300 HEMOGLOBIN DISORDERS SECTION V of these patients. 27 A former childhood disorder is increasingly a disease of adults. Widespread screening of individuals from populations where β-thalassemia trait is prevalent has enabled prenatal diagnosis of affected fetuses. In Greek American and Italian American communities, the number of affected infants has sharply decreased. The number of children with thalassemia in the United States is rising however as a result of the increase in the number of Americans of Southeast Asian ancestry. These more recent arrivals to the country have not yet achieved the cohesive approach to thalassemia that proved so successful in other American communities. Until this goal is reached, thalassemia will remain an important issue in American medicine (Tables 14-3 and 14-4). References 1 Cooley TB, Witwer ER, Lee P. 1927. Anemia in children with splenomegaly and peculiar changes in bones: Report of cases. Am J Dis Child 34:347–355. 2 Olivieri NF. 1999. The beta-thalassemias. N Engl J Med 341:99–109. 3 Wasi P, Winichagoon P, Baramee T, Fucharoean S. 1982. Globin chain synthesis in het- erozygous and homozygous hemoglobin E. Hemoglobin 6:75–78. 4 Fairbanks VF, Gilchrist GS, Brimhall B, Jereb JA, Goldston EC. 1979. Hemoglobin E trait revisited: A cause of microcytosis and erythrocytosis. Blood 53:109–115. 5 Fairbanks VF, Oliveros R, Brandabur JH, Willis RR, Fiester RF. 1980. Homozygous hemoglobin E mimics b-thalassemia minor without anemia or hemolysis: Hematologic, functional and biosynthetic studies of first North American cases. Am J Hematol 8:109– 121. 6 Rees DC, Styles L, Vichinsky EP, Clegg JB, Weatherall DJ. 1998. The hemoglobin E syndromes. Ann N Y Acad Sci 850:334–343. 7 Fucharoen S, Ketvichit P, Pootrakul P, Siritanaratkul N, Piankijagum A, Wasi P. 2000. Clinical manifestation of beta thalassemia/hemoglobin E disease. J Pediatr Hematol Oncol 22:552–557. 8 Chui DH, Fucharoen S, Chan V. 2003. Hemoglobin H disease: Not necessarily a benign disorder. Blood 101:791–800. 9 Chen FE, Ooi C, Ha SY, et al. 2000. Genetic and clinical features of hemoglobin H disease in Chinese patients. N Engl J Med 343:544–550. 10 Scopinaro F, Banci M, Vania A, et al. 1993. Radioisotope assessment of heart damage in hypertransfused thalassaemic patients. Eur J Nucl Med 20:603–608. 11 Lattanzi F, Bellotti P, Picano E, et al. 1993. Quantitative ultrasonic analysis of myocardium in patients with thalassemia major and iron overload. Circulation 87:748–754. 12 Prennell DJ, Bland JM. 2003. Deferiprone versus desferrioxamine in thalassemia, and T2 ∗ validation and utility. Lancet 361:182–184. 13 Strickland GT, Elhefni H, Salman T, et al. 2002. Role of hepatitis C infection in chronic liver disease in Egypt. Am J Trop Med Hyg 67(4):436–442. 14 Darwish MA, Faris R, Darwish N, et al. 2001. Hepatitis C and cirrhotic liver disease in the Nile delta of Egypt: A community-based study. Am J Trop Med Hyg 64:147–153. 15 Seeff LB, Hoofnagle JH. 2003. Appendix: The National Institutes of Health Consensus Development Conference Management of Hepatitis C 2002. Clin Liver Dis 7:261–287. REFERENCES 301 16 Gupta S, Bent S, Kohlwes J. 2003. Test characteristics of alpha-fetoprotein for detecting hepatocellular carcinoma in patients with hepatitis C. A systematic review and critical analysis. Ann Intern Med 139:46–50. 17 McCord JM. 1993. Human disease, free radicals, and the oxidant/antioxidant balance. Clin Biochem 26:351–353. 18 Farber JL. 1994. Mechanisms of cell injury by activated oxygen species. Environ Health Perspect 102:17–24. 19 Enright HU, Miller WJ, Hebbel RP. 1992. Nucleosomal histone protein protects DNA from iron-mediated damage. Nucleic Acids Res 20:3341–3346. 20 Bonkovsky, HL. 1991. Iron and the liver. Am J Med 301:32–43. 21 Link G, Konijn AM, Hershko C. 1999. Cardioprotective effect of alpha-tocopherol, ascor- bate, deferoxamine, and deferiprone: Mitochondrial function in cultured, iron-loaded heart cells. J Lab Clin Med 133:179–188. 22 Aldouri MA, Wonke B, Hoffbrand AV, et al. 1990. High incidence of cardiomyopathy in beta-thalassaemia patients receiving regular transfusion and iron chelation: Reversal by intensified chelation. Acta Haematol 84:113–117. 23 Olivieri NF, Nathan DG, MacMillan JH, et al. 1994. Survival in medically treated patients with homozygous beta-thalassemia. N Engl J Med 331:574–578. 24 Davis B, J. Porter. 2000. Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in high-risk beta-thalassemia. Blood 95(4):1229–1236. 