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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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Jayesh Sheth and Aadhira Nair
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It should be noted that there exists clinical variation which can be
attributed to the presence of different underlying mutations. The observed
abnormalities may vary from mild ataxia of limbs and trunk to dystonia
and dysarthria. In some, seizures may be absent while others may show
increased levels of creatine phosphokinase levels.
A late infantile form alternatively known as the B1 variant is also
observed which is more or less similar to the infantile form in clinical
features. These individuals show normal Hex-A activity with 4MUG
substrate but reduced activity when natural or 4MUGS substrate is
employed. A specific mutation R178H in the α-subunit of Hex-A has been
reported from Portugal and Mediterranean region to be associated with this
variant [50, 51].
9.2. Juvenile Subacute Form
These patients show 0.5% of the normal Hex-A activity. The
deterioration of gait and posture is seen in early stages which progresses
with age. The involvement of the brain may or may not be present with
dementia and chronic organic mental syndrome seen in some. It is
observed that 50% of the affected individuals show cerebellar atrophy. The
vision status may be normal but optic atrophy and retinitis pigmentosum
can be seen later with disease progression. These patients generally die by
the age of 15 years [52]. Smith et al., (2012) reported 2/6 children to have
visual deterioration in his study. Also, hypertonia and increased myotatic
reflexes were reported in 50% and 80% of the early-juvenile TSD cases
respectively. Increased myotatic reflex was also a major finding in juvenile
SD cases (75%) in their study. A review study on juvenile SD cases by
Hendriksz et al., (2004) demonstrated that maximum patients were
presented with a mixture of cerebellar and pyramidal signs-ataxia
combined with hyperreflexia, particularly of the lower limbs with
progression of disease. Also, bowel and bladder problems were
encountered by patients, sometimes with diarrhea followed by constipation
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and with urinary incontinence indicating autonomic dysfunction rather than
a simple cognitive decline [53].
9.3. Late-Adult Onset Form
The clinical features in these patients are recorded in later years
particularly early adolescence, generally after 10 years of age. These
patients show less than 10% of the normal enzyme activity usually in the
range of 2-4%. There is slow progression in neurologic defects leading to
lack of coordination, hand tremors, progressive dystonia, dysarthria,
dyskinesia, ataxia, lower motor neuron disease (LMD) with proximal
muscle wasting, cramping and weakness. It is found that almost one third
of the patients present with psychosis. The psychiatric abnormalities
include a range of conditions from acute hebephrenic schizophrenia,
agitation, delusions to hallucinations, paranoia and recurrent depression
but, dementia being uncommon. There is evidence indicating GM2
gangliosidosis adult patients presented with heterogeneous phenotype [54].
There are also reports of GM2 gangliosidosis adult patients mimicking
symptoms suggesting spinal muscular atrophy [55, 56].
The review study on GM2 gangliosidosis adult patients by Masingue et
al., 2020 has effectively differentiated the clinical features into 4
categories: 1) initial LMD-cause of proximal lower limb weakness and
progressing to upper limbs; 2) cerebellar ataxia; 3) psychiatric disorder or
psychosis (only in TSD patients); 4) a complex clinical picture combining
all the above 3 phenotypes. Another highlight of the study is the
determination of key parameters in order to differentiate adult TSD and SD
cases, although they have majority similarities. A noteworthy finding was
observation of sensory neuropathy in 69% of SD cases as compared to only
10% TSD cases. Generally, at the molecular level, these patients show
presence of compound heterozygous status for the alleles, common
mutations being Arg505Glu for SD and Gly269Ser for TSD cases [57]. We
have also identified adult onset TSD in a male child of 12 yrs. of age with
a novel compound heterozygous mutation (c.1496G > A/c.902T > G) in
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HEXA gene with a less than 10% of HEX-A enzyme activity in leukocytes.
This child was normal till 5 year of age, thereafter developed gait ataxia,
slurred speech, tremors in hand, absent cherry red spot with an MRI
showing mild cortical atrophic changes. It is highly likely that many cases
of adult onset TSD might have been missed due to heterogeneous
phenotype.
10. PATHOLOGY
The widespread accumulation of GM2 gangliosides inside the
lysosomes of neuronal cells give rise to structures known as membranous
cytoplasmic bodies (MCBs). These can be detected immunohisto-
chemically using antibodies. However, the path from ganglioside
accumulation to progressive neurodegeneration and death is yet to be
defined. Initial studies on brains of affected individuals have shown
neuronal depletion in cerebral cortex and demyelination in cerebral white
matter. The disease progression is suggested to be a result of a primary
consequence followed by a secondary one. There are ongoing studies to
understand and simplify this pathway. The studies till date support the
accumulation of ganglioside in the neurons as a primary cause of neuronal
damage leading to subsequent demyelination and inflammation.
