Ibrahim et al.

Orphanet Journal of
Orphanet Journal of Rare Diseases (2023) 18:52
https://doi.org/10.1186/s13023-023-02637-1 Rare Diseases

RESEARCH Open Access

Biochemical and mutational analyses

of HEXA in a cohort of Egyptian patients
with infantile Tay‑Sachs disease. Expansion
of the mutation spectrum
Doaa M. A. Ibrahim1, Ola S. M. Ali2, Hala Nasr1, Ekram Fateen3* and Alice AbdelAleem1*   

Abstract
Background Tay-Sachs disease (TSD), an autosomal recessively inherited neurodegenerative lysosomal storage
disease, reported worldwide with a high incidence among population of Eastern European and Ashkenazi Jewish
descent. Mutations in the alpha subunit of HEXA that encodes for the β-hexosaminidase-A lead to deficient enzyme
activity and TSD phenotype. This study is the first to highlight the HEXA sequence variations spectrum in a cohort of
Egyptian patients with infantile TSD.
Results This study involved 13 Egyptian infant/children patients presented with the infantile form of TSD, ten of the
13 patients were born to consanguineous marriages. β-hexosaminidase-A enzyme activity was markedly reduced in
the 13 patients with a mean activity of 3 µmol/L/h ± 1.56. Sanger sequencing of the HEXA’ coding regions and splicing
junctions enabled a detection rate of ~ 62% (8/13) in our patients revealing the molecular defects in eight patients;
six homozygous-mutant children (five of them were the product of consanguineous marriages) and two patients
showed their mutant alleles in heterozygous genotypes, while no disease-causing mutation was identified in the
remaining patients. Regulatory intragenic mutations or del/dup may underlie the molecular defect in those patients
showing no relevant pathogenic sequencing variants or in the two patients with a heterozygous genotype of the
mutant allele. This research identified three novel, likely pathogenic variants in association with the TSD phenotype;
two missense, c.920A > C (E307A) and c.952C > G (H318D) in exon 8, and a single base deletion c.484delG caus-
ing a frameshift E162Rfs*37 (p.Glu162ArgfsTer37) in exon 5. Three recurrent disease-causing missense mutations;
c.1495C > T (R499C), c.1511G > A(R504H), and c.1510C > T(R504C) in exon 13 were identified in five of the eight
patients. None of the variants was detected in 50 healthy Egyptians’ DNA. Five variants, likely benign or of uncertain
significance, S3T, I436V, E506E, and T2T, in exons 1, 11,13, & 1 were detected in our study.
Conclusions For the proper diagnostics, genetic counseling, and primary prevention, our study stresses the impor-
tant role of Next Generation Sequencing approaches in delineating the molecular defect in TSD-candidate patients
that showed negative Sanger sequencing or a heterozygous mutant allele in their genetic testing results. Interest-
ingly, the three recurrent TSD associated mutations were clustered on chromosome 13 and accounted for 38% of

*Correspondence:
Ekram Fateen
[email protected]
Alice AbdelAleem
[email protected]
Full list of author information is available at the end of the article

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Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 2 of 9

the HEXA mutations detected in this study. This suggested exon 13 as the first candidate for sequencing screening
in Egyptian patients with infantile TSD. Larger studies involving our regional population are recommended, hence
unique disease associated pathogenic variations could be identified.
Keywords Infantile Tay-Sachs disease, HEXA gene, β-hexosaminidase-A enzyme, HEXA mutation spectrum, Egyptian
patients with TSD, Biochemical analysis of HexA-enzyme, Molecular diagnostic, Rare neurodegenerative diseases,
Sanger sequencing

