- Email: [email protected]
A New Exon 9 Glucose-6-phosphate Dehydrogenase Mutation (G6PD “Rehovot”) in a Jewish Ethiopian Family with Variable Phenotypes
Iancovici-Kidon et al.
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
A New Exon 9 Glucose-6-phosphate Dehydrogenase Mutation (G6PD “Rehovot”) in a Jewish Ethiopian Family with Variable Phenotypes Submitted 10/09/00 (Communicated by E. Beutler, M.D., 10/09/00)
Mona Iancovici-Kidon,1 Daliah Sthoeger,1 Ayala Abrahamov,2 Baruch Volach,3 Ernest Beutler,4 Terri Gelbart,4 and Yigal Barak1 ABSTRACT: Hereditary nonspherocytic hemolytic anemia (HNSHA) is a rare manifestation of glucose-6phosphate dehydrogenase (G6PD) gene mutations, caused mainly by mutations located in exon 10 of the G6PD gene and less commonly by mutations in other parts of the gene. A new, exon 9, single-base mutation representing a T 3 C transition at cDNA nucleotide 964 was found in three brothers and their carrier mother of Jewish Ethiopian descent. Biochemical characterization of the resultant protein was not performed. Though clinical manifestations included HNSHA in all cases, the severity of hemolysis and the transfusion requirement differed markedly. Severe congenital neutropenia (Kostmann’s syndrome)—a disorder never reported before in conjunction with G6PD deficiency—was observed in one case. Levels of white blood cell G6PD activity of the three patients were 0 –5% of normal controls. Neutrophil oxidative and bactericidal activities were inherently impaired in the patient with Kostmann’s syndrome, but were well preserved in his two siblings. © 2000 Academic Press
INTRODUCTION
hemolytic anemia with varying severity, associated in one case with severe congenital neutropenia.
Hereditary nonspherocytic hemolytic anemia (HNSHA) is a relatively rare clinical manifestation of glucose-6-phosphate dehydrogenase (G6PD) gene mutations. Most known mutations associated with this type (class 1) disease are located in the exon 10 area of the G6PD gene, probably related to the substrate receptor, NADPH binding site, or in the region around the glucose-6-phosphate binding site at lysine 205 (1, 2). In contrast to the more benign, prevalent forms associated with favism, these panethnic, rare mutations may cause significant morbidity due to chronic hemolytic anemia. As a rule, no other hematological cell lines are involved (2). We describe a new single-base substitution in exon 9 of the G6PD gene in a Jewish Ethiopian family, expressed as hereditary nonspherocytic
MATERIALS AND METHODS Patients A.D. is a 7-year-old, firstborn son of healthy unrelated parents of Jewish Ethiopian descent. After a normal pregnancy, uncomplicated delivery and perinatal course, G6PD deficiency was found on a routine qualitative neonatal screening test. His medical history is complicated by a focal convulsive disorder diagnosed at the age of 2 years, treated with carbamazepine, and by attention deficit hyperactivity syndrome. He has a moderate chronic hemolytic anemia, with Hb ranging 8.6 –9.2 g/dl, a reticulocyte count ranging
Correspondence and reprint requests to: Yigal Barak, Department of Pediatrics, Kaplan Medical Center, Rehovot 76100, Israel. Fax: 972-8-9411942. E-mail: [email protected]. 1 Department of Pediatrics, Kaplan Medical Center, Rehovot, Israel. 2 Shaare Tzedek Medical Center, Jerusalem, Isreal. 3 Meir Hospital, Kfar Saba, Israel. 4 Scripps Research Institute, La Jolla, CA 92037. 1079-9796/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved
567
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
10 –14% and a normal leukocyte cell count and function. To date, he has never required transfusion or hospitalization. A.A., the second son, born after a normal pregnancy and delivery, presented at 4 days of age with gram-negative septicemia and acute hemolytic anemia, later identified as due to G6PD deficiency. In his first year of life the patient developed recurrent invasive, presumably bacterial infections accompanied by fever and severe granulocytopenia, and progressive hemolytic anemia requiring frequent packed red blood cell transfusions. The bone marrow examination revealed a paucity of cells in the granulocyte lineage together with a maturation arrest at the promyelocyte–myelocyte stage. Erythropoiesis was found to be moderately increased and other cell lines were normal. The peripheral blood and the bone marrow findings were interpreted as compatible with the diagnosis of severe congenital neutropenia (Kostmann’s syndrome). Treatment with rhG-CSF (Neupogen, Amgen) resulted in increased absolute neutrophil counts (ANC) only at markedly increased G-CSF doses and only for those periods of time when treatment was received with regularity. Nevertheless, rhG-CSF treatment reduced significantly the number and severity of infections and hospitalizations, but no changes in hemolysis or in transfusion requirements were observed. A treatment trial of antioxidant therapy (vitamin E), also failed to effect any improvement in the transfusion requirement. At the age of 3 years the patient died at home due to an overwhelming bacterial infection, with no granulocytes at the sites of infection, on postmortem examination. A.E., the youngest child of this Jewish Ethiopian family, is a 1.5-year-old male, who was severely jaundiced at birth and received two exchange transfusions on the first day of life. Following this, his hemoglobin rose from 12.3 to 17 g/dl, reticulocyte count changed from 28.8 to 20%, bilirubin dropped from 22.8 to 10 mg/dl and LDH decreased from 14,400 to 2356 /liter. Leukocyte, absolute neutrophil and platelet counts were consistently normal. He was later diagnosed as being G6PD deficient. In his first 18 months of life, while on continuous folic acid and vitamin E
Iancovici-Kidon et al.
