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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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Table 1. (Continued)
Patient ID
Hex-A activity (MUGS)
(nmol/hr/mg)
= (x)
Total Hex activity
(MUG)
= (y)
Hex A %
=(x/y) X 100
Genotypes
Zygosity
Reference
T6
8.1
1301.5
0.62
In4:c.459+4A > C
Homozygous
Sheth et al., 2014
T7
9.0
1807
0.50
In4:c.460-1G > A
Homozygous
Mistri et al., 2019
T8
12.5
2499.9
0.50
In9:c.1073+1G > A
Ex2:c.316C > T (p.Q106X)
Compound
Heterozygous
Akli et al., 1991
Akerman et al, 1997
T9
5.2
1000
0.52
Ex13:c.1495C > T (p. R499C)
Homozygous
Mules et al., 1992
T10
9.6
1682.2
0.57
Ex6:c.616G > C (p. V206L)
Homozygous
Mistri et al., 2019
T11
7.5
1958.8
0.38
Ex6:c.637T > C (p.Y213H)
Homozygous
Mistri et al., 2019
T12
5.6
1280.1
0.44
Ex1:c.2T. > C (p. M1T)
Homozygous
Harmon et al., 1993
T13
1.2
Hex-A%
Mother: 59%
Father: 62%
-
-
NA
Mother - Ex5:c.509G > A (p. R170Q)
Father - Ex5:c.509G > A (p. R170Q)
NA
Father: Heterozygous
Mother: Heterozygous
Nakona et al., 1990
T14
1.5
1502
0.10
Ex12:c.1360G > C (p. G454R)
Homozygous
Mistri et al., 2019
T15
2.3
-
-
In2:c.347-1G > A
Homozygous
Mistri et al., 2019
T16
24.0*
1481.0
1.6
NA
Mother – Ex12:c.1349delC (p.
A450VfsX3)
Father – Ex12:c.1349delC (p.
A450VfsX3)
NA
Father: Heterozygous
Mother: Heterozygous
Mistri et al., 2019
T17
3.5
801.3
0.43
Ex8:c.964G > T (p. D322Y)
Ex11:c.1259G > A (p. W420X)
Compound
Heterozygous
Mistri et al., 2011
Mistri et al., 2019
T20
1.53
NA
-
Ex13:c.1424C > G (p. P475R)
Homozygous
Mistri et al., 2019
T22
2.5
752.4
0.33
Ex8:c.982T > G (p. C328G)
Homozygous
Mistri et al., 2019
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Patient ID
Hex-A activity (MUGS)
(nmol/hr/mg)
= (x)
Total Hex activity
(MUG)
= (y)
Hex A %
=(x/y) X 100
Genotypes
Zygosity
Reference
T24
2.6
-
-
Ex7:c.770T > C (p. F257S)
Homozygous
Mistri et al., 2019
T27
0.5*
-
-
Ex13 :c.1444G > T (p. E482X)
Homozygous
Mistri et al., 2019
T28
2.0
466
Ex7:c.754C > T (p. R252C)
Homozygous
Mistri et al., 2019
T30
1.0*
-
-
Ex1:c.53delG (p. G18DfsX82)
Homozygous
Mistri et al., 2019
T32
0.74*
Ex8:c.964G > T (p. D322Y)
Homozygous
Mistri et al., 2011
T33
3.8
394.0
Ex1:c.26C > A (p.S9X)
Homozygous
Mistri et al., 2019
T34
6.2
2585.3
0.24
Ex11:c.1278insTATC (p. Y427IfsX5)
Homozygous
Myerowitz and Costigan,
1988
T35
0.00
833.2
0.00
Ex11:c.1278insTATC (p. Y427IfsX5)
Homozygous
T36
9.3
2250.3
0.4
Ex11:c.1278insTATC (p. Y427IfsX5)
Homozygous
T37
2.5
1890.0
0.13
Ex11:c.1278insTATC (p. Y427IfsX5)
Ex8:c.964 G > A (p. D322N)
Compound
Heterozygous
Myerowitz and Costigan,
1988
Mistri et al., 2011
Sheth et al., 2014
T38
14.5
1999.9
0.35
Ex8:c.964 G > T (p. D322Y)
Homozygous
Mistri et al., 2011
T39
8.9
2254.3
0.39
Ex8:c.964 G > T (p. D322Y)
Homozygous
Mistri et al., 2011
T40
8.3
1612.9
0.5
Ex8:c.964 G > T (p. D322Y)
Homozygous
Mistri et al., 2011
T41
HLC
Mother: 60.7%
Father: 63.9%
-
-
Mother – Ex13:c.1454 G > A/ - (p.