25 Lucarelli G, Galimberti M, Polchi P, et al. 1993. Marrow transplantation in patients with thalassemia responsive to iron chelation therapy. N Engl J Med 329:840–844. 26 Lucarelli G, Galimberti M, Giardini C, et al. 1998. Bone marrow transplantation in tha- lassemia. The experience of Pesaro. Ann N Y Acad Sci 850:270–275. 27 Pearson HA, Giardina P, Cohen A. 1996. The changing profile of thalassemia major. Pedi- atrics 97:352–356. This page intentionally left blank SECTION VI Enzymopathies Copyright © 2008 by The McGraw-Hill Companies, Inc. Click here for terms of use. This page intentionally left blank CHAPTER 15 G6PD DEFICIENCY CLASSIFICATION OF G6PD DEFICIENCY SYNDROMES 306 CLASS I G6PD DEFICIENCY 307 CLASS II G6PD DEFICIENCY 308 CLASS III G6PD DEFICIENCY 309 CLASS IV AND V G6PD VARIANTS 311 LABORATORY MANIFESTATIONS OF G6PD DEFICIENCY 311 G6PD AND RED CELL METABOLISM 313 THE MOLECULAR BIOLOGY OF G6PD DEFICIENCY 316 DIAGNOSIS OF G6PD DEFICIENCY 317 Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common ery- throcyte enzyme defect, affecting over 400 million people. The initial descriptions of the disorder arose in the wake of peculiar outbreaks of hemolytic anemia in military personnel in the Pacific theater during World War II following prophylactic treatment with the antimalarial drug Primaquine. 1 A number of unusual attributes characterized the hemolytic episodes and whetted interest in the relationship between Primaquine and hemolysis. Although the problem involved a significant number of people, only a minority of the group who used the drug were affected. The episodes of hemolysis were self-limiting, with a sometimes explosive early phase followed by spontaneous recovery. After the initial hemolytic episode, susceptible people could continue Pri- maquine treatment without further problem. A break of several months in Primaquine exposure saw a recrudescence in hemolytic sensitivity. The demographics of the problem also were unusual. The hemolytic episodes occurred almost exclusively in people of African heritage, pointing to an ethnic com- ponent of the susceptibility. The familial pattern of drug sensitivity reinforced belief 305 Copyright © 2008 by The McGraw-Hill Companies, Inc. Click here for terms of use. 306 ENZYMOPATHIES SECTION VI TABLE 15-1 CLINICAL CLASSIFICATION OF G6PD SYNDROMES Residual G6PD Class Activity (% normal) Clinical Characteristics I <20 Chronic nonspherocytic hemolytic anemia (CNSHA). Neonatal jaundice. II <10 Severe episodic hemolysis often related to drugs or other oxidant exposure. Fava bean mediated hemolysis. Neonatal jaundice. III 10–60 Episodic hemolysis often related to drug exposure. Hemolysis with infections. Neonatal jaundice in premature infants. IV 90–100 No clinical symptoms. V >100 No clinical symptoms. in the genetic nature of the disorder. Furthermore, men were the almost sole victims of Primaquine-induced hemolysis, indicating an X-linked pattern of inheritance. In retrospect the episodes of hemolysis were a result of a form of G6PD deficiency that originated in sub-Saharan Africa and spread to the New World as part of the African diaspora driven by the slave trade. Further investigation uncovered the existence of many other types of G6PD defi- ciencies with varying manifestations and severity. Over time, more than 400 descrip- tions of mutations in the gene encoding G6PD appeared in the literature. 2 While some of the mutations, such as those affecting African Americans, are common, most are extremely rare. Also, the clinical characteristics of the G6PD deficiency varied sig- nificantly. The WHO devised a classification that divided G6PD deficiency into five groups, depending on the clinical manifestations (Table 15-1). The strikingly different presentations, clinical characteristics, and manifestations seen within these subgroups reflect the degree to which the G6PD enzyme levels fall below the normal range. CLASSIFICATION OF G6PD DEFICIENCY SYNDROMES Table 15-1 outlines the five clinical classes of G6PD variant syndromes as categorized by the WHO. The Class I G6PD deficiency phenotype derives from a marked erythro- cyte enzyme deficiency. The extreme sensitivity of these cells to oxidant stress pro- duces ongoing hemolysis with a moderately severe chronic nonspherocytic hemolytic anemia (CNSHA), with an associated marked reticulocytosis as the most common CHAPTER 15 G6PD DEFICIENCY 307 manifestation. Class II syndromes exhibit extreme enzyme deficiency in which G6PD values average less than 10% of the normal