therapy, A.E. has had a steady-state hemoglobin level in the 10 to 11 g/dl range and reticulocyte counts of 6.7–19%. He thrives and develops normally, and except for one occasion, during an acute viral infection, he has never required further blood transfusions. Mutation Analysis DNA was isolated from peripheral blood leukocytes by standard methods. All exons of the G6PD gene in the patient were amplified by the polymerase chain reaction (PCR) using previously described oligonucleotide primers (3). The PCR consisted of 1 g genomic DNA, 33.5 mM Tris–HCl, pH 8.8, 8.3 mM (NH4)2SO4, 3.35 mM MgCl2, 85 g/ml bovine serum albumin, 0.2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 5% dimethyl sulfoxide, and 1.5 U Taq polymerase. PCR was performed for 30 cycles consisting of 93°C for 30 s, 58°C for 30 s, and 72°C for 30 s followed by a 7-min 72°C extension. PCR products were then purified with a QIAquick PCR purification kit (Qiagen Inc. Chatsworth, CA). The purified PCR products were sequenced on an Applied Biosystems Inc. (Foster City, CA) automatic sequencer. The DNA of subsequent family members was amplified and sequenced only in the region of interest (exon 9). Neutrophil Function Assays Polymorphonuclear leukocytes (PMNs) were isolated from heparinized blood by dextran sedimentation, layering, and erythrocyte lysis, as described by Boyum (4, 5). Superoxide production by PMNs was measured by the reduction of superoxide dismutase (SOD)-inhibitable ferricytochrome c, by the method reported by Weisbart et al. (6). Stimulants with differing modes of action were used to define the sites of alteration in signal transduction of superoxide generation. Bactericidal activity of PMNs was expressed as the decrease in the number of viable bacteria, after incubation with PMNs in the presence of autologous and homologous serum, as described by Clawson and Repine (7). The log decrease in 568
Iancovici-Kidon et al.
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
bactericidal activity with a fluctuating level of white blood cells G6PD activity, 0 –5% of normal controls.
TABLE 1 Neutrophil Functions Stimulation
A.A.
A.D.
A.E.