W485X/ -)
Father – Ex13:c.1454 G > A/ -(p.
W485X/ -)
Father: Heterozygous
Mother: Heterozygous
Sheth et al., 2014
T42
10.7
HLC
Mother: 58%
Father: 57%
1785.0
-
Mother – Ex13:c.1454 G > A/ - (p.
W485X/ -)
Father – Ex13:c.1454 G > A/ -(p.
W485X/ -)
Father: Heterozygous
Mother: Heterozygous
Sheth et al., 2014
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Table 1. (Continued)
Patient ID
Hex-A activity (MUGS)
(nmol/hr/mg)
= (x)
Total Hex activity
(MUG)
= (y)
Hex A %
=(x/y) X 100
Genotypes
Zygosity
Reference
T43
12.4
2129.0
0.58
Ex10:c.1121 A > C (p. Q374P)
Homozygous
Sheth et al., 2014
T44
10.8
1273.2
0.84
Ex14:c.1528 C > T (p. R510X)
Homozygous
Sheth et al., 2014
T45
6.2
1721.0
0.36
Ex7:c.788 C > T (p. T263I)
Homozygous
Sheth et al., 2014
T46
7.0
2602.7
0.27
Ex5:c.524A > C (p. D175A)
Ex7:c.805G > C (p. G269R)
Compound
Heterozygous
Sheth et al., 2014
T47
9.6
1589.0
0.6
In7:c.805+1 G > C
Homozygous
Mistri et al., 2012
T48
0.00
1781.0
0.0
In4:c.459+5 G > A
Homozygous
Sheth et al., 2014
T49
12.8
2050.0
0.6
Ex8:c.898_905delTTCATGAG (p.
F300HfsX21)
Homozygous
Sheth et al., 2014
T50
7.9
2185.7
0.36
Ex12:c.1385 A > T/ ? (p. E462V/ ?)
-
Mistri et al., 2011
Sheth et al., 2014
Sheth et al., 2018
T51
#
HLC
Mother: 53.8%
Father: 58.8%
-
-
Mother – Ex5: c.532 C > T/ - (p.
R178C/-)
Father – [?/ -] (?/ -)
-
Sheth et al., 2014
T52
10.5
1978.3
0.53
NF
-
-
T53
8.3
1859.0
0.44
NF
-
-
*Enzyme study carried out at other centers;
#
DNA of index case is not available.
Normal range for Hex-A - 80 to 410 nmol/hr./mg and for total-Hex-723 to 2700 nmol/hr./mg.
HLC-heat labile activity in carrier parents.
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Table 2. Clinical, biochemical and molecular details of the Indian patients with Tay–Sachs disease
Patient
ID
Age at
diagnosis
(Months/Sex)
Native
State
Cosanguinity
Hex-A
activity
(MUGS)
(nmol/hr/mg)
= (x)
Total Hex
activity
(MUG)
= (y)
ψ
Hex A
%
= (x/y)
X 100
Genotypes
Phenotypes
Nucleotide level
(Allele from
Father/
Allele from
Mother)
Protein level
(Allele from
Father/
Allele from
Mother)
1
18/M
Gujarat
No
0.9
1292.1
0.07
Exon-2-3del/
Exon-2-3del
Not identified/
Not identified
Regression of milestone, cherry red
spot, abnormal muscle tone,
hyperacusis, seizures, abnormal
MRI
2
14/M
Gujarat
No
1.05
Not done
-
Exon-2-3del/
Exon-2-3del
Not identified/
Not identified
Regression of milestone, cherry red
spot, poor vision, abnormal muscle
tone, hyperacusis, seizures,
abnormal MRI, abnormal EEG
3
12/F
Gujarat
No
3.8
2185.7
0.17
c.1385A >
T/Exon-1
deletion
p.E462V/ Not
identified
Regression of milestone, cherry red
spot, abnormal muscle tone,
seizures, hyperacusis, hearing
impairment
4
13/F
Gujarat
No
2.5
1635
0.15
c.1385A >
T/Exon-1
deletion
p.E462V/ Not
identified
Regression of milestone, hypotonia,
hyperacusis, cherry red spot,
abnormal MRI
5
13/M
Karnataka
Yes
1.78
2198.2
0.08
c.1527-2 A >
T/Exon-1
duplication
Not identified/
Not identified
Regression of milestone, cherry red
spot, hypotonia
Normal total-hexosaminidase values using MUG substrate in our controls –723 to 2700 nmol/hr/mg protein and normal Hex-A activity using MUGS substrate- 80
to 410 nmol/hr/mg. ψ The MUG/MUGS ratio for Hex A is 3.7:1 [11].
