American Journal of Hematology 1:35-44 (1976)
CLINICAL, HEMATOLOGIC AND BIOSYNTHETIC STUDIES IN SICKLE CELL-Po-THALASSEMIA:A Comparison With Sickle Cell Anemia Martin H. Steinberg, M. D., and Bernard J. Dreiling, M. D. The Hematology Research Laboratory, Jackson VA Hospital, and Department of Medicine, Division o f Hematology, University of Mississippi School o f Medicine, Jackson, Mississippi
The diseases commonly confused with sickle cell anemia include sickle cell 0-thalassemia in which synthesis of PA-chains are completely suppressed (HbS/3'-thalassemia). We obtained hematologic measurements and studied globin biosynthesis in five patients with this disorder and compared the results with those obtained in five patients with "mild" sickle cell anemia and seven individuals with sickle cell-P-thalassemia having hemoglobin A levels of 20-30% (HbS;O+-thalassemia). A distinction between HbS-Po-thalassemia and sickle cell anemia was not always possible on clinical, hematologic, or electrophoretic grounds. Thalassemia heterozygotes had hypochromia and microcytosis, not generally a feature of sickle cell anemia, although overlap of values did exist. The ratio of CY to non-a, or a to PS-chains in sickle cell anemia approximated unity, whereas patients with HbSP'-thalassemia had a deficit of 0-chain production relative to that of the a-chain. The differentiation of HbS-Po-thalassemia and sickle cell anemia can be best made on the basis of family or biosynthetic study. We estimated the regional prevalence of HbS-Po-thalassemia to be 1:23,000 of the black population. Key words: thalassemia, hemoglobin S, sickle hemoglobin, hemolysis
INTRODUCTION Marked variability exists in the clinical course of patients with sickle cell anemia (1,2). This may be due, in part, to electrophoretic and hematologic mimics of this disease (2-4). Among the conditions which may be confused with sickle cell anemia is sickle cell 0-thalassemia in which there is total suppression of PA globin synthesis (HbS-0'thalassemia) ( 5 ) . Absence of PA chain mRNA has been found in this disorder (6). In this phenocopy of sickle cell anemia, hemoglobin (Hb) A is absent and clinical symptoms may be minimal, the latter perhaps related to a low mean corpuscular HbS concentration (7). We report our studies of globin biosynthesis, as well as hematologic and clinical findings in HbS-Po-thalassemia. We have compared these findings to those noted in a Address reprint requests to M. H. Steinberg, M.D., VA Hospital, 1500 E. Woodrow Wilson, Jackson, Mississippi 392 16.
35
0 1976 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N . Y. 10011
36
Steinberg and Dreiling
group of patients with “mild” sickle cell anemia as well as a group of individuals with HbS(3-thalassernia in which some PA chain is produced (HbS- P’-thalassemia). Our findings are also contrasted with data previously published by other investigators. MATERIALS AND METHODS
Routine hematologic techniques were used (8). Erythrocyte indices were determined using a Coulter model S electronic cell counter. Electrophoresis at pH 8.4, with Tris-EDTA-borate buffer, was done in polyacrylamide gels, the hemoglobin fractions were eluted from the gels, and the fractions were quantified spectrophotometrically (9). HbS levels were taken as the difference between total Hb concentration and levels of Hbs A, A 2 , and F. Electrophoresis at pH 6.1, sodium citrate-citric acid buffer, was done in agar gels (10). HbF levels were measured by alkali denaturation (9) and the distribution of HbF in erythrocytes was determined by the acid elution technique (9). Globin chain biosynthesis was estimated by measuring the incorporation of l4 C-leucine into the newly synthesized PA, ps, y, and a chains of peripheral blood reticulocytes. 4-6 ml of blood were incubated with dextrose and l4 C-leucine at 37°C in a metabolic incubator for time intervals of 15 min-2 hrs (1 1). The globin was separated and resolved into its constituent chains on columns of carboxymethyl cellulose using a sodium phosphate gradient in 8 M urea-mercaptoethanol buffer, pH 6.7(12). Fractions were collected and 1 ml aliquots were mixed with 15 ml of Aquasol and counted in a liquid scintillation spectrometer at 40% efficiency for 14C. The total radioactivity incorporated into each globin peak was used to compute synthesis ratios. RESULTS Clinical Findings
A. HbS-0’-thalassemia. This 28-yr old man was entirely well. He was employed as an X-ray technician, and had no pain crises and normal growth and development. Physical examination was normal, save for splenomegaly. Serum iron was normal. This 49-yr old man had rare episodes of bone and joint pain. He had one attack of B. pneumonia and’a probable pulmonary infarction. A cholecystectomy had been done in the past. Cardiac evaluation showed aortic and mitral insufficiency on the basis of rheumatic heart disease. Aortic valve surgery was successfully undertaken. C. This patient was 29 yr old and suffered from occasional pain crises requiring hospitalization. He had a probable pulmonary infarction and a cholecystectomy had been done. The spleen was not enlarged. His mother had sickle cell trait. D. This 45-yr old woman had no symptoms of sickling diseases. She had two normal pregnancies and deliveries. Her examination was normal except for splenomegaly. One daughter had sickle cell trait and another, (3-thalassemia trait. This patient was a 21-yr old woman who suffered from bone, joint and abdominal E. pains which occurred frequently but were not severe or disabling. Physical examination was normal except for long thin fingers and a soft ejection systolic murmur. Iron and iron-binding capacity were normal. The mother had sickle cell trait. F. HbS-P+-thalassemia. A 22-yr old student was well until he had a mild painful crisis at age 21. At that time he recalled having occasional knee pains every 3-4 mo. His physical examination was normal.
