British Journal ofHaematology, 1976, 34, 5s.
Unbalanced Globin Chain Synthesis in Erythroid Precursor Cells of Heterozygous a-Thalassaemia MARTIN H. STEINBERG, MARYCOLEMAN AND BERNARD DREILING
Special Hematology Laboratory, VA Hospital and Departmerzt of Medicine, University of Mississippi School of Mediciize, Jackson, Mississippi (Received 27 December 1975; accepted f o r publicatiorz 3 February 1976) SUMMARY. Globin biosynthesis was studied in both erythroid precursors and reticulocytes of three individuals with heterozygous a-thalassaemia. In contrast to the finding of equal or nearly equal a and P chain synthesis in the marrow of patients with heterozygous 1-thalassaemia previously examined, our studies showed equal degrees of unbalanced globin synthesis in both reticulocytes and nucleated erythroid cells of a-thalassaemia heterozygotes. Greater stability and less susceptibility to proteolysis of the excess /J-chain formed in a-thalassaemia may explain our findings. A characteristic of thalassaemia syndromes is unbalanced globin chain synthesis in reticulocytes (Weatherall & Clegg, 1972). In homozygous 8-thalassaemia, nucleated erythroid cells have a deficit in the synthesis of p-chains relative to a-chains (Braverman & Bank, 1969); however, a and /J chain synthesis ratios may be balanced (Schwartz, 1970) or approach unity (Clegg & Weatherall, 1972) in the marrow of /J-thalassaemia heterozygotes. Equal or nearly equal a and /J chain production should be of considerable advantage to the developing erythrocyte, as cellular damage by precipitates of uncombined globin chains is in part responsible for the ineffective erythropoiesis and haemolysis typifying many of the thalassaemias. Bone marrow synthesis studies have been presented in only a single case of haemoglobin H disease (Shchory & Ramot, 1972). In this instance a-chain synthesis was impaired in marrow as well as peripheral blood with a greater deficit found in studies of the bone marrow. To assess the biosynthetic capacity of nucleated erythroid precursors in mild heterozygous a-thalassaemia, we studied three patients with this disorder. In two, globin synthesis was assessed in I h incubations of unfractionated bone marrow as well as in 72 h suspension cultures of nucleated cells, This latter technique was employed to obviate the influence of reticulocyte globin synthesis and to evaluate the persistence of the thalassaemic defect over several days in culture. In an additional individual, fractionated marrow cells were studied. In contrast to heterozygous P-thalassaemia, globin synthesis was imbalanced in both the nucleated red cells and reticulocytes of our subjects.
METHODS Informed consent for these studies was obtained from three black patients with sickle cell trait*-thalassaemia and all controls. Standard haematological techniques were used. Haenioglobin concentration and mean corpuscular volume wcrc measured with a Coulter Model S Correspondence: Dr M. H. Steinberg, VA Hospital, Jackson, Mississippi 39216,U.S.A.
55
56
M. H. Steinberg, M. Coleman and B. Dreiling
cell counter. Haemoglobin electrophoresis and quantification of haemoglobin fractions present were done using polyacrylamidc gel electrophoresis (Willard et al, 1973). Venous blood, anticoagulated with heparin (50 units/ml) was centrifuged at 20 ooo rpm for 2 h at 4°C. The top I ml of cells was washed twice with balanced salts solution and incubated for I h at 37°C under 5% C 0 2 with 25 PCi of [3H]leucine, in a medium supporting protein synthesis but lacking this amino acid (Boyer et al, 1968). Bone marrow was aspirated from the posterior iliac crest into syringes rinsed with heparin (100 unitslml). One thousand cell differential counts were done on slides prepared prior to centrifugation. Following centrifugation at 3000 rpm for 10 min the marrow was washed thrice in cold balanced salts solution and incubated under conditions similar to those used for reticulocytes. In one patient, bone marrow was fractionated on a bovine serum albumin (BSA) gradient by the method of Wood & Stamatoyannopoulos (1975) and globin synthesis measured on the toy fractions of cnriched erythroid precursors. Following incubations, cells were washed three times with iced balanced salts solution, lysed with 4 vo1o.001~M KCl. After centrifugation at 13 500 rpm for 20 niin globin was prepared by acid-acetone precipitation and resolvcd into its constituent chains by carboxymethylcellulose column chromatography (Clegg ct al, 1966). Fractions of 4 ml were collected and protein from those tubes containing the peaks of each chain was precipitated in 500 g/l trichloroacetic acid (TCA), washed with 50 g/l TCA, dissolved in a tissue solubilizer, added to 10 nil PPO-POPOP and counted in a liquid scintillation spectrometer. Nucleated marrow cells ( z x 1 0 ~ were ) cultured in a medium of NCTC-109, 400 g/l autologous serum, IOO units penicillin, 