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A case of type 2 GM1-gangliosidosis with long survival
A Case of Type 2 GMr-Gangliosidosis with Long Survival Koichi Kikuchi, MD, Ryoji Minami, MD, Toru Kudoh, MD, Toru Nakao, MD and Satoshi Tsugawa, MD
An ll-year-old boy with type 2 GMrgangliosidosis was presented, providing further evidence for the clinical and biochemical heterogeneity of the disease. The patient had several characteristics of type 2 GM1-gangliosidosis, but was different from so-called typical type 2 GM1-gangliosidosis from the point of view of survival and the degree of GM1-ganglioside accumulation. GM1-gangliosidosis was diagnosed by absence of {3-galactosidase activity in leukocytes and th e parents had the enzyme levels of heterozygotes. However, the amount of the brain GM1-ganglioside was accumulated to a less degree in comparison with that of typical type 2 GM1-gangliosidosis, though the activity of GM1-(3-galactosidase in the brain was deficient to the same degree as in the typical case. Kikuchi K, Minami R, Kudoh T, Nakao T, Tsugawa S. A case of type 2 GM1-gangliosidosis with long survival. Brain Dev 1982;4: 153-6
GM1-gangliosidosis is defined as a hereditary metabolic disease with generalized storage of galactose containing derivatives caused by a deficiency of {3-galactosidase [1]. Two types of GM1-gangliosidosis, type 1 and 2 are classified on clinical manifestations [2] . In addition to those clinical types, many variants including adults have been described
[3-5] .
Some patients, once diagnosed as variants of GM1-gangliosidosis on the basis of a {3galactosidase deficiency in leukocytes or cultured fibroblasts, have been found to have a sialidase deficiency [6, 7] .
From the Department of Pediatrics, Otaru National Sanatorium, Otaru (KK, ST): Department of Pediatrics, Sapporo Medical College, Sapporo (RM, TK, TN). Received for pUblication: August 1, 1981. Accepted for publication: February 27,1982. Key words: Type 2 GMl·gangliosidosis, GM1-fJgalactosidase, fJ-galactosidase, GMl·ganglioside.
Correspondence address: Dr. Koichi Kikuchi, Department of Pediatrics, Otaru National Sanatorium, Nagahashi 3-24-1, Otaru 047, Japan.
In this report , biochemical investigations on an autopsy case of type 2 GM1-gangliosidosis who survived onto the age of 11 years were described. Report of the Patient An 11-year-old boy was thought to be the product of an uncomplicated term pregnancy. He appeared to develop normally during the first year of life. At 16 months of age , he lost his ability to stand and had frequent generalized convulsive seizures. He deteriorated gradually and was no longer interested in his surroundings. He was admitted to our hospital at 6 years of age because of his psychomotor retardation. His facial appearance was normal and hepatosplenomegaly was not present. No cherry red spot was seen in the maculae. Radiographic fmdings revealed beaking of the first lumbar vertebrae and prominent lumbar kyphosis. Cytoplasmic vacuoles were presented in 15% of the peripheral lymphocytes. Occasional histiocytic storage cells were also present in the bone marrow aspirate . {3-Galactosidase
Table 1 GMI-(3- and 4MU-(3-galactosidase activities in brain and liver Liver
Brain 4MU Present case 0.02 (11 yrs 2 mos) Typical type 2GMlgangliosidosis (2yrs 9 mos) Controls * (n =4)
0.03
4MU
8
0.48
9
11
0.20
21
25 ± 5 92 ± 19 98 ± 25 204 ± 47 (19-30) (66-110) (78-126) (151-265)
Activities are expressed as nanomoles per mg protein per hour. * mean ± SD, ( ) range.
activity was assayed in leukocytes and he was diagnosed as type 2 GMI 'gangliosidosis. By age 7 he was gradually spastic and died suddenly at 11 years of age because of bronchial obstruction by ineffective swallowing at an other hospital.