37
Sickle Cell (3' -Thalassemia
G. A 38-yr old woman was seen at the time of her first pregnancy. She was healthy but had siblings with typical sickle cell anemia. No abnormalities were found on physical , examination. H. A 33-yr old man was asymptomatic except for fleeting polyarthralgias. Physical examination was normal. I. A 43-yr old man was retired from the U. S. Army where a diagnosis of sicklethalassemia was made shortly before retirement. He had joint pains and occasional chest or abdominal pain. The physical examination was normal.. J. This patient was 15 mo old and a son of K. He has had normal growth and development to date, and has not had symptoms or signs referable to sickle cell disease. The spleen was palpable 3 cm below the left costal margin. K. This 40-yr old man had a left nephrectomy for hematuria in 1961. There was no history of pain crises and the spleen was not enlarged. L. This patient, son of K, was 10.5 yrs. old. He had occasional abdominal and bone pains which were not severe. His physical examination was normal. M. Sickle Cell Anemia. This man was a 28-yr old truck driver who had rare episodes of bone pain. He was hospitalized once for pneumonia and on another occasion for hepatitis. On examination he was 73" tall and weighed 167 lb. N. This patient was 27 yr old and had mild bone and joint pains about twice yearly. He was employed part-time. His physical examination was normal. 0. This patient was a 24-yr old woman, hospitalized once for a pain crisis. She had one normal pregnancy. She was 62" tall and weighed 125 lbs and her examination was normal, except for an ejection systolic heart murmur. P. This 42-yr old woman was well except for rare episodes of hip pain. She had one normal pregnancy and one miscarriage. A cholecystectomy had been done in the past. On examination she had no abnormalities. This patient was 28 yr old and had never suffered from pain crises. He recently Q. developed bilateral lower leg ulcers. The examination was otherwise normal. Hematologic Values (Table I )
Significant differences (P < 0.05) existed between the mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of both HbS-0-thalassemia groups and patients with sickle cell anemia. The reticulocyte counts in sickle cell anemia and HbS-Po thalassemia were similar (P > 0.05). A significant difference in Hb concentration or hematocrit (Hct) was not present (P > 0.05) between HbS-Do-thalassemia and sickle cell anemia. The mean corpuscular hemoglobin concentration (MCHC) was similar in thalassemia patients but lower in HbS-(3' -thalassemia than in sickle cell anemia (P < 0.05). The peripheral blood film in sickle cell anemia showed frequent sickled cells, Howell-Jolly bodies, Pappenheimer bodies, and occasional target cells. In HbS-0" thalassemia fewer sickled cells were seen, hypochromia and microcytosis were present, and target cells were plentiful. Howell-Jolly and Pappenheimer bodies were also noted. Electrophoresis Values (Table I I )
Hb fractions in HbS-(3'-thalassemia did not differ (P > 0.05) from the levels present in the five patients with sickle cell anemia. A minimal and just significant (P = 0.05) difference in mean corpuscular HbS concentration (MC [HbS] C) was present between
38
Steinberg and Dreiling
d c.l
V W
N +I
b,
m +I
m
m
m N
r+I
r-
M
d
'9 m +I
r-
d N M
N
+I
W
. r LL
39
Sickle Cell 0'-Thalassemia
m
c
2
m
t-
40
Steinberg and Dreiling
sickle cell anemia and HbS-Po-thalassemia, with values of 32.1 f 1.2% and 30.3 k 1.8%, respectively. In this small series, significant differences in HbAz levels were not detected in the comparison between HbSQO -thalassemia and sickle cell anemia; however, HbAz levels in HbS-0''-thalassemia were higher (P < 0.05) than those found in the patients with sickle cell anemia. Biosynthetic Data (Tables Ill and IV)
The synthesis of a and non-a chains in sickle cell anemia was balanced with a mean a:noniY ratio of 1 .OO 0.05. Incorporation of I4C-leucine was linear for both a and p chains over a 2-hr period. Beyond this there was a fall in the rate of labeling which was more marked for a than for P chain. The a:PSratio in HbS-0'-thalassemia was 1.83 0.90 with a range of 1.27-3.17. The a:non& ty) ratios in three individuals were 1.24, 1.42, and 2.87. In two patients with this condition previously reported in the literature, a:non-a ratios of 1.5 and 2.3 were found (13). No significant difference (P > 0.05) existed between a:PSsynthesis ratio in HbSPo-thalassemia and the a : n o n a ratio in HbS-b'-thalassemia.
*
*
DISCUSSION
0-thalassemia syndromes are clinically heterogeneous( 14). The existence of multiple alleles is apparent when heterozygotes for both fl-thalassemia and HbS are examined (7,15). In HbS-P-thalassemia, HbA may be absent, or may comprise 7-15% or 20-30% TABLE 111. Globin Subunit Synthesis in Sickle Cell P-Thalassemia and Sickle Cell Anemia Patient
HbS-Po -Thalassemia
HbS-P+-Thalassemia
Sickle Cell Anemia
f
1.42 2.87
1S.D.
F G H I J* K" L* Mean f lS.D.
M
Q Mean r 1S.D.
