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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
132
effect involvement in case of these disorders. Considering this data,
carrier screening and prenatal diagnosis holds promise for proper
management and prevention of the disease. The current diagnostics
strategy includes enzyme assay and molecular diagnosis. Establishing a
genotype-phenotype relationship will also be essential from the point of
view of patient prognosis and evaluating success of various therapies.
The different treatment strategies explored include enzyme replacement
therapy, substrate reduction therapy as well as gene therapy. With
advancements in technology, the primary aim will be to provide timely
and adequate diagnosis, its prevention through prenatal study followed by
efficient therapy that aids in successful survival of the patients.
1. INTRODUCTION
Gangliosides are glycosphingolipids with presence of one or more
sialic acid groups. The accumulation of GM2 gangliosides
(monosialoganglioside2) in the central nervous system results in
development of a severe neurological condition known as GM2
gangliosidosis. This includes a group of inherited metabolic disorders due
to deficient β-hexosaminidase activity or absence of GM2 activator protein
[1, 2]. Tay-Sachs disease (TSD) and Sandhoff disease (SD) included under
GM2 gangliosidoses, are caused due to mutations present in HEXA and
HEXB gene that encode α and β subunits of β-hexosaminidase-A
respectively [3, 4], whereas mutations in GM2A gene results in another
GM2 gangliosidoses- GM2 activator protein deficiency [1]. Tay-Sachs
disease is the most common of the three forms presented with an incidence
of 1/100,000 live births [5]. This frequency is higher in Ashkenazi Jewish
population with 1/4000 [2]. Also, among the Lysosomal storage disorders
in India, Tay-Sachs disease is one of the common lipid storage disorders
[6, 7]. The mode of inheritance of these disorders is autosomal recessive
emphasizing the need for the correct diagnosis in an index case and
ensuring its prevention in subsequent pregnancies. The severity of the
condition and its age of onset are highly determined by the residual
enzyme activity. Accordingly, the condition is categorized as infantile
acute, juvenile sub-acute and adult chronic forms based on the age when
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the first symptoms are noticed. The commonly observed symptoms vary
from infantile form presented with seizures, neuro-regression, muscle
weakness, hyperacusis and cherry red spot on fundus to an adult form with
progressive dementia and gait disturbances [7, 8]. There are reports of over
199 disease causing mutations in HEXA gene as per the Human Gene
Mutation Database (HGMD) as assessed on 23
rd
April 2020 (http://www.
hgmd.cf.ac.uk/). Certain mutations are present in specific ethnicities like
c.1421+1G > C, c.1274_1277dupTATC and c.805G > A are the most
commonly prevalent in 98% of Ashkenazi Jews and 35% non-Ashkenazi
Jews population [9–11] and c.1385A > T (E462V) and c.964G > T
(D322Y) mutation in the India Gujarati ethnic population [3, 6, 12].
However, such studies have not been carried out for all the different
ethnicities. The presence of particular mutations in certain communities
denote the effect of founder mutation and this knowledge will be beneficial
from the point of view of carrying out carrier screening in those
populations followed by precise genetic counseling and prenatal diagnosis
for prevention of the disease. At present, the therapeutic approach for GM2
gangliosidosis is not providing a permanent cure but is based on reducing
symptom effects or delaying the progression in case of adult forms.
2. BRIEF HISTORY
Tay-Sachs disease was first identified by a British ophthalmologist-
Warren Tay in 1881 in an infant with severe neurological dysfunction,
aged 1 year. The distinct clinical feature observed was the presence of
symmetrical changes in the yellow spot in each eye- now known as the
cherry-red spot. However, the original patient died at 18 months and later,
Tay found another family member of the patient affected with the same
condition denoting its heritable nature. He gave a detailed clinical report of
the same in 1884. Independently, an American neurologist, Bernard Sachs
recorded a similar finding in a child with arrested cerebral development
and was the first to publish a brain cortical pathology of the disease in
1887 and hence the combined terminology is given to the condition- Tay-
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Jayesh Sheth and Aadhira Nair
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Sachs disease. Later, Ernst Klenk in 1942 discovered the accumulation of
an unknown glycosphingolipid in the brains of these patients and coined
the term- ganglioside as they were abundantly found in ganglion cells.
However, it took two decades for identification of the main storage
material in the brains of TSD patients- GM2 ganglioside by Svennerholm.
Further studies by Makita and Yamakawa in 1969 helped in elucidating its
structure [2].