Materials and Methods Leukocytes were isolated from heparinized peripheral blood [8]. Brain and liver tissues from patients with GMl-gangliosidosis were obtained at autopsy and control tissues were
60
Peak I
60
obtained post mortem from neurologically normal patients of similar ages. All specimens were frozen immediately after autopsy and stored at _70o e until use. Leukocytes were suspended in distilled water at oDe and subjected to sonication for 1 min (Insonator Model 200M, Kubota). The sonicate was used as an enzyme source. Liver and brain tissues were homogenized in a Teflon homogenizer in an aliquot of distilled water at oDe, sonicated for 3 min and centrifuged at 100,000 x g for 60 min. The supernatant was used as an enzyme source. The activity of /3-galactosidase was assayed at the optimal condition, using 4"methylumbelliferyl (4MU) /3-D-galactopyranoside (Koch-Light) as substrate according to the method previously described [9]. The activity of GMI-/3-galactosidase was assayed with GMl-ganglioside specifically tritiated in the terminal galactose, as reported previously [10] . The content of protein was determined by the method of Lowry et al [11] , using bovine serum albumin as a standard. For the gel filtration pattern of 4MV-/3galactosidase activities, liver samples were filtrated through a column (1.8 x 93 cm) of Sephadex G-150 with 0.05 M phosphate buffer (pH 7.0) and fractions of 1.5 ml were collected at a flow rate of 4.5 ml/hr. The extraction and separation of brain
Peak II
120
154 Brain & Development, Vol 4, No 2,1982
Fig 1 Gel filtration of liver 4MU-(3-galactosidase. The enzyme source was applied to a Sephadex G-I50 bed (1.8 x 93 cm) and eluted with 50 mM phosphate buffer (pH 7.0). Activity was assayed according to the method in the text. ISO c--o: control,_: present case,~ : typical Elution volume (ml) type 2 GMI-gangliosidosis.
Table 2 Composition of gray matter gangliosides
Brain
TotalNANA (%dry wt.)
Distribution of NANA (%) GM] GDlb GDla
GTl
GM2
GM3
Presen t case (11 yrs 2 mos)
0.65
4.9
6.1
15.6
65 .2
4.7
3.5
TYpical type 2 GMl-gangliosidosis (2 yrs 9 mos)
1.62
6.3
5.3
9.8
73.2
1.7
3.7
Control 1
0.46
20.5
14.6
41.6
19.0
2.5
1.8
Control 2
0.36
18.7
10.3
43.2
22.3
3.6
1.9
gangliosirles were carried out by the method of Folch et al [12]. Thin layer chromatography on silica gel G (Merck) was used to quantify individual gangliosides. The solvent system used was chloroform: methanol : 2.5 N ammonia (60:40: 9) [13]. Resorcinol reagent was used for the visualization of gangliosides on TLC and the determination of N-acetymeuraminic acid (NANA) content in the brains [14]. Results A deficiency of t3-galactosidase activity was found in the propositus and the parents had carrier levels of the enzyme in leukocytes. This case was diagnosed as type 2 GMlgangliosidosis and the parents were heterozygotes. Enzymatic results of brain and liver are shown in Table 1. 4MU and GMl-t3-galactosidase activities were profoundly deficient in both tissues as were those in the typical case. Fig 1 shows the elution patterns of t3-galactosidase on Sephadex G-lS0. Three distinct fractions of 13galactosidase activity were observed in the control liver. In this case only fraction III was detected , while a very small amount of fraction I and III were observed in the typical case. Therefore, this case was thought to belong to type 2 GM1-gangliosidosis on the basis of enzymatic findings [15] . The ganglioside pattern in the brain was similar to that in the typical case (Table 2). However, the amount of GMl-ganglioside was slightly increased as compared with that of normal controls and was about 40% of that in the typical case.
Discussion In type 2 GMl-gangliosidosis, symptoms begin usually at about 1 year of age and the average life span varies between 3 and 10 years [16] . Although our patient survived onto the age of 11 years or even more if his swallowing had been effective, the patient appeared to have type 2 GMl-gangliosidosis. The amount of brain GMl-ganglioside from the patient was slightly increased compared with that from normals, but was reduced in comparison with that of a typical case. Therefore, the accumulation of the brain GM1ganglioside seemed to progress more slowly, though the activity of t3-galactosidase was deficient in the brain to the same degree as in the typical case. We could not satisfactorily explain the discrepancy between the residual t3-galactosidase activity and the amount of GM1-ganglioside accumulated in the brain. Recently , Lowden et al also reported a patient with type 2 GM1-gangliosidosis who survived until age 17 years and resembled our patient in view of clinical features and biochemical findings [17] . Therefore it was suggested that the degree of GM1-ganglioside accumulated might not always correlate with the reduction of GM1-t3-galactosidase activity and there was considerable clinical and biochemical heterogeneity within type 2 GMl-gangliosidosis. References 1. Okada S, O'Brien JS. Generalized gangliosidosis: beta galactosidase deficiency. Science 1968;160: 1002-4. 2. Derry DM, Fawcett JS, Andermann F, Wolfe LS. Late infantile systemic lipidosis, major monosialogangliosidosis, delineation of two types.