Introduction of them, TSD is caused by one of three common muta-

Tay-Sachs disease (TSD) or GM2 gangliosidosis variant B tions; c.1277_1278 ins TATC in exon 11, 1421 + 1 G > C
(MIM: 272800) is a rare autosomal recessive neurodegen- transition at the splice junction of exon/intron 12 and
erative lysosomal storage disorder that is fatal in infancy. c.805 G > A (p.G269S) [12]. Non-Ashkenazi TSD patients
TSD is caused by mutations in the alpha-subunit HEXA exhibited rare mutations distributed all over the HEXA
gene (MIM# 606869) of β-hexosaminidase-A enzyme gene [13, 14].
leading to deficiency of the enzyme activity and TSD In the present study, we were aiming to reveal the
phenotype. The main pathology of the disease is caused HEXA mutations’ spectrum and genotypes in Egyptian
by the progressive accumulation of GM2 ganglioside and patients with the infantile form of TSD. Bidirectional
associated glycosphingolipids in the lysosomes, mainly direct Sanger sequencing analysis of the entire coding
that of the neurons promoting progressive neurodegen- region and splice junctions of the gene was our imple-
eration [1]. The clinical presentation is classified accord- mented approach.
ing to the age of disease- onset into infantile, juvenile, and
adult forms. The most severe form is the classical infan-
tile TSD, which presents initially with motor weakness, Subjects and methods
increased startle response at 3–5 months of age, and is Subjects and samples collection
invariably associated with macular red spots. Neurologi- Twenty-three unrelated individuals were enrolled in this
cal signs become increasingly evident with the loss of ear- study. Patients were ascertained based on the prelimi-
lier achieved motor milestones, impaired vision, deafness, nary clinical findings of early onset progressive neurode-
feeding problems and seizures. Further deterioration ends generative disease associated with seizures, hypotonia,
in an unresponsive vegetative state and death at 2–4 years loss of earlier acquired motor skills, cherry red spots,
of age. This fatal phenotype is common to all patients and maybe loss of vision. The confirmatory criterion
with deficient β-hexosaminidase-A activity. Later-onset for inclusion as TSD was the reduced measured HeX-A
TSD phenotypes are more clinically variable and are usu- enzyme activity. Patients were referred by the biochemi-
ally divided into juvenile and adult forms [2]. Recently, cal genetics department at the National Research Centre
an adeno-associated virus (AAV) gene therapy clinical (NRC) in Egypt. Enrolled subjects are subdivided into 3
trial to establish safety dosage and provide infantile TSD groups: the first group included 13 patients with HeX-A
patients derived data has shown promising results of a positive assay [enzyme activity was below 5 µmol/L/h],
time-limited disease stabilization [3]. TSD disease showed the second group involved 5 patients clinically presented
a particularly high incidence in the Ashkenazi Jewish with neurodegenerative manifestations but with normal
population with a carrier frequency of approximately 1 HeX-A enzyme assay [enzyme activity was in the normal
in 25 compared to 1/250–300 in most other populations. range], and a third group of 5 normal controls. Patients
Also, the incidence of patients affected by the disease is were clinically classified as infantile TSD phenotype,
estimated to be 1 in 3600 live births in Ashkenazi Jew- according to the disease’s age of onset. The study was
ish versus 1 in 360,000 in other populations worldwide approved by the Institutional Ethics committee of NRC.
[4, 5]. Population-based carrier screening programs in Written informed consent was obtained from partici-
the United States and Canada reduce the incidence of pants or their legal guardians.
TSD by more than 90% in these populations [6, 7].
To date > 220 HEXA mutations were identified in the
Enzyme assay
Tay-Sachs mutation database [8, 9], HEXA mutations
Fresh peripheral blood samples were collected on EDTA
may impact the protein’s structure, folding, and/or trans-
from each participant, a specific lab code was given
port, which ultimately leads to functional impairment
for each blood tube. A standard fluorimetric method
[10, 11]. Ethnic-related common gene mutations have
using the specific sulphated fluorogenic substrate,
been reported in Ashkenazi Jews patients, in 94–98%
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 3 of 9