0.5 mg streptomycin and 0.3 units Step 111 erythropoietin (Connaught Medical Research Laboratories), by a modification of the method of Krantz ct al (1963). After 48 h incubation at 37°C under 5% C 0 2 , 7 pCi [3H]leucine was added and the incubation continued for an additional 24 h. Triplicate cultures were pooled, 60 mg of 'cold' haemoglobin AS haemolysate added, and globin prepared and treated as outlined above. The biosyntlietic ratios are reported as the ratios of the total radioactivity incorporated into each chain. RESULTS Haematological data from our patients are shown in Table I. They were suspected of having a-thalassaemia in addition to sickle cell trait by noting the absence of anaemia, microcytosis and lower than usual levels of haemoglobin S, without evidence of iron deficiency. Patient I had two children, a son with sickle cell trait, microcytosis and an a//? ratio in reticulocytes of 0.78 and a daughter with normal haemoglobin, mild microcytosis and an ct/P synthesis ratio of 0.85. Patient 2 had a son with a haematological picture similar to his; sickle cell trait, microcytosis and an a/P ratio in reticulocytes of 0.78. The wives of both probands were normal. Family studies were not done in the third patient. Biosynthesis studies, measuring thc incorporation of ['Hlleucine into newly synthesized globin chains showed unbalanced a and p chain synthesis in reticulocytes, I h marrow incubations and 72 h marrow cultures (Table 11). Reticulocyte globin synthesis was studied on two or three occasions over a 1-2 year period in patients I and 2. In patient I , ./PA + Ps ratios of 0.70 and 0.66 were obtained, and in patient 2 ratios of 0.70, 0.76 and 0.75 were found. The a/PA+PSratios in triplicate 72 h
Globin Synthesis in a- Thalarsaenzia
57
marrow cultures in patient I were 0.72, 0.74 and 0.67 and in patient 2, 0.76, 0.75 and 0.66. Reticulocytes, coiicentrated from peripheral blood and cultured in a similar fashion synthesized little globin after a 72 h incubation. Differential counts of the marrow of patient I showed that 37.6% of the nucleated cells were erythroid. These consisted of 6% pronormoblasts, 10% early iiorinoblasts (basophilic), 25% intermediate normoblasts (polychromatophilic) aiid 59% late normoblasts (orthochromatic). Patient 2 had 29.9% erythroid precursors in his marrow with 2% pronormoblasts, 6% early, 26% intermediate and 66% late normoblasts. Results of a single experiment for each patient are presented in Table 11. In patient 3 , an aliquot of I h whole marrow incubation with a5 PCi [3H]leucine was fractioned on a BSA gradient (Wood & Stamatoyannopoulos, 1975). The top of this gradient, corresponding to fractions 4 and 5 of Wood & Stamatoyaniiopoulos (1975)~was harvested aiid found to contain 53% erythroid precursors with 8% pronormoblasts, 4% early, 7% intermediate and TABLE I. Haenlatological data in sickle cell trait-a-thalassaemia*
Pufienf I 2
3
H6 (gin!) M C V ( f ! ) MCH(pg) Reticrilocyfes (%) 16.3 14.2 13.0
75 70 72
25.9 22.9 23.7
H6A (%) H6S (%) 73.1 67.8 71.9
1.1
0.8
0.7
24.1 29.8 25.4
* Bone marrow iron stores were normal in all patients studied. TABLE 11. Biosynthetic data from peripheral blood and bone marrow of sickle cell trait-a-thalassaeniia ~
~ ~ _ _ _ _
Pufirnt I*
Patient
2*
60830 61689 25084 0.70
15987 15277 7508 0.70
309950 335917 157880 0.62
100110
88614 44644 0.75
Putietzf 3 t
39679 45232 19642 0.61
17242 I4744 9356 0.72
* 25 pCi [3H]leucine was used for reticulocyte and I 11 marrow incubation. t Reticulocyte incubations were done using 25 fiCi 13H]leucine and 40 mg of protein were chromatographed. Marrow incubations used 50 pCi [3H]leucine and 15 mg ofprotein were chrornatographed.
HbA, (%) 3 .o 2.9 2.7
M. H. Steinberg, M. Coleman and B. Dreiling
58
41% late normoblasts. The haemoglobin in these cells was purified by gel filtration using
Sephadex G-IOOafter the addition of cold carrier haemolysate (Wood & Stamatoyaiinopoulos, 197s). The .//IA + /Is synthesis ratio in purified Hb from this fraction was 0.69. Fig I shows the results of a typical 72 h marrow culture in the presence of erythropoietin. The ratio ofa to P chains in reticulocytes and short-term marrow incubations of individuals with haemoglobin A is close to unity. Five control patients with sickle cell trait had an a / P A + P Sratio in marrow of o.ggfo.10 (mean -I I SD), while this ratio in reticulocytes of 10patients with sickle cell trait was 1.01 & 0.02 (Steinberg et al, 1975). In other experiments using this culture system, the reticulocyte count prior to culture was 0.6-3.1%, and following 72 h in culture less than 1.0%. Normal bone marrow cultured in this system had u/P synthesis ratios of 0.98 and 1.03. The bone marrow of two patients with sickle cell trait without coexistent a-thalassaemia had a/P ratios of 0.91 and 0.95. All marrow samples studied had normal iron stores when examined using Perls’ stain.
