AIP Conference Proceedings 2331, 050027 (2021); https://doi.org/10.1063/5.0042046 2331, 050027
© 2021 Author(s).
Variant analysis for exon 2 and 5 of iduronate 2-sulfatase gene on mucopolysaccharidosis type II patients in Indonesia
Cite as: AIP Conference Proceedings 2331, 050027 (2021); https://doi.org/10.1063/5.0042046 Published Online: 02 April 2021
A. Pustimbara, N. M. Prakoso, R. Priambodo, et al.
ARTICLES YOU MAY BE INTERESTED IN
Identification of novel mutations in exon 1 of iduronate-2-sulfatase gene from mucopolysaccharidosis type II patient in Indonesia
AIP Conference Proceedings 2331, 050026 (2021); https://doi.org/10.1063/5.0042045
Novel base alterations at intron 3 of 6-pyruvoyl-tetrahydropterin synthase gene in Indonesian population
AIP Conference Proceedings 2331, 050028 (2021); https://doi.org/10.1063/5.0042047 Variant identification of exon 11 of galactosamine (N-acetyl)-6-sulfatase (GALNS) gene in mucopolysaccharidosis type IVA patients in Indonesia
AIP Conference Proceedings 2331, 050025 (2021); https://doi.org/10.1063/5.0042042
Variant Analysis for Exon 2 and 5 of Iduronate 2-Sulfatase Gene on Mucopolysaccharidosis Type II Patients in
Indonesia
A. Pustimbara
1, 4, N.M. Prakoso
1, R. Priambodo
1, Y. Ariani
1, 2, 3, S. Arianto
1, 4, Y. Pangestika
1, 4, A. Bowolaksono
4, and D. R. Sjarif
1, 2, a)1Human Genetics Research Center, Indonesian Medical Education and Research Institute (IMERI), Universitas Indonesia, Jakarta, 10430, Indonesia
2Department of Pediatric, Universitas Indonesia, RSUPN Dr. Cipto Mangunkusumo, Jakarta, 10430, Indonesia
3Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Depok, Indonesia
4Department of Biology, Faculty of Mathematics and Natural Science, Universitas Indonesia, Depok, Indonesia
a)Corresponding author: ukk.npm.idai@gmail.com
Abstract. Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is one of lysosomal storage disorder caused by the presence of pathogenic variant in IDS gene. The variant can be found in various exon locations. This research aimed to identify the presence of disease-causing variant that may occurs at exon 2 and 5 of IDS gene on MPS II patient, especially in Indonesia. Based on the previous research that has been conducted in a number of countries, exon 2 and 5 are the exons with the most number of variations. Analysis was conducted on 7 MPS II patient of Indonesian origin and 50 normal individuals as control that consist of 25 male or 25 female individuals. Analysis was done by going through steps of DNA isolation, amplification by polymerase chain reaction (PCR), visualization by electrophoresis, and sequencing. Research result shows that IDS gene from whole samples were successfully analyzed. This study discovered an adenine base deletion c.708+72delA in intron 5 of one healthy individual. The variant is novel and classified as likely benign.
INTRODUCTION
Mucopolysaccharidosis type II or Hunter syndrome is a disorder caused by the accumulation of mucopolysacharide in organs and tissues due to deficiency of iduronate 2-sulfatase enzyme. This enzyme is responsible for hydrolyzing ester bond in heparan sulfate and dermatan sulfate, which the accumulation of these compounds can cause a serious problem to organ functions [1,2]. Mucopolysaccharidosis type II is inherited as an X-linked recessive disorder and based on National MPS Society, it is estimated that the incidence of MPS II is around 1 in 100.000-150.000 male newborns in the world and some of them are found in female [3]. A lot of previous research on MPS II have been done such as in Taiwan [2] and China [1], but there are no variant data on MPS II patients from Indonesia.
Mucopolysaccharidosis type II may occur because the presence of pathogenic variant in IDS gene. There are 9 exons in this gene and some introns flanking them. There is also an IDS2 pseudogene found 80 kb downstream with highly homologue sequences and reported to be involved in recombination that appears in MPS II patient [1,4].