WBC–G6PD
0
0–5
0–5
RBC–G6PD (% of control)
0
0
0
fMLP (1.3–8.5)
1.0b
5.3
4.01
PMA (2.5–8.5) Autol serum (0.6–1.9)
0.94b
6.77
6.37
0.13b
0.75
0.6
Homol serum (0.6–1.6)
0.37b
0.73
0.69
Superoxide generation 6 (nmol O⫺ 2 /10 PMNs/min)
Bactericidal activity (log decrease of CFUa)
DISCUSSION All three patients exhibit hereditary nonspherocytic hemolytic anemia associated with an exon 9 mutation of the G6PD gene. Most mutations causing HNSHA are in exon 10 (9), a region that seems to be important in the binding of structural NADP. Among the 14 mutations that have previously been documented in exon 9, only 5 (G6PD West Virginia, Omiya, Manhattan, Nara, and Torun) are associated with HNSHA, and Nara is not a point mutation but rather a large deletion (10). One of these mutations, G6PD Manhattan, changes the glycine immediately upstream of the tyrosine that was mutated in our patients to glutamic acid. Even though all of our patients had the same mutation, the clinical presentation and expression of the disease differs remarkably in all three. The association with severe congenital neutropenia in the case of A.A. is still an unresolved question. It may be a “simple” case of coexisting morbidity with Kostmann’s syndrome (11, 12) aggravating the hemolytic anemia due to recurrent bacterial infections, adding an oxidative load on an already stressed system. The diagnosis of Kostmann’s syndrome is strongly supported in this case by the peripheral blood and the bone marrow appearance of the granulocytic series, with maturation arrest at the promyelocyte level (11). It is also supported by the definite dependency on continuous rhG-CSF treatment (13–15). No previous report of type I G6PD deficiency (hereditary nonspherocytic hemolytic anemia) in association with Kostmann’s syndrome was found in an extensive literature search. Another interesting association is the primary leukocyte defect induced by severe G6PD deficiency within leukocytes as well as erythrocytes. The neutrophil oxidative and bactericidal activity dysfunction, that remained abnormal after incubation with homologous sera, are indicative of the presence of an intrinsic cell defect, not usually associated with Kostmann’s syndrome (16). Various abnormali-
Note. In parentheses: normal range in our laboratory. a Colony-forming units. b Abnormal.
bactericidal activity was calculated and compared with that of controls. Measurement of G6PD activity was performed on PMNs obtained from the leukocyte-enriched plasma layer, after 20 s of hypotonic lysis, to eliminate contaminating red cells. The enzyme was assayed according to a previous WHO report (8). RESULTS Mutation analysis of blood DNA samples from all three brothers demonstrated a previously undescribed mutation representing a T [wng]224[/ wng] C transition at cDNA nucleotide 964. This is predicted to cause a tyrosine [wng]224[/wng] histidine substitution at amino acid 322. Biochemical characterization of the resultant protein was not performed. Analysis of maternal DNA revealed her to be heterozygous for the same exon 9 mutation. Results of the neutrophil functional analysis are summarized in Table 1. Analysis of neutrophil function in patient A.A. was performed during treatment with rhG-CSF. A.A.’s white blood cell G6PD enzyme activity levels were undetectable, and bactericidal activity was severely impaired and slightly improved but did not normalize after addition of homologous serum. The other two siblings had normal superoxide generation and 569
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
ties in neutrophil function have been associated with other forms of severe G6PD deficiency (17– 19). Not so with severe neutropenia, as seen in patient A.A. and probably not causatively associated with the “Rehovot” type mutation in the G6PD gene. The results from patients A.D. and A.E. of normal neutrophil functions with extremely low WBC G6PD enzyme activity and no increased susceptibility to infections also suggest that neutrophil dysfunction is not inherent in this G6PD gene mutation. Still it is interesting to note, that enzyme activity measured in patient A.A.’s PMNs was zero, compared to low fluctuating levels, 0 –5% of controls, measured in his sibs. It is well documented that levels equal or over 1% of white blood cells G6PD enzyme activity are sufficient for a good NADPH-oxidase response. The described functional abnormality in neutrophil function can be due at least partially to the rhGCSF treatment. Abnormal chemotaxis and oxidative burst have been described in Kostmann’s disease patients during rhG-CSF treatment (20, 21). The third and once more different presentation of severe neonatal hyperbilirubinemia and mild hemolytic anemia responsive to antioxidant therapy is consistent with previously reported cases of type I G6PD (2), but again diametrically different from the clinical presentation of the two older siblings. The existence of different G6PD DNA variants causing similar phenotypic disease is known and prevalent in the literature (1, 22–25). This report describes a reverse situation where the same mutation in the G6PD gene is associated with extremely divergent clinical phenotypes.
Iancovici-Kidon et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
REFERENCES 15.
1.
Beutler, E., Westwood, B., Melemed, A., Dal Borgo, P., and Margolis, D. (1995) Three new exon 10 glucose-6-phosphate dehydrogenase mutations. Blood Cells Mol. Dis. 21, 64 –72. 2. Beutler, E. (1994) G6PD deficiency. Blood 84, 3613– 3636. 3. Beutler, E., Kuhl, W., and Forman, L. (1991) DNA sequence abnormalities of human glucose-6-phosphate dehydrogenase variants. J. Biol. Chem. 266, 4145– 4150.