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Jayesh Sheth and Aadhira Nair
146
Figure 4. Schematic representation of HEXB gene showing all the mutations found in
SD patients from India.
6. BIOCHEMICAL DIAGNOSIS
The determination of β-hexosaminidase deficiency in the serum and
leukocytes is the basis for the biochemical diagnosis of GM2
gangliosidosis. Serum, white blood cells, cultured somatic tissues
including amniocytes and even dried blood spots on filter paper can be
used for testing hexosaminidase levels. This employs the use of a
fluorogenic substrate the-GlcNAc derivative of 4-methylumbelliferone
(4MUG). Both the isoforms-Hex-A and Hex-B can utilize this substrate
and convert it to a fluorescent product 4-methylumbelliferone. Differences
in the thermal, pH or electrochemical properties distinguishes the two
isoforms. Hex-A is heat labile at 50˚C whereas Hex-B is heat stable.
Hence, the primary assay calculates the total hexosaminidase activity using
MUG substrate. This is followed by subjecting a second serum aliquot to
heat, thereby inactivating Hex-A and reflecting only Hex-B activity. Thus,
the residual Hex-A activity is then calculated by subtracting Hex-B activity
from total hexosaminidase activity. To achieve more specificity, another
substrate 4-methylumbelliferyl-GlcNAc-6-sulfate (4MUGS) is used in
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GM2 Gangliosidosis/Tay Sachs Disease
147
addition to 4MUG, wherein only Hex-A can act on this substrate. It is
important to run assay of control enzyme activity simultaneously using
healthy subjects to determine a normal range. The above assay has few
Table 3. Review of molecularly proven cases of GM2
Gangliosidosis-AB variant
Case
Mutation
Location
Effect
Ethnicity
Reference
1
c.412T > C (p.C107R)
#
(Homozygous)
Exon 3
Reduced interaction with
Hex A
US
Black
Schroder et al.,
1991
2
c.412T > C (p.C138R)
#
(Homozygous)
Exon 3
Reduced interaction with
Hex A
US
Black
Xie et al., 1992
3
c.506G > C (p.R169P)
(Homozygous)
Exon 4
Pre-matured protein
degradation
Indian
Schroder et al.,
1993
4
c.262_264delAAG(p.88Kdel)
(Homozygous)
Exon 3
Absence of mature CRM
Saudi
Arabia
Schepers et al.,
1996
5
c.410delA (p.H137PfsX34)
(Homozygous)
Exon 3
Absence of mature CRM
Spanish
Schepers et al.,
1996
6
c.160G > T (p.E54X)
(Homozygous)
Exon 2
Absence of mRNA or
CRM
Laotian,
Hmong
Chen et al., 1999
7
c.522T > G (p.L174R)
(Homozygous)
Exon 4
Pre-matured protein
degradation
Indian
Kolodny et al.,
2008
8
c. 160G > T (p.E54X)
(Homozygous)
Exon 2
Absence of mRNA or
CRM
Hmong
Renaud et al.,
2015
9
c.164C > T (p.P55L)
(Homozygous)
Exon 2
Reduced interaction with
Hex A
Saudi
Arabia
Salih et al., 2015
10
c.472G > T (p.E158X)
(Homozygous)
Exon 4
Absence of mRNA or
CRM
Indian
Sheth et al., 2015
11
c.369_371delinsATTAA
(p.P124Lfs*48)
(Homozygous)
Exon 3
Non-sense mediated
mRNA decay
Turkey
Brackmann et al.,
2016
12
c.259G > T (p.E87X)
c.164C > T (p.P55L)
(Compound heterozygous)
Exon 3
Exon 2
Non-sense mediated
mRNA decay
Reduced interaction with
Hex A
Canada
Martins et al.,
2017
13
c.243-2A > T
(Homozygous)
Intron 3
Splicing affected
Indian
Kochumon et al.,
2017
#
The mutations identified by Schroder et al., (1991) (CYS107ARG) and Xie et al., (1992)
(CYS138ARG) are the same but derived from different amino acid numbering systems.