Several studies found that exon 2, where this pseudogene exist besides of exon 3, is one of the most exon that has higher rate of variations [5]. Apart from the existence of this pseudogene, it was known that exon 5 is also one of the exons with high numbers of variations which affected the function of IDS gene, such as substitution or frameshift.
Analysis of mutation profile for exon 2 and 5 IDS gene in MPS IVA patients in Indonesia has not been performed before and its information is still limited. Therefore this study aims to carried out mutation analysis on 7 patients previously diagnosed with MPS II originated from Indonesia.
METHOD
Clinical Sample and DNA Extraction
The blood samples of 7 MPS II patients and normal individuals were obtained from Cipto Mangunkusumo Hospital, Jakarta. The blood collecting process was carried out by healthcare professionals following the procedures from the Ethical Clearance Committee. Informed consent was signed and approved by the patients. DNA was extracted using GeneAid DNA Mini Kit GB100 following manufacturer’s extraction procedures. The purity and concentration of the extracted DNA was measured using spectrophotometer UV-Vis and stored in -20 °C freezer.
Primer Design
Two sets of primers have been designed using NCBI PrimerBlast and their secondary structure have been analyzed using IDT OligoAnalyzer. The forward primer for exon 2 IDS gene is [5’- CCTCCATCCAACCATCTGTCC-‘3] and reverse primer [5’-CCTCTAACAAGATGTCCCGC-‘3]. Forward primer for exon 5 is [5’-CGTGAAGGGCTGATTATGTGA-‘3] and reverse primer [5’- CTGGCGATGGCAGGATGTAG-‘3].
Polymerase Chain Reaction and Electrophoresis
Reagent mixture for PCR are as follow : 5.3 ȝL of nuclease free water, 0.1 ȝL of each primer forward and reverse, 2.5 ȝL of Taq Polymerase, 2 ȝL of DNA template for each tube. The PCR cycles follows MyTaq™ Mix reaction protocol with 40 cycles and undergone following stages: 95 °C for 1 min of initial denaturation, 95 °C for 15 sec of denaturation, annealing for 15 sec at 56 °C for exon 2 and 54°C for exon 5, 72 °C for 30 sec of extension, 72 °C for 10 min of final extension. PCR amplification process was done by using thermal cycler [BioRad] and visualized by 2% of agarose electrophoresis and stained by gel red.
Sequencing and Analysis
The sequencing analysis was conducted by Sanger Sequencing. The electropherogram result was analyzed using Chromas while the sequencing files were further aligned with IDS gene using BioEdit.
RESULT AND DISCUSSION
FIGURE 1. Alignment result for exon 2 of 7 MPS II patients and 50 healthy individuals to reference IDS gene. We did not identified any variations in exon 2 in patients nor healthy individuals as seen in the figure above.
Two sets of primers have been constructed in this study for exon 2 and 5. Primers are flanking the whole exons 2 and 5 coding target and according to in silico simulation does not bind to the IDS2 pseudogene. Exon 2 known as an exon that has biggest similarity with IDS2 pseudogene. The common method to avoid pseudogene amplification are using mRNA analysis. However, those analyses could not detect variant in the introns which are needed to be sequenced to complete the mutation profile of IDS gene in MPS II patients in Indonesia. Therefore, primers are designed in position that they are presumed would not bind to the pseudogene, and thus the pseudogene would not be amplified. Based on the alignment result between the reference IDS gene and samples, the homology reaches 100% similarity, therefore it was concluded that there are no variations have been detected in exon 2 (Fig. 1). The exon 5 was also succesfully amplified, but the result shows that there are no mutation occurs in exon 5.
Since there is no mutation found at exon 2 and 5 of IDS gene in patients, it shows that there is a dissimilarity with previous research from other populations. To date, there are 11 variants that have been reported at exon 2, 10 of the variants are missense and the other is a base deletion (121_123deICTC) [6,7]. On the other hand, about 17 of missense variants that occurred at exon 5 have been reported at different populations [7]. The research conducted by Isogai et al. on 43 MPS II patients in Japan reported that about 28% of variants identified in their research are concentrated at exon 5 [8].
