PRENATAL DIAGNOSIS, VOL.
12,1037-1042 (1992)
MUCOLIPIDOSIS TYPE IV: ACCUMULATION OF PHOSPHOLIPIDS AND GANGLIOSIDES IN CULTURED AMNIOTIC CELLS. A TOOL FOR PRENATAL DIAGNOSIS M. ZEIGLER, R. BARGAL, V. SURI, B. MEIDAN AND G. BACH Deparimeni of Human Genetics. Hadassah Universiry Hospilal, Jerusalem, Israel 91 120
SUMMARY Cultured amniotic fluid cells from two mucolipidosis type IV (ML1V)-affected fetuses demonstrated accumulation of phospholipids and gangliosides when compared with normal controls. Like cultured skin fibroblasts from MLIV patients, cultured amniotic cells from the affected fetuses accumulated primarily lyso phospholipids and this could be demonstrated by radioactive labelling with appropriate precursors, either inorganic phosphate or oleic acid. Furthermore, like cultured skin fibroblasts, there was significant retention of exogenously supplied GDl A ganglioside in the affected amniotic cells. This storage was previously demonstrated to be unique to MLIV and thus can be used at present as a specific procedure for prenatal diagnosis of MLIV. KEY WORDS
Mucolipidosis IV
Lysosomal storage disorders
INTRODUCTION Mucolipidosis type IV (MLIV) is a lysosomal storage disorder that is clinically characterized by psychomotor retardation and ophthalmological abnormalities including cornea opacities, retina degeneration, and strabismus (Amir et al., 1987). Most of the 70-80 patients diagnosed in the last 10 years have been of Jewish Ashkenazi origin, indicating a relatively high frequency of this disorder in this ethnic group (Bach et al., 1992). Since all the known patients are alive, the most extensive studies to characterize the nature of the stored materials have been performed in cultured skin fibroblasts. These studies indicated that phospholipids (primarily lyso compounds) and gangliosides (mono and polysialo) are the accumulating lipids in this disorder (Bach et al., 1975; Crandall et al., 1982; Bargal and Bach, 1988). Confirmation of these findings were reported in the urine extract of one patient (Caimi et al., 1982) and in a brain biopsy of another (Tellez-Nagel et af.,1976). The pattern of the accumulated lipids and the extent of this storage were shown to be unique to MLIV when compared with other lysosomal storage disorders (Zeigler and Bach, 1986; Bargal and Bach, 1988). Since the basic enzymatic defect and the gene involved in this disorder have not yet been identified, prenatal diagnosis has been restricted to electron microscopy examination of cultured amniotic cells, in which MLIV-affected fetuses presented characteristic storage organelles (Kohn et al., 1977; Arnon et al., 1986). It should Addressee for correspondence:Gideon Bach, Department of Human Genetics, Hadassah University Hospital, Jerusalem, Israel 91 120.
0 197-385 1/92/ 121037-O6$08.00 0 1992 by John Wiley & Sons, Ltd.
