From FRIGE (Foundation for Research in Genetics and
Endocrinology), Genetic Center, 20/1, Bima Nagar, Satellite, Ahmedabad
380 015, India.
This study was conducted to assess the
variability of clinical expression of Lysosomal storage disorders
(LSDs) and the selection of specific enzyme investigation to reach
the differential diagnosis. Initially 150 children in the age
range of 15 days to 13 years were screened for common metabolic
disorder and based on screening results, clinical signs and
symptoms, 30 children (4 mo-12 yr) of these were selected for the
leukocyte enzyme study. Of these 21 were confirmed to have LSDs.
The most common disorder was GM2-gangliosidosis (47.61%, 10/21)
followed by mucopolysaccharidosis (33.33%; 7/21). All showed
variable phenotypic expression. Metachro-matic leukodystrophy (MLD)
was observed in 9.5% (2/21) of children with arylsulphatase A
enzyme deficiency, while two children had shown pseudodeficiency
of arylsulphatase A. One case each of galactosialidosis and
GM1-gangliosidosis were observed. We conclude that children with
developmental delay, seizures, dysmorphic features and
organomegaly, with or without positive urinary screening for
common metabolic disorders, need to be investigated further for
LSDs. Variability of clinical expression is commonly observed in
LSDs which require further confirmation by specific leukocyte
enzyme study.
Key words: Lysosomal storage disorders (LSDs), Lysosomal
enzyme, Metachromatic leukodystrophy (MLD), GM2-gangliosidosis.
Lysosomal storage disorders (LSDs) are a group of
genetic diseases characterized by an inherited defect in the
functional expression of any of the lysosomal enzyme(1). The
resultant accumulation of substrates of lysosomal enzymes inside the
cell as well as in the blood stream causes a loss of function in one
or several crucial areas of the body and the clinical features are
very much dependent upon the rate and magnitude of accumulation of
the undegraded substances. Therefore, a wide spectrum of phenotypic
expression is observed in children with LSDs(2). These disorders are
progressive and most affected individuals appear normal at birth,
but subsequently develop severe morbidity and die after months or
years. As a result, many of the LSDs are missed or incorrectly
diagnosed. Some of the disorders are fatal in childhood or appear
during adolescence(3).
The clinical consequences of the enzyme defect
can vary, but commonly this may result in developmental defects,
mental and physical disability, defects in the immune systems,
skeletal abnormality, ophthalmic abnormality, etc.(4).
Paucity of Indian studies(5) makes reporting of
even small series of data worthwhile. Despite the lack of precise
biochemical assay, a clinician should have a high index of suspicion
leading to early diagnosis. This would facilitate genetic counseling
and possible enzyme replacement therapy (ERT).
Subjects & Methods
The present study involves initial investigation
of 150 children in the age range of 15 days to 13 years with
suspected meta-bolic disorder due to most common clinical symptoms
of milestone regression, seizures and organomegaly. All were
screened for the most common metabolic disorder by urine tests as
described elsewhere(6). Based on screening results and clinical
observations, thirty children were selected for leukocyte enzyme
study. Selection of four children with suspected MLD was based on
positive CT/MRI findings of leukoencephalopathy.
Thirteen different lysosomal enzyme studies were
carried out in leukocytes isolated from peripheral blood collected
in a heparinized vacutainer. Enzyme activity was measured using the
synthetic substrate 4-methylumbelliferrone (4MU) - fluorogenic
substrate and p-nitrocatechol sulfate (NCS) -spectrophotometric
substrate obtained from Sigma Chemicals(3). For hexosaminidase A
(Hex-A), the heat inactivation method was used(7). The enzyme
activity was expressed as nmol/h/mg of protein.
Results
Of 150 children, urine screening detected seven
children with positive Azure test (spot test) indicative for
mucopolysaccharidosis (MPS). Six children with positive reducing
substance were also selected for LSDs study due to specific
clinical signs and symptoms. Rest of the cases showed negative
findings.
The common clinical signs observed in all LSDs
were regression in milestone (78%) followed by dysmorphic features
(75%), organomegaly (60%), macrocephaly (43%), hypotonia (52.17%)
and seizures in 21% of the cases. Other signs were more specific to
the specific LSDs. Table I provides detailed clinical
description of these 21 cases.
