55
Clinica Chimica Acta, 63 (1975) 55-60 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7221
ICHIRO MATSIJDAa, SHINICNIRO ARASHIMAa, YOGO OKAa, TAKASHI MITSWYA~Aa, SATOSHI ARIGAb , TATSURO IKEUCHI’ and TOKURO ICHIDAd of Pediatrics, Hokkaido University, School of Medicine, bDepartment of Obstetrjcs and ~y~~~o~ogy, ~okkaido U~iversity~ School of Medicine, ‘Ckr~mosome Research Unit, ~okka~do university, Faculty of Science and dDepartment of~jo~~ern~t~. Hokkaido Urziversity, School of Dentistry, Sapporo fJapan) aDepartment
(Received April I., 1975)
Pregnancy from a family at risk for fucosidosis was monitored. The fetus was diagnosed as having a carrier state of the disease. G-Lfucosidase activity, however, was found to be absent in white blood cells obtained from identical twins after delivery. The diagnostic evaluation of (I) the enzyme activity in amniotic fluid and in cultivated amniotic fluid cells and {2) the presence of fucose rich compound in amniotic fluid are discussed. -._ .._----.-_-_---._~..Introduction Fucosidosis is a rare inborn error of metabolism due to the absence or extremely low activity of cr-L-fucosidase and is transmitted as an autosomal recessive trait. Typical phenotypic features are severe progressive cerebral degeneration, coarsening of facial features with Hurler like appearance and skeletal changes [ 1,2] . The possibility of the prenatal diagnosis of fucosidosis was discussed by Milunsky [33 and Hsu and Hirshhorn [4f . However, the present report, to the best of our knowledge, is the first description of monitoring pregnancy with special reference to fucosidosis.
casereport The first child of a healthy woman at age of 24, was a female child born on January 19, 1964. She was diagnosed to have fucosidosis at the age 4 2/12 153, The parents were first cousins and were found to have a heterozygous state (61 . The mother became pregnant again in April 1974 and after genetic counselling and discussion of the problems posed by fucosidosis, a sample of
amniotic fluid f12-15 ml) was obtained at 14 and 18 weeks by transabdomina~ puncture under local anesthetic after localisation of the placenta by ultrasound. After a normal pregnancy identical female twins were born.
MateriaJ and method Amnjot~c fluid was centrifuged and the supernatant was used for measu~ng the enzyme activity. The collected amniotic cells were cultured in Eagle’s Minimum Essential Medium supplemented with 20% fetal calf serum together with antibiotics. Growth was initially established in 3 Falcon dishes 25 cm2 , and routine maintenance of the culture was carried out. The first sample was contaminated with fungus, but the second sample was not. Only epithelial like cells were seen growing in the tested sample and subculture seemed to be impossible. Amniotic fluid from pregnant women tested for independent purposes (eg. Down’s syndrome, adrenogenital syndrome) and of corresponding gestational age were stored at -20°C until enzyme assay after centrifugation. One of these samples obtained at a date close to the present test case was employed as accompanying control cell culture, and fibroblast like cells were obtained. Total cell count obtained was 1.5 X 10’ in tested sample and 2.3 X 10’ in control. For the enzyme assay 0.8 ml of amniotic fluid was added to 0.2 ml of 12.5 mM ~-n~truphenol~-~-f~~oside dissolved in 2 M acetic acid sodium acetate buffer pH 5.5, or to 0.2 ml of 12.5 mM ~-nitrophe~ol~ff~~-mannoside dissolved in 2 M citrate buffer pH 5.0, The substrate mixture was incubated at 37°C for 3 hours. The enzyme reaction was terminated by adding 0.5 ml of 5 M glycine buffer pH 10.5, after which tubes were centrifuged 3000 X g for 20 minutes. Blanks were prepared in the same way, omitting the incubation step. Absorbance of p-nitrophenol of supernatant solution was read on a Hitachi spectrophotometer at a wavelength of 400 nm, The activity of ru-L-fucosidase or a-r)-mannosidase calculated as nmoles of substrate cleaved per ml of amniotic fluid per hour at 37°C. Cultivated amniotic cells were harvested with 0.25% trypsin and centrifuged 100 x g for 10 minutes. The cell pellet was rinsed twice in isotonic saline solution. The number of cells were counted under a light microscope. Gells were suspended in 0.1 ml of distilled water and sonicated for 40 set in an ice-cold 10 kilocycle sonic oscillator. The method of enzyme assay was slightly modified. A 0.02 ml of the sample was added to 0.05 ml of 12.5 mM p-nitrophenol-cx-Lfucoside dissolved in 0.5 M acetic acid/sodium acetate buffer pH 5.5 or to 0.05 ml of 12.5 mM ~-nitrop~~nol~-rn~nos~de dissolved in 0.5 M citrate buffer pH 5.0. After incubation for 15 hours at 37”C, 0.4 ml of 1 M glycine buffer pH 10.5 was added. Centrifugation and reading were the same as described above. Protein was measured by the method of Lowry et al_ [7]. Activity is expressed as nmofes per hour per 106 cells and per mg of protein. White blood cells were collected from the twin sisters at 2 months of age and the enzyme activity was measured by the method described elsewhere [6] .
