoint in G1, cells undergo DNA synthesis (S phase), during which the DNA in each chromosome is replicated, yielding two pairs of sister chromatids (2n → 4n). The process of DNA synthesis requires stringent fidelity in order to avoid transmitting errors to subsequent generations of cells. Genetic abnormalities of DNA mismatch/repair include xeroderma pigmentosum, Bloom’s syndrome, ataxia telangiectasia, and hereditary nonpolyposis colon cancer (HNPCC), among others. Many of these disorders strongly predispose to neoplasia because of the rapid acquisition of additional mutations (Chap. 101e). After completion of DNA synthesis, cells enter G2 and progress through a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the


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InternalMed_Harrison. a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the centromere, divide and migrate to opposite poles of the cell. After formation of a nuclear membrane around the two separated sets of chromatids, the cell divides and two daughter cells are formed, thus restoring the diploid (2n) state.


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InternalMed_Harrison. Assortment and Segregation of Genes During Meiosis Meiosis occurs only in germ cells of the gonads. It shares certain features with mitosis but involves two distinct steps of cell division that reduce the chromosome number to the haploid state. In addition, there is active recombination that generates genetic diversity. During the first cell division, two Chapter 82 Principles of Human Genetics Regulation of Gene Expression


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InternalMed_Harrison. Chapter 82 Principles of Human Genetics Regulation of Gene Expression FIGURE 82-2 Flow of genetic information. Multiple extracellular signals activate intracellular signal cascades that result in altered regulation of gene expression through the interaction of transcription factors with regulatory regions of genes. RNA polymerase transcribes DNA into RNA that is processed to mRNA by excision of intronic sequences. The mRNA is translated into a polypeptide chain to form the mature protein after undergoing posttranslational processing. CBP, CREB-binding protein; CoA, co-activator; COOH, carboxyterminus; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element–binding protein; GTF, general transcription factors; HAT, histone acetyl transferase; NH2, aminoterminus; RE, response element; TAF, TBP-associated factors; TATA, TATA box; TBP, TATA-binding protein.


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InternalMed_Harrison. sister chromatids (2n → 4n) are formed for each chromosome pair and there is an exchange of DNA between homologous paternal and maternal chromosomes. This process involves the formation of chiasmata, structures that correspond to the DNA segments that cross over between the maternal and paternal homologues (Fig. 82-6). Usually there is at least one crossover on each chromosomal arm; recombination occurs more frequently in female meiosis than in male meiosis. Subsequently, the chromosomes segregate randomly. Because there are 23 chromosomes, there exist 223 (>8 million) possible combinations of chromosomes. Together with the genetic exchanges that occur during recombination, chromosomal segregation generates tremendous diversity, and each gamete is genetically unique. The process of recombination and the independent segregation of chromosomes provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or


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InternalMed_Harrison. and the independent segregation of chromosomes provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or linked genes) with the presence of a disease or genetic trait (see below).


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InternalMed_Harrison. After the first meiotic division, which results in two daughter cells (2n), the two chromatids of each chromosome separate during a second meiotic division to yield four gametes with a haploid state (1n). When the egg is fertilized by sperm, the two haploid sets are combined, thereby restoring the diploid state (2n) in the zygote.


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InternalMed_Harrison. REGULATION OF GENE EXPRESSION Regulation by Transcription Factors The expression of genes is regulated by DNA-binding proteins that activate or repress transcription. The number of DNA sequences and transcription factors that regulate transcription is much greater than originally anticipated. Most genes contain at least 15–20 discrete regulatory elements within 300 bp of the transcription start site. This densely packed promoter region often contains binding sites for ubiquitous transcription factors such as CAAT box/enhancer binding protein (C/EBP), cyclic AMP response element–binding (CREB) protein, selective promoter factor 1 (Sp-1), or activator protein 1 (AP-1). However, factors involved in cell-specific expression may also bind to these sequences. Key regulatory elements may also reside at a large distance from the proximal promoter. The globin and the immunoglobulin genes, for example, contain locus control regions that are several kilobases away from the structural sequences


