British Journal of Haernntolog!j. 1992. 8 2 . 596-600
Homozygosity €or dominant form of hereditary spherocytosis DURI'.A Y T E M I ZG i ' R G E Y . G ~ L Y ~ O: Zz T U R K . SEI..\IA YORi:'KAN* A N D Hacettepe University Deprirtrtierit of Pnediatrics, Paediatric Haertiatologg Unit. arid * Hacettepe University Depnrtr?icwt qf Physiologg. Ariknrn. TurkeLl
FERIDE
CIGDEM
ALTAY
Received 1 0 March 1 9 9 2 : ocrepted f o r publication 2 5 June 199.?
Summary. A h-month-old male infant with hereditary spherocytosis (HS)who was the first child of a cousin marriage is presented. The patient had splenomegaly and severe anaemia. Examination of the peripheral blood smear revealed spherocytes and the osmotic fragility of red blood cells was greatly increased. Physical examination of the parents revealed that both parents had mild anaemia. jaundice and splenomegaly. Their peripheral blood smears showed spherocytes and a few acanthocytes. Osrncttic fragility of red blood cells of both parents were increased. Red cell membrane electrophoresis indicated a deficiency of ankyrin in the propositus: mild
dt:ficiency was also detected in both parents. Electrophoretic patterns of red cell membrane proteins suggested that the child was homozygous for the dominant form of HS associated with ankyrin deficiency, while both parents had the simple dominant form of the disease. Red blood cell transfusions were given to the patient starting at the age of 1 month until splenectomy was ptrrformed at the age of 1 year that resulted in complete h.-lematological response. This observation indicates that hornozygosity for dominant type of HS associated with ankyrin deficiency is life compatible and splenectomy may cure the anaemia.
I
m m I
@
0 0 0
riis+ory of s p l e r e c t o m i V i s + c r j o f laundice, splenomegaly and gallstones RepGrted to berorrnal
Hcmozyqote
Fig 1 . Pedigrcc of the family.
Hereditary spherocytosis ([is)is the most common gencti-
lc18h). In approximately 75%)of the cases mode of inheritance is autosomal dominant. while new mutation or the presence of an autosomal recessive inheritance has been suggested for the remainder (Agre et a], 1982, 1986). k.omozygosity for the autosomal dominant form of abnorniality has been regarded as lethal (Becker & Lux, 1985). A
cally determined. heterogeneous disorder of the red cell membrane skeletal proteins (b'eed. 1 9 i 5 : Croorn c't r i l . C'orrespondenct.: Professor c'. Altay. Dcpartrnent of t'acdiatrics. Hacettepe Children's Hospital. Ankara. Tiirkey.
596
Hereditary Spherocytosis follow-up study of a patient with homozygosity for dominant type of HS associated with ankyrin deficiency of red cell membrane indicated that the outcome of this condition may be compatible with life.
59 7
CASE REPORT
A 6-month-old male infant, the first child of a cousin marriage (Fig 1)was referred to our hospital for evaluation of severe anaemia for which blood transfusions had been previously given monthly starting at the age of 1month. Past history revealed that hyperbilirubinaemia was not observed in the neonatal period. On physical examination he was pale and the spleen was palpable 6 cm below the left costal margin. A peripheral blood smear showed spherocytes and acanthocytes (Fig 2A), and osmotic fragility of red cells in hypotonic saline was greatly increased (Fig 3 ) . The haemoglobin was 4.9 g/dl. PCV 0.14, MCV 78 fl and the reticulocyte count 4%. The total serum bilirubin was 5 1.3 pmol/l (direct bilirubin 13-7 pmol/l). The mother and father both had enlarged spleens palpable 4 cm and 5 cm below the left costal margins respectively. Peripheral blood smears from both parents showed spherocytes and occasional acanthocytes (Figs 2B and 2C). Red cell osmotic fragility was greatly increased in both (Fig 3 ) . Routine laboratory studies of the father showed that the Hb was 12 g/dl, PCV 0.3, reticulocyte count 1.2%. MCV 91 fl, total bilirubin 29 pmol/l, (direct bilirubin 7 pmol/l), ferritin 489 pg/l. In the mother haemoglobin was 9.6 g/dl, PCV 0.28, MCV 93 fl, reticulocyte count 3.2%. total bilirubin 4 1 pmol/l (direct bilirubin 11 pmol/l), ferritin 1 5 7 pg/I. Extensive analysis of the family members indicated that several members had a history of jaundice, splenomegaly, gallstones and three of them