value. Affected patients are healthy at baseline but are susceptible to fulminant and sometimes life-threatening episodes of hemolysis when exposed to certain oxidizing agents. The Class III syndromes have mild to moderate enzyme deficit. This group was the primary subject of the Primaquine studies. The Class IV G6PD deficiency syndromes show at most only mildly depressed enzyme levels and no clinical symptoms. Class V, a phenotype also without clinical manifestations, rounds out the categorization of the condition with G6PD enzymes whose activity exceeds the normal value. The G6PD deficiency syndromes show patterns of geographic and ethnic clus- tering. The Class III variety is the most prevalent form in people of African ethnic heritage, as noted earlier. Class II G6PD deficiency occurs commonly, but not exclu- sively, in people of Mediterranean background. 3 Both Class II and Class III G6PD deficiency variants are common in China and Southeast Asia. 4 Knowledge of a pa- tient’s ethnic background greatly aids in the evaluation and treatment of hemolytic episodes where G6PD deficiency is the possible culprit. CLASS I G6PD DEFICIENCY Patients who fall under this rubric typically have chronic hemolytic anemia that often is moderate in severity, thanks to a robust compensatory reticulocyte response. Class I G6PD deficiency manifests most commonly as congenital nonspherocytic hemolytic anemia. The very low G6PD levels in the erythrocytes render these cells incapable of withstanding the normal levels of oxidant stress visited upon all erythrocytes. The compensating reticulocytosis commonly ranges between 10% and 40%. Interestingly, splenomegaly is not a major manifestation of the condition. Occasional patients with congenital nonspherocytic hemolytic anemia due to G6PD deficiency develop recur- rent infections. This problem apparently reflects dampening of neutrophil-generated reactive oxygen species that are essential to the bacteriocidal capability of these phagocytes. 5,6 The marked elevation of bilirubin levels that sometimes occurs in the neonatal period raises the risk of kernicterus. Although hemolysis contributes to the high bilirubin levels, an additional factor is a neonatal deficiency of the hepatic glucuronyl transferase enzyme with consequent impairment of bilirubin metabolism. Neonatal hyperbilirubinemia often arises in association with Gilbert’s syndrome. 7–9 Supportive treatment including phototherapy or even exchange transfusions may be necessary to control hyperbilirubinemia. Most patients with CNSHA hemolytic anemia due to G6PD deficiency maintain hemoglobin levels in the range of 8–10 g/dL and compensate for anemia reasonably well. Epistatic factors may influence the degree of the anemia associated with the condition, since siblings sometimes have very disparate courses. Rarely, an anemia arises whose severity necessitates chronic transfusion to maintain a reasonable level of activity and comfort. The lack of splenic enlargement associated with congenital nonspherocytic hemolytic anemia due to Class I G6PD deficiency parallels the failure of splenectomy to benefit most such patients. 308 ENZYMOPATHIES SECTION VI TABLE 15-2 REPRESENTATIVE AGENTS CAUSING HEMOLYSIS IN G6PD DEFICIENCY ∗ Class Agents Antibiotics Sulphonamides, Co-trimoxazole (Bactrim, Septrin), Dapsone, Chloramphenicol, Nitrofurantoin, Nalidixic acid Antimalarials Chloroquine, Hydroxychloroquine, Primaquine, Quinine, Mepacrine Other drugs Aspirin, Phenacitin, Sulphasalazine, Methyldopa, pharmacological doses of vitamin C, Hydralazine, Procainamide, Phenothiazine, vitamin K, penicillamine Anthelmintics β-Naphthol, Stibophen, Niridazole Chemicals Mothballs (naphthalene), Methylene blue Foods Raw fava beans (broad beans) Infections Bacterial infection; viral infection, hepatitis ∗ The drugs and agents listed are representative examples only. Susceptibility to hemolytic episodes varies depending on the subtype of G6PD deficiency. Definitive information comes from the WHO Glucose-6- phosphate Working Group, Bull WHO 1989, 67:610. The maintenance of a hemoglobin value that is both stable and substantive depends on the brisk and ongoing production of new red cells. Any phenomenon or process that heightens hemolysis can precipitate life-threatening anemia. Even small quantities of oxidant drugs such as those listed in Table 15-2 can trigger massive hemolysis and a dramatic fall in hematocrit. Equally dangerous are events that dampen production of new erythrocytes. Parvovirus B19 