Type 2 GM1-gangliosidosis with long survival
155
Neurology 1968;18:340-8. 3. Pinsky L, Miller J, Shanfield B, Watter G, Wolfe LS. GMl-gangliosidosis in skin fibroblast culture: enzymatic differences between type 1 and 2 and observation on a third variant. Am J Hum Genet 1974;26:563-77. 4. Suzuki Y, Nakamura N, Fukuoka K, Shimada Y, Dono M. /l-galactosidase deficiency in juvenile and adult patients. Report of six Japanese cases and review of literature. Hum Genet 1977 ;36 : 219-29. 5. Sandhoff K, Christomanou H. Biochemistry and genetics of gangliosidosis. Hum Genet 1979;50: 107-43. 6. Strecker G, Michalski JC. Biochemical basis of six different type of sialidosis. FEBS Lett 1978; 85:20-4. . 7. Lowden JA, O'Brien JS. Sialidosis: a review of human neuraminidase deficiency. Am J Hum Genet 1978 ;31:1-18. 8. Boyum A. Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21:31-50 (SuppI97). 9. Orii T, Sukegawa K, Minami R, et al. Atypical Hurler syndrome without at-L-iduronidase deficiency . Tohoku J Exp Med 1976;120:113-23. 10. Kudoh T. The abnormalities of /l-galactosidase in
156 Brain & Development, Vol 4, No 2, 1982
11. 12.
13. 14. 15.
16
17.
GMl-gangliosidosis. Tohoku J Exp Med 1978; 125: 93-1 0 1. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Bioi Chem 1951;193:265-75. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Bioi Chem 1957; 226 :497-509. Suzuki K. A simple and accurate micromethod for quantitative determination of ganglioside pattern. Life Sci 1964;3:1227-33 . Svennerholm 1. Quantitative estimation of sialic acids. Biochim Biophys Acta 1957;24 : 604-11 . Orii T, Sukegawa K, Nakao T. A variant of GMlgangliosidosis type 2 and enzymic differences between GMl-gangliosidosis type 1 and 2. Tohoku J ExpMed 1975;117:99-100 . O'Brien JS. The gangliosidosis. In: Stanbury JB, Wyngaarden JB, Fredrickson DS. The metabolic basis of inherited disease, 4th ed. New York: McGraw-Hill Book Co, 1978:841-65. Lowden JA, Callahan JW, Gravel RA, Skomorowski MA, Becker L, Groves J. Type 2 GMl-gangliosidosis with long survival and neuronal ceroid lipofuscinosis. Neurology 1981 ;31 :719-24.
An ll-year-old boy with type 2 GMrgangliosidosis was presented, providing further evidence for the clinical and biochemical heterogeneity of the disease. The patient had several characteristics of type 2 GM1-gangliosidosis, but was different from so-called typical type 2 GM1-gangliosidosis from the point of view of survival and the degree of GM1-ganglioside accumulation. GM1-gangliosidosis was diagnosed by absence of {3-galactosidase activity in leukocytes and th e parents had the enzyme levels of heterozygotes. However, the amount of the brain GM1-ganglioside was accumulated to a less degree in comparison with that of typical type 2 GM1-gangliosidosis, though the activity of GM1-(3-galactosidase in the brain was deficient to the same degree as in the typical case. Kikuchi K, Minami R, Kudoh T, Nakao T, Tsugawa S. A case of type 2 GM1-gangliosidosis with long survival. Brain Dev 1982;4: 153-6
GM1-gangliosidosis is defined as a hereditary metabolic disease with generalized storage of galactose containing derivatives caused by a deficiency of {3-galactosidase [1]. Two types of GM1-gangliosidosis, type 1 and 2 are classified on clinical manifestations [2] . In addition to those clinical types, many variants including adults have been described
[3-5] .
Some patients, once diagnosed as variants of GM1-gangliosidosis on the basis of a {3galactosidase deficiency in leukocytes or cultured fibroblasts, have been found to have a sialidase deficiency [6, 7] .
From the Department of Pediatrics, Otaru National Sanatorium, Otaru (KK, ST): Department of Pediatrics, Sapporo Medical College, Sapporo (RM, TK, TN). Received for pUblication: August 1, 1981. Accepted for publication: February 27,1982. Key words: Type 2 GMl·gangliosidosis, GM1-fJgalactosidase, fJ-galactosidase, GMl·ganglioside.
Correspondence address: Dr. Koichi Kikuchi, Department of Pediatrics, Otaru National Sanatorium, Nagahashi 3-24-1, Otaru 047, Japan.