4-methylumbelliferyl-N-acetyl-β-D-glucosamine “MUG”, was based on the biochemical result of deficient

was applied to measure the enzyme activity in serum β-hexosaminidase-A enzyme activity. Affected chil-
[15]. dren died before reaching the age of 24 months except
for four patients (3, 6, 7, and 10) that survived to four
Sanger sequencing of the HEXA gene alpha‑subunit years old. Five normal control Egyptians were involved
Participants’ genomic DNA extracted from fresh periph- in checking for the common neutral single nucleotide
eral blood leukocytes using the salting out protocol [16] polymorphism detected in our patients and reported
was quantified and amplified. The 14 exons and splicing in other populations. Demographic data of the enrolled
junctions of HEXA were PCR amplified in 12 fragments subjects are shown in Table 1.
using specifically described primers [13]. Each amplicon
was purified using shrimp alkaline phosphatase and exo-
nuclease I protocol. Sanger sequencing analysis of every Biochemical findings (Hex‑A enzyme activity)

­ igDye® Terminator v3.1 kit

single stranded fragment was performed in forward and Deficient β-hexosaminidase-A (Hex-A) enzyme activity
reverse directions, using the B was confirmed in 13 patients, whereas a normal range
(Applied Biosystems). Sequencing reactions were purified of the enzyme activity was shown in five patients, conse-
and run on the 310 Genetic Analyzer (Applied Biosys- quently, the diagnosis of TSD was excluded in them.
tems). The occurrence of the sequence variations iden- β-Hexosaminidase-A activity was found mark-
tified in patients was tested in 50 de-identified healthy edly reduced in TSD patients compared to the refer-
Egyptian DNA samples applying the same procedures. ence values. The enzyme activity ranged between 0.1
and 5.6 µmol/L/h with a mean of 3 µmol/L/h ± 1.56
in the 13 TSD patients (the reference normal range is
In‑silico assessment of the novel sequence variants 50–200 µmol/L/h). A normal range of 52–90 µmol/L/h
The pathogenicity and frequency of sequence variations was detected in the five patients that were clinically
identified in this study were assessed using the available ascertained as potential TSD candidates. The values of
public databases including the HGMD [8, 9], HEXAdb, Hex-A enzyme activity as well as that of the associated
OMIM [18], and online-based prediction tools of Var- lysosomal hydrolase β-hexosaminidase; alpha and beta
Some [19] and Ensemble [20]. subunits measured in fresh leukocytes blood samples of
participants enrolled in this study are shown in Table1.

Results
Patients’ characteristics Molecular findings and mutation spectrum in TSD patients
This study included 18 unrelated Egyptian patients Sequencing analysis of HEXA α-subunit coding and
who presented with the provisional clinical diagnosis flanking regions detected 15 mutant alleles, out of the
of a neurodegenerative lysosomal disorder. Patients’ anticipated 26 alleles of the 13 TSD cases; six patients
age group falls in the range from birth to ~ 4 years. All were homozygous for the mutant alleles, five of them
patients enrolled in this study were Egyptian, and none were the product of consanguineous marriages. While
of them was of Jewish origin. Parental consanguin- two patients (P1 and P3) were heterozygous for the
ity was documented in ten of the 13 confirmed TSD mutant allele (Fig. 1a–f ), and in five TSD patients with
patients (77%). The patient’s phenotype was sugges- deficient enzyme activity, no mutation was identified.
tive of neurodegenerative infantile Tay-Sachs disease Mutations’ details are shown in Tables 1 and 2. The two
involving myoclonic seizures, progressive neurological unrelated patients, P1 and P3, in whom the second allele
impairment, brisk deep tendon reflex, deafness, and was not identified are the product of a non-consanguine-
macular degeneration, a Cherry red spot was found in ous parent and carrying the same heterozygous mutation
all our Tay Sachs affected patients. Exaggerated Star- c.1510C > T(R504C) in exon 13, as well as the common
tle reflex to noises was always the first sign noticed by polymorphic benign alleles, c.1306A > G (I436V) and
the parents at a median age of onset 7 months ± 3.2, c.1551G > A (E417E) in exons 11 and 13, respectively in
extended into infantile spasms that were usually febrile heterozygous genotypes.
at the beginning then myoclonic seizures happened Three Novel, likely pathogenic variants involve a
without fever. Rapid deterioration of all acquired devel- frameshift single nucleotide deletion, c.484delG and two
opmental skills, spasticity of the limbs, and loss of missense variants, c.952C → G (H318D) and c.920A → G
contact/interest in the surroundings associated with (E307A) first detected in the present study and were not
loss of vision were the sequence of events. All patients previously reported in any of the public databases or in
were macrocephalic. The confirmed clinical diagnosis 100 Egyptian chromosomes. Recurrent three variants,
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 4 of 9