TABLE I
Clinical Symptoms and Signs in Children with Suspected Metabolic Disorder.
| |
Disease |
Clinical
features |
GM1
Gangliosi-dosis
(1) |
GM2
Gangliosidosis
(10) |
MPS VI
(4)
|
MPS IV
(3)
|
MLD
(2)
|
Galactos-ialidosis
(1)
|
Global developmental
Delay/Milestone
regression |
√ |
√(10) |
√(4) |
|
√(2) |
√ |
Age of onset
|
4 month
|
5mo to 5 y
|
8 mo to
10 y
|
3 to 12 y
|
4.5 to 6 y
|
4 y
|
Facial
dysmorphism |
√ |
√(4) |
√(4) |
√(3) |
|
√ |
Seizures
|
√
|
√(4)
|
|
|
|
|
Macrocephaly
|
√
|
√(3)
|
√(4)
|
|
√(2)
|
|
Organomegaly
|
|
√(6)
|
√(4)
|
√(3)
|
|
√
|
Hypotonia
|
√
|
√(10)
|
√(1)
|
|
|
|
Cherry red spot
|
|
√(3)
|
|
|
|
|
Photophobia
|
|
|
|
|
|
√
|
Vomiting
|
|
√(1)
|
√(2)
|
|
|
|
Cerebral atrophy
|
|
√(1)
|
|
|
|
|
Leukoencephalopathy
|
|
|
|
|
√(2)
|
|
Skeletal dysplasia
|
|
|
|
√(3)
|
|
|
Kyphosis
|
|
|
√(4)
|
√(3)
|
|
|
Genu valgus
|
|
|
|
√(3)
|
|
|
Mental retardation
|
|
√(2)
|
|
|
|
|
Neural abnormalities
|
|
√(1)
|
|
|
|
|
Adenoid hyperplasia
|
|
|
|
√(3)
|
|
|
Additional
findings
|
No vision in
right eye,
left eye
small
|
Optical atrophy
(1)
|
Short
trunk
dwarfism-
(4)
|
Short
trunk
dwarfism
(4)
Interior
beaking of
lumbar
spine and
restriction
of joint
movement
(1)
|
Limb
deformity
and gait
distrur-
bance
(1)
Depressed
nasal
bridge
and
curved
fingers
(1)
|
White spot
on sclera
|
Values in parentheses are the number of cases and √ and - sign represent the presence and
absence of character respectively.
Table II demonstrates, reduced activity of
Hex-A and b-Hexosaminidase T (Hex-T) in 10 of 21. Of these,
four children showed positive reducing substances in urinary
screening. Depending on the variable con-centration of the enzyme
activity they were diagnosed as B-variant, Bl-variant and Sandhoff
O variant.
TABLE II
Related Enzyme Activities in Children with Lysosomal Storage Disorders.
Case
No. |
Enzyme
|
Assay values
nmol/hr/mg protein |
Diagnosis |
|
GM2gangliosidosis |
1.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
91.42
50
|
GM2gangliosidosis
|
2.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
320
Undetectable
|
B variant
|
3.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
362.64
5.7
|
B variant
|
4.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
333.33
2.35
|
B variant
|
5.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
186
24
|
Tay Sachs Variant B1
|
6.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
840.0
31.25
|
Tay Sachs Variant B1
|
7.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
872.18
26.32
|
Tay Sachs Variant B1
|
8.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
3.33
5
|
Sandhoff Variant O
|
9.
|
B-Hexosaminidase T
B-Hexosaminidase A
|
283.68
5
|
Sandhoff Variant O
|
|
10. |
B-Hexosaminidase T
B-Hexosaminidase A |
201.43
5.9 |
Sandhoff Variant O |
|
Mucopolysaccharidosis
|
11.
|
Arylsulphatase B
|
4.0
|
Maroteaux-Lamy (MPS-VI)
|
12.
|
Arylsulphatase B
|
11.11
|
Maroteaux-Lamy (MPS-VI)
|
13.
|
Arylsulphatase B
|
13.73
|
Maroteaux-Lamy (MPS-VI)
|
14.
|
Arylsulphatase B
|
Undetectable
|
Maroteaux-Lamy (MPS-VI)
|
15.
|
B-Galactosidase
|
Undetectable
|
Morquio-B Syndrome
|
16.
|
B-Galactosidase
|
Undetectable
|
Morquio-B Syndrome
|
17.
|
B-Galactosidase
|
2.27
|
Morquio-B Syndrome
|
|
Metachromatic Leucodystrophy |
18.
|
Arylsulphatase A
|
3.125
|
MLD
|
19.
|
Arylsulphatase A
|
Undetectable
|
MLD
|
|
GM1-gangliosidosis |
20.
|
B-Galactosidase
|
6.66
|
GM1-gangliosidosis
|
|
Galactosialidosis |
21
|
B-Galactosidase
|
16.92
|
Galactosialidosis
|
* Normal range: Beta-Hexosaminidase T:801 ± 190 nmol/hr/mg protein,Beta-
Hexosaminidase A: 5572%, Arylsulphatase A: 71 ± 11.4 nmol/hr/mg protein,
Arylsulphatase B: 121 ± 107 nmol/hr/mg protein, Beta-Galactosidase:
97.9 ± 19.8 in nmol/hr/mg protein.