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InternalMed_Harrison. at a large distance from the proximal promoter. The globin and the immunoglobulin genes, for example, contain locus control regions that are several kilobases away from the structural sequences of the gene. Specific groups of transcription factors that bind to these promoter and enhancer sequences provide a combinatorial code for regulating transcription. In this manner, relatively ubiquitous factors interact with more restricted factors to allow each gene to be expressed and regulated in a unique manner that is dependent on developmental state, cell type, and numerous extracellular stimuli. Regulatory factors also bind within the gene itself, particularly in the intronic regions. The transcription factors that bind to DNA actually represent only the first level of regulatory control. Other proteins—co-activators and co-repressors—interact with the DNA-binding transcription factors to generate large regulatory complexes. These complexes are subject to control by numerous


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InternalMed_Harrison. control. Other proteins—co-activators and co-repressors—interact with the DNA-binding transcription factors to generate large regulatory complexes. These complexes are subject to control by numerous cell-signaling pathways and enzymes, leading to phosphorylation, acetylation, sumoylation, and ubiquitination. Ultimately, the recruited transcription factors interact with, and stabilize, components of the basal transcription complex that assembles at the site of the TATA box and initiator region. This basal transcription factor complex consists of >30 different proteins. Gene transcription occurs when RNA polymerase begins to synthesize RNA from the DNA template. A large number of identified genetic diseases involve transcription factors (Table 82-2).


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InternalMed_Harrison. The field of functional genomics is based on the concept that understanding alterations of gene expression under various physiologic and pathologic conditions provides insight into the underlying functional role of the gene. By revealing specific gene expression profiles, this knowledge may be of diagnostic and therapeutic relevance. The large-scale study of expression profiles, which takes advantage of microarray and bead array technologies, is also referred to as transcriptomics 429 Known Genes (1260) SNPs (612,977) p22.3 p22.1p21.3 p21.1 p15.3 p15.1 p14.3 p14.1 p13 p12.3 p12.1 p11.2 q11.21 q11.22 q11.23 q21.11 p21.13 q21.3 q22.1q22.3q31.1 q31.2 q31.31 q31.33 q32.1 q36.1 q36.3 in 7q31.2 containing the CFTR gene is shown below. The CFTR gene contains 27 exons. More than 1900 mutations in this gene have been found in patients with cystic fibrosis. A 20-kb region encompassing exons 4–9 is shown further amplified to illustrate the SNPs in this region.


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InternalMed_Harrison. Chapter 82 Principles of Human Genetics FIGURE 82-4 The origin of haplotypes is due to repeated recombination events occurring in multiple generations. Over time, this leads to distinct haplotypes. These haplotype blocks can often be characterized by genotyping selected Tag single-nucleotide polymorphisms (SNPs), an approach that facilitates performing genome-wide association studies (GWAS). because the complement of mRNAs transcribed by the cellular genome is called the transcriptome. Most studies of gene expression have focused on the regulatory DNA elements of genes that control transcription. However, it should be emphasized that gene expression requires a series of steps, including mRNA processing, protein translation, and posttranslational modifications, all of which are actively regulated (Fig. 82-2).


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InternalMed_Harrison. Epigenetic Regulation of Gene Expression Epigenetics describes mechanisms and phenotypic changes that are not a result of variation in the primary DNA nucleotide sequence, but are caused by secondary modifications of DNA or histones. These modifications include heritable changes such as X-inactivation and imprinting, but they can also result from dynamic posttranslational protein modifications in response to environmental influences such as diet, age, or drugs. The epigenetic modifications result in altered expression of individual genes or chromosomal loci encompassing multiple genes. The term epigenome describes the constellation of covalent modifications of DNA and histones that impact chromatin structure, as well as non-coding transcripts that modulate the transcriptional activity of DNA. Although the primary DNA sequence is usually identical in all cells of an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell


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InternalMed_Harrison. of DNA. Although the primary DNA sequence is usually identical in all cells of an organism, tissue-specific changes in the epigenome contribute to determining the transcriptional signature of a cell (transcriptome) and hence the protein expression profile (proteome).


