Biochemical and mutational analyses of HEXA in a cohort of Egyptian patients with infantile Tay-Sachs disease. Expansion of the mutation spectrum

Tay-Sachs disease (TSD) or GM2 gangliosidosis variant B (MIM: 272800) is a rare autosomal recessive neurodegenerative lysosomal storage disorder that is fatal in infancy. TSD is caused by mutations in the alpha-subunit HEXA gene (MIM# 606869) of β-hexosaminidase-A enzyme leading to deficiency of the enzyme activity and TSD phenotype. The main pathology of the disease is caused by the progressive accumulation of GM2 ganglioside and associated glycosphingolipids in the lysosomes, mainly that of the neurons promoting progressive neurodegeneration [1]. The clinical presentation is classified according to the age of disease- onset into infantile, juvenile, and adult forms. The most severe form is the classical infantile TSD, which presents initially with motor weakness, increased startle response at 3–5 months of age, and is invariably associated with macular red spots. Neurological signs become increasingly evident with the loss of earlier achieved motor milestones, impaired vision, deafness, feeding problems and seizures. Further deterioration ends in an unresponsive vegetative state and death at 2–4 years of age. This fatal phenotype is common to all patients with deficient β-hexosaminidase-A activity. Later-onset TSD phenotypes are more clinically variable and are usually divided into juvenile and adult forms [2]. Recently, an adeno-associated virus (AAV) gene therapy clinical trial to establish safety dosage and provide infantile TSD patients derived data has shown promising results of a time-limited disease stabilization [3]. TSD disease showed a particularly high incidence in the Ashkenazi Jewish population with a carrier frequency of approximately 1 in 25 compared to 1/250–300 in most other populations. Also, the incidence of patients affected by the disease is estimated to be 1 in 3600 live births in Ashkenazi Jewish versus 1 in 360,000 in other populations worldwide [4, 5]. Population-based carrier screening programs in the United States and Canada reduce the incidence of TSD by more than 90% in these populations [6, 7].

To date > 220 HEXA mutations were identified in the Tay-Sachs mutation database [8, 9], HEXA mutations may impact the protein’s structure, folding, and/or transport, which ultimately leads to functional impairment [10, 11]. Ethnic-related common gene mutations have been reported in Ashkenazi Jews patients, in 94–98% of them, TSD is caused by one of three common mutations; c.1277_1278 ins TATC in exon 11, 1421 + 1 G > C transition at the splice junction of exon/intron 12 and c.805 G > A (p.G269S) [12]. Non-Ashkenazi TSD patients exhibited rare mutations distributed all over the HEXA gene [13, 14].

TSD patients ascertained in the present study displayed developmental delay, hypotonia, loss of motor milestones, seizures, deafness, and the presence of a cherry red spot in fundus examination. The markedly reduced Hex-A enzyme activity (mean 3 µmol/L/h ± 1.56) has confirmed the diagnosis of TSD.

This study is the first to uncover the HEXA mutations’ spectrum and correlate the β-hexosaminidase-A enzyme deficiency to the underlying HEXA molecular defects in a homogeneous population of Egyptian patients with the infantile form of TSD. The sequencing technology applied here, Sanger sequencing of the coding and splice junction regions of the gene enabled the identification of the molecular defect in ~ 62% of patients enrolled, in two of them a single mutant allele was detected. While in ~ 38%, the molecular pathology remained undiagnosed.

The characteristics of patients 1 and 3 in whom the same recurrent mutation c.1510C > T(R504C), as well as the two polymorphic benign alleles, c.1306A > G (I436V) and c.1551G > A (E417E) were all in heterozygous genotypes seem interesting. It may suggest a potential haplotype of the TSD disease-causing mutation (in exon13) and two common variants in exons 11&13. This heterozygous haplotype may result from an associated chromosomal rearrangement events. It will be interesting to investigate potential cytogenetic events in these two patients, the outcome may support a different mechanism of the molecular pathology that underlies the TSD clinical and biochemical phenotype.

The molecular defect in P8, born to a consanguineous family and had a confirmed Hex-A enzyme deficiency, remains unresolved. Three variants were detected in this patient; the heterozygous missense variant c.8G > C (S3T) that was classified as likely benign, however of a rare frequency (ƒ = 0.00046 in gnomAD exomes), and the two frequently occurring variants in exons 11&13, both were in a homozygous genotype. The same situation of the unrevealed genetic defect was encountered in four patients (P10-13) in whom only the polymorphic variants in exons 11&13 and a silent amino acid change in P11 were detected in their blood derived DNA.

To date, more than 220 HEXA mutations were reported causing different forms of TSD. Documented mutations involved single-base substitutions, small deletions, small duplications/insertions, partial gene deletions, splicing alterations, and complex gene rearrangements [8, 9, 17, 18]. Mutations detected in the infantile form of TSD are generally located in residues proximal to or involve a functionally important active site or dimer interface [21, 22].

