Identification of a novel HEXB Mutation in an Iranian Family with suspected patient to GM2‐gangliosidoses

Abstract Sandhoff disease is one of the GM2‐gangliosidoses which is caused by a mutation in the HEXB preventing the breakdown of GM2‐ganglioside. We report a novel HEXB variant in a family with a history of a dead girl with Sandhoff disease which was not found in controls.


| INTRODUCTION
The GM2-gangliosidoses are autosomal recessive metabolic diseases caused by a reduced ability to metabolize the GM2ganglioside and result in ganglioside accumulation especially in the central nervous system. 1,2 The disorders show a spectrum of clinical severity. Generally, the earlier the age of onset of clinical symptoms is associated with the more severe form of the disease. 3 The GM2-gangliosidoses are caused by mutations in the genes encoding the subunit α (HEXA gene) or subunit β (HEXB gene) of heterodimeric b-hexosaminidase A (HEX A), or its cofactor (the GM2 activator protein). 4,5 The lysosomal hydrolase, beta-hexosaminidase, has two isoenzymes: hexosaminidase A and hexosaminidase B. Hexosaminidase A is composed of α and ß subunits, whereas hexosaminidase B is only composed of ß subunits. 6,7 Sandhoff disease 8 (OMIMNo. 268800) caused by mutations of HEXB gene that contains 14 exons and spans about 40 kb of DNA on chromosome 5q13 9 in which there is a deficiency of both lysosomal hydrolase β-hexosaminidase A and β-hexosaminidase B. This deficiency causes accumulation of GM2-ganglioside especially in the neuronal cells. 4,10 In regard to the age at the onset of symptoms, three types of Sandhoff disease are distinguishable: infantile (classical Sandhoff disease), juvenile, and adult groups of patients. 2,11,12 Sandhoff disease has several symptoms which may include motor weakness, hyperacusis, blindness, mental deterioration, cherry-red spot in the macula, seizures, and optic atrophy. 13 Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder manifesting the same ophthalmic symptoms found in Sandhoff disease, such as optic atrophy and a cherry-red spot in the macula. 14,15 It is caused by mutations within the HEXA gene on chromosome 15q 23-24. 1 The aim of this study was to investigate and identify the plausible disease-causing mutation in an Iranian family with the dead suspected infant having GM2-gangliosidoses.

| Subjects
The family of this study had a history of an affected girl based on the clinical documents and family history was strongly suspected of Sandhoff or Tay-Sachs disease. The parents were healthy and first cousin. They had 3 children: two normal boys and only a girl who was born via normal delivery without any pre/postnatal insults with good Apgar score and normal birth weight and head circumference.
She was completely normal until the age of 9 months (based on her parents' claims), but thereafter they noticed she has a problem as she lost her motor skills such as turning over, sitting, and crawling. Later, she developed exaggerated startle reactions to loud noises and experienced seizures. At the age of 2 years, observing cherry-red spots by an eye specialist in her confirms the diagnosis of GM2-gangliosidoses. Despite taking different antiepileptic drugs, her seizure never gets controlled completely. After several times of hospitalization due to severe infections and fever, she died at the age of 3 years due to respiratory problems.
In this case-control study, we had two groups including parents of a case with suspected GM2-gangliosidoses and 100

Exon
Primer sequence (5′→3′) Amplicon size (bp) controls from population-matched Iranian subjects who had no disease in their family. After informed consent has been obtained from all participants, blood samples were collected. The experiments were approved by the Ethics Committee of Shahrekord University.

| DNA genotyping
Genomic DNA from peripheral blood of the parent and 100 control samples was extracted using the Diatome kit (Isogen Laboratory Russia) according to the vendor's recommended protocol. For all exons of the HEXA and HEXB gene, oligonucleotide primers were designed using primer3.0 software (Tables 1 and 2); the accuracy and efficiency of the designed primers were verified with the BLAST server (Basic Local Alignment Search Tool) (https://blast.ncbi.nlm. nih.gov/Blast.cgi). Genotyping of all the exons was carried out using polymerase chain reaction (PCR) and direct sequencing methods. The reactions were performed in 25 µL volume containing 2 µL genomic DNA, 2.5 µL (10×) PCR buffer, 0.75 µL MgCL 2 (50 mmol/L), 0.5 µL of four dNTPs (40 mmol/L), 0.5 µL of each primers (10 µmol/L) (Tables 1 and 2), and 0.25 µL Taq DNA polymerase (5 U/µL) (KBC, IRAN). Amplification was achieved by incubation in a BIOER DNA Thermal Cycler (TC-XP-G) for an initial denaturation of 5 minutes at 95°C, succeeded by 35 cycles of denaturation at 95°C for 40 seconds, annealing at 56°C for 40 seconds, and extension at 72°C for 30 seconds followed by final extension of 10 minutes at 72°C. Before sequencing analysis, PCR amplicons were assayed for size and purity by separation on 1% agarose gel, stained by DNA green viewer (Afratoos, Iran).