Table 1 Demographic, biochemical, and sequence variation characteristics in TSD Egyptian patients
Patient’s Gender Age at Age at Family Consangunity Clinical Hex A Variants’ Genotype
number enrollment disease history phenotype activity
onset
1 F 16 m 6m Absent Absent Infantile α 3.8 c.1306A>G(I436V) #
^
TSD β 2315 c.1551G>A (E417E)#
c.1510C>T(R504C) #
2 M 18 m 9m Absent Present Infantile α 3.7 c.1306A>G (I436V) ●
TSD β 2156 ^c.1551G>A (E417E)●
c.1510C>T(R504C) ●
3 F 3y 6m Absent Absent Infantile α 2.5 c.1306A>G (I436V) #
TSD β 2143 ^ c.1551G>A (E417E)#
c.1510C>T(R504C) #
4 M 18 m birth Present Absent Infantile α 2.1 c.1511G>A(R504H) ●
TSD β 1751.2
5 M 17 m 7m Absent Present Infantile α 2.5 c.1495C>T (R499C) ●
TSD β 1850
6 F 3.8 y 12 m Present Present Infantile α 0.12 c.952C>G(H318D)* ●
TSD β 1315 c.1306.A>G (I436V) ●
^c.1551G>A (E417E)●
7 M 3.1 y 12 m Present Present infantile α 1.1 c.920. A>C(E307A)* ●
TSD β 275 c.1306A>G (I436V) ●
^c.1551G>A (E417E)●

8 M 20 m 4m Absent Present Infantile α 5.6 c.8G>C (S3T) #

TSD β max c.1306A>G (I436V) ●
^c.1551G>A (E417E)●
9 M 13 m 9m Present Present Infantile α 4.2 c.484 del G(162)*●
TSD β 1412 c.1306A>G (I436V) ●
^c.1551G>A (E417E)●

10 F 3.5y 6m Absent Present Infantile α 2.6 c.1306A>G (I436V) ●

TSD β 118 ^c.1551G>A (E417E)●
11 F 18 m 7m Present Present Infantile α5 c.6A>G (T2T)* # c.1306A>G
TSD β 2125 (I436V) ●
^c.1551G>A (E417E)●
12 M 16m 9m Present Present Infantile Α 2.5 c.1306A>G (I436V) ●
TSD β 1852 ^c.1551G>A (E417E)●
13 F 13 m 5m Absent Present Infantile α 4.5 c.1306A>G (I436V) #
TSD β 1345 ^c.1551G>A (E417E)#
14-18 2F & 2.5-8y 12m-4y Absent Present in 3 Patients α 52-93 c.1306A>G (I436V)
3M patients & with β 2231to ^c.1551G>A (E417E))
absent in two neurodeg. 23000
manifest.
but
Normal
enzyme
activity
19-23 5F 20-30y nor Absent Absent Controls Normal c.1306A>G (I436V)
mal range 1518.G>A (E506E)

y: years, m: months, neurodeg: neurodegenerative, manifest: manifestations, F: female, M: male

