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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2984_Библиотеки_им_академика_М_И_Перельмана
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Karolina Pierzynowska, Estera Rintz, Lidia Gaffke et al.
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sulfate (DS) has also been reported. There are four different genetic and
biochemical conditions which are commonly called Sanfilippo syndrome
due to very similar clinical symptoms and accumulation of HS as a
primary storage material. These conditions, called subtypes A, B, C, and
D of MPS III, are caused by mutations in SGSH, NAGLU, HGSNAT, and
GNS resulting is deficiency of activity of heparan N-sulfatase, -N-
acetylglucosaminidase, acetyl-CoA: α-glucosaminide acetyltransferase,
and N-acetylglucosamine-6-sulfatase, respectively. MPS III patients are
asymptomatic, and the diagnosis is made usually at the age of about 4
years. Neurodegeneration is the major clinical problem in MPS III, and
progressive cognitive deficiency and behavioral changes are
characteristic symptoms in this disease. Currently, there is no cure for
Sanfilippo disease. However, there are intensive studies devoted to
develop effective therapeutic options for MPS III.
1. INTRODUCTION
Mutations in four different genes (SGSH, NAGLU, HGSNAT, and
GNS), coding for four enzymes involved in degradation of heparan sulfate
(HS), one of glycosaminoglycans (GAGs) (heparan N-sulfatase, -N-
acetylglucosaminidase, acetyl-CoA:-glucosaminide acetyltransferase, and
N-acetylglucosamine-6-sulfatase, respectively), cause the disease called
Sanfilippo syndrome or mucopolysaccharidosis type III. Depending on the
kind of genetic defect and deficiency of particular enzyme, four subtypes
of MPS III are distinguished and called A, B, C, and D, respectively (Table
1). Clinically, all subtypes of MPS III are similar, with only some
differences in the course of the disease which, however, need to be
confirmed if they are specific to the subtype or particular mutations.
In 1963, Sylvester Sanfilippo et al. described clinical symptoms of
mental retardation which were correlated with accumulation of HS [1]. The
newly described disease has been, therefore, identified as the third type of
mucopolysaccharidosis, and it is now known as MPS III or Sanfilippo
syndrome, after the surname of the discoverer. Most probably, the disease
of the patient described in the article by Sanfilippo et al. [1] should be
classified as MPS IIIA. Nevertheless, it is possible that another patient,
described in the literature two years before publication of the report by
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Sanfilippo et al., suffered from MPS III rather than MPS I, as suggested by
the authors [2]. Subsequent studies led to discovery of patients with HS
storage in which symptoms were similar to those described by Sanfilippo
et al. [1], but different enzymatic defects could be identified. Therefore,
various genetically and biochemically identified diseases in which HS
accumulates and is accompanied with severe mental and behavioral
disorders have been classified as subtypes of MPS III, called MPS IIIB [3],
MPS IIIC [4, 5], and MPS IIID [6].
Table 1. Subtypes of Sanfilippo disease (MPS III)
Disease
OMIM no.
Mutated gene
Deficient enzyme
Stored GAG
MPS IIIA
252900
SGSH
heparan N-sulfatase
HS (secondary DS
storage)
MPS IIIB
252920
NAGLU
-N-
acetylglucosaminidase
HS (secondary DS
storage)
MPS IIIC
252930
HGSNAT
acetylCoA:-
glucosaminide
acetyltransferase
HS (secondary DS
storage)
MPS IIID
252940
GNS
N-acetylglucosamine-6-
sulfatase
HS (secondary DS
storage)
2. DIAGNOSTICS
Despite resulting from four different genetic defects, all subtypes
(A, B, C and D) of Sanfilippo disease are characterized with accumulation
of HS in lysosomes and with similar clinical symptoms [7, 8, 9]. Therefore,
symptoms and diagnostics of MPS III will be described together for all the
disease subtypes.
2.1. Clinical Symptoms
The major clinical problems of patients suffering from Sanfilippo
disease arose from gradual neurodegeneration which results in cognitive
decline and other neurological dysfunctions. The most commonly
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occurring MPS III symptoms include cognitive decline, lack of
speech/speech delay, developmental delay, seizures, hyperactivity,
aggression-like behavior, and sleep disturbances. Each symptom can,
however, be pronounced differentially in individual patients, ranging from
mild to severe. Although somatic symptoms of MPS III are generally
milder than in other MPS types, they can still be serious dysfunctions,
significantly affecting functions of the organism. They include facial
dysmorphology, hepatomegaly, frequent ear and respiratory system
infections, scoliosis, kyphosis, lumbar lordosis, hip dysplasia, carpal tunnel
syndrome, hirsutism, and low hair line. Changes in hair morphology are
characteristic and easily visible under electron microscope. This feature
has been suggested as a diagnostic marker [10, 11]. Two patients with
Sanfilippo disease is shown in Figure 1.