Robinson and Sterling in 1968 had separated the two forms Hex-A and
Hex-B from the total β-N-acetyl-hexosaminidase (hex). It was only in
1969, when the deficiency of β-hexosaminidase activity particularly the
Hex-A form in TSD patients was discovered [13] and this is also referred
as the B variant. In 1968, while isolating the storage material from brains
of TSD patients, Konrad Sandhoff discovered additional accumulation of
globoside in visceral tissues in one of the TSD cases. This was designated
as the O variant and some of these were later called as Sandhoff disease
[14]. He attributed deficiency of both Hex-A and Hex-B in this condition.
In some of the O variant cases, there were normal levels of Hex-A and
Hex-B but absence of total Hex activity against the natural substrate. These
were then referred to as the AB variants and later in 1978 Ernst
Conzelmann discovered the deficiency of GM2-activator protein to be the
cause of this condition [2].
3. NATURE AND FUNCTION OF THE GANGLIOSIDES
Ganglioside was the term given by Klenk to neuraminic acid-
containing Glycosphingolipids (GSLs) that are abundant in ganglion cells.
Gangliosides are highly heterogeneous molecules showing a great
structural and functional diversity. Their structure consists of a ceramide
attached to a glycan chain via glycosidic bond at 1-hydroxyl position [15]
and also contains 1-4 sialic acid (N-acetylneuraminic acid, NANA)
residues [16]. The human brain has 12 different types of gangliosides, all
having basic tetra-saccharide chains to which sialic acid residues are
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GM2 Gangliosidosis /Tay Sachs Disease
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attached except GM2 and GM3 in which there is a tri-saccharide chain.
The structure of GM2 ganglioside is depicted in Figure 1.
Figure 1. Structure of GM2 ganglioside.
The primary site where gangliosides are found is in the plasma
membrane of animal cells. The hydrophobic ceramide moiety helps in their
attachment to the membrane. The cerebral gray matter has the highest
concentration of ganglioside particularly in the region of nerve endings and
dendrites. The brain ganglioside is distributed across neurons, glial cells
and other cell types [17, 18].
Gangliosides are responsible in a number of cellular processes like cell
differentiation and cell adhesion [19–21]. The quantitative and qualitative
patterns of expression of glycosphingolipids indicate their participation in
dynamic neurodevelopmental processes [22]. Gangliosides also aid in
adjusting intracellular and intranuclear calcium homeostasis [23]. One
study suggests that axonal membrane ganglioside acts as ligand for MAG
(myelin associated glycoprotein)- a oligodendrocyte protein that favors
axon-glia interactions [24]. Subsequently, a study on mice performed by
Allende and Proia in 2014 provides experimental evidence in support of
this theory. In conclusion, all the collaborative studies point out the
essential status of gangliosides for myelin function and stability [25].
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Jayesh Sheth and Aadhira Nair
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4. STRUCTURE, BIOSYNTHESIS AND UPTAKE
OF B-HEXOSAMINIDASE
Robinson and Sterling were the first to distinguish two forms of β-
hexosaminidase as acidic form A and basic form B with differences in their
sialic acid content. However, using immunohistochemical techniques,
Srivastava and Buetler gave further insights suggesting the two forms to
share a common subunit. They showed Hex-A to be a heteropolymer (αβ)
and Hex-B to be a homopolymer (ββ). The presence of another isoform
Hex-S (αα) was also predicted [26] (Figure 2).
In the year 1996, the first predicted 3D structure of the catalytic
domain of human Hex was derived using bacterial chitobiase by Tews et
al., [27]. The subunits are composed of two domains with the catalytic
domain present in domain II. The importance of dimerization of α and β
for activity in each subunit is suggested by structural studies [28].
However, there is no evidence until now of the independent monomeric
subunits possessing enzymatic activity. Further, the availability of
crystallographic structure of Hex-A, Hex-B and GM2 activator protein has
provided fine details about the effects of pathogenic mutations on the
protein structure and mechanism of catalysis [29–31]. These discoveries
with respect to structure determination of the protein has helped in its
better understanding and establishing a correlation between changes at
DNA level and that on the protein. In a study by Sheth et al., 2014,
structural protein analysis was performed using crystallographic structure
of HEXA as template for two novel missense variants found. The study
demonstrated that the missense variants disrupted the β-sheet, changing the
enzyme configuration and simultaneously destabilizing the structure [12].
Considering this, establishing a genotype-phenotype correlation is critical
to be addressed from the point of view of patient’s prognosis as well in
planning effective treatments for GM2 gangliosidosis. In this view, a study
by Ou and colleagues developed a method for establishing a genotype-
phenotype correlation for gangliosidosis mutations using in silico tools.