^c.1551G > A (E417E) corresponds to c.1518G > A (E506E) in a different transcript
Enzyme activity unit: l µmol/L/h
Normal HEXA- α subunit enzyme activity reference range = 50–200 µmol/L/h
Normal HEXA- β subunit enzyme activity reference range = 100–3500 µmol/L/h
Blue shaded rows present TSD patients with a confirmed deficient Hex-A enzyme activity, but the causative mutations were not identified
*Novel mutations
#Heterozygous variant
●Homozygous variant
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 5 of 9

Fig. 1 Sanger sequencing chromatographs of HEXA gene mutations in Egyptian patients with infantile TSD. The chromatographs showed the
mutations identified in nine patients [P1-P9] in exons 5, 8, and 13 as well as the polymorphic variations. a (P9): c.484 delG (homozygous); b (P6):
c.952C > G (P.H318D (homozygous); c (P7): c.920A > C (p.E307A) (homozygous); d (P5): c.1495C > T (p. R499C) homozygous; e (P1,2,3): c.1510C > T
(p.R504C) homozygous and heterozygous; f (P4): c.1511G > A (p.R504H) (homozygous); g (P11): c.6A > G (p.T2T). Silent rare sequence variant. h
(P8): c.8G > C (p.S3T). Polymorphic variant. i (P1-18,C1-5): c.1518.G > A (p.E506E). Silent sequence variant. j (P1-18, C1-5): c.1306.G > A (p. I436V).
Polymorphic variant. Arrows indicate the locations of the mutations in the amino-acid sequence of α-hexosaminidase A protein. *: Novel variant, c.:
c.DNA, p: protein

R499C (c.1495C → T), R504C (c.1510C → T) and R504H • The missense E307A variant: ACMG classification
(c.1511G → A) previously reported in association with is likely pathogenic conservation score = 7.9 by Phy-
TSD, were identified also in our patients. loP100, reported as damaging/deleterious/or disease-
causing in 11 pathogenicity prediction tools, and not
In‑silico pathogenicity prediction of the novel variants found in GnomAD Exomes or Genomes.

• The frameshift, c.484delG, E162Rfs*37 predicted of

a high impact on the encoded protein and result- Polymorphic variants (Fig. 1g–j)
ing in nonsense mediated decay. The c.484delG has Four HEXA polymorphic variants were identified in the
not been reported in gnomAD or other public data- present study. The c.1551A > G(E517E) and c.1306A > G
bases. (I436V) were commonly described. The c.1551A > G
• The missense H318D variant: ACMG classification is (p.Glu517 =) in exon 13, (Transcript NM_001318825.2)
likely pathogenic based on: has a GnomAD Exomes frequency of 0.97 and classi-
– The position is very highly conserved, score 9.7 by fied as benign. This variant corresponds to c.1518A > G,
phyloP100 vertebrates, not reported in GnomAD (p.Glu506 =) in transcript NM_000520.6.
exomes or genomes, 11 pathogenic prediction tools The c.1306A > G, I436V (p. Ile436Val) in exon 11,
predict this variant as damaging/deleterious/or classified as VUS in a single database submission
disease causing, and almost always missense muta- (Transcript NM_001318825.2) and as benign in mul-
tions that is not variant of uncertain significance tiple submissions (NM_000520.6). GnomAD Exomes
(VUS) in HEXA is disease causing. frequency = 0.971.
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 6 of 9

Table 2 Distribution of HEXA gene variations in Egyptian patients clinically suggestive of infantile TSD
Subjects Exon c.DNA position Amino acid change Frequency (%) Ethnic population References