The method of Tsay et al. [8] was slightly modified and used for detecting fucose rich compounds in amniotic fluid.‘ After enzyme assay the remaining two samples of amniotic fluid were combined together (22 ml). Similarly combined remaining samples from 4 different pregnant women (25 ml) were used as control. The sample was dialized against 0.25% NH4HC03 solution over night and lyophilized. The dry material was dissolved in 5 ml of 0.09 M saline solution and was precipitated with ethanol (5 volumes). This precipitate was dissolved in 0.1 N acetic acid and subjected to gel filtration on a Sephadex G-50 column (1 cm X 48 cm) and eluted with the same solution at a flow rate of 1 ml per 5 minutes, collecting 1.5 ml fractions. The column effluent was assayed for hexose by the phenolHzS04 technique and pooled into 4 major fractions in the tested sample and 3 fractions in control (Fig. 1). Aliquots of these fractions were subjected to protein determination by Lowry’s method [7] and to sugar analysis by gasliquid chromatography [9] . Enzyme and protein determination and sugar analysis were duplicated in all tests. Results and comment As shown in Table I, activity of cr-L-fucosidase in amnotic fluid of the tested case was approximately 10% of control sample, whereas that of a-Dmannosidase was elevated. The obtained cultivated amniotic cells in the tested sample showed epithelial like cells and that in control showed fibroblasts. TABLE
I
(Y-L-FUCOSIDASE AMNIOTIC
AND
a-D-MANNOSIDASE
ACTIVITIES
Amniotic
fluid
Control
FLUID
AND
CULTIVATED
Fucosidase
Mannosidase
F/M
(nmoI/mI/h)
(nmol/ml/h)
ratio
48.4
-
sample
* **
Cultivated
cells
-
48.4
5
9.6
45.5
5
9.6
41.0 Tested
IN AMNIOTIC
CELLS
3.0 5.5
10
4.1
12.0
0.25
7.8
0.71
Mannosidase
Fucosidase
F/M ratio
(nmol/l06
Control
fibroblast
cells/h)
39.2
epithelial
mnol/mg/h)
(nmol/106
134
15.5
36
cells/h)
(nmollmglh)
53
11.3
3.19
(fibroblast)*
* *
(
30-170)
(
640)
(epithelisl)**
*
(
95-720)
(
20-80)
Tested
sample,
epitbelial
* At ** ***
2.52
At
14
11.7
weeks
gestation.
18 weeks
gestation.
Cited
from
Gebrie
et al. [lo].
106
40.7
371
0.25
TABLIS
II
a-L-FUCOSIDASE
IN WHITE
BLOOD
CELL
mU/mg protein Father Mother Twins
a b
C0IltrOl
0.14 0.16 0 0 0.14--0.50
a-L-Fucosidase activity on cultured cells of test sample, when expressed by per cell number, was approximately 30% of control and, when compared on the basis of mg protein, both activities of control and tested sample were close to each other. Activity of a-D-mannosidase was again found to be elevated in tested cells. The results of control cell culture were consistent with that reported by Gebrie et al. [lo] . They described that the mean value of a-L-fucosidase activity of epithelial like cells was higher than that of fibroblast like cells, when the activity was expressed as per mg of protein, although these ranges were overlapped. The first child of the family affected with fucosidosis showed a complete absence of the enzyme activity in the liver tissue, white blood cells and cultured skin fibroblast [5] . In another genotype of the disease reported by Borrone et al. [ 111, a-L-fucosidase activity was present in the serum and white blood cells, but was below 10% of control. Therefore, although the nature of the cell culture was different in tested and control sample, it appeared that the presence of a-L-fucosidase activity in cultivated cells amounting approximately 30% of control on the basis of cell count and 80% of control on the basis of mg of protein might seem to suggest that the fetus was in a carrier state rather than being outwardly affected. After
Totai sugar 300
100
ug /ml
/’
I
l ‘\ ’ ‘%\
PI
T”bn”mber lo
h
\ ;-:-y-,
20
Fig. 1. Fractionation of carbohydrate -. tested sample.