1.62 1.73 1.95 1.61 1.18 2.02 2.09 1.74 f 0.31 1.01
a:ps 1.27 1.33 1.55 1.58 3.17 1.83 f 0.90
-
N 0 P
*Related.
a:PA
1.24
A B C D E Mean
a:non-01
7.32 5.83 4.47 4.78 4.15 8.57 6.62 5.96 r 1.63
2.08 2.46 1.52 2.84 2.19 3.37 3.06 2.50 r 0.63 1.08 0.99 1 .00 0.95 0.96 1.00 f 0.05
65 55 56 56 68
74 83.0 90.5 81 .0 81.5
92 78
14 7 .0 8.7 14.6 13.3
0 0
6* 7t 87 9t lot Mean f 1S.D. 1* 2*
HbS(%)
I* 2* 3* 4* 5t Mean f 1S.D.
*Data of Bank et al. reference 13. ?Data of Gill and Schwartz, reference 16.
HbS-pO -Thalassemia
HbS-0'-Thalassemia
HbA(%)
21 33 21 22 22
Patient
1 .5 2.3
1.5 1.5 * 0.37
1.3
2.2 1.3 1.4
1.3 1.6 1.4 1.6 1.3 1.4 f 0.15
or:non-a
5.8 5.3 4.2 5.9 6.7 5.6 i 0.92
6.6 8.5 6.2 3.4 5 .0 5.9 f 1.9
a:@
2.3
1.6
1.6 1.9 1.9 * 0.25
2.0
2.3 1.8
1.8 2.4 2.2 3.0 1.7 2.2 f 0.52
a:$
8.0 8.0
8 .0 12.2 11.6 8.7 6.8
10.4 12.1 13.1 12.2 12.5
Hb(g/dl)
TABLE IV. Subunit Synthesis in Peripheral Blood Reticulocytes of Individuals with Sickle Cell p-Thalassemia as Reported in the Literature
42
Steinberg and Dreiling
of the total Hb( 15). HbS+?'-thalassemia may easily be detected by Hb electrophoresis. However, there may be considerable difficulty distinguishing HbS-0' -thalassemia from sickle cell anemia. We performed hematologic and biosynthetic studies and followed the course of a group of patients with HbS-0'-thalassemia and have compared them to individuals with "mild" sickle cell anemia (1,2). Clinically, the HbS-0'-thalassemics had minimal disability and lacked stigmata felt to characterize sickle cell anemia. Two had splenomegaly. Their Hb levels and reticulocyte counts resembled those of the sickle cell anemia group, indicating similar degrees of erythrocyte production and destruction. The major hematologic distinction between these patients was microcytosis and hypochromia in the HbS-POthalassemics, however, overlap did exist. Although HbAz levels are most often elevated in HbS-0'-thalassemia (7), this too is variable and the wide range of HbAz levels in sickle cell anemia may at times curtail the usefulness of this measurement. Subunit synthesis has been studied in HbS-0-thalassemia (13,16). The relative rates of a:non-ar or a:Ps synthesis were similar in the three groups of HbS-P-thalassemics evaluated (1 3,16) (Table IV). We found similarly unbalanced a:non-a synthesis ratios in both groups of HbS-0thalassemics. In sickle cell anemia, the a:PS ratio was approximately unity, a finding recently confirmed by Sarup and White (17). Our patients with HbS-0'-thalassemia had mild disease and we could not correlate the degree of unbalanced globin synthesis with clinical findings. Both Braverman and co-workers (18) and Friedman et al. (19), studying 0-thalassemia in blacks, found no relationship between disease severity and impairment of 0-chain production. Bank and co-workers were unable to relate the globin synthesis deficit to disease manifestations in HbS-0-thalassemia (1 3). As both our thalassemic groups had similar MCVs and MCHs, the Ps allele in HbS-0'-thalassemia may compensate for the absence of PA production, allowing more Hb tetramer, per cell, to be produced. The HbS-Po-thalassemia patients had lower Hb levels and higher reticulocyte counts than HbS-o'-thalassemics consistent with a greater increase in erythrocyte turnover. Our patients resembled those reported from Jamaica in most respects (7,20), except for our finding of a normal MCHC in HbS-0' -thalassemia. We measured erythrocyte indices electronically, the Hct being derived from MCV and red cell count. Hematocrit measurement by this technique has greater reproducibility than the microhematocrit (2 1). Thus the electronically determined MCHC might have greater accuracy in sickle cell diseases where excessive plasma trapping may occur and spuriously raise the Hct. It is possible that the paucity of symptoms in HbS-0'-thalassemia does not result soley from a low MCHC. A marked increased in HbF was found in only one patient with HbS-0' thalassemia. Family studies indicated that (S0)O-thalassemia was unlikely. Two additional patients, one with sickle cell anemia and one with HbS-0'-thalassemia had HbF levels above 10%.In both, the distribution of HbF was discrete, excluding the presence of the African type of hereditary persistence of fetal Hb, and thalassemia was excluded in the sickle cell anemia patient by finding balanced globin synthesis. We have evaluated 62 patients initially suspected of having sickle cell anemia. Further investigation showed five with HbS-0'-thalassemia, a prevalence of 8%. Our patients are highly selected, being adults and referred from throughout our state. Motulsky (22) estimated that the incidence of sickle cell anemia at birth was 1 :725 and in the total population at risk, 1 :1875. The prevalence of HbS-0'4halassemia was 1:3333, however, an estimate for HbS-0'-thalassemia could not be made due to the difficulty of separating these patients from those with sickle cell anemia (22). The black population of Mississippi is 815,770 (1970 U. S. census), and 435 cases of sickle cell anemia may be
43
Sickle Cell Do-Thalassemia
expected. Of this number, 35 cases of HbS-Do-thalassemia should occur, a prevalence o f approximately 1:23,000 blacks. For reasons stated above these data must be interpreted with caution. HbS-flO-thalassemiamay be confused with sickle cell anemia when clinical, electrophoretic, or hematologic criteria alone are used in diagnosis. The two methods of greatest reliability in distinguishing these conditions are family studies or the measurement of relative rates of globin synthesis.
