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Carrier detection of sialidosis with partial -galactosidase deficiency by the assay of lysosomal sialidase in lymphocytes.

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Carrier Detection of Siahdosis with Partial
P-Galactosidase Deficiency by the Assay
of Lysosomal Siahdase in Lymphocytes
Shoji Tsuji, MD," Takamichi Yamada, PhD," Toshio Ariga, PhD,* Itaru Toyoshima, MD,?
Haruyasu Yamaguchi, MD,S Yoshisuke Kitahara, MD,§ Tadashi Miyatake, MD,V and Tamio Yamakawa, MD"
~
~
~
~~~
Lysosomal and plasma membrane sialidase activities in lymphocytes were studied in four patients with sialidosis with
partial 6-galactosidase deficiency, four obligate heterozygotes, and three siblings of a patient. Lysosomal sialidase
activity in homozygotes was absent, and that in heterozygotes was significantly decreased to 70% of control level. The
results indicate that carriers can be detected by the assay of lysosomal sialidase activity of lymphocytes.
Tsuji S, Yamada T, Ariga T, Toyoshima I, Yamaguchi H, Kitahara Y, Miyatake T, Yamakawa T: Carrier
detection of sialidosis with partial P-galactosidase deficiency by the assay of lysosomal sialidase
in lymphocytes. Ann Neurol 15:181-183, 1984
Sialidase deficiency has been demonstrated in cultured
skin fibroblasts of patients with sialidosis with partial pgalactosidase deficiency [4, 6-9, 11-14, 161. Carrier
detection by assay of leukocyte sialidase has been
difficult, however, because 20 to 50% of residual
sialidase activity has been observed in leukocytes or
lymphocytes in this disease [ 5 , 6 , 13, 141, and the
leukocyte sialidase activity in obligate heterozygotes
has not always shown an intermediate value [13].
We previously demonstrated that there are at least
two kinds of sialidases in lymphocytes-one in the lysosomal fraction and the other in the plasma membrane
fraction-and found the lysosomal sialidase to be absent in a patient with this disease [17). The present
study describes the lysosomal and plasma membrane
sialidase activities in lymphocytes from four patients,
four obligate heterozygotes, and three siblings of a
patient.
Patients and Methods
Findings in four Japanese patients from four different families
were analyzed. A summary of the clinical manifestations is
given in Table 1. Detailed descriptions of patients 1 and 2
have been given elsewhere [8, 181. Patient 1 developed peripheral neuropathy after the report in 1979 C8).
Leukocytes were prepared by the dextran sedimentation
method, and lymphocytes were prepared by the Ficoll-
From the *Department of Biochemistry and Metabolism, the Tokyo
Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 11 3; the tDepartment of Neurology, Brain Research Institute, Niigata University, Asahimachi-dori, Niigata 95 1;
the $Institute of Neurology and Rehabilitation, School of Medicine,
Gumma University, 3-39-22 Showa-machi, Maebashi, Gumma 37 1;
and the $Department of Neurology, Surnitomo Hospital, 5-2-2
Nakanoshima, Era-ku, Osaka 530, Japan.
Hypaque sedimentation method as reported previously [ 141.
The purity of the lymphocyte fraction was 97%.
Lysosomal and plasma membrane fractions were prepared
by discontinuous sucrose density gradient centrifugation as
reported previously [ 17).
Sialidase (EC 3.2.1.18) activity was assayed using a,2-*3Ne~Ac-lactit[~H~oI
as the substrate by the method reported
previously 1171. P-Galactosidase activity was assayed by the
method of Van Hoof and Hers [151.
Results
(3-Galactosidase and sialidase activity in leukocytes and
fibroblasts of patients 1 through 4 is shown in Table 2.
All four patients showed 4 to 23% residual pgalactosidase activity in both leukocytes and fibroblasts.
Sialidase activity in skin fibroblasts cultured from patients 1 , 3, and 4 was almost completely absent. In
contrast, leukocyte sialidase showed 43 to 65% residual activity. These results, together with the clinical
manifestations, confirm the existence of sialidosis with
partial P-galactosidase deficiency in all four patients.
Table 3 shows sialidase activity in the lysosomal and
plasma membrane fractions of lymphocytes from the
four patients, four obligate heterozygotes, and three
siblings of patient 4. As shown in the table, NeuAclactitol assay demonstrated an almost total absence of
lysosomal sialidase activity in homozygotes. In contrast,
Received Apr 19, 1983, and in revised form June 28. Accepted for
publication July 2, 1983.