Received 23 December 1991 Accepted 9 May 1992
1038
M. ZEIGLER ET AL.
be emphasized that this is a very laborious and complicated procedure requiring expertise and experience. In this paper we report lipid storage in cultured amniotic fluid cells of MLIVaffected fetuses similar to that reported in cultured skin fibroblasts, thus raising the possibility of using this technique for accurate prenatal diagnosis of MLIV. MATERIALS AND METHODS Cultured amnioticjuid cells
Amniotic fluid cells obtained by transabdominal amniocentesis at 15-17 weeks' gestation were cultured and propagated in 25 cm2 plastic tissue culture flasks (Falcon) in Earles MEM medium (Gibco) supplemented with 10 per cent fetal calf serum (Gibco) and harvested by trypsination as previously described (,Kohn et al., 1977; Arnon et ul., 1986, 1988). Cultures from two unrelated fetuses affected with MLIV (determined by electron microscopy and confirmed in the aborted fetuses) and two normal controls were used for these studies. All radioactive labelling studies were performed between the second and fifth subculture. Radioactive labelling
Intracellular phospholipids were labelled with either [3'P]phosphate (9000 Ci/mmol) or [I-'4C]oleic acid (56 mCi/mmol) (New England Nuclear, U.S.A.). The precursors were introduced under sterile conditions into confluent cultures 3-5 days following subculture. [32P]phosphatelabelling was carried out with 6 ,&/flask (12 x lo6 dpm/flask) for a 4-day pulse followed by a 4-day chase period in non-radioactive medium. The harvested cells were washed twice in physiological saline, suspended in 0.3 ml of twice-distilled H,O, and disrupted by two 10 s bursts of ultrasonication (Microson, Farmingdale, NY; micro-probe). Protein content was determined by the method of Lowry et al. (1951). Five ml of chloroform-methanol(2:1, v/v) was added and the mixture was homogenized using a glass/glass hand homogenizer. Phase partition was achieved following the addition of 0.7 ml of 0.1 M KCl. The lower organic phase was collected, dried under a N, stream, and lipids were redissolved in 0.2ml of chloroform-methanol (2: I , v/v) and applied to thin-layer chromatograms of Silica Gel 60 precoated plates (Merck, Darmstadt, Germany). Chromatography was performed in chloroform-methanol-ammonium hydroxide (65:30:4, v/v/v). The radioactive spots were visualized by autoradiography and identified with appropriate standards. The individual spots were scraped into scintillation vials and radioactivity was determined. Ganglioside loading
G D l A ganglioside was labelled in the sialic acid moiety as previously described (Zeigler and Bach, 1986), yielding a specific radioactivity of 3 x lo5 dpmfmmol. Ten nmol of the tritium-labelled ganglioside dissolved in 30 pl of ethanol was added to 4 ml of nutrient medium supplemented with 5 per cent fetal calf serum which was added 2-3 days after subculture and incubated for 2-4 days. The cultures were washed twice with physiological saline and incubated for 3 min at 37°C with
(11) Figure 1. Thin-layer chromatography of lipid extracts from cultured amniotic fluid cells labelled with [3ZP]phosphate(I) and [“C]oleic acid (11). Lanes A and E: MLIV-affected fetuses; lanes B and F: normal controls; lane C : phosphatidylcholine and phosphatidylethanolamine standards; lane D: oleic acid standard. ( I ) Lysophosphatidylcholine;(2) phosphatidylserine and phosphoinositide; (3) sphingomyelin; (4) phosphatidylcholine; ( 5 ) phosphatidylethanolamine; (6) lyso his phosphatidic acid
1040
M. ZEIGLER ET AL.
Table 1. Individual phospholipids in cultured amniotic fluid cells ~~
[32P]Phosphate dpm (mg protein-') x M LIV Controls ( n = 2) (H = 2)
Phospholipid Lyso phosphatidylcholine Phosphoinositide and phosphatidylserine Sphingomyelin Phosphatidylethanolamine Phosphatidylcholine Lyso bis phosphatidic acid
['4C]Oleic acid dpm (mgprotein- ') x MLIV Controls (n = 2) (n=2)
2.3-2'5 14Xb20.6
0.5-0.7 3.8-7.0
2643-28.5
3&4.1 31.141.4 30.8-32.0 2.1-2.6
2.6-3.9 16.7-23.5
154-21.3 535-554 235-265 31.6-39.6
13.4-18'1 0-6-0.8
166-180
8.7-9.8 85.1-90.0 18-25 406-426 16&117 9.8-1 0.0
The values represent the range of each determination as described in the Materials and Methods
Table 2. Retention of 13H]GD1Aganglioside in cultured amniotic fluid cells Cell line
cprn (mg protein- ') x
MLIV ( n = 2) Controls (n = 2)
1.7-3.4 0-2M.38