ACKNOWLEDGMENTS
We thank J. Ross, R. Harris, W. Lovell, F. Childress, S. Wells and R. Williard for their skillful laboratory assistance. This work was supported by VA Research funds MRIS 8123.
REFERENCES 1. Serjeant, G. R., Richards, R., Barbor, P. R. H., and Milner, P. F. Relatively benign sickle-cell anaemia in 60 patients aged over 30 in the West Indies. Br. Med. J. 3:86-91 (1968). 2. Steinberg, M. H., Dreiling, B. J., Morrison, F. S., and Necheles, T. F. Mild sickle cell disease; clinical and laboratory studies. JAMA 224:3 17-321 (1973). 3. Kraus, L. M., Miyaji, T., Iuchi, I., and Kraus, A. P. Characterization of aZ3gluNH, in hemoglobin Memphis. Hemoglobin Memphis/& a new variant of molecular disease. Biochemistry 5:3701-3708 (1966). 4. Ranney, H. M. Interactions of other hemoglobin variants with sickle-cell hemoglobin. N. Engl. J. Med. 283:1462-1463 (1970). 5 . Conconi, F., Bargellesi, A., Pontremoli, S., Vigi, V., Volpato, S., and Gaburro, D. Absence of pglobin synthesis and excess of a-globin synthesis in homozygous p-thalassemic subjects from the Ferrara region. Nature 217:259-260 (1968). 6. Benz, E. J., Jr., Swerdlow, P. S., and Forget, B. G. Absence of functional messenger RNA activity for beta globin chain synthesis in p'-thalassemia. Blood 45: 1-10 (1975). 7. Serjeant, G. R., Ashcroft, M. T., Serjeant, B. E., and Milner, P. F. The clinical features of sickle cell/@thalassemia in Jamaica. Br. J. Haematol. 24:19-30 (1973). 8. Dacie, J. V., and Lewis, S. M. Practical haematology. Grune & Stratton, New York (1968). 9. Williard, R.F., Lovell, W. J., Dreiling, B. J., and Steinberg, M. H. Electrophoresis of hemoglobin on polyacrylamide gels. Precise method for measurement of hemoglobin A,. Clin. Chem. 19~1082-1084 (1973). 10. Marder, V. J., and Conley, C. L. Electrophoresis of hemoglobin on agar gels; frequency of hemoglobin Din a Negro population. Bull. Johns Hopkins Hosp. 105:77-88 (1 959). 11. Kan, Y. W., Schwartz, E., and Nathan, D. G. Globin chain synthesis in the alpha-thalassemia syndromes. J. Clin. Invest. 47:2515-2522 (1968). 12. Clegg, J. B., Naughton, M. A., and Weatherall, D. J. Abnormal human hemoglobins: separation and characterization of the 01 and p chains by chromatography and the determination of two new variants, hb Chesapeake and hbJ(Bangkok). J. Mol. Biol. 19:91-108 (1966). 13. Bank, A., Dow, L. W., Farace, M. G., O'Donnell, J. V., Ford, S., and Natta, C. Changes in globin synthesis with erythroid cell maturation in sickle thalassemia. Blood 41 :353-357 (1973). 14. Weatherall, D. J., and Clegg, J. B. The Thalassaemia Syndromes. Blackwell Scientific Publications, Oxford (1972). 15. Pearson, H. A. Hemoglobin S-thalassemia syndrome in Negro children. Ann. N. Y.Acad. Sci. 165~83-92 (1969). 16. Gill, F. M., and Schwartz, E. Synthesis of globin chains in sickle p-thalassemia. J. Clin. Invest. 52:709-714 (1973). 17. Sarup, B. M., and White, J. M. The synthesis of globin peptide chains in sickle-cell disease. Br. J. Haematol. 27 :153-1 61 (1974)
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18. Braverman, A. S., McCurdy, P. R., Manos, O., and Sherman, A. Homozygous beta thalassemia in American blacks: The problem of mild thalassemia. J. Lab. Clin. Med. 81:857-866 (1973). 19. Friedman, S., Oski, F. A., and Schwartz, E. Bone marrow and peripheral blood globin synthesis in an American family with beta thalassemia. Blood 39:785-793 (1972). 20. Serjeant, G. R., and Serjeant, B. E. A comparison of erythrocyte characteristics in sickle cell syndromes in Jamaica. Br. J. Haematol. 23:205-213 (1973). 21. Britten, G. M., Brecher, G., and Johnson, C. A. Evaluation of the Coulter Model S. Am. J. Clin. Pathol. 52:679-689 (1969). 22. Motulsky, A. G. Frequency of sickling disorders in U. S. blacks. N. Engl. J. Med. 288:31-33 (1 973).