The clinical course of Sanfilippo disease is specific. Patients are
usually born asymptomatic [12, 13, 14, 15], and symptoms do not develop
for several months or a few years [16]. Delayed cognitive development and
speech deficiency are often the first clinical signals of the disease. Then,
behavioral disorders become evident, initially without any deficits in
physical performance. Progressive cognitive deterioration follows these
symptoms and hyperactivity is more and more pronounced in time.
Sleeping deficits become evident then. Aggressive-like behavior and
reduced fear are very often pronounced [17]. The behavior of patients at
this stage of the disease is similar to that of autistic children, and in fact,
MPS III is sometimes misdiagnosed as autism spectrum disorder. The late
phase of the disease consists of a decline in motor functions, dementia,
spasticity and problems with swallowing. The average life span is between
2 and 3 decades.
It is worth noting that variability of MPS III symptoms is huge
between patients. Apart from severe deterioration, reported for most of
patients, considerably milder course of the disease has been described in
the literature [18, 19]. As mentioned above, clinical symptoms of MPS III
patients might be untypical which can lead to misdiagnosis, with autism
and idiopathic developmental delay, attention deficit/hyperactivity disorder
(ADHD) being incorrectly suspected [8, 20, 21, 22, 23, 24]. However,
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since symptoms of Sanfilippo syndrome are progressive, they become
more specific at later stages of the disease [25].
Figure 1. Patients with characteristic features of Sanfilippo disease.
2.2. Genetics
Four subtypes of MPS III (A, B, C, and D) are caused by mutations in
following genes: SGSH, NAGLU, HGSNAT and GNS, respectively. These
genes code for enzymes involved in HS degradation, heparan
N-sulfatase, -N-acetylglucosaminidase, acetyl-CoA:-glucosaminide
acetyltransferase, and N-acetylglucosamine-6-sulfatase, respectively
(Table 1). Thus, detection of mutations in one of these genes is the
simplest way for genetic diagnosis of Sanfilippo disease. Numbers of
mutations reported for various subtypes of MPS III are different ranging
from about 20 in subtype D, through about 70 I in subtype C, to over 150
in subtypes A and B [26]. Recent studies indicated that apart from specific
mutations in SGSH, NAGLU, HGSNAT or GNS, expression of a large
group of genes coding for proteins involved in various cellular processes is
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considerably changed in each MPS III subtype [27, 28, 29]. Therefore, it
appears that impaired regulation of a battery of genes might contribute to
development of secondary or tertiary changes in cellular processes,
affecting physiology of cells and organs in Sanfilippo disease patients.
Such dysregulations can significantly modulate the course of the disease
due to disturbance of crucial cellular functions [30].
2.3. Stored Compounds
The primary biochemical defect in Sanfilippo disease is impaired
degradation of HS [7, 8, 9]. This defect has been demonstrated in both
animal models of MPS III and human patients [31, 32]. As the primary
storage material, HS is considered to be the main biomarker for this
disease. Nevertheless, secondary storage of dermatan sulfate (DS) [33] and
globotriaosylsphingosine (LysoGb3) [34] has been reported. Although
accumulation of DS and LysoGb3 may be important to understand details
of MPS III pathomechanism, the use of these compounds as biomarkers is
questionable.
For a quick diagnostic tests, semi-quantitative chromatographic
methods are used for rough determination of HS levels in urine, or rarely in
plasma. For more detailed studies, some more advanced methods can be
used, like liquid chromatography/tandem mass spectrometry [35] other
biochemical and biophysical approaches [36, 37, 38, 39, 40, 41, 42, 43, 44,
45]. In all subtypes of Sanfilippo disease the HS levels are similar, and any
differences are perhaps related to severity of particular mutations [45].
When making diagnostic test, it is necessary to use age-related reference
values, as it was shown that plasma and urinary HS levels decrease in age
[36]. Interestingly, correlations between levels of HS in biological material
(urine and plasma) and severity of the disease could be detected [18, 46].
Additional biomarkers of MPS III have been suggested which might
facilitate to assess severity of the disease. They include changes in hair
morphology, specific proteins, disability level, behavioral disturbances,
and cognitive skills [11, 47, 48].