3/13 (P1,P2,P3) Ex13 c.1510C > T p.R504C (Arg504Cys) 23 German, Akli et al. (1991)
g. 72,637,803 C > T CGC˃TGC​ French, Akli et al. (1993b)
Algerian, & Egyptian Paw et al. (1991)
Tanaka et al. (1994)
This report
1/13 (P4) Ex13 c.1511G > A p.R504H (Arg504His) 7.7 Assyria, Lebanes, Americn, Paw et al. (1990a)
g. 72,637,802 G > A CGC˃CAC​ & Egyptian This report
1/13 (P5) Ex13 c.1495C > T P.R499C (Arg499Cys) 7.7 Slavic, Irish, English, Pol- Akli et al. (1993b);
g. 72,637,818 C > T CGT˃TGT​ ish, & Egyptian Mules et al. (1992b);
Paw et al. (1990);
This report
1/13 (P6) Ex8 c.952C > G p.H318D 7.7 Egyptian Novel. This report
g. 72,641,454 C > G (Hist318Asp) {A different amino acid
CAT˃GAT​ change (H318R) reported
at the same codon}
1/13 (P7) Ex8 c.920A > C p.E307A 7.7% Egyptian Novel. This report. A dif-
g. 72,641,486 A > C (glu 307ala) ferent amino acid change
GAA˃GCA​ (E307K) reported at the
same codon
1/13 (P9) Ex5 c.484del G p. E162Rfs*37 7.7 Egyptian Novel. This report
g. 72,645,495 delG
11/13 TSD patients, 5/5 Ex11 c.1306A > G p.I436V 91 Egyptian, Black American, Mules et al. (1992b)
patients with normal g.72638892A > G (Ile 436 Val) & mixed ethnic This report
Hex-A enzyme & 5/5 ATA˃GTA​
controls
11/13 TSD patients, 5/5 Ex13 c.1551A > G p. E417 = (Glu417 =) 91 Egyptian, Paw et al. (1991);
patients with normal g.72637795 G > A GAA˃GAG​ German, & multiethnic Akli et al. (1993b); Tanaka
HEXA enzyme & 5/5 {c.1518A > G} {P. E506 =} et al. (1994);
controls This report
1/13 (P8) Ex1 c.8G > C p.S3T 7.7 Egyptian, Ashenazi J. Cecchi et al. (2019);
g.72668306G > C (Ser3Thr) &mixed ethnic This report
AGC˃ACC​
1/13 (P11) Ex 1 c.6A > G P.T2T Egyptian, This report; a ClinVar
g. 72,668,308 ACA > ACG​ No data for the ClinVar submission
submission

The c.6A > G (T2T) silent substitution in exon 1, Discussion

was newly reported in this study in association with TSD patients ascertained in the present study displayed
TSD. There was a single clinical testing submission of developmental delay, hypotonia, loss of motor mile-
T2T on CliniVar, but no assertion criteria were pro- stones, seizures, deafness, and the presence of a cherry
vided, and the TSD-affected status was unknown. red spot in fundus examination. The markedly reduced
The frequency of this variant is very rare; in gno- Hex-A enzyme activity (mean 3 µmol/L/h ± 1.56) has
mAD exomes = 0.0000319, in gnomAD genomes confirmed the diagnosis of TSD.
ƒ = 0.000004. This study is the first to uncover the HEXA mutations’
The fourth variant, c.8G > C (S3T) classified as spectrum and correlate the β-hexosaminidase-A enzyme
likely benign; the position is not conserved (phy- deficiency to the underlying HEXA molecular defects
loP100 = 0.297), while predicted as damaging in only in a homogeneous population of Egyptian patients with
two prediction tools. However, it is of the rare alleles, the infantile form of TSD. The sequencing technology
its allele frequency in gnomAD exomes ƒ = 0.00046, applied here, Sanger sequencing of the coding and splice
and in gnomAD genomes ƒ = 0.000255. The S3T has junction regions of the gene enabled the identification
never been reported as homozygous in databases, of the molecular defect in ~ 62% of patients enrolled, in
only as heterozygous with a normal enzymatic assay two of them a single mutant allele was detected. While
association. in ~ 38%, the molecular pathology remained undiagnosed.
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 7 of 9