infection, for instance, shuts off erythropoiesis for several days, creating an anemia of life-threatening severity. CLASS II G6PD DEFICIENCY Favism is a striking phenomenon that occurs in many people with Class II G6PD de- ficiency where spectacular and occasionally life-threatening hemolysis follows con- sumption of raw, uncooked fava beans (Vicia fava). These innocent legumes of the broad bean family are common throughout the world and are nutritional staples for millions of people. The form of G6PD deficiency common in the Mediterranean ren- ders red cells particularly susceptible to hemolysis after consumption of raw fava beans. The syndrome has been known for centuries without, of course, an under- standing of its basis. In some Mediterranean countries, hospitals geared up each year during planting or harvesting of the beans for the expected influxes of people stricken CHAPTER 15 G6PD DEFICIENCY 309 with favism. In some regions, such as Sardinia, the economic and social burden of favism was enormous. The particulars of the syndrome are puzzling and suggest that factors other than exposure to the bean contribute to or modify the degree of hemolysis. Perhaps the most curious aspect of favism is that not every exposure to the beans produces hemolysis in people with the Mediterranean variety of G6PD deficiency. Some people consume fava beans for years without difficulty and then are felled by a hemolytic episode. Others are so sensitive to the active agent in fava beans that hemolysis can develop after mere exposure to pollen from the plant. Interestingly, hemolysis is more common in children than in adults, with as many as three-quarters of episodes occurring in children between the ages of 2 and 10 years. As many as 48 unremarkable hours commonly pass following ingestion of the beans. The child then develops lethargy, sometimes in conjunction with confusion and a mild fever. Nausea, abdominal pain, and diarrhea are other nonspecific manifesta- tions. A more telling feature (in retrospect) is back pain, which commonly accompa- nies acute hemolytic episodes of any cause. The alarming and illuminating aspect of the illness that usually brings the child to medical attention is the passage of dark red or brown urine. Physical examination at this point reveals frank jaundice, pallor, and tachycardia. Moderately severe anemia quickly follows, often with the hemoglobin falling to range of 4–7 g/dL. Although extremely severe cases that threaten circulatory collapse sometimes demand transfusion therapy, 10 most children recover without this intervention. The hemoglobin level usually returns to normal in 4–6 weeks. The precise basis of hemolysis induced by fava beans in people with the Mediter- ranean variety of G6PD deficiency is unclear. The likely mediators are vicine and convicine, constituents in the beans that are metabolized to compounds capable of generating reactive oxygen species. The extreme deficiency of G6PD leaves the red cells compromised and very susceptible to oxidant damage and consequent hemol- ysis. The delay in the onset of manifestations following consumption of fava beans probably represents the time required to convert the latent compounds to their active metabolites. Favism is only one clinical manifestation of Class II G6PD deficiency. People with the condition are also susceptible to oxidizing compounds and drugs such as those listed in Table 15-2. Neonatal jaundice with possible kernicterus is another issue in Class II G6PD deficiency. The problem of hyperbilirubinemia in the newborn reflects both decreased hepatic Conjugation of bilirubin as well as hemolysis. An interesting neonatal syndrome sometimes designated “Greek Baby Jaundice” is common in newborns from Greece and the islands of the Aegean Sea, characterized by extreme hyperbilirubin and a risk of kernicterus. Affected babies can be shown to have Type II G6PD deficiency; however, unknown environmental factors must also be operative, because the syndrome is not seen with any frequency in children of Greek families who have emigrated to North America or Australia. CLASS III G6PD DEFICIENCY Class III G6PD deficiency is associated with only a modest depression of the ery- throcyte enzyme level (Table 15-1). The most commonly encountered variety of [...]