Novel homozygous HEXB mutation identified in a consanguineous Iranian pedigree with Sandhoff disease

Background Sandhoff disease is a rare neurodegenerative and autosomal recessive disorder, characterized by a defect in ganglioside metabolism. It is caused by mutations in the HEXB gene for the β-subunit of β-N-acetyl hexosaminidase. Results In the present study, an Iranian 14- month -old girl with an 8- month history of unsteady walking and involuntary movements is described. Biochemical testing showed defects in the normal activity of beta-hexosaminidase protein. Following sequencing of HEXB gene, a novel homozygous p.A278V mutation was identified in the patient’s DNA. Conclusions The p.A278V mutation is pathogenic because of amino acid change and changing in biochemical activity. this mutation has not been reported previously, but based on In silico analysis and structural analysis, was predicted to be disease causing.


Introduction
Sandhoff disease (SD, OMIM 268800), is an autosomal recessive neurodegenerative disorder that was initially described by Konrad Sandhoff in1968. It is occurred due to the mutation in the beta subunit of hexosaminidase [1] [2]. HEXA and HEXB are two major Hex isoenzymes. b-HEXA consisting of a/ß heterodimer subunits, whereas b-Hex B is composed of ß /ß homodimer subunits. Mutations affecting HEXB gene result in Sandhoff disease, so both HexA and HexB isoenzyme activities are decreased or absent [3]. The HEXB isoenzyme is a catalytic enzyme accountable for degrading the GM2 ganglioside [4]. HEXB gene which is about 40 kb long, contains 14 exons and located on chromosome 5q13 [5]. The prevalence of Sandhoff disease is 1 in 384000 live births and many different pathogenic variants have been reported in patients with Sandhoff disease in the Human Gene Mutation Database (HGMD at http://www.hgmd.org/). They consist of 43 missense/nonsense, 3small insertions, 19 small deletions, 6 gross deletions and 18 splicing mutation [5]. The most prevalent mutation is a 16 kb deletion at the 5' end of the gene. This deletion leads to loss of exon 1-5 and the HEXB promoter [6].
Some of them have been reported as the most common mutation in some populations. The c.171delG (p.W57CfsX6) deletion was found in accounting for 21.4% of the alleles in Spain and showed the infantile form of the disease [7]. Abnormality of HEXB results in a deficiency of hexosaminidase A and B that are responsible for lysosomal accumulation of GM2 gangliosides and linked glycolipids, 3 mostly in neurons, describing neurological symptoms in this disease [8] [9]. Classic infantile, late infantile, juvenile and adult late onset are different forms of Sandhoff disease [2]. Each category has its feature in severity and age at the onset of disease [10]. There are several signs which may include Weakness, blindness, progressive mental and motor deterioration, macrocephaly [11].The infantile form of Sandhoff disease is manifested by the early onset of signs, which usually occurs within the first 6 months of life [10]. In the classic form of Sandhoff disease, hexosaminidase activity is absent [12]. The juvenile form of Sandhoff disease indicates symptoms between the ages of 4 and 6 years [13]. Symptoms include ataxia, motor skills regression, learning disorders and some autistic features [14]. The adult form of Sandhoff disease usually is characterized by neurologic symptoms in early childhood [15]. The disorder in adult-onset patients affects, mainly the motor neuron, cerebellar and autonomic functions [16]. In current work we are going to evaluate that weather this mutation has any effect on the function of HEX B or not.

Clinical finding
The clinical feature of child at first presentation was developmental delay with a mean age at presentation of 4 months. Cognitive decline was attained after 9 months of age. After 10 months of age, there was regression in speech. The child complained of seizure, truncal hypotonia and lower limbs spasticity. Physical examination and then ultrasonography revealed no splenomegaly and hepatomegaly. Ophthalmologic examination revealed cherry red spots in both macular areas in patient. The lysosomal enzymatic activities indicated reduction of β-hexosaminidase B (HEXB) activity. A novel homozygous p.A278V mutation was identified in the patient. Brain magnetic resonance imaging (MRI) indicated signal changes over the bilateral thalami, bilateral cerebral white matter and left putamen.