- 4200 - 3600 - 3000 - 2400 I.’
- I800 - 1200
H
a
0-4
r
- 600
Froct ion
FIGI . 72 h bone narrow culture from paticnt z in the presence of 0.3 units erythropoietin. The (-) and cpin of [3H]leuci~~e (0) are depicted for /?*,bs and a chains and illustrate decreased incorporation of label into the a-chain when coinpared to the PAand Bs chain.
DISCUSSION When an apparent balance in globin chain synthesis was first noted in the bone marrow of individuals with lieterozygous P-thalassaeniia, the reasons why nucleated erythroid cells differed from reticulocytes in this regard were not clear. Suppression of a-chain production, and enhanced synthesis of /I-chain by the non-thalassaemic P-chain allele in erythroid precursors, were proposed as possible mechanisms for balanced synthesis (Kan ct al, 1972). Clegg & Weatherall (1967) presented evidence for a non-globin protein contaminating the P-globin peak and spuriously returning towards normal the a/P synthesis ratio. Later, Weatherall &
Globin Synthesis in a-Thalassaenzia
59
Clegg (1972) showed that there was degradation of free cr-chain in 8-thalassaemic marrow. More recently, Wood & Stamatoyannopoulos (1975) demonstrated catabolism of excessive a-chain in 8-thalassaemic marrow which was most prominent in the least mature erythroid precursors. Chalevelakis et al(1975) found that balanced globin synthesis resulted from nonglobin proteins contaminating the 8-chain peak as well as destruction of excessive free a-chain. These findings, taken with the demonstration by Pritchard et a1 (1975) that there is a deficit of 8-globin m RNA in heterozygous8-thalassaemic marrow, indicate that the apparent balance in globin synthesis is the result of both technical artifact and proteolytic events occurring during the post-translational phase of protein synthesis. In the nucleated red cells of our patients, the a//?" + 8' ratios were significantly less than unity and similar to those found in peripheral blood. Several observations may account for this finding. There may be greater stability in a putative pool of P-chains likely to be present in a-thalassaemia (Clegg & Weatherall, 1967) with less tendency towards precipitation or proteolysis. I n this regard, the existence of a relatively stable 84 tetramer, HbH, may make excess 8-chain resistant to proteolysis. In P-thalassaemia, a stable cr4 molecule does not appear to exist; therefore the pool of free a-chain may be susceptible to proteolysis. In our patients we were unable to demonstrate HbH by electrophoresis or the Brilliant Cresyl Blue technique (Steinberg et a / , 1975). Similar biosynthesis ratios in reticulocytes and nucleated erythroid cells of our patients argue against the presence of significant amounts of leucocyte non-globin protein chromatographing with thc 8-chain peak under our conditions of study. The 72 h marrow cultures must largely reflect globin synthesis in nucleated erythrocytes. Necheles et al(1965) showed a marked fall in the number of reticulocytes during culture over a 72 h period with reticulocyte haemoglobin synthesis only I-2% of that found in nucleated cells. W e found little globin synthesis in peripheral blood reticulocytes cultured for 72 h. The reticulocyte count of bone marrow cultures after 72 h incubation was less than 1.0%. Globin produced during the last 24 h of our cultures must therefore measure biosynthetic capabilities of maturing nucleated erythroid cells. The greater radioactivity of globin prepared from I h marrow incubations as compared to reticulocyte incubations must also reflect the greater synthetic capacity of the nucleated cells in this system. In addition, enriched nucleated red cells, in the absence of reticulocytes, showed synthesis ratios similar to those found in whole marrow cultures. Finally, if our studies with unfractionated marrow chiefly measured reticulocyte synthesis, it would be expected that heterozygous8-thalassaemic marrow, studied under conditions similar to ours (Schwartz, 1970), would have shown synthesis ratios identical to those found in reticulocytes. Unbalanced globin synthesis in the bone marrow was not associated with clinically significant alterations in the blood, and haemolysis, anaemia or ineffective erythropoiesis was not present. Additional studies will be needed to uncover the reasons why nucleated erythroid cells of heterozygous a-thalassaemia appear to differ from those of heterozygous P-thalassaemia when globin Synthesis is estimated but the hypothesis mentioned above is amenable to testing and may provide additional information on the cellular controls of globin synthesis. The effect of the coexistent gene for haemoglobin S upon globin synthesis in patients with a-thalassaeniia is not known. In family studies we have found lesser degrees of c( to P chain imbalance in reticulocytes of simple heterozygotes with a-tlialassaemia than in mixed heterozygotes with haemoglobin S (Steinberg & Adams, 1975). The a to 8 chain Synthesis ratio in