Address reprint requests to Dr Tsuji.
181
there were no significant differences in sialidase activity
in plasma membrane fractions of patients and controls.
In obligate heterozygotes, lysosomal sialidase activity
was significantly decreased to 70% of the level in controls; plasma membrane fractions showed normal levels
of activity. In one of three siblings, lysosomal sialidase
activity decreased to 70% of that of controls; in the
other two siblings lysosomal sialidase showed normal
activity.
Table 1 . Clinical Manifestations in Four Patients
Patient No.
Characteristic
1
2
3
4
Age, sex
Age at onset
Ethnic origin
Consanguinity
52, M
18
Japanese
44, M
34
Japanese
27, M
24
Japanese
-
+
44, M
30
Japanese
IQ
Cherry-red
spots
Seizures
Cerebellar
ataxia
Myoclonus
Pyramidal
signs
Peripheral
neuropath y
Hearing loss
Coarse face
Height (cm)
Vertebral
d ysplasia
Angiokeratoma
Corneal
clouding
Inguinal
hernia
Vacuolated
lymphocytes
Urinary excretion of
sialyl oligosaccharides
73
81
79
66
+
+
+
+
+
+
+
-
-
+
+
+
-
+
+
+
-
+
+
-
+
-
+
-
-
-
+
+
+
+
+
160
165
170
160
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
Discussion
As we have stated, in patients with partial pgalactosidase deficiency, sialidase deficiency has been
demonstrated only in cultured skin fibroblasts 14, 6-9,
11-14, 161, and 20 to 50%1residual activity has been
found in leukocytes 15, 6, 13, 141.
Our findings demonstrate that lymphocyte lysosomal
sialidase activity in homozygotes is absent and that in
heterozygotes is significantly decreased in our study to
70% of control level. The results indicate that carrier
detection may be possible by the assay of lysosomal
sialidase activity in lymphocytes. The results also may
indicate the difficulty in detecting carriers by assay of
leukocyte sialidase activity 1131: the concomitant
plasma membrane sialidase and lysosomal sialidase
activity.
Recently Hoogeveen and colleagues [21 demonstrated that sialidase activity in cultured skin fibroblasts
in this disease can be raised to the normal level by the
addition of a corrective factor to the culture medium.
Restoration of sialidase activity after somatic cell hybridization of fibroblasts of this disease with those of
mucolipidosis I, which is considered a primary sialidass
deficiency, suggest that the basic biochemical defect
may be different in the two diseases [3, 101. It is proposed that the primary genetic defect in this disease is
in the posttranslational step and not in the structural
genetic coding for sialidase f 11. The present data show
lysosomal siahdase activity in heterozygotes to be decreased to 70% of the normal control level. We cannot
-
Table 2. P-Galactosidase and Sialiduse Activity in kukorytes and Cultured Skin Fibroblasts"
p-Galactosidase
Patient No.
Leukocytes
1
9.2
19.0
3.9
22.3
95.0 & 27.2
(n = 168)
2
3
4
Controlsb
Sialidase
Fibroblasts
36.3
. . .
37.3
29.4
397.7
179.0
(n = 8 )
*
"Expressed as nanomoles per milligram of protein per hour.
"Values are expressed as means -+ standard errors of the mean.
182 Annals of Neurology
Vol 1 5
No 2 February 1984
Leukocytes
Fibroblasts
0.96
1.05
0.89
1.34
2.06 ? 0.15
(n = 3 )
0.16
. . .
0.29
0.88
16.3 2 5.0
(n = 3)
Table 3. Lysosoma/ and Plajma Membrane Sinlidase
Actiuity in Homozygotes, Obligate Heterozygotes,
Siblings of a Patient, and Control Subjects"
Subject
Lysosomal
Fraction
Plasma
Membrane
Patient 1
Patient 2
Patient 3
Patient 4
Mother of patient 2
Daughter of patient 3
Mother of patient 4
Father of patient 4
1st sibling of patient 4
2nd sibling of patient 4
3rd sibling of patient 4
Controls (n = 8)
0.20
0.17
0.19
0.21
5.26b
4.87b
4.95b
5.04b
6.91
7.00
5.22"
7.29
3.73
3.70
3.22
3.61
4.17
4.03
4.21
4.06
4.25
4.14
4.17
4.41
* 0.26'
* 0.40'
"Expressed as nanomoles per milligram of protein per hour.