... (Tables 1 5-4 and 1 5-5 ) References 1 2 Beutler E 199 4 G6PD deficiency Blood 84:3613–3636 Beutler E 199 2 The molecular biology of G6PD variants and other red cell enzyme defects Annu Rev Med 43:47– 59 320 ENZYMOPATHIES 3 4 5 6 7 8 9 10 11 12 13 14 SECTION VI Cappellini MD, Martinez di Montemuros F, De Bellis G, Debernardi S, Dotti C, Fiorelli G 199 6 Multiple G6PD mutations are associated with a clinical and. .. Reprod Med 43:713–715 Esen UI, Olajide F 199 8 Pyruvate kinase deficiency: An unusual cause of puerperal jaundice Int J Clin Pract 52:3 49 350 Boivin P, Galand C, Hakim J, Kahn A 197 5 Acquired red cell pyruvate kinase deficiency in leukemias and related disorders Enzyme 19: 294 – 299 Kornberg A, Goldfarb A 198 6 Preleukemia manifested by hemolytic anemia with pyruvate-kinase deficiency Arch Intern Med 146:785–786... and HS red cells The upper row of the diagram highlights the cytoskeleton in the cells, showing the reduction associated with HS The middle row shows that both normal and HS red cells initially exist as biconcave discs The relatively acidic and hypoglycemic splenic environment stresses both cells The cytoskeleton deficit of the HS cells results in membrane loss and the formation of spherocytic red cells,... the red cell membrane cytoskeleton A complex latticework of proteins whose dominant members are α and β spectrin exists immediately below the red cell lipid bilayer Ankyrin links the cytoskeleton to the membrane by forming a bridge between β spectrin and the intrinsic membrane protein, band 3 Actin and protein 4.2 are also prominent members of the red cell cytoskeleton (From Lux SE, Palek J 199 5 Disorders. .. 110 :99 3 99 7 Kugler W, Laspe P, Stahl M, Schroter W, Lakomek M 199 9 Identification of a novel promoter mutation in the human pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia Br J Haematol 105: 596 – 598 Kanno H, Fujii H, Wei DC, et al 199 7 Frame shift mutation, exon skipping, and a twocodon deletion caused by splice site mutations account for pyruvate kinase deficiency Blood 89: 4213–4218... downward spiral due to oxidant injury dooms the G6PD-deficient erythrocytes Heinz bodies also adhere to the red cell membrane where they disrupt architecture, promote oxidation and cross-linking of lipids and proteins, and impair function of membrane-associated enzymes including ion channels Cross-links of membrane structures produce rigid red cells with impaired ability to pass through capillaries CHAPTER... dysfunction, and increased susceptibility to infections Blood 100:1026–1030 Iancovici-Kidon M, Sthoeger D, Abrahamov A, et al 2000 A new exon 9 glucose-6phosphate dehydrogenase mutation (G6PD “Rehovot”) in a Jewish Ethiopian family with variable phenotypes Blood Cells Mol Dis 26:567–571 Kaplan M, Renbaum P, Levy-Lahad E, Hammerman C, Lahad A, Beutler E 199 7 Gilbert syndrome and glucose-6-phosphate dehydrogenase... leading to potassium loss and cell shrinkage Pathological levels of calcium accumulate in the cells and activate the Gardos channel with consequent further loss of cell potassium The shrunken, crenated cells become rigid and are quickly cleared from the circulation by reticuloendothelial cells CHAPTER 16 325 PYRUVATE KINASE DEFICIENCY Embden-Meyerhof Glycolytic Pathway Phosphoenol-pyruvate 2 ADP Pyruvate... deficiency, but this intervention remains unconventional22 (Tables 1 6-1 and 1 6-2 ) References 1 2 3 4 5 6 7 Dacie JV, Mollison PL, Richardson N, Selwyn JG, Shapiro L 195 3 Atypical congenital hemolytic anemia Q J Med 22: 79 91 Tanaka KR, Valentine WN, Miwa S 196 2 Pyruvate kinase (PK) deficiency hereditary non-spherocytic hemolytic anemia Blood 19: 267–277 Beutler E, Gelbart T 2000 Estimating the prevalence of... hemolysis creating a potentially life-threatening situation 316 ENZYMOPATHIES Red Cell G6PD Level 100 % Normal (G6PD B) SECTION VI Red Cell Life Span G6PD A− "Protecctive" G6PD Level G6PD Mediterranean 0% Time (days) 140 FIGURE 15–4 Schematic representation of the time-dependent decay of red cell G6PD Normal G6PD (G6PD B) decays naturally over the life of the red cell but maintains a level sufficient . approach (Tables 1 5-4 and 1 5-5 ). References 1 Beutler E. 199 4. G6PD deficiency. Blood 84:3613–3636. 2 Beutler E. 199 2. The molecular biology of G6PD variants and other red cell enzyme defects. Annu. Hershko C. 199 9. Cardioprotective effect of alpha-tocopherol, ascor- bate, deferoxamine, and deferiprone: Mitochondrial function in cultured, iron-loaded heart cells. J Lab Clin Med 133:1 79 188. 22 Aldouri. Several bite cells appear on the smear along with other nonspecific shape anomalies. Small bulges exist along the edge of some of the cells. (From Kapff CT and Jandl JH. 199 1. Blood: Atlas and Sourcebook