The deficient enzyme activity of arylsulfatase B
was observed in MPS VI and B galactosidase activity was found to be
almost absent in MPS IV (Table III). Of four children
suspected for MLD, two had shown very low activity of arylsulphatase
A (ARSA) confirming MLD, while two children had moderately reduced
activity of ARSA (43.10 and 40.0 nmol/h/mg protein respectively)
with only positive finding of leukoencephalopathy in MRI. In the
absence of other clinical symptoms and signs they were diagnosed as
pseudo deficiency for ARSA. One child with GMI-gangliosidosis had
reduced activity of b-galactosidase while one with moderately
low activity of b-galactosidase was diagnosed as probable
galactosialidosis. Though in latter case confirmative enzyme
activity of neuraminidase was not carried out. Both these cases had
positive reducing substance on urine screening.
Discussion
Although individually rare, lysosomal storage
disorders constitute a significant burden on society and an
important health problem. High prevalence of 8 per 100,000 births
has been reported in the population of British Columbia (20% of
confirmed metabolic disorders(6). Estimate of such prevalence is not
possible in present study due to small number of cases. Nonetheless
it is important to observe that the most common LSDs observed were
GM2-gangliosidosis in 47.61% (10/21) of cases and MPS in 33.33%
(7/21), with variable clinical expression. Similar observation was
made by Beek(2). He observed a broad phenotypic spectrum in MPS-I
(Hurler-Scheie disease), Gaucher disease, several forms of
GM2-gangliosidosis and the different manifestations of Beta-galactosidase
deficiency (GM1-gangliosidosis and Morquio disease type B). It is
not yet clearly understood why the same enzyme defect can manifest
variable clinical expression. However, different mutation in the
same gene could explain the different phenotypic expression in
individuals with an identical enzyme deficiency(2). The residual
activity of the affected enzyme and the turnover rate of their
substrate also determine the severity of the lysosomal storage
disorder(8). It is also likely that a specific activator protein
modulates specific enzyme activity, as has been observed in Tay-Sachs
due to hexosaminidase enzyme activity(9).
Two children with MLD had very low to
undetectable activity of ARSA that depends very much on the type of
mutation in the respective gene(l 0). None of our patients were
investigated for mutations in the ASA gene. Moderately low activity
of ARSA in two children in absence of specific clinical signs except
leukoencephalopathy were considered to have pseudodeficiency of ARSA.
Alternatively, they could be heterozygous for ASA deficiency. Barth
et al.(11) have shown mutations in a healthy population
responsible for pseudo deficiency of ARSA GM1-gangliosidosis in a
child aged 4 months was found to have about 12% of the normal
activity of beta-galactosidase. Sopelsa et al. (12) made
similar observation in such cases. Similarly, low beta-galactosidase
activity with normal activities of other leucocyte enzyme was
observed in a 4 year child and based on clinical observations he was
diagnosed with galactosialidosis, although further confirmation by
neuraminidase assay was not available in our center.
We observe from this study that milestone
regression, mental handicap, dysmorphic features, seizures, abnormal
fundus findings and organomegaly are the clinical features leading
to the suspicion for LSDs. Variability in clinical expression always
need further confirmation by specific lysosomal enzyme. Urine
metabolic screening has very little role except for MPS. While the
presence of reducing substance seems to be by chance and likely to
be due to high carbohydrate diet in those children. In children with
suspected MLD, a high index of suspicion coupled with clinical
observation and an imaging technique such as CT scan or MRI provide
clue for further confirmation. Once the confirmed diagnosed is made
in the proband, the prenatal diagnosis in subsequent pregnancies
provide great relief to the parent (13,14).
Acknowledgement
Our sincere thanks to Dr. Varsha Tripathi, Dr.
Bhairavi Shah, Dr. Rekha Bhavsar, Dr. Nidhish Nanavaty and Dr.
Ashish Bavdekar for referring patients, to Dr. Anthony Fensom for
guidance and help during the course of investigation and Dr. Ashok
Vaidya for constructive suggestions in manuscript writing.
Contributors: JS and PP have carried out
technical work and manuscript preparation; FS has given technical
guidance in leucocyte separation and critical evaluation of the
manuscript, RS has provided clinical information and guidance.
Funding: None.
Competing interests: None stated.