1 per cent fatty acid-free bovine albumin (Sigma Chemicals, St Louis, MO, U.S.A.) dissolved in physiological saline to remove gangliosides attached to the outer cell surface. The cultures were then harvested, washed with physiological saline, and gangliosides were extracted and analysed as previously described (Zeigler and Bach, 1986). All the determinations were repeated twice. RESULTS The distribution of radioactive lipids following pulse and chase labelling of the cultured amniotic fluid cells with [32P]phosphateand [14C]oleic acid is presented in Figures l(1) and 1(11), respectively. Quantitative measurement of the radioactivity of the various lipids is presented in Table 1. The retention of exogenously supplied E3H]GD1A in the cultured amniotic fluid cells is presented in Table 2. DISCUSSION Since MLIV is one of the few lysosomal storage disorders in which the basic metabolic defect and/or the gene involved has not yet been elucidated, the diagnosis, particularly prenatal diagnosis in high-risk families, is not readily available. At
MUCOLIPIDOSIS TYPE IV
1041
present, the only procedure used for these purposes is electron microscopy, which requires carefully monitored culture conditions and expertise in interpretation; hence it is performed at only one centre in the world (Arnon et al., 1986). The great demand for prenatal diagnosis of MLIV in high-risk families emphasizes the need for alternative accurate and simpler procedures. The biochemical abnormalities found in cultured amniotic cells of MLIV-affected fetuses can be used as a diagnostic tool. Like cultured fibroblasts, the cultured amniotic cells of the two MLIV-affected fetuses demonstrated the specific accumulation of both phospholipids and gangliosides. The phospholipids that accumulated to the largest extent were, as in cultured fibroblasts (Crandall et al., 1982; Bargal and Bach, 1988), the lyso compounds and, to a lesser extent, phosphatidylethanolamine and phosphatidylcholine, while sphingomyelin was not stored. This storage has been observed by labelling either with the fatty acid or the phosphorus precursors, thus precluding an obvious direction of investigation for the basic metabolic defect causing this accumulation. The retention of GDlA was similar to that observed in cultured fibroblasts of MLIV patients (Zeigler and Bach, 1986). As already mentioned, this storage distinguishes MLIV from all other lysosomal storage disorders and can easily be followed by radioactive labelling with appropriate precursors or the introduction of the radioactively labelled lipid substrate itself. Until an unambiguous enzymatic or molecular technique for the diagnosis of MLIV is available, these analyses, together with electron microscopy, should be adopted for this diagnosis. ACKNOWLEDGEMENT
This research was supported by the Mucolipidosis TV Foundation.
REFERENCES Amir, N., Zlotogora, J., Bach, G. (1987). Mucolipidosis type IV: clinical spectrum and natural history, Pedialrics, 79,953-959. Amon, J., Ornoy, A., Bach, G. (1986). Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders. A methodological study, Prenat. Diagn., 6,35 1-36], Amon, J., Ornoy, A., Bach, G. (1988). Cultured conditions found to minimize false positive diagnosis of lysosomal storage disorders in-vivo, In-vifro Cell Dev. B i d . , 24, 1159-1 164. Bach, G., Cohen, M.M., Kohn, G . (1975). Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV, Biochem. Biophys. Rex Commun., 66, 1483-1 490. Bach, G . , Zlotogora, J., Zeigler, M. (1992). Lysosomal storage disorders among Jews. In: Bonne-Tamir, B. (Ed.). Genetic Diversity Among Jews, Oxford: Oxford University Press, 301-304. Bargal, R., Bach, G. (1988). Phospholipid accumulation in mucolipidosis IV cultured fibroblasts, J . Inher. Metab. Dis., 11, 144-150. Caimi, L., Tettamanti, G., Berra, B., Sale, F.O., Borrone, C., Durand, P., Martin, J.J. (1982). Mucolipidosis IV, a sialolipidosis due to ganglioside sialidase deficiency, J. Inher. Metab. Dis., 5,2 18-224. Crandall, B.F., Phillipart, M., Brown, W.J., Bluestone, D. (1982). Mucolipidosis IV, Am. J. Med. Genet., 12,301-308. Kohn, G.,Livni, N., Ornoy, A., Sekeles, E., Beyth, Y., Legurn, C., Bach, G., Cohen, M.M. (1977). Prenatal diagnosis of mucolipidosis IV by electron microscopy, J . Pedintr., 90, 62-66.
1042
M. ZEIGLER ET AL.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the fohn phenol reagent, J. Biol. Chem., 193,265-275. Tellez-Nagel, I., Rapin, I., Iwamoto, R., Johnson, A.B., Norton, W.T., Nitowsky, H. (1976). Mucolipidosis IV: Clinical, ultrastructural, histochemical and chemical studies of a case, including brain biopsy, Arch. NeuroI., 33,828-835. Zeigler, M., Bach, G . (1986). Internalization of exogenous gangliosides in cultured skin fibroblasts for the diagnosis of mucolipidosis type IV, Clin. Chim. Acta, 157,183-190.