CLINICAL, HEMATOLOGIC AND BIOSYNTHETIC STUDIES IN SICKLE CELL-Po-THALASSEMIA:A Comparison With Sickle Cell Anemia Martin H. Steinberg, M. D., and Bernard J. Dreiling, M. D. The Hematology Research Laboratory, Jackson VA Hospital, and Department of Medicine, Division o f Hematology, University of Mississippi School o f Medicine, Jackson, Mississippi
The diseases commonly confused with sickle cell anemia include sickle cell 0-thalassemia in which synthesis of PA-chains are completely suppressed (HbS/3'-thalassemia). We obtained hematologic measurements and studied globin biosynthesis in five patients with this disorder and compared the results with those obtained in five patients with "mild" sickle cell anemia and seven individuals with sickle cell-P-thalassemia having hemoglobin A levels of 20-30% (HbS;O+-thalassemia). A distinction between HbS-Po-thalassemia and sickle cell anemia was not always possible on clinical, hematologic, or electrophoretic grounds. Thalassemia heterozygotes had hypochromia and microcytosis, not generally a feature of sickle cell anemia, although overlap of values did exist. The ratio of CY to non-a, or a to PS-chains in sickle cell anemia approximated unity, whereas patients with HbSP'-thalassemia had a deficit of 0-chain production relative to that of the a-chain. The differentiation of HbS-Po-thalassemia and sickle cell anemia can be best made on the basis of family or biosynthetic study. We estimated the regional prevalence of HbS-Po-thalassemia to be 1:23,000 of the black population. Key words: thalassemia, hemoglobin S, sickle hemoglobin, hemolysis
INTRODUCTION Marked variability exists in the clinical course of patients with sickle cell anemia (1,2). This may be due, in part, to electrophoretic and hematologic mimics of this disease (2-4). Among the conditions which may be confused with sickle cell anemia is sickle cell 0-thalassemia in which there is total suppression of PA globin synthesis (HbS-0'thalassemia) ( 5 ) . Absence of PA chain mRNA has been found in this disorder (6). In this phenocopy of sickle cell anemia, hemoglobin (Hb) A is absent and clinical symptoms may be minimal, the latter perhaps related to a low mean corpuscular HbS concentration (7). We report our studies of globin biosynthesis, as well as hematologic and clinical findings in HbS-Po-thalassemia. We have compared these findings to those noted in a Address reprint requests to M. H. Steinberg, M.D., VA Hospital, 1500 E. Woodrow Wilson, Jackson, Mississippi 392 16.
35
0 1976 Alan R. Liss, Inc., 150 Fifth Avenue, New York, N . Y. 10011
36
Steinberg and Dreiling
group of patients with “mild” sickle cell anemia as well as a group of individuals with HbS(3-thalassernia in which some PA chain is produced (HbS- P’-thalassemia). Our findings are also contrasted with data previously published by other investigators. MATERIALS AND METHODS
Routine hematologic techniques were used (8). Erythrocyte indices were determined using a Coulter model S electronic cell counter. Electrophoresis at pH 8.4, with Tris-EDTA-borate buffer, was done in polyacrylamide gels, the hemoglobin fractions were eluted from the gels, and the fractions were quantified spectrophotometrically (9). HbS levels were taken as the difference between total Hb concentration and levels of Hbs A, A 2 , and F. Electrophoresis at pH 6.1, sodium citrate-citric acid buffer, was done in agar gels (10). HbF levels were measured by alkali denaturation (9) and the distribution of HbF in erythrocytes was determined by the acid elution technique (9). Globin chain biosynthesis was estimated by measuring the incorporation of l4 C-leucine into the newly synthesized PA, ps, y, and a chains of peripheral blood reticulocytes. 4-6 ml of blood were incubated with dextrose and l4 C-leucine at 37°C in a metabolic incubator for time intervals of 15 min-2 hrs (1 1). The globin was separated and resolved into its constituent chains on columns of carboxymethyl cellulose using a sodium phosphate gradient in 8 M urea-mercaptoethanol buffer, pH 6.7(12). Fractions were collected and 1 ml aliquots were mixed with 15 ml of Aquasol and counted in a liquid scintillation spectrometer at 40% efficiency for 14C. The total radioactivity incorporated into each globin peak was used to compute synthesis ratios. RESULTS Clinical Findings
A. HbS-0’-thalassemia. This 28-yr old man was entirely well. He was employed as an X-ray technician, and had no pain crises and normal growth and development. Physical examination was normal, save for splenomegaly. Serum iron was normal. This 49-yr old man had rare episodes of bone and joint pain. He had one attack of B. pneumonia and’a probable pulmonary infarction. A cholecystectomy had been done in the past. Cardiac evaluation showed aortic and mitral insufficiency on the basis of rheumatic heart disease. Aortic valve surgery was successfully undertaken. C. This patient was 29 yr old and suffered from occasional pain crises requiring hospitalization. He had a probable pulmonary infarction and a cholecystectomy had been done. The spleen was not enlarged. His mother had sickle cell trait. D. This 45-yr old woman had no symptoms of sickling diseases. She had two normal pregnancies and deliveries. Her examination was normal except for splenomegaly. One daughter had sickle cell trait and another, (3-thalassemia trait. This patient was a 21-yr old woman who suffered from bone, joint and abdominal E. pains which occurred frequently but were not severe or disabling. Physical examination was normal except for long thin fingers and a soft ejection systolic murmur. Iron and iron-binding capacity were normal. The mother had sickle cell trait. F. HbS-P+-thalassemia. A 22-yr old student was well until he had a mild painful crisis at age 21. At that time he recalled having occasional knee pains every 3-4 mo. His physical examination was normal.
