34 3.Globular Proteins The higher oxygen affinity of Hb F facilitates the transfer of al crculion os the pacenta to 2.Hemoglobin A2(Hb A2):Hb A2 is a minor component of normal adult hemoglobin,first appearing shortly before birth and,ulti Pg0e9130agl6o0 siologic conditions Hh a is tion being dependent on the plasma concentration of a particular Glucose hexose.The most abundant form of glycosylated hemoglobin is H ot the N-te the B-n snee ncraamountsof ar ondin with diabetes mellitus,because their Hb A has contact with highe ns duning the 120 me of these cel Hemoglobin A. III.ORGANIZATION OF THE GLOBIN GENES Figure3.15 g from ge ne tdnenaasaditonofglucose es.which direct the synthesis of the different alobin chain t is turally organized into gene families and also how they are expressed. A.a-Gene family The genes lies)located on two different chromosomes (Figure 3.16).The ncluster on chromosom 6 onta wo genes Globin gene famillies also contain globin-like genes that are not Chromom Two copes of thei mbin y3 m。med Choom Figure 3.16 Organization of the globin gene families
The higher oxygen affinity of Hb F facilitates the transfer of oxygen from the maternal circulation across the placenta to the RBCs of the fetus. 2. Hemoglobin A2 (Hb A2): Hb A2 is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting about 2% of the total hemoglobin. It is composed of two α-globin chains and two δ-globin chains (α2δ2, see Figure 3.13). 3. Hemoglobin A1c (HbA1c): Under physiologic conditions, Hb A is slowly and nonenzymically glycosylated, the extent of glycosylation being dependent on the plasma concentration of a particular hexose. The most abundant form of glycosylated hemoglobin is Hb A1c. It has glucose residues attached predominantly to the NH2 groups of the N-terminal valines of the β-globin chains (Figure 3.15). Increased amounts of Hb A1c are found in RBCs of patients with diabetes mellitus, because their Hb A has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of this phenomenon in assessing average blood glucose levels in persons with diabetes.) III. ORGANIZATION OF THE GLOBIN GENES To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobins, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed. A. α-Gene family The genes coding for the α-globin-like and β-globin-like subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Figure 3.16). The α-gene cluster on chromosome 16 contains two genes for the α-globin chains. It also contains the ζ gene that is expressed early in development as a component of embryonic hemoblobin. [Note: Globin gene famillies also contain globin-like genes that are not expressed (that is, their genetic information is not used to produce globin chains). These are called pseudogenes.] 34 3. Globular Proteins Figure 3.16 Organization of the globin gene families. ζ α α2 1 ε δ Gγ β Hb Gower 1 Hb A Hb F Hb A2 α2 α2β2 δ α2 2 γ2 ζ2ε2 Hemoglobins are formed by combinations of α-globin-like chains and β-globin-like chains α-Globin-like genes (Chromosome 16) β-Globin-like genes (Chromosome 11) Aγ Two copies of the α-globin gene are designated α1 and α2. Each can provide α-globin chains that combine with β-globin chains. Figure 3.15 Nonenzymic addition of glucose to hemoglobin. CH2OH HCOH HCH HCOH HOCH CO NH NH NH2 NH2 CH2OH HCOH HCO HCOH HOCH HCOH CH2OH HCOH HCH HCOH HOCH CO Glucose Hemoglobin A1c Hemoglobin A 168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 34
IV.Hemoglobinopathies B.B-Gene family a single gene for the b-alobin chain is located on chromosome 11 (see Figure 3.16).There are an additional four B-globin-like genes the(which. nd that codes for the aloin chain found in the mino adult hemoglobin Hb A2. C.Steps in globin chain synthesis Exon 1 Ex The RNA produced by transcription is actuallya precursor of the messenger RNA(mRNA)that is used as a template for the synthe- reiches oRN hed from a linear manner.The resulting mature mRNA enters the cvtosol heeis98neiCnbmon6nansaeaDr29Aamg8aeiha r3n431 IV.HEMOGLOBINOPATHIES Hemoglobinopathies have traditionally been defined as a family of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synth of I insufficient quantities of norma lassemia syndromes are representative hemoglobinopathies that can have severe em n an h sequencelg0edby decreased production of normal hemoglobin (quantitative hem globinopathy). A.Sickle cell anemia(hemoglobin S disease) of gobin chain inrie lod isorde tation)in the e for primarily in th African recessive disorder.It occurs in individuals who have inherited two mutant genes (one from each parent)that code for synthesis of the B chains of B-globin chan until sufficient Hb F has been replaced by Hb S so that sickling can occur (see be le cell anemia is erize by lne becinnin nrt mia p284)and incr eptibility to infec