16.
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Boyum, A. (1968) Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and Ficoll as erythrocyte-aggregating agents. Scand. J. Clin. Lab. Invest. Suppl. 97, 31–50. Boyum, A. (1968) A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1g gravity field. Scand. J. Clin. Lab. Invest. Suppl. 97, 51–76. Weisbart, R. H., Golde, D. W., Clark, S. C., Wong, G. G., and Gasson, J. C. (1985) Human granulocyte– macrophage colony-stimulating factor is a neutrophil activator. Nature 314, 361–363. Clawson, C. C., and Repinem, J. E. (1976) Quantitation of maximal bactericidal capability in human neutrophils. J. Lab. Clin. Med. 88, 316 –327. Betke, K., et al. (1967) Standardization of procedures for the study of glucose-6-phosphate dehydrogenase. Report of a WHO Scientific Group, 366, WHO Technical Report Series. Beutler, E. (1991) Glucose-6-phosphate dehydrogenase (G6PD) deficiency. N. Engl. J. Med. 324, 169 – 174. Hirono, A., Kuhl, W., Gelbart, T., Forman, L., Fairbanks, V. F., and Beutler, E. (1989) Identification of the binding domain for NADP⫹ of human glucose-6phosphate dehydrogenase by sequence analysis of mutants. Proc. Natl. Acad. Sci. USA 86, 10015–10017. Parmley, R. T., Ogawa, M., Darby, C. P., Jr., and Spicer, S. S. (1975) Congenital neutropenia: Neutrophil proliferation with abnormal maturation. Blood 46, 723–734. Amato, D., Freedman, M. H., and Saunders, E. F. (1976) Granulopoiesis in severe congenital neutropenia. Blood 47, 531–538. Kobayashi, M., Yumiba, C., Kawaguchi, Y., et al. (1990) Abnormal responses of myeloid progenitor cells to recombinant human colony-stimulating factors in congenital neutropenia. Blood 75, 2143–2149. Hestdal, K., Welte, K., Lie, S. O., Keller, J. R., Ruscetti, F. W., and Abrahamsen, T. G. (1993) Severe congenital neutropenia: Abnormal growth and differentiation of myeloid progenitors to granulocyte colony-stimulating factor (G-CSF) but normal response to G-CSF plus stem cell factor. Blood 82, 2991–2997. Guba, S. C., Sartorm, C. A., Hutchinson, R., Boxer, L. A., and Emerson, S. G. (1994) Granulocyte colonystimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia. Blood 83, 1486 –1492. Roesler, J., Emmendorffer, A., Elsner, J., Zeidler, C., Lohmann-Matthes, M., and Welte, K. (1991) In vitro functions of neutrophils induced by treatment with rhG-CSF in severe congenital neutropenia. Eur. J. Haematol. 46, 112–118.
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Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
Lohmann-Matthes, M. L., and Welte, K. (1992) Altered function and surface marker expression of neutrophils induced by rhG-CSF treatment in severe congenital neutropenia. Eur. J. Haematol. 48, 10 –19. 22. Beutler, E. (1996) G6PD: Population genetics and clinical manifestations. Blood Rev. 10, 45–52. 23. Beutler, E., Vulliamy, T., and Luzzatto, L. (1996) Hematologically important mutations: Glucose-6phosphate dehydrogenase. Blood Cells Mol. Dis. 22, 49 –56. 24. Mason, P. J., Sonati, M. F., MacDonald, D., et al. (1995) New glucose-6-phosphate dehydrogenase mutations associated with chronic anemia. Blood 85, 1377–1380. 25. Cappellini, M. D., Martinez, d. M., De Bellis, G., Debernardi, S., Dotti, C., and Fiorelli, G. (1996) Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean. Blood 87, 3953–3958.
17.