12,1037-1042 (1992)
MUCOLIPIDOSIS TYPE IV: ACCUMULATION OF PHOSPHOLIPIDS AND GANGLIOSIDES IN CULTURED AMNIOTIC CELLS. A TOOL FOR PRENATAL DIAGNOSIS M. ZEIGLER, R. BARGAL, V. SURI, B. MEIDAN AND G. BACH Deparimeni of Human Genetics. Hadassah Universiry Hospilal, Jerusalem, Israel 91 120
SUMMARY Cultured amniotic fluid cells from two mucolipidosis type IV (ML1V)-affected fetuses demonstrated accumulation of phospholipids and gangliosides when compared with normal controls. Like cultured skin fibroblasts from MLIV patients, cultured amniotic cells from the affected fetuses accumulated primarily lyso phospholipids and this could be demonstrated by radioactive labelling with appropriate precursors, either inorganic phosphate or oleic acid. Furthermore, like cultured skin fibroblasts, there was significant retention of exogenously supplied GDl A ganglioside in the affected amniotic cells. This storage was previously demonstrated to be unique to MLIV and thus can be used at present as a specific procedure for prenatal diagnosis of MLIV. KEY WORDS
Mucolipidosis IV
Lysosomal storage disorders
INTRODUCTION Mucolipidosis type IV (MLIV) is a lysosomal storage disorder that is clinically characterized by psychomotor retardation and ophthalmological abnormalities including cornea opacities, retina degeneration, and strabismus (Amir et al., 1987). Most of the 70-80 patients diagnosed in the last 10 years have been of Jewish Ashkenazi origin, indicating a relatively high frequency of this disorder in this ethnic group (Bach et al., 1992). Since all the known patients are alive, the most extensive studies to characterize the nature of the stored materials have been performed in cultured skin fibroblasts. These studies indicated that phospholipids (primarily lyso compounds) and gangliosides (mono and polysialo) are the accumulating lipids in this disorder (Bach et al., 1975; Crandall et al., 1982; Bargal and Bach, 1988). Confirmation of these findings were reported in the urine extract of one patient (Caimi et al., 1982) and in a brain biopsy of another (Tellez-Nagel et af.,1976). The pattern of the accumulated lipids and the extent of this storage were shown to be unique to MLIV when compared with other lysosomal storage disorders (Zeigler and Bach, 1986; Bargal and Bach, 1988). Since the basic enzymatic defect and the gene involved in this disorder have not yet been identified, prenatal diagnosis has been restricted to electron microscopy examination of cultured amniotic cells, in which MLIV-affected fetuses presented characteristic storage organelles (Kohn et al., 1977; Arnon et al., 1986). It should Addressee for correspondence:Gideon Bach, Department of Human Genetics, Hadassah University Hospital, Jerusalem, Israel 91 120.
0 197-385 1/92/ 121037-O6$08.00 0 1992 by John Wiley & Sons, Ltd.
