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Hemoglobin H disease in two Iranian families
CLINICA
CHIMICA
HEMOGLOBIN
ACTA
H DISEASE
381
IN TWO IRANIAN
FAMILIES
SUMMARY
Two Iranian families with hemoglobin H disease are described. In the first family two children have Hb H disease and the parents and a third child show signs of a thalassemia minor. In the second family also two members have Hb H disease, but the offsprings are normal.
Hemoglobin H disease first discovered by Rigas’ in Greece has been observed in different parts of the world. It is an unusual abnormal hemoglobin characterized by the presence of an electrophoretically fast moving hemoglobin and inclusion body formation in the erythrocyte when incubating the red cells with Brilliant Cresyl Blue. In this communication two families with hemoglobin H disease are reported. It is noteworthy to indicate that both families come from the northern part of Iran (Caspian sea coast) where hemoglobinopathies are frequently observed2. The propositus of one of these families presented with repeated subdural hematoma, which could be attributed to infarction by the precipitation of denatured hemoglobin H in the arterioles. Another case of Hb H disease has been reported by Aghaies in an Iranian Jewish family. MATERIALS
AND
METHODS
Routine hematologic~ examinations were performed by standard technique@. Alk~i-resistant hemoglobin was estimated by the method of Singer et &.Q. Inclusion bodies in erythrocyte were demonstrated by incubating a few drops of the patient’s blood in I ml of filtered 1% Brilliant Cresyl Blue in 0.85% NaCl and 3.5% sodium citrate for 20 min at 37” and examining the dried smears with oil immersion. Heat denaturation was performed by the method of Dacie. Hemoglobin electrophoresis was performed on cellulose acetate according to Graham and Grunbaums. Starch gel electrophoresis was performed in a discontinuous buffer system as previously describede, paper electrophoresis at pH 6.5 on Whatman 3 mm in a Shandon Vertical Apparatus. Finger print of purified fractions was performed according to IngramlO and Baglionill in a Michl type tank12. C&n. Chim. Acta, 20
(1968) 381-385
RAHBAH
Fig. 1. Starch gel electrophorcsis of hemolysate 2 : hemoglobin A-H; 3 : hemoglobin -1-S.
Case histories The propositus
of the first family
of propositus.
I
: hemoglobin
M. Gh. is a boy of II
4. J. (Iran) (ref. 2)
years presenting
;
with
anemia and splenomegalia. The erythrocyte morphology showed hypochromia, anisocytosis, poikilocytosis and microcytosis. R.B.C. 3800000, Hb 450/i, reticulocyte 3 y/o,osmotic fragility was reduced in hypotonic salt solution, foetal hemoglobin was
less than z~/o. Electrophoresis of the patient hemolysate on cellulose acetate and starch gel (Fig. I) showed a fast moving hemoglobin in addition to normal hemoglobin A which was estimated to be approximately 12% of total hemoglobins. The electrophoretic position of the fast moving hemoglobin at pH 8.6 was on
Fig. 2. Paper electrophoresis migrates to the cathode.
at pH 6.5. Note that hemoglobin
H remains at the origin and Hh X
HEMOGLOBIK
Fig.
H
3, Inclusion
DISEASE
body
383
formation
in the red cells after incubation
hemoglobin I or H. For the differentiation, was characteristic of Hb H. In this condition
in Brilliant
Cresyl
Blue
paper electrophoresis at pH 6.5 (Fig. 2) the hemoglobin H remains at the origin
and all other hemoglobins (i.e. Hb I) migrate to the cathode. On incubation of red cells with Brilliant Cresyl Blue many red cells containing inclusion bodies were observed (Fig. 3) and the heat denaturation was positive after I h at 50’. Thus, the diagnosis of hemoglobin H disease was confirmed. Other members of the family were investigated and the following results were
Fig. 4. Fingerprint
of purified
hemoglobin
H.