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InternalMed_Harrison. Mechanistically, DNA and histone modifications can result in the activation or silencing of gene expression (Fig. 82-7). DNA methylation involves the addition of a methyl group to cytosine residues. This is FIGURE 82-5 Copy number variations (CNV) encompass relatively large regions of the genome that have been duplicated or deleted. Chromosome 8 is shown with CNV detected by genomic hybridization. An increase in the signal strength indicates a duplication, a decrease reflects a deletion of the covered chromosomal regions. of the two X chromosome copies present in females.


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InternalMed_Harrison. FIGURE 82-6 Crossing-over and genetic recombination. During chiasma formation, either of the two sister chromatids on one chromosome pairs with one of the chromatids of the homologous chromosome. Genetic recombination occurs through crossing-over and results in recombinant and nonrecombinant chromosome segments in the gametes. Together with the random segregation of the maternal and paternal chromosomes, recombination contributes to genetic diversity and forms the basis of the concept of linkage.


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InternalMed_Harrison. usually restricted to cytosines of CpG dinucleotides, which are abundant throughout the genome. Methylation of these dinucleotides is thought to represent a defense mechanism that minimizes the expression of sequences that have been incorporated into the genome such as retroviral sequences. CpG dinucleotides also exist in so-called CpG islands, stretches of DNA characterized by a high CG content, which are found in the majority of human gene promoters. CpG islands in promoter regions are typically unmethylated, and the lack of methylation facilitates transcription.


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InternalMed_Harrison. Histone methylation involves the addition of a methyl group to lysine residues in histone proteins (Fig. 82-7). Depending on the specific lysine residue being methylated, this alters chromatin configuration, either making it more open or tightly packed. Acetylation of histone proteins is another well-characterized mechanism that results in an open chromatin configuration, which favors active transcription. Acetylation is generally more dynamic than methylation, and many transcriptional activation complexes have histone acetylase activity, whereas repressor complexes often contain deacetylases and remove acetyl groups from histones. Other histone modifications, whose effects are incompletely characterized, include phosphorylation and sumoylation. Lastly, noncoding RNAs that bind to DNA can have a significant impact on transcriptional activity.


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InternalMed_Harrison. Physiologically, epigenetic mechanisms play an important role in several instances. For example, X-inactivation refers to the relative silencing of one The inactivation process is a form of dosage compensation such that females (XX) do not generally express twice as many X-chromosomal gene products as males (XY). In a given cell, the choice of which chromosome is inactivated occurs randomly in humans. But once the maternal or paternal X chromosome is inactivated, it will remain inactive, and this information is transmitted with each cell division. The X-inactive specific transcript (Xist) gene encodes a large noncoding RNA that mediates the silencing of the X chromosome from which it is transcribed by coating it with Xist RNA. The inactive X chromosome is highly methylated and has low levels of histone acetylation.


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InternalMed_Harrison. Epigenetic gene inactivation also occurs on selected chromosomal regions of autosomes, a phenomenon referred to as genomic imprinting. Through this mechanism, a small subset of genes is only expressed in a monoallelic fashion. Imprinting is heritable and leads to the preferential expression of one of the parental alleles, which deviates from the usual biallelic expression seen for the majority of genes. Remarkably, imprinting can be limited to a subset of tissues. Imprinting is mediated through DNA methylation of one of the alleles. The epigenetic marks on imprinted genes are maintained throughout life, but during zygote formation, they are activated or inactivated in a sex-specific manner (imprint reset) (Fig. 82-8), which allows a differential expression pattern in the fertilized egg and the subsequent mitotic divisions. Appropriate expression of imprinted genes is important for normal development and cellular functions. Imprinting defects and uniparental disomy, which is the


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InternalMed_Harrison. egg and the subsequent mitotic divisions. Appropriate expression of imprinted genes is important for normal development and cellular functions. Imprinting defects and uniparental disomy, which is the inheritance of two chromosomes or chromosomal regions from the same parent, are the cause of several developmental disorders such as Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman’s syndrome, and Prader-Willi syndrome (see below). Monoallelic loss-of-function mutations in the GNAS1 gene lead to Albright’s hereditary osteodystrophy (AHO). Paternal transmission of GNAS1 mutations leads to an isolated AHO phenotype (pseudopseudohypoparathyroidism), whereas maternal transmission leads to AHO in combination with hormone resistance to parathyroid hormone, thyrotropin, and gonadotropins (pseudohypoparathyroidism type IA). These phenotypic differences are explained by tissue-specific imprinting of the GNAS1 gene, which is expressed primarily from the maternal allele in the