Exon 2 and 5 of IDS gene have become exons with the highest number of variants, especially in European populations, however the result seems to be different in the case for Southeast Asian populations. A number of countries have reported variants in IDS gene from MPS II patients, such as Thailand, Philippines, and Vietnam.
Based on the research on MPS II patients in Thailand by Keeratichamroen et al., the most variants on MPS II patients in Thailand are found at exon 9 which accounted for 27% from the total variants they reported [9].
Meanwhile, report on MPS II patients in Vietnam reveals that around 33% of variants are concentrated on exon 8 [10]. Mutation analysis of IDS gene on MPS II patients from Philippines shows that 47% of the variants are located at exon 9 [11]. It is therefore concluded that the number of variants in exon 2 and 5 in MPS II patients in Southeast Asian, including Indonesia are different from European populations.
However, we have identified a single base deletion c.708+72delA at the intron 5 in a healthy individual (Fig. 2).
According to the variant classification guidelines published by the American College of Medical Genetics and Genomics (ACMG), we classified this variant as likely benign since this variant was only found in a normal individual and this variant was predicted does not couse any alterations in gene products [12].
FIGURE 2. Alignment result for exon 5 of 7 MPS II patients and 50 heathy individuals. A single base deletion c.708+72delA has been identified in N29.
CONCLUSION
In conclusion, we did not identified any variation in exon 2 and 5 of IDS gene which would be classified as disease causing variant in MPS II patients in Indonesia. However, we report a single base deletion c.708+72delA in intron 5 of a normal individual. Further analysis is needed to be carried out to seek pathogenic variants in another exons of IDS gene in MPS II patients in Indonesia.
ACKNOWLEDGMENT
This research was funded by Direktorat Riset dan Pengabdian Masyarakat FKUI 2018. We thanked the team of Human Genetic Research Center, IMERI FK UI for all the cooperation and technical assistance.
REFERENCES
1. H. Zhang, J. Li, X. Zhang, Y. Wang, W. Qiu, J. Ye, et al., PloS ONE. 6(8), p. e22951 (2011).
2. H. Y. Lin, S. P. Lin, C. K. Chuang, D. M. Niu, M. R. Chen, F. Tsai, et al., American Journal of Medical Genetics 194A, pp. 960-964 (2004).
3. National MPS Society, (2011). [Online]. Available from: http://mpssociety.org/mps/mps-ii/. [Accessed August 8, 2018].
4. S. Bunge, C. Steglich, C. Zuther, M. Beck, C. P. Morris, E. Schwinger, et al., Human Molecular Genetics 2(11), pp.1871-1875 (1993).
5. M. L. Bondeson, H. Malmgren, N. Dahl, B. M. Carlberg, and U. Petterson, European Journal of Human Genetics 3, pp. 219-227 (1993).
6. C. H. Kim, H. Z. Hwang, S. M. Song, K. H. Paik, E. K. Kwon, K. B. Moon, et al., Human Mutation 21(4), pp.
449-450 (2003).
7. Uniprot, New York: National Center of Biotechnology Information, (2018). [Online]. Available from:
https://www.uniprot.org/uniprot/P22304. [Accessed August 2018].
8. K. Isogai, K. Sukegawa, S. Tomatsu, T. Fukao, X. Q. Song, Y. Yamada, S. Fukuda, T. Orii, and N. Kondo, Journal of Inherited Metabolic Disease 21, pp. 60-70 (1998).
9. S. Keeratichamroen, et al., Journal of Inherited Metabolic Disease 31, pp. 303-311 (2008).
10. L. T. T. Hang, et al., Annals of Translation Medicine 3, p. 53 (2015).
11. M. A. D. Chiong, D. M. Canson, M. A. R Abacan, M. M. P. Baluyot, C. P. Codero, and C. L. T Silao, Orphanet Journal of Rare Diseases 12(7), pp. 1-11 (2017).
12. S. Richards, et al., Genetics in Medicine 17, pp. 405-423 (2015).