Biochemical enzyme assay
The biochemical enzyme assay showed a deficiency of both hexosaminidases A(0.19nmol/ml/min) and B(0.08nmol/ml/min), compatible with a diagnosis of Sandhoff disease (Table 1).
We identified a novel c.833C>T (p.A278V) homozygous mutation in exon 7 of the HEXB gene. Both parents were heterozygous for this mutation (Fig.1).
To predict the possible effects of the new mutation on the structure and function of HEXB protein, the p.A278V mutation was analyzed using SIFT and PolyPhen (Table 2). Here, SIFT result indicated that p.A278V mutation was predicted as deleterious, with SIFT scores of -3.735. Based on the PolyPhen score, the p.A278V mutation was found as "Probably Damaging" to protein structure and function, with a score of 1.000. The structure of the region around Ala 278 was shown in Fig.2, using the PyMOL molecular graphics program (14).
We compared mutant and wild protein structures, using YASARA software, difference of 3D structure was 0.5434 Å. The interaction energy of mutant protein to GDL (-0.09 kcal/mol) ( Table 3) (Figure 4), was obviously lower than wild protein to GDL (-196.35 kcal/mol). We also compared interaction energy of wild and mutant protein to NAG, EDO (Table 3).

Discussion
Sandhoff disease is a rare metabolic disease and results from unusual accumulation of gangliosides leads to progressive deterioration of the central nervous system. Sandhoff disease individuals have missing or decreased activity of beta-hexosaminidase A and beta-hexosaminidase B [29].It can be detected by making a lysosomal enzyme assay to show activity of the hexosaminidase A and B enzymes [30].
There is genetic testing for determination of specific pathogenic mutations that is existent in the beta subunit of hexosaminidase (HEXB) to corroborate the diagnosis, on the HEXB mutations.
To date, some Sandhoff disease in Iranian population with pathogenic variants have been reported. A>G, c.550delT, c.1597C>T, c.1752delTG and the most common findings (50%) was a 16 kb deletion including the promoter and exons 1-5. This mutation was not observed in healthy controls [10].
Other studies have identified pathogenic missense mutation c.821T N A (p.Val274Glu; Homozygous) in exon 7 of the HEXB gene, lead to unusual enzyme activity [5]. infantile-onse Sandhoff disease that was the explanation for the disease [33].
Seven novel mutations on the HEXB gene in French Sandhoff patients were recognized and their pathogenicity was confirmed by in silico analysis [8]. Before that, Macarena Gomez-Lira et al Gene therapy may be a hopeful treatment modality, but is unlikely to be available for years. Given the magnitude of carrier burden in recessive diseases and the lower cost of diagnostic genetic tests compared with treating, diagnostic screening of family members may be useful.

Case Report
A 14 months old girl was referred to Endocrinology and Metabolism Research Institute (EMRI/TUMS) with psychomotor regression from 7 months age. She was born after a full-term pregnancy with head circumference of 33 cm and birth weight of 3.200 Kg from a consanguineous marriage (first cousin couple) and positive family history (the previous male child died at 18 months age with the same phenotype).

β-Hexosaminidase activity assay
The activity of β-Hexosaminidase was measured via dried blood spots (DBSs) on filter.
The blood samples from patient was collected and spotted on filter paper and placed in 3 mm diameter. After addition of elution liquid and substrate solution then incubate of the samples at 37°C , the enzyme activity quantified by comparing the amount of hydrolyzed product with a calibrator [17] [18].

Mutational Analysis of HEXB Gene
Genomic DNA of the patient was extracted from peripheral blood leukocytes according to the standard procedure using FlexiGene DNA Kit (QIAGEN). The exonic and flanking intronic sequences of the HEXB gene were amplified by PCR and examined for specificity via 1.5% agarose gel electrophoresis. The PCR products were sequenced on an ABI 3700 sequencer (Kosar Company, Tehran) and compared with the wild-type HEXB sequence (NM_000521.3).

In Silico Analysis
To predict the possible effects of the new mutation on the structure and function of HEXB protein, the sequence alteration were assessed by the in silico prediction algorithms SIFT and Polyphen-2 [19] [20].

Structural Analysis
The structures of Beta-hexosaminidase subunit beta was carried out using molecular modeling tools server [21].the sequences and 3D shapes of hex b as well as ligand were acquired from protein databank (PDB) [22].The crystallographic structure of molecules was removed for the existence of water atoms and other unnecessary additional chains using UCSF Chimera 1.8.1 [23] [24]. Protein homology modeling of the newly-identified mutated HEXB protein, p.A278V, was carried out using the online Swiss-Prot server for automated modeling [25].protein structure prediction server, RaptorX, was also used for homology based 3D structure prediction [26].
x-ray crystallography was obtained to better understand the structural effects caused by mutations in Beta-hexosaminidase subunit beta (Accession code: 1o7A) [27].protein-ligand docking were

Availability of data and materials
The activity of β-Hexosaminidase was measured via dried blood spots (DBSs) on filter.
The sequence alteration was assessed by the in silico prediction algorithms SIFT and Polyphen-2.
15 Figure 3 Multiple protein alignment highlighting the evolutionary conservation of Alanin 278 in the human beta-hexosaminidase subunit beta amino acid sequences. Figure 4 interaction of mutant protein to GDL.