had undergone splenectomy (Fig 1). During the following 5 months red blood cell transfusions were given monthly to the patient in order to increase Hb level to 9 g/dl from 3-4 g/dl. At the age of 11 months his Hb was 5 g/dl, reticulocyte count 6.1% and serum ferritin was 412 pg/l. Splenectomy was performed at the age of 12 months. The patient did not require any further blood transfusions during the 2 years follow-up period. The Hb was stable at 12 g/dl, and the reticulocyte count 0.6%. Red cell membrane proteins of the patient and his parents were analysed electrophoretically 10 months after splenectorny. Red cell ghosts were prepared by hypotonic lysis as described previously (Fortes et al, 1973). Sodium dodecyl sulphate polyacrylamide gel electrophoreses (SDS-PAGE) were performed according to the methods established by Fairbanks et aJ (1971). Gels were stained with Coomassie blue and scanned on a densitometer (Beckman Appraise TM). The ankyrin/band 3 (Ank/B3) and spectrin/band 3 (Sp/B3) ratios were calculated by weighing a specific area of each protein on the densitometric scanning obtained with diluted samples. The electrophoretic study of the red cell membrane proteins showed ankyrin deficiency in the patient and mild deficiency in the parents (Figs 4 and 5). DISCUSSION
Fig 2. Peripheral blood smears: (A) patient: (B) mother: (C) father.
The severity of the anaemia observed in the propositus suggested the presence of homozygosity for the recessive type of hereditary spherocytosis (Agre et al, 1982). However, the patient could be either heterozygous or homozygous for the
598
Feridr D i m rt a1
100
80
8 ?-
-2
60
0
E 0
I
40
Father
20
Mother Probond
--€I--
-0-
I
0 0.85 0.80
0.70
0.60
0.50
0.40
0.30
I
I
0.20
I
I
0.10
Saline (o/o W / V )
Fig 3. Incubated red cell osmotic fraglllty curves of patient and his parents, shaded area indicates normal range.
Fig 4.SDS-PAGE of red cell membrane proteins showing deficiency of ankyrin in the patient and milder deficiency in the parents as compared with controls. ( A ) Control for the patient: ( H ) patient: ( C ) mother: ID) father: ( E l control for the parents.
Hereditary Spherocytosis
599
3
Fig 5. Densitometric protiles of lanes. The ratios were calculated by weighing a specific area of each protein in the densitometric tracing obtained with diluted samples. At least five determinations were made for each and values were expressed as mean fSD. (A) Patient (Ank/B3: 0.121 f0.004, Sp/B3: 1.08f0.04): (B) mother (Ank/B3: 0.155*0.010. Sp/B3: 1.06f0.05): (C) father (Ank/B3: 0.161 f0.008, Sp/B3: 1.04&0.04): (D) control (Ank/B3: 0.212 f0.006, Sp/B3: 1.04*0.06). 1: t[ spectrin; 2: /lspectrin: 2.1: ankyrin: 2,2: ankyrin: 2.3: ankyrin: 3: band 3 .
dominant form of the disease, since both parents have overt signs of HS. The presence of ankyrin deficiency in red cell membrane electrophoresis in the propositus and milder ankyrin deficiency in the affected parents (Figs 4 and 5) with decreased Ank/B3 and normal Sp/B3 ratios gave the impression that the patient is homozygous for a dominant type HS associated with ankyrin deficiency. However, we believe that further gene studies in this family are needed for a more definite diagnosis of ankyrin deficiency. A deletion in the short arm of chromosome 8 or a translocation between chromosome 8 and 12 or 3 has been shown to be associated with some cases of dominant form of hereditary spherocytosis with ankyrin deficiency (Kimberling et ul, 1978: Basset al, 1983; Chilcote et aI, 1987 :Costa et al, 1990). Karyotype analysis of our patient revealed no abnormality. This observation supports previously published findings that molecular pathology causing ankyrin deficiency is heterogeneous as reported previously (Lux et al, 1990; Iolascan et al, 1991). Clinical and haematological findings of affected parents and other affected family members (Fig 1) indicated that molecular pathology resulting in HS in this family is probably a mild one. However, homozygosity of this condition was associated with severe anaemia. This observation indicates that homozygosity for at least one of the determinants of the autosomal dominant type of HS most probably
associated with ankyrin deficiency is compatible with life and severe anaemia is responsive to splenectomy.