B. β-Gene family A single gene for the β-globin chain is located on chromosome 11 (see Figure 3.16). There are an additional four β-globin-like genes: the ε gene (which, like the ζ gene, is expressed early in embryonic development), two γ genes (Gγ and Aγ that are expressed in Hb F), and the δ gene that codes for the globin chain found in the minor adult hemoglobin Hb A2. C. Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of red cell precursors, where the DNA sequence encoding the gene is transcribed. The RNA produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence, and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.17. A more detailed description of protein synthesis is presented in Chapter 31, p. 431.) IV. HEMOGLOBINOPATHIES Hemoglobinopathies have traditionally been defined as a family of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (Hb S), hemoglobin C disease (Hb C), hemoglobin SC disease (Hb S + Hb C), and the thalassemia syndromes are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thal assemias are caused by decreased production of normal hemoglobin (quantitative hemo - globinopathy). A. Sickle cell anemia (hemoglobin S disease) Sickle cell anemia, the most common of the red cell sickling diseases, is a genetic disorder of the blood caused by a single nucleotide alteration (a point mutation) in the gene for β-globin. It is the most common inherited blood disorder in the United States, affecting 80,000 Americans. It occurs primarily in the AfricanAmerican population, affecting one of 500 newborn African-American infants in the United States. Sickle cell anemia is a homozygous, recessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the β chains of the globin molecules. [Note: The mutant β-globin chain is designated βS, and the resulting hemoglobin, α2βS 2, is referred to as Hb S.] An infant does not begin showing symptoms of the disease until sufficient Hb F has been replaced by Hb S so that sickling can occur (see below). Sickle cell anemia is characterized by lifelong episodes of pain (“crises”), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in early childhood. [Note: The lifetime of an IV. Hemoglobinopathies 35 Figure 3.17 Synthesis of globin chains. 5' Transcription Splicing Translation mRNA precursor NUCLEUS CYTOSOL Hemoglobin mRNA Exon 1 Exon 2 Exon 3 Intron 2 α-Globin and β-globin gene families contain three exons (coding regions) separated by two noncoding introns. Intron 1 168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 35
36 3.Globular Proteins erythrocyte in sickle cell anemia is less than 20 days,compared with m days for normal RBCs;hence,the anemia.]Other symp de ac rome,stroke,spl asi and cell gene.The blood cells of such heterozygotes contain both Hb S They usually do not ValHsPo-clu-cLys w 1.Amino acid substitution in Hb s 8 chains:a molecule of Hb s Hb A contains two normal a-globin chains and two mutant B-globin chains(B),in which glutamate at position six has been replaced re,d rng ele of Hb s is a result of the absence of the negatively charged es in the thus rendering Hb S I RBCs is routine Valpro-valLys sickle cell trait and sickle cell disease.] Hb S 2.Sickling and tissue anoxia:The replacement of the charged gluta- the nonpol ar valine forms a protrusion on the B-globir cule in the 20)At 8mo6owhem8go8nsh8ome2es9n3aehe1Rec969mnga network of fibrous polymers that stiffen and distort produ the supply of oxygen leads en deprivation in the tissue,ca HbC Ihe anoxia so leads to is 7.5 网bsm re3.18 squeezing through the microvasculature like Hb A-containing RBCs,sickled cells hav a decreased ability to deform and esse ave dit 3.Variables that increase sickling:The extent of sickling and.there Anode fore.the severity of disease is s enhanced by any variable that Hb S in the deoxy st Hb A te(thats nonpressurized plane increased pco. decreased H.dehvdra tion,and an increased concentration of 2.3-BPG in erythrocytes. 