RAHBAR
384
obtained. The father of the propositus, 52 years old, shows a mild anemia, microcytosis and rare red cells with inclusion bodies were observed. No hemoglobin H could be detected on electrophoresis of the father’s hemolysate. The mother and brother of the propositus were apparently healthy but hypochromia and microcytosis were seen. In peripheral blood smears, very few erythrocytes with inclusion bodies were found, but no hemoglobin H could be detected on electrophoretic examination of their hemolysates. The youngest child of the family, 2 years old, had anemia and splenomegalia and red cell morphology showed hypochromia, anisocytosis, poikilocytosis and microcytosis and many red cells with inclusion bodies were observed; hemoglobin H in the same amount as in the propositus was detected on electrophoresis. Heat denaturation was also positive and 2% of alkali-resistant hemoglobin was estimated in the child’s hemolysate. No attempt was made to investigate the co-existence of Bart’s hemoglobin in the hemolysate. The propositus of the second family N.E., aged 38 years, was admitted to the Department of Neuro-surgery with the diagnosis of subdural hematoma which has recurred three times in the last four years. The examination of the patient for coagulation disorders was negative but he presented a mild hemolysis and moderate splenomegalia. Peripheral blood examination showed microcytosis and anisocytosis hypochromia. Incubation of the red cells with Brilliant Cresyl Blue showed characteristic inclusion bodies in almost all the red cells. Osmotic fragility of the red cells was reduced. Hemoglobin electrophoresis revealed a fast moving fraction at pH 8.6, approx. 20% of total hemoglobin was identified to be Hb H. Electrophoresis at pH 6.5 and fingerprint of the purified abnormal fraction was characteristic for Hb H. The patient was hospitalized under special care and treated with good recovery after two months. The patient has three children. The examination of the children for Hb H was negative but a younger brother of the propositus, aged 30, showed inclusion bodies in red cells and Hb H in electrophoretic examination in the same concentration without any other symptom. DISCUSSION
The definition of thalassemia is the failure to produce enough normal hemoglobin A (ct.&) and this failure may be due to inability to produce either cc or t!lpolypeptide chain. In the /? thalassemia the production of /I chain is suppressed, and in the homozygous state both loci for /I chain synthesis are affected. In the cc thalassemia the synthesis of a chain is suppressed; in cc thalassemia minor one locus is affected. The inhibition of tc chain production is compensated by other loci and enough a chain is produced so that no symptoms are caused, and the carriers of the tc thalassemia minor are clinically symptomless. As Ingram and Stretton7 suggested, the inhibition of tc chain synthesis would be reflected not only in inhibition of Hb A production, but also in the synthesis of minor hemoglobin components. In the case of Hb H disease, the abnormal hemoglobin is composed of four p polypeptide chains’. It is related to a thalassemia, representing a relative excess of @ chain production. The excess /? chains form tetramers (&). Hemoglobin Bart’s, a tetramer composed only of y chain (rl), is also related to Clin. Chim. Acta,
20 (1968) 381-385
HEMOGLOBIN
H
385
DISEASE
a deficiency of CIchain production. The inheritance of Hb H disease is not only by simple co-dominance, but a third silent gene in combination with a thalassemia gene is suggested to be involved for revealing the Hb H disease*. This is in accord with the observation of the parents of our patients with Hb H disease, where no appreciable quantity of Hb H could be detected in the parents’ hemolysate, but two of the children showed significant quantities of Hb H. The recurrent subdural hemorrhage in the propositus of the second family, a young man of 38, may be due to infarction caused by denatured Hb H. Infarction of the other organs in patient with Hb H disease has been reported elsewhere13. REFERENCES I 2 3 4 5 6
7 8 g IO II
12 13
D. A. RIGAS. R. D. KOLER AND E. E. OSGOOD, 1. Lab. Clin. Med., 47 (1~56) 51. S. RAHBAR, D. BEALE, W. A. ISAAC AND H. LE~MANN, Brit. Med. j.; i (;967)-674. M. M. WINTROBE, Clinical Haematology, 5th, ed., Lea & Febiger, Philadelphia, 1961. K. SINGER, A. I. CHERNOFF AND L. SINGER, Blood, 6 (1951) 413. I. L. GRAHAM AND B. W. GRUNBAUM, Am. I. Clin. Pathol.. 39 (1963) 567. S. RAHBAR, Acta Biochim. Iranica. 3 (1965) 54. V. M. INGRAM AND A. 0. W. STRETTON. Nature, 184 (1959) 1903. H. LEHMANN, in Man’s Hemoglobins, North Holland, Amsterdam, 1966. ESTER AGHAIE, Acta Med. Iranica, 8 (1965) 84. V. M. INGRAM, Biochim. Biophys. Acta, 28 (1958) 539. C. BAGLIONI, Biochim. Biophys. Acta, 48 (1961) 392. H. MICHL, Monatsh., 82 (1951) 489. T. F. NECHELES. M. CATES, R. G. SHEEHAN AND H. J. MEYER, Blood, 28 (1966) 501. Clin. Chim. Acta, 20 (1968) 381~385
CHIMICA
HEMOGLOBIN
ACTA
H DISEASE
381
IN TWO IRANIAN
FAMILIES
SUMMARY
Two Iranian families with hemoglobin H disease are described. In the first family two children have Hb H disease and the parents and a third child show signs of a thalassemia minor. In the second family also two members have Hb H disease, but the offsprings are normal.