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InternalMed_Harrison. (pseudohypoparathyroidism type IA). These phenotypic differences are explained by tissue-specific imprinting of the GNAS1 gene, which is expressed primarily from the maternal allele in the thyroid, gonadotropes, and the proximal renal tubule. In most other tissues, the GNAS1 gene is expressed biallelically.


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InternalMed_Harrison. Abbreviations: CREB, cAMP responsive element–binding protein; HNF, hepatocyte nuclear factor; PML, promyelocytic leukemia; RAR, retinoic acid receptor; SRY, sex-determining region Y; VHL, von Hippel–Lindau. In patients with isolated renal resistance to parathyroid hormone (pseu-It is caused by mutations in the MECP2 gene, which encodes a methyldohypoparathyroidism type IB), defective imprinting of the GNAS1 binding protein. The ensuing aberrant methylation results in abnormal gene results in decreased Gsα expression in the proximal renal tubules. gene expression in neurons, which are otherwise normally developed. Rett’s syndrome is an X-linked dominant disorder resulting in devel-Remarkably, epigenetic differences also occur among monozygotic opmental regression and stereotypic hand movements in affected girls. twins. Although twins are epigenetically indistinguishable during the


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InternalMed_Harrison. FIGURE 82-7 Epigenetic modifications of DNA and histones. Methylation of cytosine residues is associated with gene silencing. Methylation of certain genomic regions is inherited (imprinting), and it is involved in the silencing of one of the two X chromosomes in females (X-inactivation). Alterations in methylation can also be acquired, e.g., in cancer cells. Covalent posttranslational modifications of histones play an important role in altering DNA accessibility and chromatin structure and hence in regulating transcription. Histones can be reversibly modified in their amino-terminal tails, which protrude from the nucleosome core particle, by acetylation of lysine, phosphorylation of serine, methylation of lysine and arginine residues, and sumoylation. Acetylation of histones by histone acetylases (HATs), e.g., leads to unwinding of chromatin and accessibility to transcription factors. Conversely, deacetylation by histone deacetylases (HDACs) results in a compact chromatin structure


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InternalMed_Harrison. acetylases (HATs), e.g., leads to unwinding of chromatin and accessibility to transcription factors. Conversely, deacetylation by histone deacetylases (HDACs) results in a compact chromatin structure and silencing of transcription.


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InternalMed_Harrison. early years of life, older monozygotic twins exhibit differences in the overall content and genomic distribution of DNA methylation and histone acetylation, which would be expected to alter gene expression in various tissues.


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InternalMed_Harrison. In cancer, the epigenome is characterized by simultaneous losses and gains of DNA methylation in different genomic regions, as well as repressive histone modifications. Hyperand hypomethylation are associated with mutations in genes that control DNA methylation. Hypomethylation is thought to remove normal control mechanisms that prevent expression of repressed DNA regions. It is also associated with genomic instability. Hypermethylation, in contrast, results in the silencing of CpG islands in promoter regions of genes, including tumor-suppressor genes. Epigenetic alterations are considered to be more easily reversible compared to genetic changes, and modification of the epigenome with demethylating agents and histone deacetylases is being explored in clinical trials. Several organisms have been studied extensively as genetic models, including M. musculus (mouse), D. melanogaster (fruit fly),


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InternalMed_Harrison. Several organisms have been studied extensively as genetic models, including M. musculus (mouse), D. melanogaster (fruit fly), C. elegans (nematode), S. cerevisiae (baker’s yeast), and E. coli (colonic bacterium). The ability to use these evolutionarily distant organisms as genetic models that are relevant