Among the six disease-causing mutations identified in this study, three were originally detected in our patients [E307A, H318D, and E162Rfs*37] and another three have been previously reported [R504C, R504H, and R499C] in different ethnic populations pointing out their diversity. [23‐28]. Mutations in exon 13 [R504C, R504H, and R499C] constitute 38.4% of the mutations detected in the present cohort of Egyptian patients (5/13) with infantile TSD, whereas missense mutations in exon 8 [H318D and E307A] present 15.4%. The nucleotide deletion in exon 5 [c.484delG] constitutes 7.7%. These sequencing findings lead us to suggest HEXA-exon13 as the first candidate for molecular screening in Egyptian patients with infantile TSD.

The novel missense c.952C > G mutation (H318D) in exon 8, homozygous in P6, falls within residues 192–402 of the enzyme’s alpha subunit. These residues were found to be involved in the hydrolysis of the GM2-ganglioside [29]. Histidine residue is the most common amino acid involved in protein active sites [30]. His318 residue is located within four residues forming D322, a potential active site. A mutation at this site leads to abnormal protein retention and degradation within the endoplasmic reticulum [31]. A missense mutation involving  the same codon H318 was reported in non-Jewish TSD carrier from Germany; however, the substitution was for Arginine H318R [32]. The 2nd novel mutation in exon 8, glutamate substitution for alanine (E307A) at codon 920 was homozygous in P7. Glutamate is a negatively charged, polar amino acid that is frequently involved in protein active sites. Alanine is a non-polar small-size residue with a very short non-reactive side chain. Substitution of a small side chain for a large one can be damaging [30]. Studies that investigated the HEXA mutations affecting the α-subunit candidate active site residues, reported the occurrence of severe impairments of the enzyme catalytic activity, which was found to interfere with α-subunit maturation, when introducing substitution of E307 to another residue [33]. A missense mutation at the same 307codon exchanging amino acid glutamate into lysine (E307K) was  previously reported in association with TSD, further supporting the pathogenicity of E307A [34]. The homozygous base pair deletion, c.484 delG, the first base in codon 162 (gAG) in exon 5 of P9, originally described here, leads to a frameshift and premature stop codon and apparently early mediated decay of the encoded protein.

The S3T missense heterozygous variant detected in P8 is positioned as part of the residues of α-subunit that seems to have a role in the enzyme activity against the specific sulfated substrate 4MUGS. S3T has been detected in a screening study for TSD carriers [35]. Earlier studies demonstrated these two domain-residues of α1-191 and α 403-529 to account for enzyme activity against 4MUGS [29]. However, other investigators pointed residues 1–131 as not being involved in binding the negatively charged substrates, instead, they suggested residues 132–283 as the α-subunit negatively charged substrates-binding site [36].

The recurrent R499C mutation was identified as homozygous in P5, the substitution is likely to interrupt the specific disulfide bond and disrupt the three-dimensional structure of the enzyme [37]. The other two mutations R504H and R504C affect the ability of the alpha subunit to dimerize.

R504C was detected as heterozygous in P1 & P3 and homozygous in P2. R504H was homozygous in P4. R504 of the α-subunit directly binds to D494 of the β-subunit in the α-β heterodimer model. Substitution of R504 to C or H causes a disruption of that binding leading to conformational changes in the dimer interface [38]. Frequent recurrence of Arg to His/Cys substitution at positions 499 and 504 is probably a consequence of a mutagenic hot spot of the CpG dinucleotides [29, 33].

The polymorphic variants I436V and E506E were detected in 91% of our patients and control subjects. I436V was frequently reported in normal American black population [14, 39], and also found to occur at high frequency among Ethiopian Jews (heterozygotes frequency: 26.1%), while at a lower frequency in Jewish from eight populations, Iraq, Syria, Tunis, Morocco, Libya, Yemen, Persia and Eastern Europe (1.5–7.0%) [40].

In this study, 77% of patients were the product of consanguineous marriages. The cConsanguinity rate in Egypt reported to be above 30% throughout the last 40 years [41]. In an earlier biochemical study, 58% of Egyptian patients with GM2 gangliosidosis were diagnosed as Tay Sachs disease by enzymatic assay. Parental consanguinity was present in 73.3% of these cases. [42]. Consanguinity is a principal factor in increasing the incidence of rare autosomal recessive diseases among the Arab population. The incidence of Tay-Sachs disease has been markedly reduced by more than 90% since the introduction of carrier screening and counseling in Jewish population in comparison to the non-Jewish who is now at 3–4-fold higher frequency [6, 7].

The HEXA mutations R504C, R504H, and R499C are confirmed as being not specific to the Jewish population but of a diverse background. The novel mutations E307A, H318D, and E162Rfs*37 may be specific to Egyptian or Arab TSD patients, hence additional studies in Arab and Middle East populations are highly recommended for the delineation of unique ethnic-related disease-causing variants. The findings of this study suggest Sanger sequencing of HEXA-exon 13, as the first screening mutational target in suspected TSD Egyptian patients. Mutation identification in a proband will be the basic measure in the prevention of TSD recurrence in Egyptian families, the reason the NGS should be considered in patients with negative Sanger sequencing results.