Gray, G. R., Klebanoff, S. J., Stamatoyannopoulos, G., et al. (1973) Neutrophil dysfunction, chronic granulomatous disease, and non-spherocytic haemolytic anaemia caused by complete deficiency of glucose-6phosphate dehydrogenase. Lancet 2, 530 –534. 18. Ardati, K. O., Bajakian, K. M., and Tabbara, K. S. (1997) Effect of glucose-6-phosphate dehydrogenase deficiency on neutrophil function. Acta Haematol. 97, 211–215. 19. Yeung, C. Y., Wan, T. S., and Tam, A. Y. (1999) Neutrophil function in glucose-6-phosphate dehydrogenase deficient neonates. J. Paediatr. Child Health 35, 324 –325. 20. Weston, B., Todd, R. F., Axtell, R., et al. (1991) Severe congenital neutropenia: Clinical effects and neutrophil function during treatment with granulocyte colony-stimulating factor. J. Lab. Clin. Med. 117, 282–290. 21. Elsner, J., Roesler, J., Emmendorffer, A., Zeidler, C.,
571
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
A New Exon 9 Glucose-6-phosphate Dehydrogenase Mutation (G6PD “Rehovot”) in a Jewish Ethiopian Family with Variable Phenotypes Submitted 10/09/00 (Communicated by E. Beutler, M.D., 10/09/00)
Mona Iancovici-Kidon,1 Daliah Sthoeger,1 Ayala Abrahamov,2 Baruch Volach,3 Ernest Beutler,4 Terri Gelbart,4 and Yigal Barak1 ABSTRACT: Hereditary nonspherocytic hemolytic anemia (HNSHA) is a rare manifestation of glucose-6phosphate dehydrogenase (G6PD) gene mutations, caused mainly by mutations located in exon 10 of the G6PD gene and less commonly by mutations in other parts of the gene. A new, exon 9, single-base mutation representing a T 3 C transition at cDNA nucleotide 964 was found in three brothers and their carrier mother of Jewish Ethiopian descent. Biochemical characterization of the resultant protein was not performed. Though clinical manifestations included HNSHA in all cases, the severity of hemolysis and the transfusion requirement differed markedly. Severe congenital neutropenia (Kostmann’s syndrome)—a disorder never reported before in conjunction with G6PD deficiency—was observed in one case. Levels of white blood cell G6PD activity of the three patients were 0 –5% of normal controls. Neutrophil oxidative and bactericidal activities were inherently impaired in the patient with Kostmann’s syndrome, but were well preserved in his two siblings. © 2000 Academic Press
INTRODUCTION
hemolytic anemia with varying severity, associated in one case with severe congenital neutropenia.
Hereditary nonspherocytic hemolytic anemia (HNSHA) is a relatively rare clinical manifestation of glucose-6-phosphate dehydrogenase (G6PD) gene mutations. Most known mutations associated with this type (class 1) disease are located in the exon 10 area of the G6PD gene, probably related to the substrate receptor, NADPH binding site, or in the region around the glucose-6-phosphate binding site at lysine 205 (1, 2). In contrast to the more benign, prevalent forms associated with favism, these panethnic, rare mutations may cause significant morbidity due to chronic hemolytic anemia. As a rule, no other hematological cell lines are involved (2). We describe a new single-base substitution in exon 9 of the G6PD gene in a Jewish Ethiopian family, expressed as hereditary nonspherocytic
MATERIALS AND METHODS Patients A.D. is a 7-year-old, firstborn son of healthy unrelated parents of Jewish Ethiopian descent. After a normal pregnancy, uncomplicated delivery and perinatal course, G6PD deficiency was found on a routine qualitative neonatal screening test. His medical history is complicated by a focal convulsive disorder diagnosed at the age of 2 years, treated with carbamazepine, and by attention deficit hyperactivity syndrome. He has a moderate chronic hemolytic anemia, with Hb ranging 8.6 –9.2 g/dl, a reticulocyte count ranging
Correspondence and reprint requests to: Yigal Barak, Department of Pediatrics, Kaplan Medical Center, Rehovot 76100, Israel. Fax: 972-8-9411942. E-mail: [email protected]. 1 Department of Pediatrics, Kaplan Medical Center, Rehovot, Israel. 2 Shaare Tzedek Medical Center, Jerusalem, Isreal. 3 Meir Hospital, Kfar Saba, Israel. 4 Scripps Research Institute, La Jolla, CA 92037. 1079-9796/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved
567
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