ACKNOWLEDGMENTS The authors are very grateful to Dr M. Mikkelson at J.F. Kennedy Institute Department of Genetics, Glostrup. Denmark, and to Dr E. Tuncbilek at Hacettepe IJniversity Department of Genetics for performing karyotype analyses in the patient. REFERENCES A g e , P.. Asimos. A., Casella, J.F. & McMillan, C. (1986)Inheritance pattern and clinical response to splenectomy as a reflection of erythrocyte spectrin deficiency in hereditary spherocytosis. New EnglundJuurnul ofMedicine, 315, 1579-1583. Agre, P., Orringer, E.P. & Bennett. V. (1982) Deficient red ccll spectrin in severe, recessively inherited spherocytosis. N e w England Journal ufMedicine, 306, 1155-1161. Rass,E.B.,Smith.S.W..Stevenson.R.E.&Rosse, W.F.(1983)Further evidence for location of the spherocytosis gene on chromosome 8. Annuls oflnternal Medicine, 99, 192-193. Becker, P.S. & Lux, S.E. (1985) Hereditary spherocytosis and related disorders. Clinical Haernatulugy, 14, 1 5-4 3.
600
Feride Duru et a1
Chiicote. R.R.. LeReau. M.M.. Dampier. C.. Pergament. E., Verlinsky. Y . . Mohandas. N., Frischer. H. & Rowley. J.D. (1987) Association of red cell spherocytosis with deletion of the short arm of chromosome 8. Blood. 69. 156-1 59. Costa. F.F.. Agre. P., Watkins. P.C.. Winkeimann, J.C., John. K.M., Lux. S.E. & Forget, B.G. ( 1 9 9 0 ) Linkage of dominant hereditary spherocytosis to the gene for the erythrocyte membrane skeleton protein ankyrin. Xew Englarid /oitrrinl of Medicine. 323. 10461050. Croom. R.D.. 111, McMillan. C . L i ' . , Orringer. 1i.P. & Sheldon. G.F. ( 1986) Hereditary spherocytosis: recent experience and current concepts of pathophysiology. .4ririals of Surgerg. 2 0 3 . 34-39. Fairbanks. G..Steck. T.L. 8 M'allach. D.F.H. ( 1 9 7 1 ) Electrophoretic analysis of major polypeptides of human erythrocyte membrane. Bioc~hemistr.~~. 10, 2 6 0 6 - 2 h l i . Fortes. F.A.. Ellory. J.C. & Lelv. \'.I, ( 197 3 1 Suramin: a potent ATPase inhibitor which acts on the insidc surface of the sodium pump. Hiochitriica et Ijioplii/sicct A ru. 3 1 8 . 2 6 - 2 7 2 .
Icilascam. A,. Giudice. E.M.. Camaschella. C.. Pinto, L., Nobili. B., Perrotta. S. & Cutillo. S . (1991) Ankyrin deficiency in dominant hereditary spherocytosis: report of three cases. British Journal of Haematology. 78, 5 5 1 - 5 5 4 . Kimberling. W.J.. Taylor. R.A., Chapman, R.G. & Lubs. H.A. (1978) Linkage and gene localization of hereditary spherocytosis. Blood, 52. 859-867. LJX.S.E.. Tse. W.T.. Menninger. J.C., John, K.M.. Harris, P., Shaky. 0.. Chilcote. R.R.. Marchesi. S.L.. Watkins. P.C., Bennett, V, Mcfntosh. S . . Collin, F.S.. Francke. U.. Ward, D.C. & Forget, B.G. ( 1990) Hereditary spherocytosis associatcd with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature, 345, 736-i39. V.'ced. R.I. ( 1 9753 Hereditary spherocytosis: a review. Archives of Irirernnl Medicine. 1 3 5 , 1316-1 323.