Hemoglobin H disease first discovered by Rigas’ in Greece has been observed in different parts of the world. It is an unusual abnormal hemoglobin characterized by the presence of an electrophoretically fast moving hemoglobin and inclusion body formation in the erythrocyte when incubating the red cells with Brilliant Cresyl Blue. In this communication two families with hemoglobin H disease are reported. It is noteworthy to indicate that both families come from the northern part of Iran (Caspian sea coast) where hemoglobinopathies are frequently observed2. The propositus of one of these families presented with repeated subdural hematoma, which could be attributed to infarction by the precipitation of denatured hemoglobin H in the arterioles. Another case of Hb H disease has been reported by Aghaies in an Iranian Jewish family. MATERIALS
AND
METHODS
Routine hematologic~ examinations were performed by standard technique@. Alk~i-resistant hemoglobin was estimated by the method of Singer et &.Q. Inclusion bodies in erythrocyte were demonstrated by incubating a few drops of the patient’s blood in I ml of filtered 1% Brilliant Cresyl Blue in 0.85% NaCl and 3.5% sodium citrate for 20 min at 37” and examining the dried smears with oil immersion. Heat denaturation was performed by the method of Dacie. Hemoglobin electrophoresis was performed on cellulose acetate according to Graham and Grunbaums. Starch gel electrophoresis was performed in a discontinuous buffer system as previously describede, paper electrophoresis at pH 6.5 on Whatman 3 mm in a Shandon Vertical Apparatus. Finger print of purified fractions was performed according to IngramlO and Baglionill in a Michl type tank12. C&n. Chim. Acta, 20
(1968) 381-385
RAHBAH
Fig. 1. Starch gel electrophorcsis of hemolysate 2 : hemoglobin A-H; 3 : hemoglobin -1-S.
Case histories The propositus
of the first family
of propositus.
I
: hemoglobin
M. Gh. is a boy of II
4. J. (Iran) (ref. 2)
years presenting
;
with
anemia and splenomegalia. The erythrocyte morphology showed hypochromia, anisocytosis, poikilocytosis and microcytosis. R.B.C. 3800000, Hb 450/i, reticulocyte 3 y/o,osmotic fragility was reduced in hypotonic salt solution, foetal hemoglobin was
less than z~/o. Electrophoresis of the patient hemolysate on cellulose acetate and starch gel (Fig. I) showed a fast moving hemoglobin in addition to normal hemoglobin A which was estimated to be approximately 12% of total hemoglobins. The electrophoretic position of the fast moving hemoglobin at pH 8.6 was on
Fig. 2. Paper electrophoresis migrates to the cathode.
at pH 6.5. Note that hemoglobin
H remains at the origin and Hh X
HEMOGLOBIK
Fig.
H
3, Inclusion
DISEASE
body
383
formation
in the red cells after incubation
hemoglobin I or H. For the differentiation, was characteristic of Hb H. In this condition
in Brilliant
Cresyl
Blue
paper electrophoresis at pH 6.5 (Fig. 2) the hemoglobin H remains at the origin
and all other hemoglobins (i.e. Hb I) migrate to the cathode. On incubation of red cells with Brilliant Cresyl Blue many red cells containing inclusion bodies were observed (Fig. 3) and the heat denaturation was positive after I h at 50’. Thus, the diagnosis of hemoglobin H disease was confirmed. Other members of the family were investigated and the following results were
Fig. 4. Fingerprint
of purified
hemoglobin
H.