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Jayesh Sheth and Aadhira Nair
148
limitations such as in case of pregnant women or those taking oral
contraceptives pills (OCPs) a circulating heat-stable hexosaminidase P is
synthesized. This can falsely decrease the calculated serum activity of
hexosaminidase A relative to that of total hexosaminidase. This is why
carrier status in pregnant women or those taking OCPs should be assessed
in leukocytes, as opposed to serum. Another limitation is due to the effect
of some mutations imparting thermal stability to Hex-A. As a result, using
the heat inactivation assay will give false positive results.
The testing for the AB variant TSD with activator deficiency involves
using a labeled GM2 ganglioside rather than a chromogenic substrate. It
has been demonstrated that presence of two alleles-R247W and R249W do
not lead to disease conditions in tested individuals as they can process
normal GM2 ganglioside substrate. But, when tested with 4MUGS
substrate, they show reduced activity. Hence these alleles are termed as
pseudo deficiency alleles.
7. CARRIER SCREENING FOR TSD
Though lysosomal enzymes show overlapping activity in leucocytes
hence carrier identification cannot be confirmed by enzymes study.
Nonetheless, for TSD, serum or plasma level can be used as a marker for
carrier screening.
TSD carrier screening in certain populations like Ashkenazi Jewish,
French Canadian, or Cajun descents (ACOG, 2005) has been
recommended by The American Congress of Obstetrics and Gynecologists.
However, due to multiethnic and endogamous marriages, carrier screening
using enzyme study has become challenging. In our center we have a
limited experience of carrier screening by enzymes study from plasma or
serum, while study by Lazarin et al., (2013) have shown that, nearly 40%
of carriers of diseases/mutations most prevalent in the Ashkenazi Jewish
population for TSD, did not report Jewish ancestry [43]. Recent study by
Strom et al., (2013) suggests that performance by enzymatic assays may
not be optimal in the general population [44]. Therefore, in the era of NGS,
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GM2 Gangliosidosis/Tay Sachs Disease
149
identification of the risk allele in the ethnicity or population remains the
gold standard for carrier screening of Gm2 gangliosidosis.
8. PRENATAL DIAGNOSIS OF GM2 GANGLIOSIDOSIS
With the limited availability of therapeutic option; prenatal diagnosis
(PD) remains the only preventable measure for storage disorders. In our
study of one hundred forty pregnancies having confirmed diagnosis of
LSDs in index case, prenatal diagnosis was carried out by enzymes study
during antenatal period from uncultured and/or cultured chorionic villus
(CV/CCV) and cultured amniotic fluid (CAF) cells. In seven pregnancies,
molecular analysis was additionally carried out where mutation was known
in an index case. Table 4 depicts the biochemical and molecular diagnosis
of the above-mentioned prenatal samples of our study. Of 140 pregnancies,
60 (42.9%) were diagnosed as affected, 13 (9.3%) had an intermediate
enzyme activity and 67 (47.8%) had normal enzyme activity. Results were
confirmed in 83 cases whereas 57 cases were lost from the follow-up. In
one case, enzyme b-galactose-6 sulphate sulphatase specific for Morquio-
A disorder [mucopolysaccharidosis-IVA (MPS-IVA)] had shown 30%
reduced activity in CV cells and the case was diagnosed as carrier for
MPS-IVA while it delivered an affected child. Further molecular analysis
in seven cases that included six with Tay-Sachs and one with Gaucher
disease, confirmed the results obtained by enzymatic study during PD. PD
of LSDs can be carried out by enzymes study from CV/CCV and CAF
with an accuracy of molecular method. However, in cases of MPS and
mucolipidosis, Amniotic fluid (AF) is preferred over CV/CCV. In addition,
special care is needed while interpreting enzyme results encompassing
carrier status and they need to be further evaluated by molecular studies
[7, 45].