The characteristics of patients 1 and 3 in whom the present cohort of Egyptian patients (5/13) with infantile
same recurrent mutation c.1510C > T(R504C), as well as TSD, whereas missense mutations in exon 8 [H318D and
the two polymorphic benign alleles, c.1306A > G (I436V) E307A] present 15.4%. The nucleotide deletion in exon 5
and c.1551G > A (E417E) were all in heterozygous geno- [c.484delG] constitutes 7.7%. These sequencing findings
types seem interesting. It may suggest a potential hap- lead us to suggest HEXA-exon13 as the first candidate for
lotype of the TSD disease-causing mutation (in exon13) molecular screening in Egyptian patients with infantile
and two common variants in exons 11&13. This hete- TSD.
rozygous haplotype may result from an associated chro- The novel missense c.952C > G mutation (H318D) in
mosomal rearrangement events. It will be interesting exon 8, homozygous in P6, falls within residues 192–402
to investigate potential cytogenetic events in these two of the enzyme’s alpha subunit. These residues were found
patients, the outcome may support a different mecha- to be involved in the hydrolysis of the GM2-ganglioside
nism of the molecular pathology that underlies the TSD [29]. Histidine residue is the most common amino acid
clinical and biochemical phenotype. involved in protein active sites [30]. His318 residue
The molecular defect in P8, born to a consanguineous is located within four residues forming D322, a potential
family and had a confirmed Hex-A enzyme deficiency, active site. A mutation at this site leads to abnormal pro-
remains unresolved. Three variants were detected in tein retention and degradation within the endoplasmic
this patient; the heterozygous missense variant c.8G > C reticulum [31]. A missense mutation involving the same
(S3T) that was classified as likely benign, however of a codon H318 was reported in non-Jewish TSD carrier
rare frequency (ƒ = 0.00046 in gnomAD exomes), and the from Germany; however, the substitution was for Argi-
two frequently occurring variants in exons 11&13, both nine H318R [32]. The 2nd novel mutation in exon 8, glu-
were in a homozygous genotype. The same situation of tamate substitution for alanine (E307A) at codon 920 was
the unrevealed genetic defect was encountered in four homozygous in P7. Glutamate is a negatively charged,
patients (P10-13) in whom only the polymorphic variants polar amino acid that is frequently involved in protein
in exons 11&13 and a silent amino acid change in P11 active sites. Alanine is a non-polar small-size residue
were detected in their blood derived DNA. with a very short non-reactive side chain. Substitution of
HEXA is the only gene, up to date, described in asso- a small side chain for a large one can be damaging [30].
ciation with TSD, this highlights the importance of NGS Studies that investigated the HEXA mutations affecting
technology, WGS, for the detection of the full spectrum the α-subunit candidate active site residues, reported the
of the associated disease-causing variants involving occurrence of severe impairments of the enzyme cata-
intragenic, deep intronic or copy number variations in lytic activity, which was found to interfere with α-subunit
TSD patients that proved mutations’ negative in Sanger maturation, when introducing substitution of E307 to
sequencing. NGS will also facilitate the differential diag- another residue [33]. A missense mutation at the same
nosis promoted by the notable clinical overlap seen in the 307codon exchanging amino acid glutamate into lysine
clinic among infants and children presented with a phe- (E307K) was previously reported in association with
notype suggestive of neurometabolic or neurodegenera- TSD, further supporting the pathogenicity of E307A
tive storage diseases. [34]. The homozygous base pair deletion, c.484 delG, the
To date, more than 220 HEXA mutations were reported first base in codon 162 (gAG) in exon 5 of P9, originally
causing different forms of TSD. Documented mutations described here, leads to a frameshift and premature
involved single-base substitutions, small deletions, small stop codon and apparently early mediated decay of the
duplications/insertions, partial gene deletions, splic- encoded protein.
ing alterations, and complex gene rearrangements [8, 9, The S3T missense heterozygous variant detected in
17, 18]. Mutations detected in the infantile form of TSD P8 is positioned as part of the residues of α-subunit
are generally located in residues proximal to or involve a that seems to have a role in the enzyme activity against
functionally important active site or dimer interface [21, the specific sulfated substrate 4MUGS. S3T has been
22]. detected in a screening study for TSD carriers [35]. Ear-
Among the six disease-causing mutations identified in lier studies demonstrated these two domain-residues
this study, three were originally detected in our patients of α1-191 and α 403-529 to account for enzyme activ-
[E307A, H318D, and E162Rfs*37] and another three have ity against 4MUGS [29]. However, other investigators
been previously reported [R504C, R504H, and R499C] pointed residues 1–131 as not being involved in binding
in different ethnic populations pointing out their diver- the negatively charged substrates, instead, they suggested
sity. [23–28]. Mutations in exon 13 [R504C, R504H, and residues 132–283 as the α-subunit negatively charged
R499C] constitute 38.4% of the mutations detected in the substrates-binding site [36].
Ibrahim et al. Orphanet Journal of Rare Diseases (2023) 18:52 Page 8 of 9