1,
L’
k
__*--_
30 compounds
40 in amniotic
fluid on Sephadex
G-50. _ -
- - -. control:
59
TABLE
III
FRACTIONATION
OF
Fraction
AMNIOTIC
FLUID
Total
Sugar
CARBOHYDRATE
(pg/mg
ON
SEPHADEX
G-50
protein)
protein (mg)
Fucose
Mannose
Galactose
Hexosamine
NANA
Pre I Patient
20.7
0.24
0.15
0.44
0.87
0.72
I
Control
54.4
0.63
1.12
1.58
8.40
0.35
II
Tested sample Control
79.3 193.8
2.66 1.59
1.24 0.76
1.84 0.80
59.9
1.62
0.59
1.30
1;
*
III
Control
96.2
0.05
0.05
0.11
*
*
89.0
0.05
0.17
0.08
*
*
Tested
sample
Tested
sample
* Since
duplicated
results
did
not
coincide,
figures
were
26.4 *
2.03 1;
omitted.
this study was concluded, epithelial like cells were cultivated from the amniotic fluid of a pregnant woman tested for Down’s syndrome and the enzyme activities were measured as listed in Table I. Even when this result is taken into account, similar trends are suspected. This diagnosis, however, was incorrect, since the enzyme activity was absent in white blood cells obtained from identical twins after delivery as shown in Table II. Residue of amniotic fluid was used for detecting fucose rich compounds and, as was shown in Fig. 1 and Table III, fucose, hexosarnine and NANA contents were apparently increased in the peak 1 fraction of the tested sample. Retrospectively, our observation indicates that the calculated ratio of ol-L-fucosidase to cu-D-mannosidase activity is much more reliable to give a correct diagnosis as compared with cr-L-fucosidase activity per cell counts or mg protein, since, as shown in Table I, the ratio of these two activities in cultivated cells was significantly low in tested sample. a-L-Fucosidase activity and the ratio of these two enzyme activities in amniotic fluid also appeared to be useful. In other words, LX-D-mannosidase activity may be of use as an internal standard. For further confirmation of the diagnosis detecting fucose rich compounds in amniotic fluid might be useful and the entire procedure will require much less time than performing cell culture. The reasons for presence of enzyme activity in amniotic fluid and cell culture is obscure at present, although contamination from the maternal side might be possible. Acknowledgement This investigation of Public Welfare.
was supported
by a Research
Grant from the Ministry
References 1
Durand,
2
Loeb, Acta
P.. Borrone, H.,
24,
Tondeur, 519-537
C. and M..
Della
Jonniax,
Cella.
G. (1969)
G.,
Mockel-Pohl,
J. Pediatr. S.
and
75,
665-674
Vamos-Hurwitz.
E.
(1969)
Helv.
Pediatr.
60
3
Milunsky.
A.
Thomas 4
Hsu.
5
Matsuda,
L.Y.E.
Med.
(1973)
Publisher, and I..
109,
6
Matsuda.
7
Lowry,
8
Tsay,
The
prenatal
diagnosis
of
hereditary
disorders,
Springfield.
Ill..
Charles.
C. and
P. 106. Hirshhorn.
Arashima,
K.
S.,
(1974)
Anakura.
Life M.,
Sci.
Ege.
14, A.
2311-2336 and
Hayata,
1. (1973)
Fucosidosis,
Tohoku,
J. Exp.
4148 I., Arashima,
O.H., G.G..
9
Bolton,
C.,
10
Gebrie,
A.B..
11
Borrone,
C..
S..
Anakura,
Rosebrough, Dawson,
Clamp,
G. and J.R.
Melancon. Gatti,
N.J.,
R.,
and
M. and
Far,
Matalon. Hough.
S..
Ryan.
Trias,
X.
A.L. R.
Oka. and
(1974)
L. (1965) C. and
and
(1973)
Clin. R.J.
J. Pediatr. Biochem.
Nadler,
Durrand,
Y.
Randall.
H.C.
P. (1974)
84.
J. 96, (1972)
Chim.
(1951)
Acta
J. Biol.
48,
9-13
Chem.
193.
265-275
865-868 5C Am.
J. Pediatr.
J. Obstetr. 84,
727-730
Gynec.
114.