10.1. Accumulation of GM2 Ganglioside and Other Glycolipids
and Its Implicated Effects
Studies have shown the presence of the unacylated metabolite lyso-
GM2 (sialylgangliotriaosylsphingosine) in the brain and plasma of patients
and animal models of GM2 gangliosidosis to have toxic effects. Also, there
is evidence regarding disruption of the endosomal transport and sorting
inside the lysosomes due to sphingolipid accumulation. Another
manifestation due to accumulation of storage material is the impairment in
normal fusion of autophagosome and lysosomes. This results in the
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formation of inclusion bodies containing aggregates of polyubiquitinated
proteins [26]. Suzuki and colleagues demonstrated presence of α-
synuclein- a naturally expressed protein in neurons and glia in human
lipidoses [58] and SD mice brains [59]. The protein is reported in young
SD animal models and the amount is shown to increase with age and
disease severity [60]. Also, recent study shows truncation of α-synuclein
by the inflammasome protein caspase-I causing its accumulation and
subsequently, neurotoxicity [61].
10.2. Demyelination and Neuroinflammation
A histological evidence of severe and extensive demyelination along
with a 50-80% biochemical decrease in cerebrosides was obtained from the
white matter studies on GM2 gangliosidosis by Haberland and colleagues.
The studies also proposed a link between failure in myelination and
reduced cerebroside synthesis [62]. Another study aimed to determine the
expression of oligodendrocyte-specific genes in SD mice found their
downregulation in all regions of the brain at an early age. The mRNA
expression of a key enzyme Uridine diphosphate (UDP) galactose-
ceramide transferase, involved in galacto-cerebroside biosynthesis was
observed to be reduced at all ages (5-19 weeks), and even before the
development of other disease signs [60].
The innate inflammatory response is chronically stimulated in case of
neurodegenerative disorders causing overexpression of cytokines and
proapoptotic molecules resulting in severe neuronal damage. The studies
using expression analysis of cerebral cortex from GM2 gangliosidosis
patients showed inflammation to be a cause of neuronal cell death [63].
The inflammatory response being a dynamic process involves several
molecules, many of them having overlapping functions. A study by Hayase
et al., 2010 showed a significantly increased level of tumour necrosis
factor-α (TNF-α) in the cerebrospinal fluid of TSD patients, thereby
inducing inflammatory response, which may contribute to disease
progression [64]. In order to study the effect of inflammation in GM2
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gangliosidosis, several investigators targeted the elements like Macrophage
inflammatory protein 1α (MIP-1α) [65], TNF-α [66]. Utz et al., 2015
demonstrated the association of increased levels of five possible
inflammatory biomarkers- ENA-78, MCP-1, MIP-1α, MIP-1β and TNFR2
with infantile gangliosidosis. The observations from these manipulation
studies denote a modest improvement in lifespan with better understanding
of the disease features in SD mouse [67].
10.3. Neuronal Death and Degeneration
The neuronal death in GM2 gangliosidosis was investigated by Huang
et al., 1997 and Wada et al., 2000, wherein they identified TUNEL positive
cells in human and murine brains, which is considered a signature of
apoptotic demise [68, 69]. However, no such observation was made by
Sargeant and colleagues in SD mouse brains. A decrease in neuronal
numbers with age in Ventroposterior Medial and Ventroposterior Lateral
(VPM/VPL) nuclei of the thalamus was observed along with increase in
activated microglia. The lateral vestibular nucleus of the brain stem
showed abundant cell loss and a significant low number of cells in the
ventral spinal cord [70]. It is noteworthy that neuronal dysfunction is
widespread and demonstrable from a young age even before disease signs
are visible as compared to neuronal loss; however, this is not universal in
SD mouse [60]. All the investigations indicate neuronal dysfunction to be
potential in disease manifestations characterizing GM2 gangliosidosis.
10.4. Imaging Studies
Magnetic resonance imaging (MRI) studies have been carried out in
GM2 gangliosidosis patients. However, they do not clearly distinguish
between the different forms of the condition. Generally, extensive T2-
wieghted hyperintensities in the cerebral white matter, basal ganglia,
thalamus, pyramidal tract and cerebellum are seen in MRI of the patients
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with infantile form (26). The changes in intensity/density likely suggest
accumulation of calcium or GM2 ganglioside in the cerebral white matter.