The recurrent R499C mutation was identified as Acknowledgements

We express our sincere thanks to the families of TSD patients for their
homozygous in P5, the substitution is likely to interrupt participation in this study. Authors acknowledge Prof. Dr. Maha S. Zaki for her
the specific disulfide bond and disrupt the three-dimen- appreciated support in referring one of the families enrolled in this study.
sional structure of the enzyme [37]. The other two muta-
Author contributions
tions R504H and R504C affect the ability of the alpha Study concept and design: all authors. Cases referral: EF. Acquisition, analysis,
subunit to dimerize. and interpretation of data: DI, OA, HN, EF, and AA. Drafting of the manuscript:
R504C was detected as heterozygous in P1 & P3 and DI. Critical revision of the manuscript: AA. All authors read and approved the
final manuscript.
homozygous in P2. R504H was homozygous in P4. R504
of the α-subunit directly binds to D494 of the β-subunit Funding
in the α-β heterodimer model. Substitution of R504 to C Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
or H causes a disruption of that binding leading to con- (EKB). This work was supported, in part, by fund from National Research Cen-
formational changes in the dimer interface [38]. Frequent tre, Egypt to Doaa Ibrahim.
recurrence of Arg to His/Cys substitution at positions 499
Availability of data and materials
and 504 is probably a consequence of a mutagenic hot Sequencing and biochemical data generated in this study are available upon
spot of the CpG dinucleotides [29, 33]. a reasonable request made to the corresponding authors.
The polymorphic variants I436V and E506E were
detected in 91% of our patients and control subjects. Declarations
I436V was frequently reported in normal American black
Ethics approval and consent to participate
population [14, 39], and also found to occur at high fre- The Research Ethics Committee of the National Research Centre, Egypt has
quency among Ethiopian Jews (heterozygotes frequency: approved the present study and the informed consent.
26.1%), while at a lower frequency in Jewish from eight
Consent for publication
populations, Iraq, Syria, Tunis, Morocco, Libya, Yemen, Participants or legal guardians have consented for the publication.
Persia and Eastern Europe (1.5–7.0%) [40].
In this study, 77% of patients were the product of con- Competing interests
The authors declared that no conflict of interest exists.
sanguineous marriages. The cConsanguinity rate in Egypt
reported to be above 30% throughout the last 40 years Author details
[41]. In an earlier biochemical study, 58% of Egyptian 1
Department of Medical Molecular Genetics, Human Genetics and Genome
Research Institute, National Research Centre, Cairo, Egypt. 2 Department
patients with GM2 gangliosidosis were diagnosed as Tay of Biochemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt.
Sachs disease by enzymatic assay. Parental consanguinity 3
Department of Biochemical Genetics, Human Genetics and Genome
was present in 73.3% of these cases. [42]. Consanguinity Research Institute, National Research Centre, Cairo, Egypt.
is a principal factor in increasing the incidence of rare Received: 18 October 2022 Accepted: 6 February 2023
autosomal recessive diseases among the Arab popula-
tion. The incidence of Tay-Sachs disease has been mark-
edly reduced by more than 90% since the introduction
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