4.Treatment:The rapy involves ade quate hydration,analgesics apy pres ohoresi Intermittent transtusions with packed red cells reduce the risk of stroke,but the benefits must be weighed against the complications of transtusion,whi (he of eutically useful b ating levels of Hb F.which decreases RBC sickling.This leads to decreased frequency of painful crises and reduces mortality
erythrocyte in sickle cell anemia is less than 20 days, compared with 120 days for normal RBCs; hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Heterozygotes, representing 1 in 12 African-Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both Hb S and Hb A. These individuals have sickle cell trait. They usually do not show clinical symptoms and can have a normal life span. 1. Amino acid substitution in Hb S β chains: A molecule of Hb S contains two normal α-globin chains and two mutant β-globin chains (βS), in which glutamate at position six has been replaced with valine (Figure 3.18). Therefore, during electrophoresis at alkaline pH, Hb S migrates more slowly toward the anode (positive electrode) than does Hb A (Figure 3.19). This altered mobility of Hb S is a result of the absence of the negatively charged glutamate residues in the two β chains, thus rendering Hb S less negative than Hb A. [Note: Electrophoresis of hemoglobin obtained from lysed RBCs is routinely used in the diagnosis of sickle cell trait and sickle cell disease.] 2. Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the β-globin that fits into a complementary site on the β chain of another hemoglobin molecule in the cell (Figure 3.20). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of fibrous polymers that stiffen and distort the cell, producing rigid, misshapen erythrocytes. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated Hb S. [Note: The mean diameter of RBCs is 7.5 μm, whereas that of the microvasculature is 3–4 μm. Instead of squeezing through the microvasculature like Hb A–containing RBCs, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls, and so have difficulty moving through small vessels.] 3. Variables that increase sickling: The extent of sickling and, therefore, the severity of disease is enhanced by any variable that increases the proportion of Hb S in the deoxy state (that is, reduces the affinity of Hb S for oxygen). These variables include decreased oxygen tension as a result of high altitudes or flying in a nonpressurized plane, increased pCO2, decreased pH, dehydration, and an increased concentration of 2,3-BPG in erythrocytes. 4. Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and trans - fusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed red cells reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload (hemosiderosis), bloodborne infections, and immunologic complications. Hydroxyurea, an antitumor drug, is therapeutically useful because it increases circulating levels of Hb F, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality. 36 3. Globular Proteins Figure 3.18 Amino acid substitutions in Hb S and Hb C. Val . His . Leu . Thr . Pro . Glu . Glu . Lys 1 2 3 4 5 6 7 8 C C H O COOHb A Hb S Val . His . Leu . Thr . Pro . Val . Glu . Lys CH2 CH2 1 2 3 4 5 6 7 8 Val . His . Leu . Thr . Pro . Lys . Glu . Lys C C O H Hb C CH2 CH2 CH2 CH2 NH3 + 1 2 3 4 5 6 7 8 C H N C O CH H3C CH3 H H N H N Anode Cathode Figure 3.19 Diagram of hemoglobins A, S, and C after electrophoresis. Power source at the start of electro- Hemoglobins are negatively charged and migrate toward the anode. Hb S Hb C Hb A 168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 36