| RESULTS
The patient's family pedigree is shown in Figure 1. The control group included an equal number of men and women. First of all, since the symptoms exhibited by the dead girl were more likely to be signs of sandhoff disease, therefore, we started from HEXB gene which has 14 exons. Second, it was PolyPhen 2.0 Sequence and structure-based method that predicts the influence of amino acid substitution on the structure and function of proteins. The output levels of probably damaging and possibly damaging were classified as deleterious (≤0.5) and the benign level being classified as tolerated (≥0.5).
Adzhubei et al ( 21 ) better to sequence firstly the exons which were more likely affected by mutation (s) and impact protein function that in turn lead to a severe form of the disease. Third, by considering the HEXB gene mutations in the GM2-gangliosidoses database (http://data.mch.mcgill.ca/gm2-gangl iosid oses), we found that among the 14 exons of the gene, 4 exons including 7, 11, 13, and 14 have more mutations to take into account for further analysis. Fourth, the literature review has also revealed that among these 4 exons, mutations in exon 7 is more related to the acute form of GM2 gangliosidosis which is a devastating disease. 5 Fifth, investigation in the three-dimensional structure of beta-hexosaminidase β-subunit (HEXB) showed that exon 7 has less distance with the enzyme catalytic site and actually it is located in the vicinity of the enzyme active site. 25,26 Considering all the above reasons (first to fifth), we decided to begin our analysis from exon 7. PCR was performed for exon 7 of HEXB gene for all the samples including the parent's of the dead girl and the controls ( Figure 2). Sequence analysis of exon 7 of HEXB revealed heterozygote C → T substitution in both parent of the dead suspected girl having GM2-gangliosidoses. Sequences of the parent (Figure 3A, B) and one of the controls have been shown in Figure 3C.
After mutation was found in exon 7 HEXB, other exons of HEXB and HEXA were also sequenced, but no mutation was detected in any of the other HEXB exons and HEXA exons.
C → T substitution resulted in amino acid change at 278 position leading to Ala to Val substitution. This alteration was detected to be deleterious and damaging with in silico tools (SIFT, PROVEAN, Polyphen-2, I-Mutant3.0, PHD-SNP) ) Table 4). Project Hope (http://www.cmbi.ru.nl/hope/) revealed that the wild-type and mutant amino acids differ in their size. The wild-type residue was buried in the core of the protein. The mutant residue is bigger and probably will not fit ( Figure 4).
Inspecting the position of the mutation in the NCBI database (https://www.ncbi.nlm.nih.gov/) revealed that c.833 C>T was not a reported single nucleotide polymorphism. The results of this survey are shown in ( Figure 5). Furthermore, the position of c.833 C>T mutation corresponded to the nucleotide number 74713567 and its flanking SNPs is shown in Figure 6.

| DISCUSSION
In this study, we reported a novel variant in an Iranian family with a history of a dead girl with Sandhoff disease which was not found in 100 controls. The c.833C>T is a novel mutation in exon 7 of HEXB that causes a substitution of Alanine 278 with Valine. Both parents were heterozygous (CT) for this mutation, and all of the control samples were homozygous (CC) in this position.
SIFT, PROVEAN, PolyPhen, and PhD-SNP predicted that this mutation is causing disease and classified this mutation in a dangerous group of mutations. The I-mutant analysis showed that this mutation is reducing the stability of the protein. We used 5 in silico servers because the accuracy of the in silico techniques for determining the effect (s) of mutation can be increased by combining different computational methods. 27 HOPE simulates the structural variation between mutant and wild-type residues. The wild-type residue is located in a region annotated in UniProt to form an α-helix. The mutation converts the wild-type residue to a residue that does not prefer α-helixes as a secondary structure (Figure 4).
Although Sandhoff  Argentina. Studies have revealed that mutations occur all along this gene. 5 Very few studies have been done in Iran 28 on this group of diseases, and common mutations of the disease in the Iranian population are yet to be determined. Identification of common mutations in Iranian families appears essential especially for families who have a history of the disease, for genetic counseling, prenatal diagnosis, and prevention of neonatal with the disease.
HEXB gene is located on 5q13 and includes 14 exons. 1 Up to now, for HEXB gene 88 mutations in the human mutation database have been registered. 29 Several mutations have been detected in HEXB so far which causes an infantile form of the disorder; the most common mutation among them is a 16 kb deletion at the 5' end of the gene that no mRNA or β-protein produce. 4 Some mutations have been found in the exon 7 of HEXB including a nonsense mutation, c.850C˃T, which generated a stop codon at codon 284 30 in which all of the cell lines originated from the Kennedy Institute, Baltimore, MD; a 4-bp deletion in exon 7 (delCTTT) causes early stop codon at 273 31 in Argentinean patients with Sandhoff disease and a single base-pair deletion in exon 7 that change the codon for Gly-258, GGA, and to GA 28 in the patient with Caucasian/ Indian ancestry. Finally, A missense mutation, c.821T>A, which causes p.Val274Glu 33 has been reported in an Iranian child with juvenile Sandhoff disease.

| CONCLUSION
We concluded that c.833 C>T mutation carries out almost all criteria for pathogenic mutation such as changing amino acid at the conserved location and not found in nonpatient controls. Functional studies need to confirm the results.

ACKNOWLEDGMENTS
Not applicable.

CONFLICT OF INTEREST
The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS
FMM: participated in the design and coordination of the research study, performed experiments, and analyzed the data; FA: participated in the design and coordination of the research study, performed experiments, analyzed the data, and wrote the initial manuscript; PN: participated in the design and coordination of the research study and revised the manuscript for submission; MG: participated in the design and coordination of the research study, provided some samples, assisted in the clinical analysis, performed some experiments, and analyzed the related data; MEB: participated in the design and coordination of the research study, supervised the study, wrote the initial manuscript, and revised the manuscript for submission; all authors have seen and confirmed the final format of the manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
The experiments were approved by the Ethics Committee of Shahrekord University.

CONSENT FOR PUBLICATION
Written informed consent was obtained from the patient parents' for publication of their individual details and accompanying images in this manuscript. The consent form is held by the authors.