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InternalMed_Harrison. Maternal somatic cell Paternal somatic cell its functional consequences. Some mutations may be lethal, others are less deleterious, and some may confer an evolutionary advantage. Mutations can occur in the germline (sperm or oocytes); these can be transmitted to progeny. Alternatively, mutations can occur during embryogenesis or in somatic tissues. Mutations that occur during development lead to mosaicism, a situation in which tissues are composed of cells with different genetic constitutions. If the germline is mosaic, a mutation can be transmitted to some progeny but not others, which sometimes leads to confusion in assessing the pattern of inheritance. Somatic mutations that do not affect cell survival can sometimes be detected because of variable phenotypic effects in tissues (e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events may also influence gene


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InternalMed_Harrison. (e.g., pigmented lesions in McCune-Albright syndrome). Other somatic mutations are associated with neoplasia because they confer a growth advantage to cells. Epigenetic events may also influence gene expression or facilitate genetic damage. With the exception of triplet nucleotide repeats, which can expand (see below), mutations are usually stable.


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InternalMed_Harrison. Mutations are structurally diverse—they can involve the entire genome, as in triploidy (one extra set of chromosomes), or gross numerical or structural alterations in chromosomes or individual genes (Chap. 83e). Large deletions may affect a portion of a gene or an entire gene, or, if several genes are involved, they may lead to a contiguous gene


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InternalMed_Harrison. Zygote syndrome. Unequal crossing-over between homologous genes can result in fusion gene mutations, as illustrated by color blindness. Mutations involving single nucleotides are referred to as point mutations. Substitutions are called transitions if a purine is replaced by another purine base (A ↔ G) or if a pyrimidine is replaced by another pyrimidine (C ↔ T). Changes from a purine to a pyrimidine, or vice versa, are referred to as transversions. If the DNA sequence change occurs in a coding region and alters an amino acid, it is called a missense mutation. Depending on the functional consequences of such a missense mutation, amino acid substitutions in different regions of the protein can lead to distinct


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InternalMed_Harrison. Inactive Methylated pat mat Active Unmethylated FIGURE 82-8 A few genomic regions are imprinted in a parent-specific fashion. The unmethylated chromosomal regions are actively expressed, whereas the methylated regions are silenced. In the germline, the imprint is reset in a parent-specific fashion: both chromo-somes are unmethylated in the maternal (mat) germline and methylated in the paternal (pat) germline. In the zygote, the resulting imprinting pattern is identical with the pattern in the somatic cells of the parents.


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InternalMed_Harrison. to human physiology reflects a surprising conservation of genetic pathways and gene function. Transgenic mouse models have been particularly valuable, because many human and mouse genes exhibit similar structure and function and because manipulation of the mouse genome is relatively straightforward compared to that of other mammalian species. Transgenic strategies in mice can be divided into two main approaches: (1) expression of a gene by random insertion into the genome, and (2) deletion or targeted mutagenesis of a gene by homologous recombination with the native endogenous gene (knock-out, knock-in). Previous versions of this chapter provide more detail about the technical principles underlying the development of genetically modified animals. Several databases provide comprehensive information about natural and transgenic animal models, the associated phenotypes, and integrated genetic, genomic, and biologic data (Table 82-1).


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InternalMed_Harrison. TRANSMISSION OF GENETIC DISEASE Origins and Types of Mutations A mutation can be defined as any change in the primary nucleotide sequence of DNA regardless of phenotypes. Mutations can occur in all domains of a gene (Fig. 82-9). A point mutation occurring within the coding region leads to an amino acid substitution if the codon is altered (Fig. 82-10). Point mutations that introduce a premature stop codon result in a truncated protein. Large deletions may affect a portion of a gene or an entire gene, whereas small deletions and insertions alter the reading frame if they do not represent a multiple of three bases. These “frameshift” mutations lead to an entirely altered carboxy terminus. Mutations in intronic sequences or in exon junctions may destroy or create splice donor or splice acceptor sites. Mutations may also be found in the regulatory sequences of genes, resulting in reduced or enhanced gene transcription.