In this report , biochemical investigations on an autopsy case of type 2 GM1-gangliosidosis who survived onto the age of 11 years were described. Report of the Patient An 11-year-old boy was thought to be the product of an uncomplicated term pregnancy. He appeared to develop normally during the first year of life. At 16 months of age , he lost his ability to stand and had frequent generalized convulsive seizures. He deteriorated gradually and was no longer interested in his surroundings. He was admitted to our hospital at 6 years of age because of his psychomotor retardation. His facial appearance was normal and hepatosplenomegaly was not present. No cherry red spot was seen in the maculae. Radiographic fmdings revealed beaking of the first lumbar vertebrae and prominent lumbar kyphosis. Cytoplasmic vacuoles were presented in 15% of the peripheral lymphocytes. Occasional histiocytic storage cells were also present in the bone marrow aspirate . {3-Galactosidase
Table 1 GMI-(3- and 4MU-(3-galactosidase activities in brain and liver Liver
Brain 4MU Present case 0.02 (11 yrs 2 mos) Typical type 2GMlgangliosidosis (2yrs 9 mos) Controls * (n =4)
0.03
4MU
8
0.48
9
11
0.20
21
25 ± 5 92 ± 19 98 ± 25 204 ± 47 (19-30) (66-110) (78-126) (151-265)
Activities are expressed as nanomoles per mg protein per hour. * mean ± SD, ( ) range.
activity was assayed in leukocytes and he was diagnosed as type 2 GMI 'gangliosidosis. By age 7 he was gradually spastic and died suddenly at 11 years of age because of bronchial obstruction by ineffective swallowing at an other hospital.
Materials and Methods Leukocytes were isolated from heparinized peripheral blood [8]. Brain and liver tissues from patients with GMl-gangliosidosis were obtained at autopsy and control tissues were
60
Peak I
60
obtained post mortem from neurologically normal patients of similar ages. All specimens were frozen immediately after autopsy and stored at _70o e until use. Leukocytes were suspended in distilled water at oDe and subjected to sonication for 1 min (Insonator Model 200M, Kubota). The sonicate was used as an enzyme source. Liver and brain tissues were homogenized in a Teflon homogenizer in an aliquot of distilled water at oDe, sonicated for 3 min and centrifuged at 100,000 x g for 60 min. The supernatant was used as an enzyme source. The activity of /3-galactosidase was assayed at the optimal condition, using 4"methylumbelliferyl (4MU) /3-D-galactopyranoside (Koch-Light) as substrate according to the method previously described [9]. The activity of GMI-/3-galactosidase was assayed with GMl-ganglioside specifically tritiated in the terminal galactose, as reported previously [10] . The content of protein was determined by the method of Lowry et al [11] , using bovine serum albumin as a standard. For the gel filtration pattern of 4MV-/3galactosidase activities, liver samples were filtrated through a column (1.8 x 93 cm) of Sephadex G-150 with 0.05 M phosphate buffer (pH 7.0) and fractions of 1.5 ml were collected at a flow rate of 4.5 ml/hr. The extraction and separation of brain
Peak II
120
154 Brain & Development, Vol 4, No 2,1982
Fig 1 Gel filtration of liver 4MU-(3-galactosidase. The enzyme source was applied to a Sephadex G-I50 bed (1.8 x 93 cm) and eluted with 50 mM phosphate buffer (pH 7.0). Activity was assayed according to the method in the text. ISO c--o: control,_: present case,~ : typical Elution volume (ml) type 2 GMI-gangliosidosis.
Table 2 Composition of gray matter gangliosides
Brain
TotalNANA (%dry wt.)
Distribution of NANA (%) GM] GDlb GDla
GTl
GM2
GM3
Presen t case (11 yrs 2 mos)
0.65
4.9
6.1
15.6
65 .2
4.7
3.5
TYpical type 2 GMl-gangliosidosis (2 yrs 9 mos)
1.62
6.3
5.3
9.8
73.2
1.7
3.7
Control 1
0.46
20.5
14.6
41.6
19.0
2.5
1.8
Control 2
0.36
18.7
10.3
43.2
22.3
3.6
1.9
gangliosirles were carried out by the method of Folch et al [12]. Thin layer chromatography on silica gel G (Merck) was used to quantify individual gangliosides. The solvent system used was chloroform: methanol : 2.5 N ammonia (60:40: 9) [13]. Resorcinol reagent was used for the visualization of gangliosides on TLC and the determination of N-acetymeuraminic acid (NANA) content in the brains [14]. Results A deficiency of t3-galactosidase activity was found in the propositus and the parents had carrier levels of the enzyme in leukocytes. This case was diagnosed as type 2 GMlgangliosidosis and the parents were heterozygotes. Enzymatic results of brain and liver are shown in Table 1. 4MU and GMl-t3-galactosidase activities were profoundly deficient in both tissues as were those in the typical case. Fig 1 shows the elution patterns of t3-galactosidase on Sephadex G-lS0. Three distinct fractions of 13galactosidase activity were observed in the control liver. In this case only fraction III was detected , while a very small amount of fraction I and III were observed in the typical case. Therefore, this case was thought to belong to type 2 GM1-gangliosidosis on the basis of enzymatic findings [15] . The ganglioside pattern in the brain was similar to that in the typical case (Table 2). However, the amount of GMl-ganglioside was slightly increased as compared with that of normal controls and was about 40% of that in the typical case.