Received 23 December 1991 Accepted 9 May 1992
1038
M. ZEIGLER ET AL.
be emphasized that this is a very laborious and complicated procedure requiring expertise and experience. In this paper we report lipid storage in cultured amniotic fluid cells of MLIVaffected fetuses similar to that reported in cultured skin fibroblasts, thus raising the possibility of using this technique for accurate prenatal diagnosis of MLIV. MATERIALS AND METHODS Cultured amnioticjuid cells
Amniotic fluid cells obtained by transabdominal amniocentesis at 15-17 weeks' gestation were cultured and propagated in 25 cm2 plastic tissue culture flasks (Falcon) in Earles MEM medium (Gibco) supplemented with 10 per cent fetal calf serum (Gibco) and harvested by trypsination as previously described (,Kohn et al., 1977; Arnon et ul., 1986, 1988). Cultures from two unrelated fetuses affected with MLIV (determined by electron microscopy and confirmed in the aborted fetuses) and two normal controls were used for these studies. All radioactive labelling studies were performed between the second and fifth subculture. Radioactive labelling
Intracellular phospholipids were labelled with either [3'P]phosphate (9000 Ci/mmol) or [I-'4C]oleic acid (56 mCi/mmol) (New England Nuclear, U.S.A.). The precursors were introduced under sterile conditions into confluent cultures 3-5 days following subculture. [32P]phosphatelabelling was carried out with 6 ,&/flask (12 x lo6 dpm/flask) for a 4-day pulse followed by a 4-day chase period in non-radioactive medium. The harvested cells were washed twice in physiological saline, suspended in 0.3 ml of twice-distilled H,O, and disrupted by two 10 s bursts of ultrasonication (Microson, Farmingdale, NY; micro-probe). Protein content was determined by the method of Lowry et al. (1951). Five ml of chloroform-methanol(2:1, v/v) was added and the mixture was homogenized using a glass/glass hand homogenizer. Phase partition was achieved following the addition of 0.7 ml of 0.1 M KCl. The lower organic phase was collected, dried under a N, stream, and lipids were redissolved in 0.2ml of chloroform-methanol (2: I , v/v) and applied to thin-layer chromatograms of Silica Gel 60 precoated plates (Merck, Darmstadt, Germany). Chromatography was performed in chloroform-methanol-ammonium hydroxide (65:30:4, v/v/v). The radioactive spots were visualized by autoradiography and identified with appropriate standards. The individual spots were scraped into scintillation vials and radioactivity was determined. Ganglioside loading
G D l A ganglioside was labelled in the sialic acid moiety as previously described (Zeigler and Bach, 1986), yielding a specific radioactivity of 3 x lo5 dpmfmmol. Ten nmol of the tritium-labelled ganglioside dissolved in 30 pl of ethanol was added to 4 ml of nutrient medium supplemented with 5 per cent fetal calf serum which was added 2-3 days after subculture and incubated for 2-4 days. The cultures were washed twice with physiological saline and incubated for 3 min at 37°C with
(11) Figure 1. Thin-layer chromatography of lipid extracts from cultured amniotic fluid cells labelled with [3ZP]phosphate(I) and [“C]oleic acid (11). Lanes A and E: MLIV-affected fetuses; lanes B and F: normal controls; lane C : phosphatidylcholine and phosphatidylethanolamine standards; lane D: oleic acid standard. ( I ) Lysophosphatidylcholine;(2) phosphatidylserine and phosphoinositide; (3) sphingomyelin; (4) phosphatidylcholine; ( 5 ) phosphatidylethanolamine; (6) lyso his phosphatidic acid
1040
M. ZEIGLER ET AL.
Table 1. Individual phospholipids in cultured amniotic fluid cells ~~
[32P]Phosphate dpm (mg protein-') x M LIV Controls ( n = 2) (H = 2)
Phospholipid Lyso phosphatidylcholine Phosphoinositide and phosphatidylserine Sphingomyelin Phosphatidylethanolamine Phosphatidylcholine Lyso bis phosphatidic acid
['4C]Oleic acid dpm (mgprotein- ') x MLIV Controls (n = 2) (n=2)
2.3-2'5 14Xb20.6
0.5-0.7 3.8-7.0
2643-28.5
3&4.1 31.141.4 30.8-32.0 2.1-2.6
2.6-3.9 16.7-23.5
154-21.3 535-554 235-265 31.6-39.6
13.4-18'1 0-6-0.8
166-180
8.7-9.8 85.1-90.0 18-25 406-426 16&117 9.8-1 0.0
The values represent the range of each determination as described in the Materials and Methods
Table 2. Retention of 13H]GD1Aganglioside in cultured amniotic fluid cells Cell line
cprn (mg protein- ') x
MLIV ( n = 2) Controls (n = 2)
1.7-3.4 0-2M.38