314-320
Clinica Chimica Acta, 63 (1975) 55-60 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7221
ICHIRO MATSIJDAa, SHINICNIRO ARASHIMAa, YOGO OKAa, TAKASHI MITSWYA~Aa, SATOSHI ARIGAb , TATSURO IKEUCHI’ and TOKURO ICHIDAd of Pediatrics, Hokkaido University, School of Medicine, bDepartment of Obstetrjcs and ~y~~~o~ogy, ~okkaido U~iversity~ School of Medicine, ‘Ckr~mosome Research Unit, ~okka~do university, Faculty of Science and dDepartment of~jo~~ern~t~. Hokkaido Urziversity, School of Dentistry, Sapporo fJapan) aDepartment
(Received April I., 1975)
Pregnancy from a family at risk for fucosidosis was monitored. The fetus was diagnosed as having a carrier state of the disease. G-Lfucosidase activity, however, was found to be absent in white blood cells obtained from identical twins after delivery. The diagnostic evaluation of (I) the enzyme activity in amniotic fluid and in cultivated amniotic fluid cells and {2) the presence of fucose rich compound in amniotic fluid are discussed. -._ .._----.-_-_---._~..Introduction Fucosidosis is a rare inborn error of metabolism due to the absence or extremely low activity of cr-L-fucosidase and is transmitted as an autosomal recessive trait. Typical phenotypic features are severe progressive cerebral degeneration, coarsening of facial features with Hurler like appearance and skeletal changes [ 1,2] . The possibility of the prenatal diagnosis of fucosidosis was discussed by Milunsky [33 and Hsu and Hirshhorn [4f . However, the present report, to the best of our knowledge, is the first description of monitoring pregnancy with special reference to fucosidosis.
casereport The first child of a healthy woman at age of 24, was a female child born on January 19, 1964. She was diagnosed to have fucosidosis at the age 4 2/12 153, The parents were first cousins and were found to have a heterozygous state (61 . The mother became pregnant again in April 1974 and after genetic counselling and discussion of the problems posed by fucosidosis, a sample of
amniotic fluid f12-15 ml) was obtained at 14 and 18 weeks by transabdomina~ puncture under local anesthetic after localisation of the placenta by ultrasound. After a normal pregnancy identical female twins were born.
MateriaJ and method Amnjot~c fluid was centrifuged and the supernatant was used for measu~ng the enzyme activity. The collected amniotic cells were cultured in Eagle’s Minimum Essential Medium supplemented with 20% fetal calf serum together with antibiotics. Growth was initially established in 3 Falcon dishes 25 cm2 , and routine maintenance of the culture was carried out. The first sample was contaminated with fungus, but the second sample was not. Only epithelial like cells were seen growing in the tested sample and subculture seemed to be impossible. Amniotic fluid from pregnant women tested for independent purposes (eg. Down’s syndrome, adrenogenital syndrome) and of corresponding gestational age were stored at -20°C until enzyme assay after centrifugation. One of these samples obtained at a date close to the present test case was employed as accompanying control cell culture, and fibroblast like cells were obtained. Total cell count obtained was 1.5 X 10’ in tested sample and 2.3 X 10’ in control. For the enzyme assay 0.8 ml of amniotic fluid was added to 0.2 ml of 12.5 mM ~-n~truphenol~-~-f~~oside dissolved in 2 M acetic acid sodium acetate buffer pH 5.5, or to 0.2 ml of 12.5 mM ~-nitrophe~ol~ff~~-mannoside dissolved in 2 M citrate buffer pH 5.0, The substrate mixture was incubated at 37°C for 3 hours. The enzyme reaction was terminated by adding 0.5 ml of 5 M glycine buffer pH 10.5, after which tubes were centrifuged 3000 X g for 20 minutes. Blanks were prepared in the same way, omitting the incubation step. Absorbance of p-nitrophenol of supernatant solution was read on a Hitachi spectrophotometer at a wavelength of 400 nm, The activity of ru-L-fucosidase or a-r)-mannosidase calculated as nmoles of substrate cleaved per ml of amniotic fluid per hour at 37°C. Cultivated amniotic cells were harvested with 0.25% trypsin and centrifuged 100 x g for 10 minutes. The cell pellet was rinsed twice in isotonic saline solution. The number of cells were counted under a light microscope. Gells were suspended in 0.1 ml of distilled water and sonicated for 40 set in an ice-cold 10 kilocycle sonic oscillator. The method of enzyme assay was slightly modified. A 0.02 ml of the sample was added to 0.05 ml of 12.5 mM p-nitrophenol-cx-Lfucoside dissolved in 0.5 M acetic acid/sodium acetate buffer pH 5.5 or to 0.05 ml of 12.5 mM ~-nitrop~~nol~-rn~nos~de dissolved in 0.5 M citrate buffer pH 5.0. After incubation for 15 hours at 37”C, 0.4 ml of 1 M glycine buffer pH 10.5 was added. Centrifugation and reading were the same as described above. Protein was measured by the method of Lowry et al_ [7]. Activity is expressed as nmofes per hour per 106 cells and per mg of protein. White blood cells were collected from the twin sisters at 2 months of age and the enzyme activity was measured by the method described elsewhere [6] .