10 –14% and a normal leukocyte cell count and function. To date, he has never required transfusion or hospitalization. A.A., the second son, born after a normal pregnancy and delivery, presented at 4 days of age with gram-negative septicemia and acute hemolytic anemia, later identified as due to G6PD deficiency. In his first year of life the patient developed recurrent invasive, presumably bacterial infections accompanied by fever and severe granulocytopenia, and progressive hemolytic anemia requiring frequent packed red blood cell transfusions. The bone marrow examination revealed a paucity of cells in the granulocyte lineage together with a maturation arrest at the promyelocyte–myelocyte stage. Erythropoiesis was found to be moderately increased and other cell lines were normal. The peripheral blood and the bone marrow findings were interpreted as compatible with the diagnosis of severe congenital neutropenia (Kostmann’s syndrome). Treatment with rhG-CSF (Neupogen, Amgen) resulted in increased absolute neutrophil counts (ANC) only at markedly increased G-CSF doses and only for those periods of time when treatment was received with regularity. Nevertheless, rhG-CSF treatment reduced significantly the number and severity of infections and hospitalizations, but no changes in hemolysis or in transfusion requirements were observed. A treatment trial of antioxidant therapy (vitamin E), also failed to effect any improvement in the transfusion requirement. At the age of 3 years the patient died at home due to an overwhelming bacterial infection, with no granulocytes at the sites of infection, on postmortem examination. A.E., the youngest child of this Jewish Ethiopian family, is a 1.5-year-old male, who was severely jaundiced at birth and received two exchange transfusions on the first day of life. Following this, his hemoglobin rose from 12.3 to 17 g/dl, reticulocyte count changed from 28.8 to 20%, bilirubin dropped from 22.8 to 10 mg/dl and LDH decreased from 14,400 to 2356 /liter. Leukocyte, absolute neutrophil and platelet counts were consistently normal. He was later diagnosed as being G6PD deficient. In his first 18 months of life, while on continuous folic acid and vitamin E
Iancovici-Kidon et al.
therapy, A.E. has had a steady-state hemoglobin level in the 10 to 11 g/dl range and reticulocyte counts of 6.7–19%. He thrives and develops normally, and except for one occasion, during an acute viral infection, he has never required further blood transfusions. Mutation Analysis DNA was isolated from peripheral blood leukocytes by standard methods. All exons of the G6PD gene in the patient were amplified by the polymerase chain reaction (PCR) using previously described oligonucleotide primers (3). The PCR consisted of 1 g genomic DNA, 33.5 mM Tris–HCl, pH 8.8, 8.3 mM (NH4)2SO4, 3.35 mM MgCl2, 85 g/ml bovine serum albumin, 0.2 mM of each dNTP (dATP, dCTP, dGTP, and dTTP), 5% dimethyl sulfoxide, and 1.5 U Taq polymerase. PCR was performed for 30 cycles consisting of 93°C for 30 s, 58°C for 30 s, and 72°C for 30 s followed by a 7-min 72°C extension. PCR products were then purified with a QIAquick PCR purification kit (Qiagen Inc. Chatsworth, CA). The purified PCR products were sequenced on an Applied Biosystems Inc. (Foster City, CA) automatic sequencer. The DNA of subsequent family members was amplified and sequenced only in the region of interest (exon 9). Neutrophil Function Assays Polymorphonuclear leukocytes (PMNs) were isolated from heparinized blood by dextran sedimentation, layering, and erythrocyte lysis, as described by Boyum (4, 5). Superoxide production by PMNs was measured by the reduction of superoxide dismutase (SOD)-inhibitable ferricytochrome c, by the method reported by Weisbart et al. (6). Stimulants with differing modes of action were used to define the sites of alteration in signal transduction of superoxide generation. Bactericidal activity of PMNs was expressed as the decrease in the number of viable bacteria, after incubation with PMNs in the presence of autologous and homologous serum, as described by Clawson and Repine (7). The log decrease in 568
Iancovici-Kidon et al.
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
bactericidal activity with a fluctuating level of white blood cells G6PD activity, 0 –5% of normal controls.
TABLE 1 Neutrophil Functions Stimulation
A.A.
A.D.
A.E.