1 per cent fatty acid-free bovine albumin (Sigma Chemicals, St Louis, MO, U.S.A.) dissolved in physiological saline to remove gangliosides attached to the outer cell surface. The cultures were then harvested, washed with physiological saline, and gangliosides were extracted and analysed as previously described (Zeigler and Bach, 1986). All the determinations were repeated twice. RESULTS The distribution of radioactive lipids following pulse and chase labelling of the cultured amniotic fluid cells with [32P]phosphateand [14C]oleic acid is presented in Figures l(1) and 1(11), respectively. Quantitative measurement of the radioactivity of the various lipids is presented in Table 1. The retention of exogenously supplied E3H]GD1A in the cultured amniotic fluid cells is presented in Table 2. DISCUSSION Since MLIV is one of the few lysosomal storage disorders in which the basic metabolic defect and/or the gene involved has not yet been elucidated, the diagnosis, particularly prenatal diagnosis in high-risk families, is not readily available. At
MUCOLIPIDOSIS TYPE IV
1041
present, the only procedure used for these purposes is electron microscopy, which requires carefully monitored culture conditions and expertise in interpretation; hence it is performed at only one centre in the world (Arnon et al., 1986). The great demand for prenatal diagnosis of MLIV in high-risk families emphasizes the need for alternative accurate and simpler procedures. The biochemical abnormalities found in cultured amniotic cells of MLIV-affected fetuses can be used as a diagnostic tool. Like cultured fibroblasts, the cultured amniotic cells of the two MLIV-affected fetuses demonstrated the specific accumulation of both phospholipids and gangliosides. The phospholipids that accumulated to the largest extent were, as in cultured fibroblasts (Crandall et al., 1982; Bargal and Bach, 1988), the lyso compounds and, to a lesser extent, phosphatidylethanolamine and phosphatidylcholine, while sphingomyelin was not stored. This storage has been observed by labelling either with the fatty acid or the phosphorus precursors, thus precluding an obvious direction of investigation for the basic metabolic defect causing this accumulation. The retention of GDlA was similar to that observed in cultured fibroblasts of MLIV patients (Zeigler and Bach, 1986). As already mentioned, this storage distinguishes MLIV from all other lysosomal storage disorders and can easily be followed by radioactive labelling with appropriate precursors or the introduction of the radioactively labelled lipid substrate itself. Until an unambiguous enzymatic or molecular technique for the diagnosis of MLIV is available, these analyses, together with electron microscopy, should be adopted for this diagnosis. ACKNOWLEDGEMENT
This research was supported by the Mucolipidosis TV Foundation.
REFERENCES Amir, N., Zlotogora, J., Bach, G. (1987). Mucolipidosis type IV: clinical spectrum and natural history, Pedialrics, 79,953-959. Amon, J., Ornoy, A., Bach, G. (1986). Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders. A methodological study, Prenat. Diagn., 6,35 1-36], Amon, J., Ornoy, A., Bach, G. (1988). Cultured conditions found to minimize false positive diagnosis of lysosomal storage disorders in-vivo, In-vifro Cell Dev. B i d . , 24, 1159-1 164. Bach, G., Cohen, M.M., Kohn, G . (1975). Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV, Biochem. Biophys. Rex Commun., 66, 1483-1 490. Bach, G . , Zlotogora, J., Zeigler, M. (1992). Lysosomal storage disorders among Jews. In: Bonne-Tamir, B. (Ed.). Genetic Diversity Among Jews, Oxford: Oxford University Press, 301-304. Bargal, R., Bach, G. (1988). Phospholipid accumulation in mucolipidosis IV cultured fibroblasts, J . Inher. Metab. Dis., 11, 144-150. Caimi, L., Tettamanti, G., Berra, B., Sale, F.O., Borrone, C., Durand, P., Martin, J.J. (1982). Mucolipidosis IV, a sialolipidosis due to ganglioside sialidase deficiency, J. Inher. Metab. Dis., 5,2 18-224. Crandall, B.F., Phillipart, M., Brown, W.J., Bluestone, D. (1982). Mucolipidosis IV, Am. J. Med. Genet., 12,301-308. Kohn, G.,Livni, N., Ornoy, A., Sekeles, E., Beyth, Y., Legurn, C., Bach, G., Cohen, M.M. (1977). Prenatal diagnosis of mucolipidosis IV by electron microscopy, J . Pedintr., 90, 62-66.
1042
M. ZEIGLER ET AL.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the fohn phenol reagent, J. Biol. Chem., 193,265-275. Tellez-Nagel, I., Rapin, I., Iwamoto, R., Johnson, A.B., Norton, W.T., Nitowsky, H. (1976). Mucolipidosis IV: Clinical, ultrastructural, histochemical and chemical studies of a case, including brain biopsy, Arch. NeuroI., 33,828-835. Zeigler, M., Bach, G . (1986). Internalization of exogenous gangliosides in cultured skin fibroblasts for the diagnosis of mucolipidosis type IV, Clin. Chim. Acta, 157,183-190.