They also conducted 3D structural analysis to determine the mechanism
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from which a particular mutation causes GM2 gangliosidosis. The study
also proposed a model for predicting the disease subtypes of autosomal
recessive disorders based on the presence of mutant alleles [32].
Figure 2. Catabolism of the GM2 ganglioside by the β-hexosaminidase system.
The biosynthesis and uptake of β-hexosaminidase has been elucidated
efficiently by the experimental studies of William Sly, Stuart Kornfeld and
Elizabeth Neufeld. The major discovery was identification of mannose-6-
phosphate as recognition tags for the correct targeting of nascent proteins
to the lysosomes [33]. The principal process steps of protein modification
in its pathway to the lysosome involve following steps: 1) β-
hexosaminidase isozymes first appear in a precursor form; 2) Mannose 6-
phosphate (M6P) motifs are attached to these precursors, and 3) after
traversing the Golgi and on reaching the lysosome, the precursors are
converted into mature forms of smaller molecular mass [26]. In case of the
complex Hex-A, the two subunit chains α and β are synthesized
independently in the endoplasmic reticulum (ER). This is followed by
modifications involving acquisition of N-linked oligosaccharides and
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Jayesh Sheth and Aadhira Nair
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intrachain disulphide bonds, attachment of M6P moieties and loss of signal
peptide. The precursor enzyme with the help of M6P receptors is
transported to Golgi and subsequently to the lysosomes. Inside the
lysosomes, they are proteolytically processed to their mature forms [33]. A
noteworthy demonstration by the studies of D’Azzo is that the association
of β-chain with α-chain not only imparts the latter with a catalytic activity
but also helps in its transportation to the lysosomes [34].
5. GENE DEFECT CAUSING THE DISEASE
The enzymatic hydrolysis of GM2 ganglioside inside the lysosomes by
β-hexosaminidase requires the association of GM2 with a substrate specific
cofactor- GM2 activator protein encoded by GM2A gene. It is a small
glycolipid binding protein that forms a water-soluble complex with GM2
ganglioside. There are two isoenzymes for β-hexosaminidase namely Hex-
A and Hex-B. Hex-A is a heterodimer consisting of α and β subunits
encoded by HEXA and HEXB respectively. A number of studies and
considerable effort was involved in cloning and characterization of all
three genes. In, 1984 Myerowitz and Proia were the first to isolate cDNA
clones of α-subunit of Hex-A [35]. The human HEXA gene is located on
chromosome 15q23-q24 with 14 exons and 13 introns, spanning a region
of approximately 35 kb. The coding sequence consists of 528 amino acids
including a 22 amino acid signal sequence peptide at the N-terminus [36].
The fact that human HEXB gene also encompasses 14 exons and the
intron/exon boundaries are conserved between the two genes in 12/13
introns is noteworthy [37]. It is localized to chromosome 5q12 spanning a
region of 40 kb.
The comparison of the α-subunit of French-Canadian population with
the available cDNA probes at DNA level revealed presence of a large 5’
deletion in the α-chain gene. This deletion spans a region of 7.6 kb
encompassing part of intron-1, all of exon-1 and extends 2 kb upstream
that has the putative promoter region. This 7.6 kb deletion is seen in almost
70% infantile TSD cases in French Canadian population. In case of
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Ashkenazi Jewish population, a 4 bp insertion (c.1278insTATC) within
exon-11 and a G > A transversion at the 5’splice site of intron 12 accounts
for 95% of infantile TSD alleles [28] and also commonly seen in 20.58%
of the Indian infantile TSD patients mainly Muslims [6]. The 4 bp insertion
leads to an early termination codon resulting in deficiency of α-subunit
mRNA [9]. The p. Gly269Ser mutation results in adult-onset disease [38].
The resultant mutant Hex-A enzyme due to sequence alteration at a
particular position will have change in its properties like inefficient
biochemical activity or impairment of substrate binding at catalytic site.
The structure of the enzyme and its ability to interact with its substrate and
/or activator is influenced by the conformational changes due to missense
mutations [28, 39].
Figure 3. Schematic representation of HEXA gene showing all the mutations found in
TSD patients from India.
A study on 34 Indian families affected with TSD detected 25 variants,
out of which 15 were novel [6]. The variants p. D322Y, p. E462V and
c.1278insTATC were the most commonly seen with 5.88%, 14.7% and
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Jayesh Sheth and Aadhira Nair
140
20.58% respectively. All the patients were presented with infantile form of
the disease. Table 1 depicts an overall picture of the Tay-Sachs patients
highlighting the key clinical and molecular details as observed in India.
