|Year : 2019 | Volume
| Issue : 3 | Page : 38-42
Identification of a deletion variant in exon 9 of iduronate-2-sulfatase gene in patients with type II mucopolysaccharidosis
Rizky Priambodo1, Yulia Ariani2, Damayanti Rusli Sjarif3
1 Human Genetic Research Center, Indonesian Medical Education and Research Institute, Universitas Indonesia, Jakarta, Indonesia
2 Human Genetic Research Center, Indonesian Medical Education and Research Institute; Department of Pediatric, Faculty of Medicine, Dr. Cipto Mangunkusumo National Hospital; Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
3 Human Genetic Research Center, Indonesian Medical Education and Research Institute; Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
|Date of Web Publication||14-Jan-2020|
Damayanti Rusli Sjarif
Komplek Depnaker RT.008/002, Jl. Empang Tiga Dalam No. 13, Pejaten Timur, Jakarta Selatan, 12510, Jakarta
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disorder caused by mutation of the iduronate-2-sulfatase (IDS) gene, which is located on chromosome X. The profile of IDS gene at exon 9 has not been previously studied in Indonesian patients. The objective of this study was to detect and analyze mutations in exon 9 of the IDS gene in MPS II patients from Indonesia. Subjects and Methods: DNA from 10 MPS II patients from Indonesia was analyzed along with 50 healthy individuals, both male and female, which formed the control group. DNA isolation, polymerase chain reaction amplification, electrophoresis, and sequencing were performed for the analysis. Results: The IDS gene was successfully analyzed for all samples. A DNA base deletion at position c.1549delATC was found in an MPS II patient. Conclusions: This IDS gene variant is novel. Further research would be necessary to detect other IDS gene variants, with mutations at other exons, in the Indonesian MPS II patients.
Keywords: Exon 9, iduronate sulfatase, Indonesia, mucopolysaccharidosis II
|How to cite this article:|
Priambodo R, Ariani Y, Sjarif DR. Identification of a deletion variant in exon 9 of iduronate-2-sulfatase gene in patients with type II mucopolysaccharidosis. J Nat Sc Biol Med 2019;10, Suppl S1:38-42
|How to cite this URL:|
Priambodo R, Ariani Y, Sjarif DR. Identification of a deletion variant in exon 9 of iduronate-2-sulfatase gene in patients with type II mucopolysaccharidosis. J Nat Sc Biol Med [serial online] 2019 [cited 2020 Jan 23];10, Suppl S1:38-42. Available from: http://www.jnsbm.org/text.asp?2019/10/3/38/275602
| Introduction|| |
Mucopolysaccharidosis (MPS) is a lysosomal storage disease caused by the deficiency of enzymes involved in the molecular degradation process of mucopolysaccharide, more commonly referred to as glycosaminoglycan (GAG). The deficiency of these enzymes disrupts GAG metabolism such that it accumulates in the lysosomes of various body tissues. MPS II is caused by iduronate 2-sulfatase enzyme deficiency (I2S) encoded by the IDS gene. The I2S enzyme plays a role in the metabolic process of GAG by catalyzing the hydrolysis of heparan sulfate and dermatan sulfate. The accumulation of dermatan sulfate and heparan sulfate in the lysosomes due to I2S enzyme deficiency leads to various progressive disorders, such as skeletal and cardiac abnormalities, organomegaly, hernia, hearing difficulty, and occasional neurological impairment.