60
M. H. Steinberg, M . Coleman and B. Dreiling
two such individuals was 0.90 and 0.87, and the daughter of patient 2 in this present study had a ratio of 0.85. The reasons underlying this observation are not clear. These values are similar to those obtained by Scliwartz & Atwatcr (1972) in many of their black patients with a-thalassaemia. The examination of globin synthesis in nuclcated erythroid precursors of simple heterozygous subjects should be of interest. ACKNOWLEDGMENT
This work was supported by VA Rcsearch Funds MRIS 8123-04. REFERENCES BOYER,S.H., CROSBY,E.F. & NOYES,A.N. (1968) Hemoglobin switching in non-anemic sheep. I. Mediation by plasma from anemic animals. Johns Hopkins Medical Journal, 123, 85. BRAVERMAN, A.S. & BANK,A. (1969) Changing rates of globin chain synthesis during erythroid cell maturation in thalassemia. Journal of Molecular Biology, 42, 57. CHALEVELAKIS, G., CLEGG, J.B. & WEATHERALL, D.J. (1975) Imbalanced globin chain synthesis in heterozygous 8-thalassemic bone marrow. Proceedings oj’ the National Academy of Sciences ofthe United Slates of America, 72, 3 8 5 3 . CLEGG, J.B., NAUGHTON, M.A. & WEATHERALL, D.J. (1966) Abnormal human haemoglobins. Separation and characterization of the a and 8 chains by chromatography, and the determination of two new variants, Hb Chesepeake and Hb J (Bangkok). Journal ofMolecular Biology, 19,91. CLEGG,J.B. & WEATHERALL, D.J. (1967) Haemoglobin synthesis in a-thalasaeniia (haemoglobin H disease). Nature, 215, 1241. CLEGG, J.B. & WEATHERALL, D.J. (1972) Haemoglobin synthesis during erythroid maturation in /Ithalassaemia. Nature: N ew Biology, 240, 190. KAN,Y.W., NATHAN, D.G. & LODISH,H.F. (1972) Equal synthesis of a- and 8-globin chains in erythroid precursors in heterozygous /?-thalassemia. Journal of Clinical Investigation, 51,1906. KRANTZ, S.B., GALLIEN-LARTIQUE, 0.& GOLDWASSER, E. (1963) The effect of erythropoietin upon heme synthesis by marrow cells in vitro. Journal of Biological chemistry, 238, 4085. NECHELES, T.F., SHEEHAN, R.G. & MEYER,H.J. ( I 965) Effect of erythropoietin and oxygen tension on in vitro synthesis of hemoglobin A and F by adult
human bone marrow. Proceedings of the Society f o r Experimental Biology and Medicine, 119,1207. PRITCHARD, J., HILLMAN, D.G., GLASS, J. & FORGET, B.G. (1975) Deficiency of Fglobin chain messenger RNA (m-RNA) in nucleated red precursors from heterozygous 8-thalassemia (hetero-p-thal) marrow. 18th Annual Meeting, American Society of Hematology, Dallas (Abstract), p 96. SHCHORY, M. & RAMOT,B. (1972) Globin chain synthesis in the inarrow and reticulocytes of beta thalassemia, hemoglobin H disease, and beta delta thalassemia. Blood, 40, 10s. SCHWARTZ, E. (1970) Heterozygous beta thalassemia: balanced globin synthesis in bone marrow cells. Science, 167, 1513. SCHWARTZ, E. & ATWATER, J. (1972) a-Thalassemia in the American Negro.]ournal of Clinical Invertigation, 51,412. M.H. & ADAMS, J.G. (1975) a-Thalassemia STEINBERG, in patients with hemoglobins S and C: family and biosynthetic studies. (Abstract). Clinical Research, 23,407A. B.J. STHNBERG, M.H., ADAMS, J.G., 111 & DREILING, (1975) Alpha thalassaemia in adults with sickle-cell trait. British Jocrrnal of Haematology, 30, 31. WEATHERALL, D.J. & CLEGG, J.B. (1972) The Thalassaemia Syndromes, 2nd edn, p 374. Blackwell Scientific Publications, Oxford. WILLARD,R.F., LOVELL,W.J., DREILING,B.J. & STEINBERG, M.H. (1973) Electrophoresis of hemoglobin on polyacrylamide gels: precise method for measurement of hemoglobin A*. Clinical Chemistry, 19,1082. WOOD,W.G. & STAMATOYANNOPOULOS, G. (1975) Globin synthesis in fractionated normoblasts of 8thalassemia heterozygotes. Journal of Clinical Investigation, 55, 567.