The method of Tsay et al. [8] was slightly modified and used for detecting fucose rich compounds in amniotic fluid.‘ After enzyme assay the remaining two samples of amniotic fluid were combined together (22 ml). Similarly combined remaining samples from 4 different pregnant women (25 ml) were used as control. The sample was dialized against 0.25% NH4HC03 solution over night and lyophilized. The dry material was dissolved in 5 ml of 0.09 M saline solution and was precipitated with ethanol (5 volumes). This precipitate was dissolved in 0.1 N acetic acid and subjected to gel filtration on a Sephadex G-50 column (1 cm X 48 cm) and eluted with the same solution at a flow rate of 1 ml per 5 minutes, collecting 1.5 ml fractions. The column effluent was assayed for hexose by the phenolHzS04 technique and pooled into 4 major fractions in the tested sample and 3 fractions in control (Fig. 1). Aliquots of these fractions were subjected to protein determination by Lowry’s method [7] and to sugar analysis by gasliquid chromatography [9] . Enzyme and protein determination and sugar analysis were duplicated in all tests. Results and comment As shown in Table I, activity of cr-L-fucosidase in amnotic fluid of the tested case was approximately 10% of control sample, whereas that of a-Dmannosidase was elevated. The obtained cultivated amniotic cells in the tested sample showed epithelial like cells and that in control showed fibroblasts. TABLE
I
(Y-L-FUCOSIDASE AMNIOTIC
AND
a-D-MANNOSIDASE
ACTIVITIES
Amniotic
fluid
Control
FLUID
AND
CULTIVATED
Fucosidase
Mannosidase
F/M
(nmoI/mI/h)
(nmol/ml/h)
ratio
48.4
-
sample
* **
Cultivated
cells
-
48.4
5
9.6
45.5
5
9.6
41.0 Tested
IN AMNIOTIC
CELLS
3.0 5.5
10
4.1
12.0
0.25
7.8
0.71
Mannosidase
Fucosidase
F/M ratio
(nmol/l06
Control
fibroblast
cells/h)
39.2
epithelial
mnol/mg/h)
(nmol/106
134
15.5
36
cells/h)
(nmollmglh)
53
11.3
3.19
(fibroblast)*
* *
(
30-170)
(
640)
(epithelisl)**
*
(
95-720)
(
20-80)
Tested
sample,
epitbelial
* At ** ***
2.52
At
14
11.7
weeks
gestation.
18 weeks
gestation.
Cited
from
Gebrie
et al. [lo].
106
40.7
371
0.25
TABLIS
II
a-L-FUCOSIDASE
IN WHITE
BLOOD
CELL
mU/mg protein Father Mother Twins
a b
C0IltrOl
0.14 0.16 0 0 0.14--0.50
a-L-Fucosidase activity on cultured cells of test sample, when expressed by per cell number, was approximately 30% of control and, when compared on the basis of mg protein, both activities of control and tested sample were close to each other. Activity of a-D-mannosidase was again found to be elevated in tested cells. The results of control cell culture were consistent with that reported by Gebrie et al. [lo] . They described that the mean value of a-L-fucosidase activity of epithelial like cells was higher than that of fibroblast like cells, when the activity was expressed as per mg of protein, although these ranges were overlapped. The first child of the family affected with fucosidosis showed a complete absence of the enzyme activity in the liver tissue, white blood cells and cultured skin fibroblast [5] . In another genotype of the disease reported by Borrone et al. [ 111, a-L-fucosidase activity was present in the serum and white blood cells, but was below 10% of control. Therefore, although the nature of the cell culture was different in tested and control sample, it appeared that the presence of a-L-fucosidase activity in cultivated cells amounting approximately 30% of control on the basis of cell count and 80% of control on the basis of mg of protein might seem to suggest that the fetus was in a carrier state rather than being outwardly affected. After
Totai sugar 300
100
ug /ml
/’
I
l ‘\ ’ ‘%\
PI
T”bn”mber lo
h
\ ;-:-y-,
20
Fig. 1. Fractionation of carbohydrate -. tested sample.