The iduronate 2-sulfatase (IDS) gene is 28 kb in length and consists of 9 exons which encode 550 amino acid residues that constitute the structure of the active I2S enzyme. Previous studies have reported on various types of mutations present in the IDS gene of MPS II patients. To date, 521 types of IDS gene mutations have been reported. These include point mutations (51.8%), small deletions (18%), splicing changes (9%), large deletions (7.5%), small insertions (8.3%), complex rearrangements (2.7%), indel mutations (1.9%), and large insertions/duplications (0.8%). In Indonesian patients, several IDS gene mutations have been reported. These mutations were located in exons 4, 6, and 9, and in introns 3 and 4. Exon 4 contained 21 novel mutations, including silent or missense mutation. A 14-bp novel insertion was found in exon 6 of the IDS gene from an Indonesian patient, while one novel silent mutation in exon 9 was detected in another Indonesian patient., The discovery of novel mutations from Indonesian patients is still possible, as the mutations described are still limited. Therefore, this study was aimed to analyze the mutations in exon 9 of the IDS gene in Indonesian patients.
| Subjects and Methods|| |
DNA samples were obtained from the whole blood of 10 MPS type II patients and 50 healthy individuals (non-MPS type II patients) at Cipto Mangunkusumo Hospital, Jakarta. The blood collection process was carried out by a health-care professional, who followed the procedure based on ethical approval of the Ethical Clearance Committee. Informed consent was signed and approved by the participants whose blood samples were collected. DNA was extracted from the samples according to the salting-out method. The extracted DNA was quantified to determine its concentration and purity using a spectrophotometer (Thermo Scientific). The primers for the exon 9 region were constructed using NCBI Primer BLAST. The results of primer design are shown in [Table 1]. Polymerase chain reaction (PCR) (Applied Biosystems) was performed to amplify the exon 9 of IDS gene using the specific primers that were constructed. The following PCR procedure was followed based on the instructions provided in the PCR Master Mix kit (Bioline): initial denaturation at 95°C for 60 s; denaturation at 95°C for 15 s, annealing with gradient to optimize the amplification process (50°C, 52°C, 54°C, 56°C, 58°C, and 60°C) for 15 s; and extension at 72°C for 30 s. The process from denaturation to annealing was repeated up to 40 cycles. The process was followed by 10 min at 72°C for the postextension. The 780-bp PCR product was visualized on a 1.8% agarose gel. The PCR products were sent to 1st Base Sequencing Services in Singapore for Sanger sequencing. All the sequence data were verified with the NCBI database, and mutations were identified using BioEdit software [Carlsbad, CA].
|Table 1: Primer and conditions for polymerase chain reaction amplification of iduronate-2-sulfatase exon 9|
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| Results|| |
The concentration and purity of extracted DNA is listed in [Table 2]. The DNA concentrations ranged from 257.20 ng/μL to 2384 ng/μL and DNA purity from 1.82 to 1.89 (A260/A280 ratio). Nucleic acid purity is acceptable when the A260/A280 ratio ranges between 1.8 and 2.0. The results indicated that the DNA obtained was pure enough to be used in the amplification process.
The optimization for amplification was performed using a PCR gradient process, which included six different temperatures ranging from 50°C to 60°C. The results of the primer optimization process are shown in [Figure 1]. A single DNA band appeared under all temperature conditions. This indicated that the specific annealing temperature for exon 9 could be 60°C because the DNA bands still appeared at the maximum gradient temperature. Another interpretation is that both the primers could anneal specifically to the target region in the IDS gene. The amplification of exon 9 yielded a 780-bp product. The amplification results are shown in [Figure 2].
|Figure 1: Gel image of polymerase chain reaction optimization for exon 9 at six different temperatures. The expected band was obtained in the range between 50°C and 60°C, with no multiple bands. The annealing temperature can be selected as 60°C (M: 1 kb DNA ladder from Geneaid)|
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|Figure 2: Gel image of amplification after optimization process for each sample at exon 9. A 780-bp fragment was observed (M: 1 kb DNA ladder from Geneaid; 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; 6: P6; 7: P7; 8: P8; 9: P9; and 10: P10)|
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The sequencing results are shown as an electropherogram in [Figure 3]. The figure shown the sequence that is ready to be aligned using the BioEdit software. The alignment was performed using the IDS gene from NCBI as a template. The results of the sequence alignment are shown in [Figure 4]. A mutation at c.1549delATC at exon 9 was detected in one of the patients [Figure 4]a, while the other patients showed no mutation in exon 9 of the IDS gene. This c.1549delATC mutation has not been reported before, as listed in [Table 3]. This mutation involves the deletion of three nucleotides – adenine, thymine, and guanine. Sequence alignment [Figure 4]b revealed a change in the amino acid sequence, with the deletion of an isoleucine (frameshift mutation).