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InternalMed_Harrison. Certain DNA sequences are particularly susceptible to mutagenesis. Successive pyrimidine residues (e.g., T-T or C-C) are subject to the formation of ultraviolet light–induced photoadducts. If these pyrimidine dimers are not repaired by the nucleotide excision repair pathway, mutations will be introduced after DNA synthesis. The dinucleotide C-G, or CpG, is also a hot spot for a specific type of mutation. In this case, methylation of the cytosine is associated with


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InternalMed_Harrison. FIGURE 82-9 Point mutations causing β thalassemia as example of allelic heterogeneity. The β-globin gene is located in the globin gene cluster. Point mutations can be located in the promoter, the CAP site, the 5’-untranslated region, the initiation codon, each of the three exons, the introns, or the polyadenylation signal. Many mutations introduce missense or nonsense mutations, whereas others cause defective RNA splicing. Not shown here are deletion mutations of the β-globin gene or larger deletions of the globin locus that can also result in thalassemia. ▼, promoter mutations; *, CAP site; •, 5’UTR; 1 , initiation codon; ♦, defective RNA processing; ✦, missense and nonsense Chapter 82 Principles of Human Genetics A, Poly A signal.


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InternalMed_Harrison. Chapter 82 Principles of Human Genetics A, Poly A signal. an enhanced rate of deamination to uracil, which is then replaced with thymine. This C → T transition (or G → A on the opposite strand) accounts for at least one-third of point mutations associated with polymorphisms and mutations. In addition to the fact that certain types of mutations (C → T or G → A) are relatively common, the nature of the genetic code also results in overrepresentation of certain amino acid substitutions.


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InternalMed_Harrison. Polymorphisms are sequence variations that have a frequency of at least 1%. Usually, they do not result in a perceptible phenotype. Often they consist of single base-pair substitutions that do not alter the protein coding sequence because of the degenerate nature of the genetic code (synonymous polymorphism), although it is possible that some might alter mRNA stability, translation, or the amino acid sequence (nonsynonymous polymorphism) (Fig. 82-10). The detection of sequence variants poses a practical problem because mutations is much greater in the male germline than the female germline, in which rates of aneuploidy are increased (Chap. 83e). Thus, the incidence of new point mutations in spermatogonia increases with paternal age (e.g., achondrodysplasia, Marfan’s syndrome, neurofibromatosis). It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are


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InternalMed_Harrison. It is estimated that about 1 in 10 sperm carries a new deleterious mutation. The rates for new mutations are calculated most readily for autosomal dominant and X-linked disorders and are ~10−5−10−6/locus per generation. Because most monogenic diseases are relatively rare, new mutations account for a significant fraction of cases. This is important in the context of genetic counseling, because a new mutation can be transmitted to the affected individual but does not necessarily imply that the parents are at risk to transmit the disease to other children. An exception to this is when the new mutation occurs early in germline development, leading to gonadal mosaicism.


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InternalMed_Harrison. it is often unclear whether it creates 433 a mutation with functional consequences or a benign polymorphism. In this situation, the sequence alteration is described as variant of unknown significance (VUS).


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InternalMed_Harrison. mutation rates Mutations represent an important cause of genetic diversity as well as disease. Mutation rates are difficult to determine in humans because many mutations are silent and because testing is often not adequate to detect the phenotypic consequences. Mutation rates vary in different genes but are estimated to occur at a rate of ~10−10/bp per cell division. Germline mutation rates (as opposed to somatic mutations) are relevant in the transmission of genetic disease. Because the population of oocytes is established very early in development, only ~20 cell divisions are required for completed oogenesis, whereas spermatogenesis involves ~30 divisions by the time of puberty and 20 cell divisions each year thereafter. Consequently, the probability of acquiring new point 1 bp Deletion with frameshift


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InternalMed_Harrison. FIGURE 82-10 A. Examples of mutations. The coding strand is shown with the encoded amino acid sequence. B. Chromatograms of sequence analyses after amplification of genomic DNA by polymerase chain reaction.