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2.4. Biochemistry
To distinguish between particular MPS III subtypes, measurement of
activities of four enzymes, heparan N-sulfatase, -N-acetyl-
glucosaminidase, acetyl-CoA:-glucosaminide acetyltransferase, and N-
acetylglucosamine-6-sulfatase, is used as the primary biochemical assay.
As a standard, determination of these activities in extracts from leukocytes
or cultured fibroblasts is used [49].
More advanced biochemical analyses of enyzmes may be crucial to
understand genotype-phenotype correlations. For example, determination
of the heparan N-sulfatase crystal structure facilitated prediction of
severity of MPS IIIA [50]. Recommendations on laboratory diagnostics of
MPS III include determination of HS levels in urine and/or plasma, then
measurement the enzyme activity, followed by identification of mutations
[51]. In addition, a computational algorithm also be useful in predicting
effects of mutations at the protein level [52].
2.5. Radiology
In MPS III patients, neurological symptoms predominate while
somatic changes are relatively mild relative to other MPS types.
Nevertheless, radiological examination of patients may facilitate
monitoring of the disease course and predicting severity. In fact,
radiological studies indicated various abnormalities in structures of
different organs in patients suffering from Sanfilippo syndrome. The most
important ones are listed below.
Changes in teeth of MPS III patients consist of obliterated pulp
chambers and root canals [53]. Osteonecrosis accompanied with femoral
head and hip dysplasia may occur [54]. Tachypnea was demonstrated in
some patients [55], as was precocious puberty in boys [56]. Changes in the
brain include diffuse hypomyelination with thinning of the corpus
callosum [57], which can suggest a possible mechanism of sleeping
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problems [58], compromised blood-brain-barrier integrity [59], and stunted
growth [60].
3. CRUCIAL ROLE OF HEPARAN SULFATE
IN MPS III PATHOGENESIS
Because mutations causing Sanfilippo syndrome occur in genes coding
for enzymes involved in degradation of HS (Table 1), lysosomal storage of
this GAG is the primary cellular defect in this disease. Single catabolic
pathway of HS degradation. However, the storage itself (and resultant
dysfunction of lysosomes) is only partially responsible for appearance of
MPS III symptoms. Recent studies demonstrated that non-degraded
extracellular HS, as well as secondary effects of the storage, may
contribute significantly to development of various dysfunction.
When normal neural cells were exposed to exogenous soluble HS
fragments, activation of integrin-based focal adhesions could be observed
which modulated the attachment of cells to the extracellular matrix [61].
Abnormal cell polarization and improperly oriented migration were also
observed in these cells [61]. Moreover, accumulation of HS was suggested
to be responsible for enhanced oxidative stress, microglial activation, and
neurodegeneration [62].
It is also worth mentioning that the primary HS storage leads to a
network of pathological cellular processed which result in secondary
storage various compounds, including DS (another GAG), gangliosides,
ceramides, galactosylceramides and sphingomyelin [63, 33, 34]. It is likely
that these secondary effects induce cellular stress responses, leading to
global changes in gene expression regulation which cause further
disturbances in different cellular processes [27, 28, 29, 30], and subsequent
neuroinflammation and neurodegeneration [64].
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4. ANIMAL MODELS
As in many genetic diseases, the most commonly used animal models
in studies on Sanfilippo disease are mice. In the MPS IIIA mouse model,
symptoms resembling MPS III patients are evident, including
hyperactivity, rapid exploratory behavior, and reduced fear [65]. It was
suggested that neuronopathy of these animals might be related to the
observed impaired exocytosis [66]. Unexpectedly, significant differences
were noted between male and female MPS IIIA mice [65], contrary to
patients suffering from Sanfilippo syndrome A.
In the MPS IIIB mouse model, neuropathology was detected in both
central and peripheral systems [67]. Symptoms resembles those occurring
in human patients, including hyperactivity, progressive impairment in
sensory functions and reduced response to pain stimulation [67, 68].
In the MPS IIIC mouse model, neuroinflammation and neuro
degeneration have been described [69]. Moreover, dysmorphology of
mitochondria was evident, and impaired energy metabolism was measured
in cells [69].
Apart from mice, emu is employed as another animal model for MPS
IIIB [70]. Symptoms observed in the birds well resemble those in humans
and mice, indicating that the emu mode can also be useful in studies on this
subtype of Sanfilippo disease [70].