Homozygosity €or dominant form of hereditary spherocytosis DURI'.A Y T E M I ZG i ' R G E Y . G ~ L Y ~ O: Zz T U R K . SEI..\IA YORi:'KAN* A N D Hacettepe University Deprirtrtierit of Pnediatrics, Paediatric Haertiatologg Unit. arid * Hacettepe University Depnrtr?icwt qf Physiologg. Ariknrn. TurkeLl
FERIDE
CIGDEM
ALTAY
Received 1 0 March 1 9 9 2 : ocrepted f o r publication 2 5 June 199.?
Summary. A h-month-old male infant with hereditary spherocytosis (HS)who was the first child of a cousin marriage is presented. The patient had splenomegaly and severe anaemia. Examination of the peripheral blood smear revealed spherocytes and the osmotic fragility of red blood cells was greatly increased. Physical examination of the parents revealed that both parents had mild anaemia. jaundice and splenomegaly. Their peripheral blood smears showed spherocytes and a few acanthocytes. Osrncttic fragility of red blood cells of both parents were increased. Red cell membrane electrophoresis indicated a deficiency of ankyrin in the propositus: mild
dt:ficiency was also detected in both parents. Electrophoretic patterns of red cell membrane proteins suggested that the child was homozygous for the dominant form of HS associated with ankyrin deficiency, while both parents had the simple dominant form of the disease. Red blood cell transfusions were given to the patient starting at the age of 1 month until splenectomy was ptrrformed at the age of 1 year that resulted in complete h.-lematological response. This observation indicates that hornozygosity for dominant type of HS associated with ankyrin deficiency is life compatible and splenectomy may cure the anaemia.
I
m m I
@
0 0 0
riis+ory of s p l e r e c t o m i V i s + c r j o f laundice, splenomegaly and gallstones RepGrted to berorrnal
Hcmozyqote
Fig 1 . Pedigrcc of the family.
Hereditary spherocytosis ([is)is the most common gencti-
lc18h). In approximately 75%)of the cases mode of inheritance is autosomal dominant. while new mutation or the presence of an autosomal recessive inheritance has been suggested for the remainder (Agre et a], 1982, 1986). k.omozygosity for the autosomal dominant form of abnorniality has been regarded as lethal (Becker & Lux, 1985). A
cally determined. heterogeneous disorder of the red cell membrane skeletal proteins (b'eed. 1 9 i 5 : Croorn c't r i l . C'orrespondenct.: Professor c'. Altay. Dcpartrnent of t'acdiatrics. Hacettepe Children's Hospital. Ankara. Tiirkey.
596
Hereditary Spherocytosis follow-up study of a patient with homozygosity for dominant type of HS associated with ankyrin deficiency of red cell membrane indicated that the outcome of this condition may be compatible with life.
59 7
CASE REPORT
A 6-month-old male infant, the first child of a cousin marriage (Fig 1)was referred to our hospital for evaluation of severe anaemia for which blood transfusions had been previously given monthly starting at the age of 1month. Past history revealed that hyperbilirubinaemia was not observed in the neonatal period. On physical examination he was pale and the spleen was palpable 6 cm below the left costal margin. A peripheral blood smear showed spherocytes and acanthocytes (Fig 2A), and osmotic fragility of red cells in hypotonic saline was greatly increased (Fig 3 ) . The haemoglobin was 4.9 g/dl. PCV 0.14, MCV 78 fl and the reticulocyte count 4%. The total serum bilirubin was 5 1.3 pmol/l (direct bilirubin 13-7 pmol/l). The mother and father both had enlarged spleens palpable 4 cm and 5 cm below the left costal margins respectively. Peripheral blood smears from both parents showed spherocytes and occasional acanthocytes (Figs 2B and 2C). Red cell osmotic fragility was greatly increased in both (Fig 3 ) . Routine laboratory studies of the father showed that the Hb was 12 g/dl, PCV 0.3, reticulocyte count 1.2%. MCV 91 fl, total bilirubin 29 pmol/l, (direct bilirubin 7 pmol/l), ferritin 489 pg/l. In the mother haemoglobin was 9.6 g/dl, PCV 0.28, MCV 93 fl, reticulocyte count 3.2%. total bilirubin 4 1 pmol/l (direct bilirubin 11 pmol/l), ferritin 1 5 7 pg/I. Extensive analysis of the family members indicated that several members had a history of jaundice, splenomegaly, gallstones and three of them had undergone splenectomy (Fig 1). During the following 5 months red blood cell transfusions were given monthly to the patient in order to increase Hb level to 9 g/dl from 3-4 g/dl. At the age of 11 months his Hb was 5 g/dl, reticulocyte count 6.1% and serum ferritin was 412 pg/l. Splenectomy was performed at the age of 12 months. The patient did not require any further blood transfusions during the 2 years follow-up period. The Hb was stable at 12 g/dl, and the reticulocyte count 0.6%. Red cell membrane proteins of the patient and his parents were analysed electrophoretically 10 months after splenectorny. Red cell ghosts were prepared by hypotonic lysis as described previously (Fortes et al, 1973). Sodium dodecyl sulphate polyacrylamide gel electrophoreses (SDS-PAGE) were performed according to the methods established by Fairbanks et aJ (1971). Gels were stained with Coomassie blue and scanned on a densitometer (Beckman Appraise TM). The ankyrin/band 3 (Ank/B3) and spectrin/band 3 (Sp/B3) ratios were calculated by weighing a specific area of each protein on the densitometric scanning obtained with diluted samples. The electrophoretic study of the red cell membrane proteins showed ankyrin deficiency in the patient and mild deficiency in the parents (Figs 4 and 5). DISCUSSION
Fig 2. Peripheral blood smears: (A) patient: (B) mother: (C) father.
The severity of the anaemia observed in the propositus suggested the presence of homozygosity for the recessive type of hereditary spherocytosis (Agre et al, 1982). However, the patient could be either heterozygous or homozygous for the
598
Feridr D i m rt a1
100
80
8 ?-
-2
60
0
E 0
I
40
Father
20
Mother Probond
--€I--
-0-
I
0 0.85 0.80
0.70
0.60
0.50
0.40
0.30
I
I
0.20
I
I
0.10
Saline (o/o W / V )
Fig 3. Incubated red cell osmotic fraglllty curves of patient and his parents, shaded area indicates normal range.
Fig 4.SDS-PAGE of red cell membrane proteins showing deficiency of ankyrin in the patient and milder deficiency in the parents as compared with controls. ( A ) Control for the patient: ( H ) patient: ( C ) mother: ID) father: ( E l control for the parents.
Hereditary Spherocytosis
599
3
Fig 5. Densitometric protiles of lanes. The ratios were calculated by weighing a specific area of each protein in the densitometric tracing obtained with diluted samples. At least five determinations were made for each and values were expressed as mean fSD. (A) Patient (Ank/B3: 0.121 f0.004, Sp/B3: 1.08f0.04): (B) mother (Ank/B3: 0.155*0.010. Sp/B3: 1.06f0.05): (C) father (Ank/B3: 0.161 f0.008, Sp/B3: 1.04&0.04): (D) control (Ank/B3: 0.212 f0.006, Sp/B3: 1.04*0.06). 1: t[ spectrin; 2: /lspectrin: 2.1: ankyrin: 2,2: ankyrin: 2.3: ankyrin: 3: band 3 .
dominant form of the disease, since both parents have overt signs of HS. The presence of ankyrin deficiency in red cell membrane electrophoresis in the propositus and milder ankyrin deficiency in the affected parents (Figs 4 and 5) with decreased Ank/B3 and normal Sp/B3 ratios gave the impression that the patient is homozygous for a dominant type HS associated with ankyrin deficiency. However, we believe that further gene studies in this family are needed for a more definite diagnosis of ankyrin deficiency. A deletion in the short arm of chromosome 8 or a translocation between chromosome 8 and 12 or 3 has been shown to be associated with some cases of dominant form of hereditary spherocytosis with ankyrin deficiency (Kimberling et ul, 1978: Basset al, 1983; Chilcote et aI, 1987 :Costa et al, 1990). Karyotype analysis of our patient revealed no abnormality. This observation supports previously published findings that molecular pathology causing ankyrin deficiency is heterogeneous as reported previously (Lux et al, 1990; Iolascan et al, 1991). Clinical and haematological findings of affected parents and other affected family members (Fig 1) indicated that molecular pathology resulting in HS in this family is probably a mild one. However, homozygosity of this condition was associated with severe anaemia. This observation indicates that homozygosity for at least one of the determinants of the autosomal dominant type of HS most probably
associated with ankyrin deficiency is compatible with life and severe anaemia is responsive to splenectomy.