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InternalMed_Harrison. unequal crossinG-over Normally, DNA recombination in germ cells occurs with remarkable fidelity to maintain the precise junction sites for the exchanged DNA sequences (Fig. 82-6). However, mispairing of homologous sequences leads to unequal crossover, with gene duplication on one of the chromosomes and gene deletion on the other chromosome. A significant fraction of growth hormone (GH) gene deletions, for example, involve unequal crossing-over (Chap. 402). The GH gene is a member of a large gene cluster that includes a GH variant gene as well as several structurally related chorionic somatomammotropin genes and pseudogenes (highly homologous but functionally inactive relatives of a normal gene). Because such gene clusters contain multiple homologous DNA sequences arranged in tandem, they are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased


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InternalMed_Harrison. are particularly prone to undergo recombination and, consequently, gene duplication or deletion. On the other hand, duplication of the PMP22 gene because of unequal crossing-over results in increased gene dosage and type IA Charcot-Marie-Tooth disease. Unequal crossing-over resulting in deletion of PMP22 causes a distinct neuropathy called hereditary liability to pressure palsy (Chap. 459).


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InternalMed_Harrison. Glucocorticoid-remediable aldosteronism (GRA) is caused by a gene fusion or rearrangement involving the genes that encode aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1), normally arranged in tandem on chromosome 8q. These two genes are 95% identical, predisposing to gene duplication and deletion by unequal crossing-over. The rearranged gene product contains the regulatory regions of 11β-hydroxylase fused to the coding sequence of aldosterone synthetase. Consequently, the latter enzyme is expressed in the adrenocorticotropic hormone (ACTH)–dependent zona fasciculata of the adrenal gland, resulting in overproduction of mineralocorticoids and hypertension (Chap. 406).


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InternalMed_Harrison. Gene conversion refers to a nonreciprocal exchange of homologous genetic information. It has been used to explain how an internal portion of a gene is replaced by a homologous segment copied from another allele or locus; these genetic alterations may range from a few nucleotides to a few thousand nucleotides. As a result of gene conversion, it is possible for short DNA segments of two chromosomes to be identical, even though these sequences are distinct in the parents. A practical consequence of this phenomenon is that nucleotide substitutions can occur during gene conversion between related genes, often altering the function of the gene. In disease states, gene conversion often involves intergenic exchange of DNA between a gene and a related pseudogene. For example, the 21-hydroxylase gene (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to


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InternalMed_Harrison. (CYP21A2) is adjacent to a nonfunctional pseudogene (CYP21A1P). Many of the nucleotide substitutions that are found in the CYP21A2 gene in patients with congenital adrenal hyperplasia correspond to sequences that are present in the CYP21A1P pseudogene, suggesting gene conversion as one cause of mutagenesis. In addition, mitotic gene conversion has been suggested as a mechanism to explain revertant mosaicism in which an inherited mutation is “corrected” in certain cells. For example, patients with autosomal recessive generalized atrophic benign epidermolysis bullosa have acquired reverse mutations in one of the two mutated COL17A1 alleles, leading to clinically unaffected patches of skin.


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InternalMed_Harrison. insertions anD Deletions Although many instances of insertions and deletions occur as a consequence of unequal crossing-over, there is also evidence for internal duplication, inversion, or deletion of DNA sequences. The fact that certain deletions or insertions appear to occur repeatedly as independent events indicates that specific regions within the DNA sequence predispose to these errors. For example, certain regions of the DMD gene, which encodes dystrophin, appear to be hot spots for deletions and result in muscular dystrophy (Chap. 462e). Some regions within the human genome are rearrangement hot spots and lead to CNVs.


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InternalMed_Harrison. errors in Dna rePair Because mutations caused by defects in DNA repair accumulate as somatic cells divide, these types of mutations are particularly important in the context of neoplastic disorders (Chap. 102e). Several genetic disorders involving DNA repair enzymes underscore their importance. Patients with xeroderma pigmentosum have defects in DNA damage recognition or in the nucleotide excision and repair pathway (Chap. 105). Exposed skin is dry and pigmented and is extraordinarily sensitive to the mutagenic effects of ultraviolet irradiation. More than 10 different genes have been shown to cause the different forms of xeroderma pigmentosum. This finding is consistent with the earlier classification of this disease into different complementation groups in which normal function is rescued by the fusion of cells derived from two different forms of xeroderma pigmentosum.