Also, the cerebral white matter shows an increased abundance of creatine
and energy rich phosphocreatine at an increased abundance than that
present in basal ganglia [71].
The late onset form is characterized by cerebellar atrophy and often
hypo density of the thalamus. A study including late onset patients
demonstrated diffused cerebellar atrophy in all cases along with reduced
levels of N-acetylaspartate in cerebellum as shown by magnetic resonance
spectroscopy [72]. Figure 6 shows the MRI scan of the brain revealing
hyper intensity and hypo intensity in T2/T1 weighted images (A) & (B)
which is usually seen in GM2 gangliosidosis patients due to GM2 activator
protein deficiency. Another study carried out to quantify brain MRI
volume during the disease progression in gangliosidosis, revealed that the
intracranial volume both, brain tissue volume and ventricular volume
increased consistently with time. The study concludes quantitative MRI to
be a robust non-invasive method for monitoring CNS changes in the
clinical course of gangliosidosis [52].
Figure 6. MRI scan of the brain showing putaminal hyperintensity and thalamic
hypointensity with some unmyelinated white matter in T2/T1 weighted images
(A)-(B), seen in a child with Gm2 activator protein deficiency.
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11. EFFECT AND SIDE EFFECT OF THERAPY
A number of therapies have been proposed with a view to improve the
lives of GM2 gangliosidosis patients. These include substrate reduction
[73], bone marrow transplantation [74], hematopoietic [75] or neural stem
cell transplantation [76], use of anti-inflammatory drugs [64],
administration of purified enzyme [77] and gene therapy helping in
restoration of dysfunctional protein expression [78].
11.1. Substrate Reduction Therapy
The main aim of this approach is to reduce the rate of glycolipid
synthesis by partially inhibiting the respective enzymes using small
molecules [79]. This idea was first proposed by Norman Radin for the
treatment of Gaucher disease [80]. Miglustat- (N-butyldeoxynojirimycin,
NB-DNJ) a small iminosugar is a competitive inhibitor of
gylcosylceramide synthase. As, glycosylceramide is the first step in the
biosynthesis of glycosphingolipids, use of these inhibitors could be a
potential target for therapy. Prior studies have shown prevention of GM2
ganglioside accumulation in TSD murine models by using miglustat
[73, 81]. A study was carried out to assay the clinical efficacy of NB-DNJ
in two TSD infantile patients. They showed a significant concentration of
NB-DNJ in their cerebrospinal fluid; however, this did not stop the
neurologic dysfunction in patients [73]. Also, there are studies reporting
the inefficiency of the drug in meeting its endpoint target in a clinical trial
of late-onset TSD patients [82, 83]. The studies also show that the major
drug related events because of miglustat in infantile GM2 patients were
abdominal discomfort and flatulence while in the juvenile group; they were
diarrhea and weight loss [84].
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11.2. Enzyme Replacement Therapy
This approach has been very effective in treatment of lysosomal
storage disorders; however, the intravenous administration restricts the
enzyme to cross the blood-brain barrier. As a result, this therapy is not
promising for prevention of CNS neurodegeneration [85, 86].
Nevertheless, there are continuous efforts for developing strategies to
synthesize and deliver enzyme efficiently. The production of recombinant
HexA enzyme using methylotrophic yeast Ogataea minuta (Om) culture, to
simultaneously express both HEXA and HEXB genes is one of the
possibilities. In order to expose mannose-6-phosphate (M6P) residues on
the N-glycans, the purified HexA was treated with α-mannosidase [77, 87].
On testing the therapeutic efficacy of the recombinant HexA in SD mice,
the noteworthy outcomes were improvement of motor function, increase of
survival rate and inhibition of the induction of MIP-1α [77]. Also, in a
study by Tropak et al., 2016, a hybrid µ subunit having the critical
characteristics of α and β subunits was created. HEK239 cells with
CRISPR deleted HEXA and HEXB genes, stably expressing the µ subunit
were used to purify the HexM µ-homodimer. The combination of HexM
with GM2A allowed the hydrolysis of a derivative of GM2 ganglioside
both in cellulo and in vitro [88]. A chimeric HexB was created by
modifying the nucleotide sequence of the human HEXB gene.
Intracerebroventricular administration of this protein to SD model mice
effectively reduced the GM2 ganglioside accumulation in parenchyma and
also restored Hexosaminidase activity in the brain [89].