IV.Hemoglobinopathies Fibers nto long.rope-lik Val His Lou-Thr Pro Glu Glu-Lys /al-His-Leu-Thr-Pro-Val-Glu-Lys B Chain B-6-Valine Fibe 5.Possible sel tforeteor damaging effec ts in the homozy sts that a sele heterozygotes for the susceptibl paras in the RE is One theory is that because these cells in individuals heterozy geascgrh8heheenho2opeba8anacraesg9 mal,the death.Figure 3.21 illustrates that in Africa,the geographic distri- anemla s sim lar to tha The mor in screening panels to allow pro antibiotic therapy to begin soon after the birth of an affected child. . Hemoglobin C disease Like Hb s.Hb c is a hemoglobin variant that has a single amino acid substitution in the sixth position of the B-globin chain(see Figure 3.18).In this case, ever,a lysine is substitute r the Hb Ct anode than Hb A or Hb S does(see Figure 3.19).]Patients homo hemoly nd anemia C.Hemoglobin SC disease Figure 3.20 memoglobin SC disease is anotner of he red cell sickling diseases. n this dise p-globin chains ve the leaingtosck6elnersents
Figure 3.20 Molecular and cellular events leading to sickle cell crisis. 3 Intracellular fibers of Hb S distort the erythrocyte. 1 Hydrophobic pocket Fiber Fibers β Chain β-6-Valine A point mutation in the DNA codes for structurally altered Hb S. 2 In the deoxygenated state, Hb S polymerizes into long, rope-like fibers. ...GTG... ...GAG... Val.His.Leu.Thr.Pro.Glu.Glu.Lys Val.His.Leu.Thr.Pro.Val.Glu.Lys Microinfarcts produce tissue anoxia, resulting in severe pain. 5 Rigid erythrocytes occlude blood flow in the capillaries. 4 α1 α2 β1 β2 α1 α2 β1 β2 α1 α2 β1 β2 α1 α2 β1 β2 IV. Hemoglobinopathies 37 5. Possible selective advantage of the heterozygous state: The high frequency of the βS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to malaria, caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for Hb S, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This fact may provide a selective advantage to hetero zygotes living in regions where malaria is a major cause of death. Figure 3.21 illustrates that in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria.The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child. B. Hemoglobin C disease Like Hb S, Hb C is a hemoglobin variant that has a single amino acid substitution in the sixth position of the β-globin chain (see Figure 3.18). In this case, however, a lysine is substituted for the glutamate (as compared with a valine substitution in Hb S). [Note: This substitution causes Hb C to move more slowly toward the anode than Hb A or Hb S does (see Figure 3.19).] Patients homo - zygous for hemoglobin C generally have a relatively mild, chronic hemolytic anemia. These patients do not suffer from infarctive crises, and no specific therapy is required. C. Hemoglobin SC disease Hemoglobin SC disease is another of the red cell sickling diseases. In this disease, some β-globin chains have the sickle cell mutation, whereas other β-globin chains carry the mutation found in Hb C disease. [Note: Patients with Hb SC disease are doubly hetero - 168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 37
38 3.Globular Proteins zygous (compound heterozygote)because both of their B-globin A evets are aonormal angugn than in s 4t0+5⊙P001 anemia in that symptoms such as painful crises are less frequent and less severe;however,there is significant clinical variability. 10-20 D.Methemoglobinemias 5-10% 11-5% Oxidation of the heme component of hemoglobin to the ferric (Fe3) state forms methemoglobin.which cannot bind oxygen.This oxida tion may be caused by the action of certain drugs, such as nitrates example,certain mutations in the o-or B-globin chain promote the formation of methemoglobin (Hb M).Furthe JH-Cylc ome bs reductase（ moglobin (Fto hemoglobin (F).leads to the accumulation of methemoglobin.[Note:The erythrocytes of newborns have approxi mately half the capacity of t se of adults o reduce m I The mether mias are characterized by"chocolate cyanosis"(a brownish-blue coloration of Figure 3.21 chocolate-colored blood,as a Distribution of sickle cell in tage the ue h and a6e B. dyspnea.In rare cases,coma and death can occur.Treatment is with n Africa methylene blue,which is oxidized as Fe3 is reduced E.Thalassemias are the most common single gene disorders in humans.Normally,syn- the a-and B- obin chains is.so that ac (Hb A).In the thala Synthesis of either the B 书 5 ions or 月■ B many nu Normal chains are produced (or B-thalassemia).or one in which some chains are synthesized,but at a reduced level (a-or B'-thalassemia).1 1.B-Thalassemias:In these disorders. of B-globin cl HbA Hb A that affect typically as NA:h point mutati chain synthesis is normal.g-Globin chains cannot form stable TY 8 Hb F tetramers and,therefore,precipitate,causing the premature death precipatat initi ture T ncrease in the B-g utations in the Therefore,individuals with B-globin gene defects have either etramers omed in B-thalassemias. -thala e only one e n gene,or assemla majo ley anemia)