The different mutations found in HEXA gene in case of Indian TSD
patients has been schematically represented in Figure 3. There have been
cases where no variant was identified in two patients and only a single
variant was identified in three patients after sequencing the coding region
of HEXA. Multiplex ligation-dependent probe amplification (MLPA) was
carried out which indicated presence of homozygous deletion of exon-2
and exon-3 in two patients, compound heterozygous deletion of exon-1 in
two patients and duplication of exon-1 in one patient [40]. The clinical,
biochemical and molecular profile of all the 5 patients is shown in Table 2.
Thus, we find all types of mutations including missense, nonsense, small
deletions, small insertions, indels, large deletions as well as duplication in
HEXA gene to be associated with infantile form of TSD.
One report from India (1) describes a case of GM2 gangliosidosis AB
variant due to a novel homozygous nonsense mutation (c.472G > T) in
exon-4 of GM2A gene. The GM2A gene is localized to human
chromosome 5q31.2 spread across a 16kb sequence with 4 exons and 3
introns and it was successfully cloned by Schröder and colleagues [41]. In
total, there are reports of only thirteen molecularly proven cases of GM2
activator deficiency from different parts of the world, indicating its rare
diagnosis. Another case of GM2 activator deficiency from India showed
presence of a homozygous mutation c.243–2A > T in intron 3 of GM2A
gene [42]. The molecular details of all the cases along with their effect on
the protein and the ethnicity in which they were reported are systematically
depicted in Table 3. These include missense, nonsense and frame-shift
mutations. The mutational profile in Indian patients with Sandhoff disease
was studied by Tamhankar, Sheth and colleagues. Their report suggests
c.850C > T (p. R248X) mutation as the most common mutation (21%)
(Figure 4) observed in children with Sandhoff disease in India. Also, they
observed a significant difference in the molecular profile in SD cases
versus TSD cases. A major proportion of indels were reported in SD cases
as compared to missense mutations predominantly seen in TSD cases [4].
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Table 1. Biochemical and molecular analysis in patients with Tay-Sachs disease
Patient ID
Hex-A activity (MUGS)
(nmol/hr/mg)
= (x)
Total Hex activity
(MUG)
= (y)
Hex A %
=(x/y) X 100
Genotypes
Zygosity
Reference
T1
6.1
2451
0.25
Ex12:c.1385A > T (p.E462V)
Homozygous
Mistri et al., 2011
Sheth et al., 2014
Sheth et al., 2018
T2
4.7
2049.7
0.23
Ex12:c.1385A > T (p.E462V)
Homozygous
T3
1.39
ND
-
Ex12:c.1385A > T (p.E462V)
Homozygous
T26
1.95
-
-
Ex12:c.1385A > T (p.E462V)
Homozygous
T29
1.46
-
Ex12:c.1385A > T (p.E462V)
Ex11:c.1278insTATC (p.Y427IfsX5)
Compound
Heterozygous
Mistri et al., 2011
Myerowitz and Costigan,
1988
T4
2.5
862
0.29
Ex11:c.1278insTATC (p.Y427IfsX5)
Homozygous
Myerowitz and Costigan,
1988
T19
0.59*
NA
-
Ex11:c.1278insTATC (p.Y427IfsX5)
Homozygous
T21
1.3*
NA
-
Ex11:c.1278insTATC (p.Y427IfsX5)
Homozygous
T34
1.47
-
Ex11:c.1278insTATC (p.Y427IfsX5)
Homozygous
T25
1.0
-
NA
Mother-
Ex11:c.1278insTATC (p.Y427IfsX5)
Father-
Ex11:c.1278insTATC (p.Y427IfsX5)
NA
Father: Heterozygous
Mother: Heterozygous
T23
1.43
432.0
Ex11:c.1278insTATC (p.Y427IfsX5)
Ex2:c.271G > T (p.E91X)
Compound
Heterozygous
Myerowitz and Costigan,
1988
Mistri et al., 2019
T31
0.4
709.2
-
Ex11:c.1259G > A (p.W420X)
Homozygous
Mistri et al., 2019
T18
1.7
424
0.4
Ex11:c.1259G > A (p.W420X)
Homozygous
T5
6.7
1525
0.44
Ex8:c.964G > T (p.D322N)
Homozygous
Mistri et al., 2011
Sheth et al., 2014
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