37
Sickle Cell (3' -Thalassemia
G. A 38-yr old woman was seen at the time of her first pregnancy. She was healthy but had siblings with typical sickle cell anemia. No abnormalities were found on physical , examination. H. A 33-yr old man was asymptomatic except for fleeting polyarthralgias. Physical examination was normal. I. A 43-yr old man was retired from the U. S. Army where a diagnosis of sicklethalassemia was made shortly before retirement. He had joint pains and occasional chest or abdominal pain. The physical examination was normal.. J. This patient was 15 mo old and a son of K. He has had normal growth and development to date, and has not had symptoms or signs referable to sickle cell disease. The spleen was palpable 3 cm below the left costal margin. K. This 40-yr old man had a left nephrectomy for hematuria in 1961. There was no history of pain crises and the spleen was not enlarged. L. This patient, son of K, was 10.5 yrs. old. He had occasional abdominal and bone pains which were not severe. His physical examination was normal. M. Sickle Cell Anemia. This man was a 28-yr old truck driver who had rare episodes of bone pain. He was hospitalized once for pneumonia and on another occasion for hepatitis. On examination he was 73" tall and weighed 167 lb. N. This patient was 27 yr old and had mild bone and joint pains about twice yearly. He was employed part-time. His physical examination was normal. 0. This patient was a 24-yr old woman, hospitalized once for a pain crisis. She had one normal pregnancy. She was 62" tall and weighed 125 lbs and her examination was normal, except for an ejection systolic heart murmur. P. This 42-yr old woman was well except for rare episodes of hip pain. She had one normal pregnancy and one miscarriage. A cholecystectomy had been done in the past. On examination she had no abnormalities. This patient was 28 yr old and had never suffered from pain crises. He recently Q. developed bilateral lower leg ulcers. The examination was otherwise normal. Hematologic Values (Table I )
Significant differences (P < 0.05) existed between the mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of both HbS-0-thalassemia groups and patients with sickle cell anemia. The reticulocyte counts in sickle cell anemia and HbS-Po thalassemia were similar (P > 0.05). A significant difference in Hb concentration or hematocrit (Hct) was not present (P > 0.05) between HbS-Do-thalassemia and sickle cell anemia. The mean corpuscular hemoglobin concentration (MCHC) was similar in thalassemia patients but lower in HbS-(3' -thalassemia than in sickle cell anemia (P < 0.05). The peripheral blood film in sickle cell anemia showed frequent sickled cells, Howell-Jolly bodies, Pappenheimer bodies, and occasional target cells. In HbS-0" thalassemia fewer sickled cells were seen, hypochromia and microcytosis were present, and target cells were plentiful. Howell-Jolly and Pappenheimer bodies were also noted. Electrophoresis Values (Table I I )
Hb fractions in HbS-(3'-thalassemia did not differ (P > 0.05) from the levels present in the five patients with sickle cell anemia. A minimal and just significant (P = 0.05) difference in mean corpuscular HbS concentration (MC [HbS] C) was present between
38
Steinberg and Dreiling
d c.l
V W
N +I
b,
m +I
m
m
m N
r+I
r-
M
d
'9 m +I
r-
d N M
N
+I
W
. r LL
39
Sickle Cell 0'-Thalassemia
m
c
2
m
t-
40
Steinberg and Dreiling
sickle cell anemia and HbS-Po-thalassemia, with values of 32.1 f 1.2% and 30.3 k 1.8%, respectively. In this small series, significant differences in HbAz levels were not detected in the comparison between HbSQO -thalassemia and sickle cell anemia; however, HbAz levels in HbS-0''-thalassemia were higher (P < 0.05) than those found in the patients with sickle cell anemia. Biosynthetic Data (Tables Ill and IV)
The synthesis of a and non-a chains in sickle cell anemia was balanced with a mean a:noniY ratio of 1 .OO 0.05. Incorporation of I4C-leucine was linear for both a and p chains over a 2-hr period. Beyond this there was a fall in the rate of labeling which was more marked for a than for P chain. The a:PSratio in HbS-0'-thalassemia was 1.83 0.90 with a range of 1.27-3.17. The a:non& ty) ratios in three individuals were 1.24, 1.42, and 2.87. In two patients with this condition previously reported in the literature, a:non-a ratios of 1.5 and 2.3 were found (13). No significant difference (P > 0.05) existed between a:PSsynthesis ratio in HbSPo-thalassemia and the a : n o n a ratio in HbS-b'-thalassemia.
*
*
DISCUSSION
0-thalassemia syndromes are clinically heterogeneous( 14). The existence of multiple alleles is apparent when heterozygotes for both fl-thalassemia and HbS are examined (7,15). In HbS-P-thalassemia, HbA may be absent, or may comprise 7-15% or 20-30% TABLE 111. Globin Subunit Synthesis in Sickle Cell P-Thalassemia and Sickle Cell Anemia Patient
HbS-Po -Thalassemia
HbS-P+-Thalassemia
Sickle Cell Anemia
f
1.42 2.87
1S.D.
F G H I J* K" L* Mean f lS.D.
M
Q Mean r 1S.D.