Unbalanced Globin Chain Synthesis in Erythroid Precursor Cells of Heterozygous a-Thalassaemia MARTIN H. STEINBERG, MARYCOLEMAN AND BERNARD DREILING
Special Hematology Laboratory, VA Hospital and Departmerzt of Medicine, University of Mississippi School of Mediciize, Jackson, Mississippi (Received 27 December 1975; accepted f o r publicatiorz 3 February 1976) SUMMARY. Globin biosynthesis was studied in both erythroid precursors and reticulocytes of three individuals with heterozygous a-thalassaemia. In contrast to the finding of equal or nearly equal a and P chain synthesis in the marrow of patients with heterozygous 1-thalassaemia previously examined, our studies showed equal degrees of unbalanced globin synthesis in both reticulocytes and nucleated erythroid cells of a-thalassaemia heterozygotes. Greater stability and less susceptibility to proteolysis of the excess /J-chain formed in a-thalassaemia may explain our findings. A characteristic of thalassaemia syndromes is unbalanced globin chain synthesis in reticulocytes (Weatherall & Clegg, 1972). In homozygous 8-thalassaemia, nucleated erythroid cells have a deficit in the synthesis of p-chains relative to a-chains (Braverman & Bank, 1969); however, a and /J chain synthesis ratios may be balanced (Schwartz, 1970) or approach unity (Clegg & Weatherall, 1972) in the marrow of /J-thalassaemia heterozygotes. Equal or nearly equal a and /J chain production should be of considerable advantage to the developing erythrocyte, as cellular damage by precipitates of uncombined globin chains is in part responsible for the ineffective erythropoiesis and haemolysis typifying many of the thalassaemias. Bone marrow synthesis studies have been presented in only a single case of haemoglobin H disease (Shchory & Ramot, 1972). In this instance a-chain synthesis was impaired in marrow as well as peripheral blood with a greater deficit found in studies of the bone marrow. To assess the biosynthetic capacity of nucleated erythroid precursors in mild heterozygous a-thalassaemia, we studied three patients with this disorder. In two, globin synthesis was assessed in I h incubations of unfractionated bone marrow as well as in 72 h suspension cultures of nucleated cells, This latter technique was employed to obviate the influence of reticulocyte globin synthesis and to evaluate the persistence of the thalassaemic defect over several days in culture. In an additional individual, fractionated marrow cells were studied. In contrast to heterozygous P-thalassaemia, globin synthesis was imbalanced in both the nucleated red cells and reticulocytes of our subjects.
METHODS Informed consent for these studies was obtained from three black patients with sickle cell trait*-thalassaemia and all controls. Standard haematological techniques were used. Haenioglobin concentration and mean corpuscular volume wcrc measured with a Coulter Model S Correspondence: Dr M. H. Steinberg, VA Hospital, Jackson, Mississippi 39216,U.S.A.
55
56
M. H. Steinberg, M. Coleman and B. Dreiling
cell counter. Haemoglobin electrophoresis and quantification of haemoglobin fractions present were done using polyacrylamidc gel electrophoresis (Willard et al, 1973). Venous blood, anticoagulated with heparin (50 units/ml) was centrifuged at 20 ooo rpm for 2 h at 4°C. The top I ml of cells was washed twice with balanced salts solution and incubated for I h at 37°C under 5% C 0 2 with 25 PCi of [3H]leucine, in a medium supporting protein synthesis but lacking this amino acid (Boyer et al, 1968). Bone marrow was aspirated from the posterior iliac crest into syringes rinsed with heparin (100 unitslml). One thousand cell differential counts were done on slides prepared prior to centrifugation. Following centrifugation at 3000 rpm for 10 min the marrow was washed thrice in cold balanced salts solution and incubated under conditions similar to those used for reticulocytes. In one patient, bone marrow was fractionated on a bovine serum albumin (BSA) gradient by the method of Wood & Stamatoyannopoulos (1975) and globin synthesis measured on the toy fractions of cnriched erythroid precursors. Following incubations, cells were washed three times with iced balanced salts solution, lysed with 4 vo1o.001~M KCl. After centrifugation at 13 500 rpm for 20 niin globin was prepared by acid-acetone precipitation and resolvcd into its constituent chains by carboxymethylcellulose column chromatography (Clegg ct al, 1966). Fractions of 4 ml were collected and protein from those tubes containing the peaks of each chain was precipitated in 500 g/l trichloroacetic acid (TCA), washed with 50 g/l TCA, dissolved in a tissue solubilizer, added to 10 nil PPO-POPOP and counted in a liquid scintillation spectrometer. Nucleated marrow cells ( z x 1 0 ~ were ) cultured in a medium of NCTC-109, 400 g/l autologous serum, IOO units penicillin, 0.5 mg streptomycin