'Significantly different from control value (p < 0.001).
'Values are expressed as means t standard errors of the mean.
determine on the basis of our present results, however,
whether the primary genetic defect here, which is different from the defect in mucolipidosis I, is in the
posttranslational processing of lysosomal sialidase or
in the structural genetic coding for the subunit of
sialidase.
Supported in part by grants for the study of intractable diseases and
neuroimmunological disorders from the Ministry of Health and Welfare of Japan.
References
1. d'Azzo A, Hoogeveen A, Reuser AJJ, et al: Molecular defect in
combined P-galactosidase and neuraminidase deficiency in man.
Proc Natl Acad Sci USA 79:4535-4539, 1982
2. Hoogeveen A, d'Azzo A, Brossmer R, Galjaard H : Correction
of combined P-galactosidase/neuraminidase deficiency in human
fibroblasts. Btochem Biophys Res Commun 103:292-300,
1981
3. Hoogeveen AT, Verheijen F W ,d'Azzo A, Galjaard H: Genetic
heterogeneity in human neuraminidase deficiency. Nature
285:500-502, 1980
4. Kobayashi T, Goto I, Tanaka Y, e t al: Biochemical comparison
of the dysmorphic type with the norrnosomatic type of sialidosis.
Clin Chim Acta 103:343-347, 1980
5. Kobayashi T, Ohta M, Goto I, et al: Adult type mucolipidosis
with P-galactosidase and sialidase deficiency: histological and
biochemical studies. J Neurol 221:137-149, 1979
6. Kuriyama M, Miyatake T, Owada M, Kitagawa T: Neuraminidase activities in sialidosis and mucolipidosis. J Neurol Sci
541181-187, 1982
7. Kuriyama M, Okada S, Tanaka Y, Umezaki H: Adult
mucolipidosis with P-galactosidase and neuraminidase deficiencies. J Neurol Sci 46:245-254, 1980
8. Miyatake T, Atsumi T , Obayashi T , et al: Adult type neuronal
storage disease with neuraminidase deficiency. Ann Neurol
6232-244, 1979
9. Miyatake T, Yamada T, Suzuki M, et al: Sialidase deficiency in
adult-type neuronal storage disease. FEBS Lett 97:257-259,
1979
10. Mueller OT, Shows TB: Human P-galactosidase and aneuraminidase deficient mucolipidosis: genetic complementation analysis of the neuraminidase deficiency. Hum Genet
60:158-162, 1982
11. Okada S, Yutaka T, Kato T, et al: A case of neuraminidase
deficiency associated with a partial P-galactosidase defect: clinical, biochemical and radiological studies. Eur J Pediatr 130:239249, 1979
12. Pallmann B, Sandhoff K, Berra B, Miyatake T: Sialidase in brain
and fibroblasts in three patients with different types of sialidosis.
Adv Exp Med Biol 125:401-414, 1980
13. Suzuki Y , Sakuraba H, Potier M, e t al: P-Galactosidaseneuraminidase deficiency in adults: deficiency of a freeze-labile
neuraminidase in leukocytes and fibroblasts. H u m Genet
58:387-389, 1981
14. Tsuji S, Yamada T, Tsutsumi A, Miyatake T Neuraminidase
deficiency and accumulation of sialic acid in lymphocytes in adult
type sialidosis with partial P-galactosidase deficiency. Ann
Neurol 11:541-543, 1982
15. Van Hoof F, Hers HG: The abnormalities of lysosomal enzymes
in mucopolysaccharidoses. Eur J Biochem 7:34-44, 1968
16. Wenger DA, Tarby TJ, Wharton C: Macular cherry-red spots
and myoclonus with dementia: coexistent neuraminidase and Pgalactosidase deficiencies. Biochern Biophys Res Commun
82~589-595, 1978
17. Yamada T, Tsuji S, Ariga T, Miyatake T: Lysosomal sialidase
deficiency in sialidosis with partial P-galactosidase deficiency.
Biochim Biophys Acta 755:106-111, 1983
18. Yamaguchi H , Okamoto K, Shooji M, et al: A case of adult type
sialidosis with partial p-galactosidase deficiency without myoclonus. Clin Neurol 23:1-8, 1983
Tsuji et al: Lymphocyte Lysosornal Sialidase
183
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carrier, detection, deficiency, galactosidase, sialidosis, lysosomal, partial, sialidase, lymphocytes, assays
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