RAHBAR
384
obtained. The father of the propositus, 52 years old, shows a mild anemia, microcytosis and rare red cells with inclusion bodies were observed. No hemoglobin H could be detected on electrophoresis of the father’s hemolysate. The mother and brother of the propositus were apparently healthy but hypochromia and microcytosis were seen. In peripheral blood smears, very few erythrocytes with inclusion bodies were found, but no hemoglobin H could be detected on electrophoretic examination of their hemolysates. The youngest child of the family, 2 years old, had anemia and splenomegalia and red cell morphology showed hypochromia, anisocytosis, poikilocytosis and microcytosis and many red cells with inclusion bodies were observed; hemoglobin H in the same amount as in the propositus was detected on electrophoresis. Heat denaturation was also positive and 2% of alkali-resistant hemoglobin was estimated in the child’s hemolysate. No attempt was made to investigate the co-existence of Bart’s hemoglobin in the hemolysate. The propositus of the second family N.E., aged 38 years, was admitted to the Department of Neuro-surgery with the diagnosis of subdural hematoma which has recurred three times in the last four years. The examination of the patient for coagulation disorders was negative but he presented a mild hemolysis and moderate splenomegalia. Peripheral blood examination showed microcytosis and anisocytosis hypochromia. Incubation of the red cells with Brilliant Cresyl Blue showed characteristic inclusion bodies in almost all the red cells. Osmotic fragility of the red cells was reduced. Hemoglobin electrophoresis revealed a fast moving fraction at pH 8.6, approx. 20% of total hemoglobin was identified to be Hb H. Electrophoresis at pH 6.5 and fingerprint of the purified abnormal fraction was characteristic for Hb H. The patient was hospitalized under special care and treated with good recovery after two months. The patient has three children. The examination of the children for Hb H was negative but a younger brother of the propositus, aged 30, showed inclusion bodies in red cells and Hb H in electrophoretic examination in the same concentration without any other symptom. DISCUSSION
The definition of thalassemia is the failure to produce enough normal hemoglobin A (ct.&) and this failure may be due to inability to produce either cc or t!lpolypeptide chain. In the /? thalassemia the production of /I chain is suppressed, and in the homozygous state both loci for /I chain synthesis are affected. In the cc thalassemia the synthesis of a chain is suppressed; in cc thalassemia minor one locus is affected. The inhibition of tc chain production is compensated by other loci and enough a chain is produced so that no symptoms are caused, and the carriers of the tc thalassemia minor are clinically symptomless. As Ingram and Stretton7 suggested, the inhibition of tc chain synthesis would be reflected not only in inhibition of Hb A production, but also in the synthesis of minor hemoglobin components. In the case of Hb H disease, the abnormal hemoglobin is composed of four p polypeptide chains’. It is related to a thalassemia, representing a relative excess of @ chain production. The excess /? chains form tetramers (&). Hemoglobin Bart’s, a tetramer composed only of y chain (rl), is also related to Clin. Chim. Acta,
20 (1968) 381-385
HEMOGLOBIN
H
385
DISEASE
a deficiency of CIchain production. The inheritance of Hb H disease is not only by simple co-dominance, but a third silent gene in combination with a thalassemia gene is suggested to be involved for revealing the Hb H disease*. This is in accord with the observation of the parents of our patients with Hb H disease, where no appreciable quantity of Hb H could be detected in the parents’ hemolysate, but two of the children showed significant quantities of Hb H. The recurrent subdural hemorrhage in the propositus of the second family, a young man of 38, may be due to infarction caused by denatured Hb H. Infarction of the other organs in patient with Hb H disease has been reported elsewhere13. REFERENCES I 2 3 4 5 6
7 8 g IO II
12 13
D. A. RIGAS. R. D. KOLER AND E. E. OSGOOD, 1. Lab. Clin. Med., 47 (1~56) 51. S. RAHBAR, D. BEALE, W. A. ISAAC AND H. LE~MANN, Brit. Med. j.; i (;967)-674. M. M. WINTROBE, Clinical Haematology, 5th, ed., Lea & Febiger, Philadelphia, 1961. K. SINGER, A. I. CHERNOFF AND L. SINGER, Blood, 6 (1951) 413. I. L. GRAHAM AND B. W. GRUNBAUM, Am. I. Clin. Pathol.. 39 (1963) 567. S. RAHBAR, Acta Biochim. Iranica. 3 (1965) 54. V. M. INGRAM AND A. 0. W. STRETTON. Nature, 184 (1959) 1903. H. LEHMANN, in Man’s Hemoglobins, North Holland, Amsterdam, 1966. ESTER AGHAIE, Acta Med. Iranica, 8 (1965) 84. V. M. INGRAM, Biochim. Biophys. Acta, 28 (1958) 539. C. BAGLIONI, Biochim. Biophys. Acta, 48 (1961) 392. H. MICHL, Monatsh., 82 (1951) 489. T. F. NECHELES. M. CATES, R. G. SHEEHAN AND H. J. MEYER, Blood, 28 (1966) 501. Clin. Chim. Acta, 20 (1968) 381~385