and 0.3 units Step 111 erythropoietin (Connaught Medical Research Laboratories), by a modification of the method of Krantz ct al (1963). After 48 h incubation at 37°C under 5% C 0 2 , 7 pCi [3H]leucine was added and the incubation continued for an additional 24 h. Triplicate cultures were pooled, 60 mg of 'cold' haemoglobin AS haemolysate added, and globin prepared and treated as outlined above. The biosyntlietic ratios are reported as the ratios of the total radioactivity incorporated into each chain. RESULTS Haematological data from our patients are shown in Table I. They were suspected of having a-thalassaemia in addition to sickle cell trait by noting the absence of anaemia, microcytosis and lower than usual levels of haemoglobin S, without evidence of iron deficiency. Patient I had two children, a son with sickle cell trait, microcytosis and an a//? ratio in reticulocytes of 0.78 and a daughter with normal haemoglobin, mild microcytosis and an ct/P synthesis ratio of 0.85. Patient 2 had a son with a haematological picture similar to his; sickle cell trait, microcytosis and an a/P ratio in reticulocytes of 0.78. The wives of both probands were normal. Family studies were not done in the third patient. Biosynthesis studies, measuring thc incorporation of ['Hlleucine into newly synthesized globin chains showed unbalanced a and p chain synthesis in reticulocytes, I h marrow incubations and 72 h marrow cultures (Table 11). Reticulocyte globin synthesis was studied on two or three occasions over a 1-2 year period in patients I and 2. In patient I , ./PA + Ps ratios of 0.70 and 0.66 were obtained, and in patient 2 ratios of 0.70, 0.76 and 0.75 were found. The a/PA+PSratios in triplicate 72 h
Globin Synthesis in a- Thalarsaenzia
57
marrow cultures in patient I were 0.72, 0.74 and 0.67 and in patient 2, 0.76, 0.75 and 0.66. Reticulocytes, coiicentrated from peripheral blood and cultured in a similar fashion synthesized little globin after a 72 h incubation. Differential counts of the marrow of patient I showed that 37.6% of the nucleated cells were erythroid. These consisted of 6% pronormoblasts, 10% early iiorinoblasts (basophilic), 25% intermediate normoblasts (polychromatophilic) aiid 59% late normoblasts (orthochromatic). Patient 2 had 29.9% erythroid precursors in his marrow with 2% pronormoblasts, 6% early, 26% intermediate and 66% late normoblasts. Results of a single experiment for each patient are presented in Table 11. In patient 3 , an aliquot of I h whole marrow incubation with a5 PCi [3H]leucine was fractioned on a BSA gradient (Wood & Stamatoyannopoulos, 1975). The top of this gradient, corresponding to fractions 4 and 5 of Wood & Stamatoyaniiopoulos (1975)~was harvested aiid found to contain 53% erythroid precursors with 8% pronormoblasts, 4% early, 7% intermediate and TABLE I. Haenlatological data in sickle cell trait-a-thalassaemia*
Pufienf I 2
3
H6 (gin!) M C V ( f ! ) MCH(pg) Reticrilocyfes (%) 16.3 14.2 13.0
75 70 72
25.9 22.9 23.7
H6A (%) H6S (%) 73.1 67.8 71.9
1.1
0.8
0.7
24.1 29.8 25.4
* Bone marrow iron stores were normal in all patients studied. TABLE 11. Biosynthetic data from peripheral blood and bone marrow of sickle cell trait-a-thalassaeniia ~
~ ~ _ _ _ _
Pufirnt I*
Patient
2*
60830 61689 25084 0.70
15987 15277 7508 0.70
309950 335917 157880 0.62
100110
88614 44644 0.75
Putietzf 3 t
39679 45232 19642 0.61
17242 I4744 9356 0.72
* 25 pCi [3H]leucine was used for reticulocyte and I 11 marrow incubation. t Reticulocyte incubations were done using 25 fiCi 13H]leucine and 40 mg of protein were chromatographed. Marrow incubations used 50 pCi [3H]leucine and 15 mg ofprotein were chrornatographed.
HbA, (%) 3 .o 2.9 2.7
M. H. Steinberg, M. Coleman and B. Dreiling
58
41% late normoblasts. The haemoglobin in these cells was purified by gel filtration using
Sephadex G-IOOafter the addition of cold carrier haemolysate (Wood & Stamatoyaiinopoulos, 197s). The .//IA + /Is synthesis ratio in purified Hb from this fraction was 0.69. Fig I shows the results of a typical 72 h marrow culture in the presence of erythropoietin. The ratio ofa to P chains in reticulocytes and short-term marrow incubations of individuals with haemoglobin A is close to unity. Five control patients with sickle cell trait had an a / P A + P Sratio in marrow of o.ggfo.10 (mean -I I SD), while this ratio in reticulocytes of 10patients with sickle cell trait was 1.01 & 0.02 (Steinberg et al, 1975). In other experiments using this culture system, the reticulocyte count prior to culture was 0.6-3.1%, and following 72 h in culture less than 1.0%. Normal bone marrow cultured in this system had u/P synthesis ratios of 0.98 and 1.03. The bone marrow of two patients with sickle cell trait without coexistent a-thalassaemia had a/P ratios of 0.91 and 0.95. All marrow samples studied had normal iron stores when examined using Perls’ stain.