1.62 1.73 1.95 1.61 1.18 2.02 2.09 1.74 f 0.31 1.01
a:ps 1.27 1.33 1.55 1.58 3.17 1.83 f 0.90
-
N 0 P
*Related.
a:PA
1.24
A B C D E Mean
a:non-01
7.32 5.83 4.47 4.78 4.15 8.57 6.62 5.96 r 1.63
2.08 2.46 1.52 2.84 2.19 3.37 3.06 2.50 r 0.63 1.08 0.99 1 .00 0.95 0.96 1.00 f 0.05
65 55 56 56 68
74 83.0 90.5 81 .0 81.5
92 78
14 7 .0 8.7 14.6 13.3
0 0
6* 7t 87 9t lot Mean f 1S.D. 1* 2*
HbS(%)
I* 2* 3* 4* 5t Mean f 1S.D.
*Data of Bank et al. reference 13. ?Data of Gill and Schwartz, reference 16.
HbS-pO -Thalassemia
HbS-0'-Thalassemia
HbA(%)
21 33 21 22 22
Patient
1 .5 2.3
1.5 1.5 * 0.37
1.3
2.2 1.3 1.4
1.3 1.6 1.4 1.6 1.3 1.4 f 0.15
or:non-a
5.8 5.3 4.2 5.9 6.7 5.6 i 0.92
6.6 8.5 6.2 3.4 5 .0 5.9 f 1.9
a:@
2.3
1.6
1.6 1.9 1.9 * 0.25
2.0
2.3 1.8
1.8 2.4 2.2 3.0 1.7 2.2 f 0.52
a:$
8.0 8.0
8 .0 12.2 11.6 8.7 6.8
10.4 12.1 13.1 12.2 12.5
Hb(g/dl)
TABLE IV. Subunit Synthesis in Peripheral Blood Reticulocytes of Individuals with Sickle Cell p-Thalassemia as Reported in the Literature
42
Steinberg and Dreiling
of the total Hb( 15). HbS+?'-thalassemia may easily be detected by Hb electrophoresis. However, there may be considerable difficulty distinguishing HbS-0' -thalassemia from sickle cell anemia. We performed hematologic and biosynthetic studies and followed the course of a group of patients with HbS-0'-thalassemia and have compared them to individuals with "mild" sickle cell anemia (1,2). Clinically, the HbS-0'-thalassemics had minimal disability and lacked stigmata felt to characterize sickle cell anemia. Two had splenomegaly. Their Hb levels and reticulocyte counts resembled those of the sickle cell anemia group, indicating similar degrees of erythrocyte production and destruction. The major hematologic distinction between these patients was microcytosis and hypochromia in the HbS-POthalassemics, however, overlap did exist. Although HbAz levels are most often elevated in HbS-0'-thalassemia (7), this too is variable and the wide range of HbAz levels in sickle cell anemia may at times curtail the usefulness of this measurement. Subunit synthesis has been studied in HbS-0-thalassemia (13,16). The relative rates of a:non-ar or a:Ps synthesis were similar in the three groups of HbS-P-thalassemics evaluated (1 3,16) (Table IV). We found similarly unbalanced a:non-a synthesis ratios in both groups of HbS-0thalassemics. In sickle cell anemia, the a:PS ratio was approximately unity, a finding recently confirmed by Sarup and White (17). Our patients with HbS-0'-thalassemia had mild disease and we could not correlate the degree of unbalanced globin synthesis with clinical findings. Both Braverman and co-workers (18) and Friedman et al. (19), studying 0-thalassemia in blacks, found no relationship between disease severity and impairment of 0-chain production. Bank and co-workers were unable to relate the globin synthesis deficit to disease manifestations in HbS-0-thalassemia (1 3). As both our thalassemic groups had similar MCVs and MCHs, the Ps allele in HbS-0'-thalassemia may compensate for the absence of PA production, allowing more Hb tetramer, per cell, to be produced. The HbS-Po-thalassemia patients had lower Hb levels and higher reticulocyte counts than HbS-o'-thalassemics consistent with a greater increase in erythrocyte turnover. Our patients resembled those reported from Jamaica in most respects (7,20), except for our finding of a normal MCHC in HbS-0' -thalassemia. We measured erythrocyte indices electronically, the Hct being derived from MCV and red cell count. Hematocrit measurement by this technique has greater reproducibility than the microhematocrit (2 1). Thus the electronically determined MCHC might have greater accuracy in sickle cell diseases where excessive plasma trapping may occur and spuriously raise the Hct. It is possible that the paucity of symptoms in HbS-0'-thalassemia does not result soley from a low MCHC. A marked increased in HbF was found in only one patient with HbS-0' thalassemia. Family studies indicated that (S0)O-thalassemia was unlikely. Two additional patients, one with sickle cell anemia and one with HbS-0'-thalassemia had HbF levels above 10%.In both, the distribution of HbF was discrete, excluding the presence of the African type of hereditary persistence of fetal Hb, and thalassemia was excluded in the sickle cell anemia patient by finding balanced globin synthesis. We have evaluated 62 patients initially suspected of having sickle cell anemia. Further investigation showed five with HbS-0'-thalassemia, a prevalence of 8%. Our patients are highly selected, being adults and referred from throughout our state. Motulsky (22) estimated that the incidence of sickle cell anemia at birth was 1 :725 and in the total population at risk, 1 :1875. The prevalence of HbS-0'4halassemia was 1:3333, however, an estimate for HbS-0'-thalassemia could not be made due to the difficulty of separating these patients from those with sickle cell anemia (22). The black population of Mississippi is 815,770 (1970 U. S. census), and 435 cases of sickle cell anemia may be
43
Sickle Cell Do-Thalassemia
expected. Of this number, 35 cases of HbS-Do-thalassemia should occur, a prevalence o f approximately 1:23,000 blacks. For reasons stated above these data must be interpreted with caution. HbS-flO-thalassemiamay be confused with sickle cell anemia when clinical, electrophoretic, or hematologic criteria alone are used in diagnosis. The two methods of greatest reliability in distinguishing these conditions are family studies or the measurement of relative rates of globin synthesis.