Discussion In type 2 GMl-gangliosidosis, symptoms begin usually at about 1 year of age and the average life span varies between 3 and 10 years [16] . Although our patient survived onto the age of 11 years or even more if his swallowing had been effective, the patient appeared to have type 2 GMl-gangliosidosis. The amount of brain GMl-ganglioside from the patient was slightly increased compared with that from normals, but was reduced in comparison with that of a typical case. Therefore, the accumulation of the brain GM1ganglioside seemed to progress more slowly, though the activity of t3-galactosidase was deficient in the brain to the same degree as in the typical case. We could not satisfactorily explain the discrepancy between the residual t3-galactosidase activity and the amount of GM1-ganglioside accumulated in the brain. Recently , Lowden et al also reported a patient with type 2 GM1-gangliosidosis who survived until age 17 years and resembled our patient in view of clinical features and biochemical findings [17] . Therefore it was suggested that the degree of GM1-ganglioside accumulated might not always correlate with the reduction of GM1-t3-galactosidase activity and there was considerable clinical and biochemical heterogeneity within type 2 GMl-gangliosidosis. References 1. Okada S, O'Brien JS. Generalized gangliosidosis: beta galactosidase deficiency. Science 1968;160: 1002-4. 2. Derry DM, Fawcett JS, Andermann F, Wolfe LS. Late infantile systemic lipidosis, major monosialogangliosidosis, delineation of two types.
Type 2 GM1-gangliosidosis with long survival
155
Neurology 1968;18:340-8. 3. Pinsky L, Miller J, Shanfield B, Watter G, Wolfe LS. GMl-gangliosidosis in skin fibroblast culture: enzymatic differences between type 1 and 2 and observation on a third variant. Am J Hum Genet 1974;26:563-77. 4. Suzuki Y, Nakamura N, Fukuoka K, Shimada Y, Dono M. /l-galactosidase deficiency in juvenile and adult patients. Report of six Japanese cases and review of literature. Hum Genet 1977 ;36 : 219-29. 5. Sandhoff K, Christomanou H. Biochemistry and genetics of gangliosidosis. Hum Genet 1979;50: 107-43. 6. Strecker G, Michalski JC. Biochemical basis of six different type of sialidosis. FEBS Lett 1978; 85:20-4. . 7. Lowden JA, O'Brien JS. Sialidosis: a review of human neuraminidase deficiency. Am J Hum Genet 1978 ;31:1-18. 8. Boyum A. Separation of leukocytes from blood and bone marrow. Scand J Clin Lab Invest 1968; 21:31-50 (SuppI97). 9. Orii T, Sukegawa K, Minami R, et al. Atypical Hurler syndrome without at-L-iduronidase deficiency . Tohoku J Exp Med 1976;120:113-23. 10. Kudoh T. The abnormalities of /l-galactosidase in
156 Brain & Development, Vol 4, No 2, 1982
11. 12.
13. 14. 15.
16
17.
GMl-gangliosidosis. Tohoku J Exp Med 1978; 125: 93-1 0 1. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Bioi Chem 1951;193:265-75. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Bioi Chem 1957; 226 :497-509. Suzuki K. A simple and accurate micromethod for quantitative determination of ganglioside pattern. Life Sci 1964;3:1227-33 . Svennerholm 1. Quantitative estimation of sialic acids. Biochim Biophys Acta 1957;24 : 604-11 . Orii T, Sukegawa K, Nakao T. A variant of GMlgangliosidosis type 2 and enzymic differences between GMl-gangliosidosis type 1 and 2. Tohoku J ExpMed 1975;117:99-100 . O'Brien JS. The gangliosidosis. In: Stanbury JB, Wyngaarden JB, Fredrickson DS. The metabolic basis of inherited disease, 4th ed. New York: McGraw-Hill Book Co, 1978:841-65. Lowden JA, Callahan JW, Gravel RA, Skomorowski MA, Becker L, Groves J. Type 2 GMl-gangliosidosis with long survival and neuronal ceroid lipofuscinosis. Neurology 1981 ;31 :719-24.