- 4200 - 3600 - 3000 - 2400 I.’
- I800 - 1200
H
a
0-4
r
- 600
Froct ion
FIGI . 72 h bone narrow culture from paticnt z in the presence of 0.3 units erythropoietin. The (-) and cpin of [3H]leuci~~e (0) are depicted for /?*,bs and a chains and illustrate decreased incorporation of label into the a-chain when coinpared to the PAand Bs chain.
DISCUSSION When an apparent balance in globin chain synthesis was first noted in the bone marrow of individuals with lieterozygous P-thalassaeniia, the reasons why nucleated erythroid cells differed from reticulocytes in this regard were not clear. Suppression of a-chain production, and enhanced synthesis of /I-chain by the non-thalassaemic P-chain allele in erythroid precursors, were proposed as possible mechanisms for balanced synthesis (Kan ct al, 1972). Clegg & Weatherall (1967) presented evidence for a non-globin protein contaminating the P-globin peak and spuriously returning towards normal the a/P synthesis ratio. Later, Weatherall &
Globin Synthesis in a-Thalassaenzia
59
Clegg (1972) showed that there was degradation of free cr-chain in 8-thalassaemic marrow. More recently, Wood & Stamatoyannopoulos (1975) demonstrated catabolism of excessive a-chain in 8-thalassaemic marrow which was most prominent in the least mature erythroid precursors. Chalevelakis et al(1975) found that balanced globin synthesis resulted from nonglobin proteins contaminating the 8-chain peak as well as destruction of excessive free a-chain. These findings, taken with the demonstration by Pritchard et a1 (1975) that there is a deficit of 8-globin m RNA in heterozygous8-thalassaemic marrow, indicate that the apparent balance in globin synthesis is the result of both technical artifact and proteolytic events occurring during the post-translational phase of protein synthesis. In the nucleated red cells of our patients, the a//?" + 8' ratios were significantly less than unity and similar to those found in peripheral blood. Several observations may account for this finding. There may be greater stability in a putative pool of P-chains likely to be present in a-thalassaemia (Clegg & Weatherall, 1967) with less tendency towards precipitation or proteolysis. I n this regard, the existence of a relatively stable 84 tetramer, HbH, may make excess 8-chain resistant to proteolysis. In P-thalassaemia, a stable cr4 molecule does not appear to exist; therefore the pool of free a-chain may be susceptible to proteolysis. In our patients we were unable to demonstrate HbH by electrophoresis or the Brilliant Cresyl Blue technique (Steinberg et a / , 1975). Similar biosynthesis ratios in reticulocytes and nucleated erythroid cells of our patients argue against the presence of significant amounts of leucocyte non-globin protein chromatographing with thc 8-chain peak under our conditions of study. The 72 h marrow cultures must largely reflect globin synthesis in nucleated erythrocytes. Necheles et al(1965) showed a marked fall in the number of reticulocytes during culture over a 72 h period with reticulocyte haemoglobin synthesis only I-2% of that found in nucleated cells. W e found little globin synthesis in peripheral blood reticulocytes cultured for 72 h. The reticulocyte count of bone marrow cultures after 72 h incubation was less than 1.0%. Globin produced during the last 24 h of our cultures must therefore measure biosynthetic capabilities of maturing nucleated erythroid cells. The greater radioactivity of globin prepared from I h marrow incubations as compared to reticulocyte incubations must also reflect the greater synthetic capacity of the nucleated cells in this system. In addition, enriched nucleated red cells, in the absence of reticulocytes, showed synthesis ratios similar to those found in whole marrow cultures. Finally, if our studies with unfractionated marrow chiefly measured reticulocyte synthesis, it would be expected that heterozygous8-thalassaemic marrow, studied under conditions similar to ours (Schwartz, 1970), would have shown synthesis ratios identical to those found in reticulocytes. Unbalanced globin synthesis in the bone marrow was not associated with clinically significant alterations in the blood, and haemolysis, anaemia or ineffective erythropoiesis was not present. Additional studies will be needed to uncover the reasons why nucleated erythroid cells of heterozygous a-thalassaemia appear to differ from those of heterozygous P-thalassaemia when globin Synthesis is estimated but the hypothesis mentioned above is amenable to testing and may provide additional information on the cellular controls of globin synthesis. The effect of the coexistent gene for haemoglobin S upon globin synthesis in patients with a-thalassaeniia is not known. In family studies we have found lesser degrees of c( to P chain imbalance in reticulocytes of simple heterozygotes with a-tlialassaemia than in mixed heterozygotes with haemoglobin S (Steinberg & Adams, 1975). The a to 8 chain Synthesis ratio in