WBC–G6PD
0
0–5
0–5
RBC–G6PD (% of control)
0
0
0
fMLP (1.3–8.5)
1.0b
5.3
4.01
PMA (2.5–8.5) Autol serum (0.6–1.9)
0.94b
6.77
6.37
0.13b
0.75
0.6
Homol serum (0.6–1.6)
0.37b
0.73
0.69
Superoxide generation 6 (nmol O⫺ 2 /10 PMNs/min)
Bactericidal activity (log decrease of CFUa)
DISCUSSION All three patients exhibit hereditary nonspherocytic hemolytic anemia associated with an exon 9 mutation of the G6PD gene. Most mutations causing HNSHA are in exon 10 (9), a region that seems to be important in the binding of structural NADP. Among the 14 mutations that have previously been documented in exon 9, only 5 (G6PD West Virginia, Omiya, Manhattan, Nara, and Torun) are associated with HNSHA, and Nara is not a point mutation but rather a large deletion (10). One of these mutations, G6PD Manhattan, changes the glycine immediately upstream of the tyrosine that was mutated in our patients to glutamic acid. Even though all of our patients had the same mutation, the clinical presentation and expression of the disease differs remarkably in all three. The association with severe congenital neutropenia in the case of A.A. is still an unresolved question. It may be a “simple” case of coexisting morbidity with Kostmann’s syndrome (11, 12) aggravating the hemolytic anemia due to recurrent bacterial infections, adding an oxidative load on an already stressed system. The diagnosis of Kostmann’s syndrome is strongly supported in this case by the peripheral blood and the bone marrow appearance of the granulocytic series, with maturation arrest at the promyelocyte level (11). It is also supported by the definite dependency on continuous rhG-CSF treatment (13–15). No previous report of type I G6PD deficiency (hereditary nonspherocytic hemolytic anemia) in association with Kostmann’s syndrome was found in an extensive literature search. Another interesting association is the primary leukocyte defect induced by severe G6PD deficiency within leukocytes as well as erythrocytes. The neutrophil oxidative and bactericidal activity dysfunction, that remained abnormal after incubation with homologous sera, are indicative of the presence of an intrinsic cell defect, not usually associated with Kostmann’s syndrome (16). Various abnormali-
Note. In parentheses: normal range in our laboratory. a Colony-forming units. b Abnormal.
bactericidal activity was calculated and compared with that of controls. Measurement of G6PD activity was performed on PMNs obtained from the leukocyte-enriched plasma layer, after 20 s of hypotonic lysis, to eliminate contaminating red cells. The enzyme was assayed according to a previous WHO report (8). RESULTS Mutation analysis of blood DNA samples from all three brothers demonstrated a previously undescribed mutation representing a T [wng]224[/ wng] C transition at cDNA nucleotide 964. This is predicted to cause a tyrosine [wng]224[/wng] histidine substitution at amino acid 322. Biochemical characterization of the resultant protein was not performed. Analysis of maternal DNA revealed her to be heterozygous for the same exon 9 mutation. Results of the neutrophil functional analysis are summarized in Table 1. Analysis of neutrophil function in patient A.A. was performed during treatment with rhG-CSF. A.A.’s white blood cell G6PD enzyme activity levels were undetectable, and bactericidal activity was severely impaired and slightly improved but did not normalize after addition of homologous serum. The other two siblings had normal superoxide generation and 569
Blood Cells, Molecules, and Diseases (2000) 26(6) December: 567–571 doi:10.1006/bcmd.2000.0334, available online at http://www.idealibrary.com on
ties in neutrophil function have been associated with other forms of severe G6PD deficiency (17– 19). Not so with severe neutropenia, as seen in patient A.A. and probably not causatively associated with the “Rehovot” type mutation in the G6PD gene. The results from patients A.D. and A.E. of normal neutrophil functions with extremely low WBC G6PD enzyme activity and no increased susceptibility to infections also suggest that neutrophil dysfunction is not inherent in this G6PD gene mutation. Still it is interesting to note, that enzyme activity measured in patient A.A.’s PMNs was zero, compared to low fluctuating levels, 0 –5% of controls, measured in his sibs. It is well documented that levels equal or over 1% of white blood cells G6PD enzyme activity are sufficient for a good NADPH-oxidase response. The described functional abnormality in neutrophil function can be due at least partially to the rhGCSF treatment. Abnormal chemotaxis and oxidative burst have been described in Kostmann’s disease patients during rhG-CSF treatment (20, 21). The third and once more different presentation of severe neonatal hyperbilirubinemia and mild hemolytic anemia responsive to antioxidant therapy is consistent with previously reported cases of type I G6PD (2), but again diametrically different from the clinical presentation of the two older siblings. The existence of different G6PD DNA variants causing similar phenotypic disease is known and prevalent in the literature (1, 22–25). This report describes a reverse situation where the same mutation in the G6PD gene is associated with extremely divergent clinical phenotypes.
Iancovici-Kidon et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
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