ACKNOWLEDGMENTS The authors are very grateful to Dr M. Mikkelson at J.F. Kennedy Institute Department of Genetics, Glostrup. Denmark, and to Dr E. Tuncbilek at Hacettepe IJniversity Department of Genetics for performing karyotype analyses in the patient. REFERENCES A g e , P.. Asimos. A., Casella, J.F. & McMillan, C. (1986)Inheritance pattern and clinical response to splenectomy as a reflection of erythrocyte spectrin deficiency in hereditary spherocytosis. New EnglundJuurnul ofMedicine, 315, 1579-1583. Agre, P., Orringer, E.P. & Bennett. V. (1982) Deficient red ccll spectrin in severe, recessively inherited spherocytosis. N e w England Journal ufMedicine, 306, 1155-1161. Rass,E.B.,Smith.S.W..Stevenson.R.E.&Rosse, W.F.(1983)Further evidence for location of the spherocytosis gene on chromosome 8. Annuls oflnternal Medicine, 99, 192-193. Becker, P.S. & Lux, S.E. (1985) Hereditary spherocytosis and related disorders. Clinical Haernatulugy, 14, 1 5-4 3.
600
Feride Duru et a1
Chiicote. R.R.. LeReau. M.M.. Dampier. C.. Pergament. E., Verlinsky. Y . . Mohandas. N., Frischer. H. & Rowley. J.D. (1987) Association of red cell spherocytosis with deletion of the short arm of chromosome 8. Blood. 69. 156-1 59. Costa. F.F.. Agre. P., Watkins. P.C.. Winkeimann, J.C., John. K.M., Lux. S.E. & Forget, B.G. ( 1 9 9 0 ) Linkage of dominant hereditary spherocytosis to the gene for the erythrocyte membrane skeleton protein ankyrin. Xew Englarid /oitrrinl of Medicine. 323. 10461050. Croom. R.D.. 111, McMillan. C . L i ' . , Orringer. 1i.P. & Sheldon. G.F. ( 1986) Hereditary spherocytosis: recent experience and current concepts of pathophysiology. .4ririals of Surgerg. 2 0 3 . 34-39. Fairbanks. G..Steck. T.L. 8 M'allach. D.F.H. ( 1 9 7 1 ) Electrophoretic analysis of major polypeptides of human erythrocyte membrane. Bioc~hemistr.~~. 10, 2 6 0 6 - 2 h l i . Fortes. F.A.. Ellory. J.C. & Lelv. \'.I, ( 197 3 1 Suramin: a potent ATPase inhibitor which acts on the insidc surface of the sodium pump. Hiochitriica et Ijioplii/sicct A ru. 3 1 8 . 2 6 - 2 7 2 .
Icilascam. A,. Giudice. E.M.. Camaschella. C.. Pinto, L., Nobili. B., Perrotta. S. & Cutillo. S . (1991) Ankyrin deficiency in dominant hereditary spherocytosis: report of three cases. British Journal of Haematology. 78, 5 5 1 - 5 5 4 . Kimberling. W.J.. Taylor. R.A., Chapman, R.G. & Lubs. H.A. (1978) Linkage and gene localization of hereditary spherocytosis. Blood, 52. 859-867. LJX.S.E.. Tse. W.T.. Menninger. J.C., John, K.M.. Harris, P., Shaky. 0.. Chilcote. R.R.. Marchesi. S.L.. Watkins. P.C., Bennett, V, Mcfntosh. S . . Collin, F.S.. Francke. U.. Ward, D.C. & Forget, B.G. ( 1990) Hereditary spherocytosis associatcd with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature, 345, 736-i39. V.'ced. R.I. ( 1 9753 Hereditary spherocytosis: a review. Archives of Irirernnl Medicine. 1 3 5 , 1316-1 323.