|Figure 3: Sequencing results shown as an electropherogram. Good peaks, without any background signals, were observed|
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|Figure 4: (a) don alignment result using BioEdit software showing the same aminn at c. 1549dlATC on P8. (b) Codon alignment result using BioEdit software showing the same amino acid at p. I517del on P8|
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| Discussion|| |
The novel mutation was found in exon 9 of the IDS gene in an MPS type II patient, while no mutation in exon 9 was detected in other patients. However, their IDS sequences were similar to the IDS gene sequence from the GenBank National Center for Biotechnology Information (NCBI). The other patients, who did not exhibit mutations in exon 9, would have a mutation in another exon of the IDS gene, or even in the splice site of the IDS gene. Although exon 9 has the highest number of reported mutations in the IDS gene, there is still a possibility that mutations would be found in other exons. This mutation has not been reported previously, whereas other mutations in exon 9 have been previously reported in the literature.,,, All the mutations in exon 9 of the IDS gene that were previously reported are missense mutations, which involve substitutions in the nucleotide bases that lead to amino acid changes. None of the reported mutation in exon 9 of the IDS gene was a deletion or frameshift mutation. In this study, the mutation site is at c.1549delATC, which deletes the codon ATC at p. Ile517. The codon exactly deletes one amino acid, which is an isoleucine. This novel mutation is a frameshift mutation. This kind of mutations leads to changes in the amino acid structure because of the loss of an amino acid. As expected, codon alteration affected the structure of the I2S enzyme. The function of I2S enzyme is to degrade the GAGs, such as heparan sulfate and dermatan sulfate. The deletion of amino acid affects the I2S enzyme function and prevents the degradation of GAG molecules. In recent years, there is evidence that even a single synonymous codon substitution (silent mutation) can bear a significant impact on gene expression levels, protein folding, and protein cellular function.,,, Limited evidence on the role of synonymous codon substitutions, particularly in slow translating regions of mRNA, and their impact on protein structure or function have been briefly summarized. It would be interesting to use bioinformatics approaches in this study. It might help to determine the secondary structure and its implication in the alteration of function. Further research is needed to determine the effect of the deletion on the protein structure and enzyme function. The pathogenic classification of the mutation is also required. In conclusion, a novel frameshift mutation at c.1549delATC was found in exon 9 of the IDS gene in an Indonesian MPS type II patient. Further study on the secondary structure of the mutant is warranted.
This article was presented at the 3rd International Conference and Exhibition on Indonesian Medical Education and Research Institute (ICE on IMERI 2018), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. The research was supported by Hibah PITTA 2018 from Direktorat Riset dan Pengabdian Masyarakat (DRPM) UI No. 5000/UN2.R3.1/HKP. 05.00/2018. We thank the staff at the Human Genetic Research Center, Indonesian Medical Education and Research Institute, Universitas Indonesia, for all their support and cooperation.
| Conclusion|| |
We found that the c.1549delATC mutation occurred in one Indonesian patients with MPS II, based on the result of sequencing analysis. This might open possibilities for further research on the screening for other mutation in such patients.
Financial support and sponsorship
This article was presented at The 3rd International Conference and Exhibition on Indonesian Medical Education and Research Institute (ICE on IMERI 2018), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia. This research was supported Direktorat Riset dan Pengabdian Masyarakat (DRPM) UI and Ministry of Research, Technology, and Higher Education of the Republic of Indonesia. We thank staff at the Human Genetic Research Center, Indonesian Medical Education and Research Institute, Universitas Indonesia, for all their support and cooperation.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]