60
M. H. Steinberg, M . Coleman and B. Dreiling
two such individuals was 0.90 and 0.87, and the daughter of patient 2 in this present study had a ratio of 0.85. The reasons underlying this observation are not clear. These values are similar to those obtained by Scliwartz & Atwatcr (1972) in many of their black patients with a-thalassaemia. The examination of globin synthesis in nuclcated erythroid precursors of simple heterozygous subjects should be of interest. ACKNOWLEDGMENT
This work was supported by VA Rcsearch Funds MRIS 8123-04. REFERENCES BOYER,S.H., CROSBY,E.F. & NOYES,A.N. (1968) Hemoglobin switching in non-anemic sheep. I. Mediation by plasma from anemic animals. Johns Hopkins Medical Journal, 123, 85. BRAVERMAN, A.S. & BANK,A. (1969) Changing rates of globin chain synthesis during erythroid cell maturation in thalassemia. Journal of Molecular Biology, 42, 57. CHALEVELAKIS, G., CLEGG, J.B. & WEATHERALL, D.J. (1975) Imbalanced globin chain synthesis in heterozygous 8-thalassemic bone marrow. Proceedings oj’ the National Academy of Sciences ofthe United Slates of America, 72, 3 8 5 3 . CLEGG, J.B., NAUGHTON, M.A. & WEATHERALL, D.J. (1966) Abnormal human haemoglobins. Separation and characterization of the a and 8 chains by chromatography, and the determination of two new variants, Hb Chesepeake and Hb J (Bangkok). Journal ofMolecular Biology, 19,91. CLEGG,J.B. & WEATHERALL, D.J. (1967) Haemoglobin synthesis in a-thalasaeniia (haemoglobin H disease). Nature, 215, 1241. CLEGG, J.B. & WEATHERALL, D.J. (1972) Haemoglobin synthesis during erythroid maturation in /Ithalassaemia. Nature: N ew Biology, 240, 190. KAN,Y.W., NATHAN, D.G. & LODISH,H.F. (1972) Equal synthesis of a- and 8-globin chains in erythroid precursors in heterozygous /?-thalassemia. Journal of Clinical Investigation, 51,1906. KRANTZ, S.B., GALLIEN-LARTIQUE, 0.& GOLDWASSER, E. (1963) The effect of erythropoietin upon heme synthesis by marrow cells in vitro. Journal of Biological chemistry, 238, 4085. NECHELES, T.F., SHEEHAN, R.G. & MEYER,H.J. ( I 965) Effect of erythropoietin and oxygen tension on in vitro synthesis of hemoglobin A and F by adult
human bone marrow. Proceedings of the Society f o r Experimental Biology and Medicine, 119,1207. PRITCHARD, J., HILLMAN, D.G., GLASS, J. & FORGET, B.G. (1975) Deficiency of Fglobin chain messenger RNA (m-RNA) in nucleated red precursors from heterozygous 8-thalassemia (hetero-p-thal) marrow. 18th Annual Meeting, American Society of Hematology, Dallas (Abstract), p 96. SHCHORY, M. & RAMOT,B. (1972) Globin chain synthesis in the inarrow and reticulocytes of beta thalassemia, hemoglobin H disease, and beta delta thalassemia. Blood, 40, 10s. SCHWARTZ, E. (1970) Heterozygous beta thalassemia: balanced globin synthesis in bone marrow cells. Science, 167, 1513. SCHWARTZ, E. & ATWATER, J. (1972) a-Thalassemia in the American Negro.]ournal of Clinical Invertigation, 51,412. M.H. & ADAMS, J.G. (1975) a-Thalassemia STEINBERG, in patients with hemoglobins S and C: family and biosynthetic studies. (Abstract). Clinical Research, 23,407A. B.J. STHNBERG, M.H., ADAMS, J.G., 111 & DREILING, (1975) Alpha thalassaemia in adults with sickle-cell trait. British Jocrrnal of Haematology, 30, 31. WEATHERALL, D.J. & CLEGG, J.B. (1972) The Thalassaemia Syndromes, 2nd edn, p 374. Blackwell Scientific Publications, Oxford. WILLARD,R.F., LOVELL,W.J., DREILING,B.J. & STEINBERG, M.H. (1973) Electrophoresis of hemoglobin on polyacrylamide gels: precise method for measurement of hemoglobin A*. Clinical Chemistry, 19,1082. WOOD,W.G. & STAMATOYANNOPOULOS, G. (1975) Globin synthesis in fractionated normoblasts of 8thalassemia heterozygotes. Journal of Clinical Investigation, 55, 567.