5. POTENTIAL THERAPEUTIC APPROACHES
Currently, no therapy is available for Sanfilippo disease [71].
However, there are various therapeutic approaches which were proposed
and which are being tested. Various therapies are at different stages of
research and tests, from in vitro studies, through experiments on animal
models, to clinical trials. Below, they are summarized briefly, with a few
most promising options discussed in somewhat more detail.
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5.1. Enzyme Replacement Therapy (ERT)
Enzyme replacement therapy (ERT) consists of administration of
human recombinant enzyme to patients lacking such protein due to
mutations in corresponding gene. Since the most commonly used method
of enzyme administration is intravenous injection, efficacy of ERT is
generally restricted to somatic tissues and organs due to inefficient
crossing the blood-brain-barrier by proteins. Therefore, serious problems
were met in applying ERT as a therapy for MPS III in which
neurodegeneration is the major problem [9].
There are various attempts to overcome this drawback of ERT. First,
intrathecal or intra-spinal enzyme injection is possible. Experiments on
animals suggested that this method of drug administration can be relatively
safe [72] and allows for effective penetration of the enzyme to the brain
[73, 74]. However, positive therapeutic effects could be observed mostly if
ERT was started early, as efficacy in animals at advanced disease stages
was low [75]. Fusion of the enzyme with a molecule that can naturally
cross the blood-brain-barrier is another approach. For example, heparan N-
sulfatase has been attached to a part of apolipoprotein B [76], α-N-
acetylglucosaminidase was fused to the receptor-binding motif of insulin-
like growth factor 2 [77, 78], and sulfamidase was bound to the insulin
receptor antibody [79]. Below, recent reports on potential ERT for MPS III
are summarized in somewhat more detail.
An approach involving the administration of an enzyme, sulfamidase,
by injection into the cerebrospinal fluid (CSF) has been tested [74].
According to the authors of that report, the key parameter is the site of
enzyme administration. Studies conducted on the MPS IIIA mouse model
involving the administration of recombinant human sulfamidase (rhSGSH)
by three different pathways (intrathecal lumbar, cisternal and ventricular)
indicated that despite the highest invasiveness, the ventricular
administration of the enzyme leads to the largest statistically significant
decreases in substrate levels and reducing microglial activation. Single
lumbar enzyme administration did not lead to reduction in substrate levels
and gave poor effects on the improvement of morphology and physiology
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of nerve tissue [74]. Not only the exact site of administration but also the
dose of the enzyme may be key to the effects of the treatment. That is why
different doses of rhSGSH infused into CSF via the cisterna magna in MPS
IIIA Huntaway dogs were tested [80]. The authors determined a dose- and
frequency-dependent reduction in HS in the CSF and deeper layers of the
cerebral cortex, improved lysosomal functionality as well as reduced
activation of microglia and other inflammatory markers. The same team
conducted a study on the effectiveness of sustained release of enzyme
(delivered by an osmotic pump) in MPS IIIA mice [81]. During these
studies, almost normalization of primary stored HS in both hemispheres of
the brain and cervical spinal cord was observed, as was a reduction in the
amount of secondary stored GM3 ganglioside markers and inflammatory
markers [81]. This approach seemed to be important because of the
limitation of frequent infusions in medical facilities in patients with MPS
IIIA. Positive research results prompted the authors to test this approach
using a SynchroMed II® pump attached to a spinal infusion cannula on the
MPS IIIA canine model [82]. These studies have shown that the use of a
slow infusion as well as the use a recombinant enzyme without pump, led
to a significant reduction in HS levels, but it was less efficient in reducing
secondary effects of the disease (activation of microglia or storage of GM3
ganglioside) [82]. One of the newest approaches is the use of chemically
modified recombinant human sulfamidase (CM-rhSulfamidase) with
disrupted glycans [83]. This enzyme exhibits reduced glycan receptor-
mediated endocytosis which may indicate non-receptor mediated uptake by
cells. CM-rhSulfamidase showed prolonged exposure in plasma and
distribution to the brain, and was present in both vascular profiles and the
brain parenchyma after intravenous administration. Studies on the mouse
model of MPS IIIA indicated that weekly intravenous administration of
this enzyme led to a decrease in HS concentration in CNS and improved
the cognitive, motor and social activities of animals. The conducted
research became the basis for the clinical trial initiated using this modified
enzyme [83].
Recombinant human heparan N-sulfatase (rhHNS) bioavailability and
efficiency experiments were also performed on the cynomolgus monkey
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