11.3. Gene Therapy
This is based on the principle of manipulating a targeted region in the
genome to rectify the gene defect. This area has been exploited,
particularly for providing solutions to neurodegenerative disorders which
are generally caused by single gene defects. In the mid-1990s, several
experiments were carried out in this view and Akli et al., 1996 was the first
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to produce TSD patient skin-derived fibroblasts expressing HEXA gene by
adenovirus transduction [90]. There were supporting studies that suggested
the intravenous co-administration of adenoviral vectors expression of both
HEXA and HEXB, preferentially transduced the liver cells in HEXA
knockout mice [91]. However, there were limitations in the successful use
of adenoviral vectors, the major reason being its inability to cross the
blood-brain barrier [92]. Secondly, there is a high risk of development of
hepatocellular carcinoma due to accumulation of the vector and
overexpression of the transgene in the liver [93]. The major challenges to
overcome the blood-brain barrier and ensure minimal side effects are the
choice of vector and delivery method of therapeutic genes [94].
Recently, adeno-associated virus (AAV) has shown a great potential in
developing approaches for GM2 gangliosidosis gene therapy [95]. These
are small 20-25nm in diameter, non-enveloped single-stranded DNA
viruses of the Parvoviridae family. Recombinant AAV (rAAV) serotypes
2/1 or 2/2 encoding the HEXA and HEXB gene cDNA when intracranially
administered to SD model mice, resulted in efficient spread of HexA and
increase in mouse survival rate without any apparent cytotoxicity due to
rAAV use [78, 96]. The inability of the AAVs to carry the cDNA of both
HEXA and HEXB genes due to their less capacity (from 2.1-4.5 kb), adds
an undesirable complexity and expense limiting its use as a treatment
option in clinic [26]. The expression of both subunits α and β is required
for the effective recovery of the secretion of the absent heterodimeric
HexA isoenzyme [89]. In this view, a self-complementary AAV9.47
(scAAV9.47-HEXM) encoding a hybrid µ subunit was designed by Tropak
et al., 2016 and its intracranial administration in the brain of TSD model
mice was found to decrease GM2 ganglioside amount. Further studies
provide evidence for long term decrease in GM2 ganglioside accumulation
in the CNS of newborn TSD model mice following intravenous
administration of scAAV9.47-HEXM with a decreased distribution of this
vector as compared to other serotypes namely AAV9, AAVrh10 or
AAVrh8 [97]. Recently, the modified human Hex µ subunit (HEXM) was
used by Ou and colleagues to treat neonatal Sandhoff mice. The plasma
MUGS and MUG analysis 4 months after the i.v. of AAV vectors, showed
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up to 144- and 17-fold of wild-type levels (n = 10, p < 0.0001),
respectively. The highlight of the study was that the MUGS and MUG
activities in the brain also increased significantly compared with untreated
Sandhoff mice (p < 0.001). Further, the reduced level of GM2 gangliosides
in multiple tissues, except the brain, of treated mice was confirmed by
HPLC-MS/MS analysis [98]. Subsequently, the therapeutic efficacy of
AAVrh8-based gene therapy on the Jacob sheep TSD model was also
demonstrated through an experimental study. They observed that following
intracranial injection of AAV, the sheep showed delay in the appearance of
symptoms and/or decrease in the acquired symptoms of the disease. The
distribution of HexA was excellent in the sheep brain when there was a co-
injection of AAVrh8-HEXA and AAVrh8-HEXB as compared to injection
with only AAVrh8-HEXA. On analyzing the sheep spinal cord, it was
observed that the HexA distribution was low in addition to decreased
activation and proliferation of microglia in the sheep brain following gene
therapy [95].
Another important criterion to be considered is the safety check on the
use of virus-based vectors as there are a number of side-effects associated
with it. Several studies have been carried out in this regard, wherein one
study shows that intracranial injection of AAVrh8-cmHexα/β led to
development of dyskinesia, ataxia and loss of dexterity in most of the
monkeys [99]. The authors also observed a sharp increase in Hex activity
in the thalamus, suggesting a probable association of severe neurotoxicity
with Hex overexpression.
11.4. Bone Marrow Transplantation
This approach has been exploited in a few experimental studies with
no considerable benefit to human patients [100] as compared to SD mouse
in which survival was improved and decrease in the rate of disease
progression was observed [101]. A study by Jacobs et al., 2005
demonstrated the application of bone marrow transplantation with the
following substrate reduction therapy (miglustat). There was increase in
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