1,
L’
k
__*--_
30 compounds
40 in amniotic
fluid on Sephadex
G-50. _ -
- - -. control:
59
TABLE
III
FRACTIONATION
OF
Fraction
AMNIOTIC
FLUID
Total
Sugar
CARBOHYDRATE
(pg/mg
ON
SEPHADEX
G-50
protein)
protein (mg)
Fucose
Mannose
Galactose
Hexosamine
NANA
Pre I Patient
20.7
0.24
0.15
0.44
0.87
0.72
I
Control
54.4
0.63
1.12
1.58
8.40
0.35
II
Tested sample Control
79.3 193.8
2.66 1.59
1.24 0.76
1.84 0.80
59.9
1.62
0.59
1.30
1;
*
III
Control
96.2
0.05
0.05
0.11
*
*
89.0
0.05
0.17
0.08
*
*
Tested
sample
Tested
sample
* Since
duplicated
results
did
not
coincide,
figures
were
26.4 *
2.03 1;
omitted.
this study was concluded, epithelial like cells were cultivated from the amniotic fluid of a pregnant woman tested for Down’s syndrome and the enzyme activities were measured as listed in Table I. Even when this result is taken into account, similar trends are suspected. This diagnosis, however, was incorrect, since the enzyme activity was absent in white blood cells obtained from identical twins after delivery as shown in Table II. Residue of amniotic fluid was used for detecting fucose rich compounds and, as was shown in Fig. 1 and Table III, fucose, hexosarnine and NANA contents were apparently increased in the peak 1 fraction of the tested sample. Retrospectively, our observation indicates that the calculated ratio of ol-L-fucosidase to cu-D-mannosidase activity is much more reliable to give a correct diagnosis as compared with cr-L-fucosidase activity per cell counts or mg protein, since, as shown in Table I, the ratio of these two activities in cultivated cells was significantly low in tested sample. a-L-Fucosidase activity and the ratio of these two enzyme activities in amniotic fluid also appeared to be useful. In other words, LX-D-mannosidase activity may be of use as an internal standard. For further confirmation of the diagnosis detecting fucose rich compounds in amniotic fluid might be useful and the entire procedure will require much less time than performing cell culture. The reasons for presence of enzyme activity in amniotic fluid and cell culture is obscure at present, although contamination from the maternal side might be possible. Acknowledgement This investigation of Public Welfare.
was supported
by a Research
Grant from the Ministry
References 1
Durand,
2
Loeb, Acta
P.. Borrone, H.,
24,
Tondeur, 519-537
C. and M..
Della
Jonniax,
Cella.
G. (1969)
G.,
Mockel-Pohl,
J. Pediatr. S.
and
75,
665-674
Vamos-Hurwitz.
E.
(1969)
Helv.
Pediatr.
60
3
Milunsky.
A.
Thomas 4
Hsu.
5
Matsuda,
L.Y.E.
Med.
(1973)
Publisher, and I..
109,
6
Matsuda.
7
Lowry,
8
Tsay,
The
prenatal
diagnosis
of
hereditary
disorders,
Springfield.
Ill..
Charles.
C. and
P. 106. Hirshhorn.
Arashima,
K.
S.,
(1974)
Anakura.
Life M.,
Sci.
Ege.
14, A.
2311-2336 and
Hayata,
1. (1973)
Fucosidosis,
Tohoku,
J. Exp.
4148 I., Arashima,
O.H., G.G..
9
Bolton,
C.,
10
Gebrie,
A.B..
11
Borrone,
C..
S..
Anakura,
Rosebrough, Dawson,
Clamp,
G. and J.R.
Melancon. Gatti,
N.J.,
R.,
and
M. and
Far,
Matalon. Hough.
S..
Ryan.
Trias,
X.
A.L. R.
Oka. and
(1974)
L. (1965) C. and
and
(1973)
Clin. R.J.
J. Pediatr. Biochem.
Nadler,
Durrand,
Y.
Randall.
H.C.
P. (1974)
84.
J. 96, (1972)
Chim.
(1951)
Acta
J. Biol.
48,
9-13
Chem.
193.
265-275
865-868 5C Am.
J. Pediatr.
J. Obstetr. 84,
727-730
Gynec.
114.
314-320