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Jayesh Sheth and Aadhira Nair
150
Table 4. Prenatal Diagnosis of Tay-Sach’s disease (TSD): Biochemical
and Molecular based Diagnosis
Sr.
No
Types of
Specimen
Hex-A activity
(nmol/h/mg protein
Diagnosis
Mutation present in
Index case
Mutation analysis in
Prenatal sample.
1.
CT cells
21.7 nmol
TSD
c.1277_1278insTATC/
c.1277_1278insTATC
c.1277_1278insTATC/
c.1277_1278insTATC
2.
CT cells
359.2
TSD
D322N/ D322N
Absent
3.
CT cells
403.5
TSD
D322N/
c.1277_1278insTATC
Absent
4.
AF cells
23.4 nmol
TSD
E114K/E114K
E114K/E114K
5.
AF cells
247.2 nmol
(50% of control mean)
TSD
D322N/ D322N
D322N/ Normal allele
(Heterozygous mutation)
6.
AF cells
650.2
TSD
W485X/ W485X
Absent
Normal Range of Hex-A: [(CT: 271–1621 nmol/h/mg protein); (AF:271-2213.2 nmol/h/mg protein)].
9. CLINICAL SYMPTOMS/PHENOTYPE
9.1. Infantile Acute Form
The infantile TSD also known as the B variant is the most severe form
causing multiple anomalies in the affected individual followed by early
death. These patients generally lack Hex-A or have only 0.1% of the
normal Hex-A enzyme activity resulting in complete discrepancy in GM2-
ganglioside breakdown. These children are born normal at birth, showing
clinical manifestations in 2-3 months of age. The first distinctive signs
include exaggerated startle response, generalized hypotonia, and failure to
follow objects visually. The gross accumulation of GM2 gangliosides in
the neuronal cells is the main reason for the neurodegeneration as well as
muscle weakness which may cause paralysis in some cases. The lipid
deposition in the bipolar ganglion cells of the retina causes the appearance
of a cherry-red spot on the macula which can be observed by fundoscopic
examination. Hyperacusis-a distinct feature can aid in an early diagnosis of
the condition. There is rapid deterioration of the nervous system leading to
difficulty in swallowing, uncontrolled seizures, spasticity, blindness and
dementia due to progression of the condition. The patients generally die by
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GM2 Gangliosidosis/Tay Sachs Disease
151
the age of 2 to 4 years [46]. Figure 5 depicts the distinct clinical phenotype
in the infantile form of TSD.
A study by Bley and colleagues on TSD and SD infantile forms stated
that the most initial symptoms were developmental arrest (83%), abnormal
startle response (65%), and reduced muscle tone (60%) [46].
Macrocephaly generally begins by the age of 18 months. The most
common neuroimaging findings are signal changes like hyperintensity or
hypointensity in the basal ganglia and cerebral white matter on T2-
weighted sequences on magnetic resonance imaging (MRI) studies [3]. A
population-based review of GM2 gangliosidosis patients in UK was carried
out by Smith and colleagues. They reported death in 40 of the 41 infantile
patients within the range of 18 months-6 year 9 months. In 53% cases,
pneumonia and/or respiratory failure was the cause of death and for the
remaining it was unspecified [47]. A study by Er et al., 2018 reported
similar findings thus, indicating pneumonia and/or respiratory failure to be
a major cause of death [48].
The infantile form of Sandhoff disease shows a similar clinical
manifestation like that of TSD, however, liver and spleen enlargement are
a key feature in SD. Presence of hypointense thalami is another significant
feature of infantile form of SD. This is due to calcium accumulation, loss
of axon and myelin in the central cortical neurons, and gliosis [49].
Figure 5. Clinical symptoms in patients with Tay-Sachs disease. A) A child showing
global developmental delay and poor social responses and the infantile form TSD
patient. B) A child with global developmental delay, hypotonia, microcephaly,
hyperacusis and cherry red spot at macula.
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