ACKNOWLEDGMENTS
We thank J. Ross, R. Harris, W. Lovell, F. Childress, S. Wells and R. Williard for their skillful laboratory assistance. This work was supported by VA Research funds MRIS 8123.
REFERENCES 1. Serjeant, G. R., Richards, R., Barbor, P. R. H., and Milner, P. F. Relatively benign sickle-cell anaemia in 60 patients aged over 30 in the West Indies. Br. Med. J. 3:86-91 (1968). 2. Steinberg, M. H., Dreiling, B. J., Morrison, F. S., and Necheles, T. F. Mild sickle cell disease; clinical and laboratory studies. JAMA 224:3 17-321 (1973). 3. Kraus, L. M., Miyaji, T., Iuchi, I., and Kraus, A. P. Characterization of aZ3gluNH, in hemoglobin Memphis. Hemoglobin Memphis/& a new variant of molecular disease. Biochemistry 5:3701-3708 (1966). 4. Ranney, H. M. Interactions of other hemoglobin variants with sickle-cell hemoglobin. N. Engl. J. Med. 283:1462-1463 (1970). 5 . Conconi, F., Bargellesi, A., Pontremoli, S., Vigi, V., Volpato, S., and Gaburro, D. Absence of pglobin synthesis and excess of a-globin synthesis in homozygous p-thalassemic subjects from the Ferrara region. Nature 217:259-260 (1968). 6. Benz, E. J., Jr., Swerdlow, P. S., and Forget, B. G. Absence of functional messenger RNA activity for beta globin chain synthesis in p'-thalassemia. Blood 45: 1-10 (1975). 7. Serjeant, G. R., Ashcroft, M. T., Serjeant, B. E., and Milner, P. F. The clinical features of sickle cell/@thalassemia in Jamaica. Br. J. Haematol. 24:19-30 (1973). 8. Dacie, J. V., and Lewis, S. M. Practical haematology. Grune & Stratton, New York (1968). 9. Williard, R.F., Lovell, W. J., Dreiling, B. J., and Steinberg, M. H. Electrophoresis of hemoglobin on polyacrylamide gels. Precise method for measurement of hemoglobin A,. Clin. Chem. 19~1082-1084 (1973). 10. Marder, V. J., and Conley, C. L. Electrophoresis of hemoglobin on agar gels; frequency of hemoglobin Din a Negro population. Bull. Johns Hopkins Hosp. 105:77-88 (1 959). 11. Kan, Y. W., Schwartz, E., and Nathan, D. G. Globin chain synthesis in the alpha-thalassemia syndromes. J. Clin. Invest. 47:2515-2522 (1968). 12. Clegg, J. B., Naughton, M. A., and Weatherall, D. J. Abnormal human hemoglobins: separation and characterization of the 01 and p chains by chromatography and the determination of two new variants, hb Chesapeake and hbJ(Bangkok). J. Mol. Biol. 19:91-108 (1966). 13. Bank, A., Dow, L. W., Farace, M. G., O'Donnell, J. V., Ford, S., and Natta, C. Changes in globin synthesis with erythroid cell maturation in sickle thalassemia. Blood 41 :353-357 (1973). 14. Weatherall, D. J., and Clegg, J. B. The Thalassaemia Syndromes. Blackwell Scientific Publications, Oxford (1972). 15. Pearson, H. A. Hemoglobin S-thalassemia syndrome in Negro children. Ann. N. Y.Acad. Sci. 165~83-92 (1969). 16. Gill, F. M., and Schwartz, E. Synthesis of globin chains in sickle p-thalassemia. J. Clin. Invest. 52:709-714 (1973). 17. Sarup, B. M., and White, J. M. The synthesis of globin peptide chains in sickle-cell disease. Br. J. Haematol. 27 :153-1 61 (1974)
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18. Braverman, A. S., McCurdy, P. R., Manos, O., and Sherman, A. Homozygous beta thalassemia in American blacks: The problem of mild thalassemia. J. Lab. Clin. Med. 81:857-866 (1973). 19. Friedman, S., Oski, F. A., and Schwartz, E. Bone marrow and peripheral blood globin synthesis in an American family with beta thalassemia. Blood 39:785-793 (1972). 20. Serjeant, G. R., and Serjeant, B. E. A comparison of erythrocyte characteristics in sickle cell syndromes in Jamaica. Br. J. Haematol. 23:205-213 (1973). 21. Britten, G. M., Brecher, G., and Johnson, C. A. Evaluation of the Coulter Model S. Am. J. Clin. Pathol. 52:679-689 (1969). 22. Motulsky, A. G. Frequency of sickling disorders in U. S. blacks. N. Engl. J. Med. 288:31-33 (1 973).