38 3. Globular Proteins Figure 3.21 A. Distribution of sickle cell in Africa expressed as a percentage of the population with disease. B. Distribution of malaria in Africa. 10–20% 5–10% 1–5% A B zygous (compound heterozygote) because both of their β-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in Hb SC disease than in sickle cell anemia, and may even be at the low end of the normal range. The clinical course of adults with Hb SC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe; however, there is significant clinical variability. D. Methemoglobinemias Oxidation of the heme component of hemoglobin to the ferric (Fe3+) state forms methemoglobin, which cannot bind oxygen. This oxidation may be caused by the action of certain drugs, such as nitrates, or endogenous products, such as reactive oxygen intermediates (see p. 148). The oxidation may also result from inherited defects, for example, certain mutations in the α- or β-globin chain promote the formation of methemoglobin (Hb M). Furthermore, a deficiency of NADH-cytochrome b5 reductase (also called NADH-met hemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe3+) to hemoglobin (Fe2+), leads to the accumulation of met hemoglobin. [Note: The erythrocytes of newborns have approximately half the capacity of those of adults to reduce methemoglobin. They are therefore particularly susceptible to the effects of met - hemoglobin-producing compounds.] The methemoglobin emias are characterized by “chocolate cyanosis” (a brownish-blue coloration of the skin and mucous membranes) and chocolate-colored blood, as a result of the dark-colored methemoglobin. Symptoms are related to the degree of tissue hypoxia, and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fe+3 is reduced. E. Thalassemias The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. As a group, they are the most common single gene disorders in humans. Normally, synthesis of the α- and β-globin chains is coordinated, so that each α-globin chain has a β-globin chain partner. This leads to the formation of α2β2 (Hb A). In the tha l assemias, the synthesis of either the α- or the β-globin chain is defective. A thalassemia can be caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one to many nucleo tides in the DNA. [Note: Each thalassemia can be classified as either a disorder in which no globin chains are produced (αo - or βo -thal assemia), or one in which some chains are synthesized, but at a reduced level (α+ - or β+ -thalassemia).] 1. β-Thalassemias: In these disorders, synthesis of β-globin chains is decreased or absent, typically as a result of point mutations that affect the production of functional mRNA; however, α-globin chain synthesis is normal. α-Globin chains cannot form stable tetramers and, therefore, precipitate, causing the premature death of cells initially destined to become mature RBCs. Increase in α2γ2 (Hb F) and α2δ2 (Hb A2) also occurs. There are only two copies of the β-globin gene in each cell (one on each chromosome 11). Therefore, individuals with β-globin gene defects have either β-thal assemia trait (β-thalassemia minor) if they have only one defective β-globin gene, or β-thalassemia major (Cooley anemia) Figure 3.22 A. β-Globin gene mutations in the β-thalassemias. B. Hemoglobin tetramers formed in β-thalassemias. Normal β β β β β β β-Thalassemia minor β-Thalassemia major β β β Each copy of chromosome 11 has only one gene for β-globin chains. α-Chain precipatate Hb A Hb F A B α β β β β β β β γ γ γ γ δ δ δ δ δ α α α α α α Hb A2 α α α α α α αα ααα αα α α 168397_P025-042.qxd7.0:03 Hemoglobin 5-20-04 2010.4.4 1:04 PM Page 38