in Indonesia

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AIP Conference Proceedings 2331, 050026 (2021); https://doi.org/10.1063/5.0042045 2331, 050026

Identification of novel mutations in exon 1 of iduronate-2-sulfatase gene from

mucopolysaccharidosis type II patient in Indonesia

Cite as: AIP Conference Proceedings 2331, 050026 (2021); https://doi.org/10.1063/5.0042045 Published Online: 02 April 2021

A. R. Widyaningrum, N. M. Prakoso, R. Priambodo, et al.

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Identification of Novel Mutations in Exon 1 of Iduronate-2- Sulfatase Gene from Mucopolysaccharidosis Type II Patient

in Indonesia

A. R. Widyaningrum

¹

, N. M. Prakoso

²

, R. Priambodo

³

, Y. A. Aswin

^{3, 4}

, C. N.

Hafifah

^{2, 3}

, and D. R. Sjarif

^{2, 3, a)}

1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, Indonesia

2Human Genetics Research Center, Indonesian Medical Education and Research Institute (IMERI), Universitas Indonesia, Jakarta, 10430, Indonesia

3Department of Pediatric, Universitas Indonesia, RSUPN Dr. Cipto Mangunkusumo, Jakarta, 10430, Indonesia

4Department of Medical Biology, Faculty of Medicine, Universitas Indonesia, Depok, Indonesia

a)Corresponding author: [email protected]

Abstract. Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked recessive lysosomal storage disorder caused by the accumulation of heparan sulfate and dermatan sulfate due to iduronate-2-sulfatase (IDS) enzyme deficiency. To detect IDS gene mutation, DNA samples are obtained from 10 MPS II patients and 50 normal individuals, then the exon 1 of IDS gene was analyzed with Sanger sequencing. Two novel mutations are found from one male patient at the site of c.22C>A (p.Arg8=) and c.54C>A (p.Ser18Arg). Both mutations are not located in the bases which are responsible as the signal peptide cleavage site. Amino acid substitution c.54C>A (p.Ser18Arg) does not change the hydrophobic characteristic as both amino acids are hydrophobic. Therefore, those mutations do not change IDS enzyme structure nor alter the signaling pathway of IDS mRNA-ribosome complex to the endoplasmic reticulum. This study of exon 1 is the first to be performed in Indonesia. The novel mutations found in this study can contribute to a single nucleotide polymorphism (SNP) database of MPS II patients from all over the world, thus it leads to a deeper understanding of this rare disease at the molecular level. Therefore, a genotype study is needed to get a full profile of MPS II patients in Indonesia.

INTRODUCTION

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked recessive lysosomal storage disorder that affects the accumulation of dermatan sulfate and heparan sulfate that is caused by iduronate-2-sulfatase (IDS) enzyme deficiency [1]. Dermatan sulfate and heparan sulfate are two types of glycosaminoglycans (GAGs) which is bind to core protein via serine residue and formed proteoglycans. Glycosaminoglycans itself can be found in the extracellular matrix [2]. Specifically, heparan sulfate is found in basement membranes [3], while dermatan sulfate is mostly found in the skin [4]. Heparan sulfate proteoglycan (HSPG) or known as a heparan sulfate that is bind to core protein, is well studied for its role ranging from cellular development to defensive mechanism against microbial invasion, meanwhile, dermatan sulfate proteoglycan (DSPG) is well-known for its role in wound repair, for example, skin wound [5].

Specifically, both heparan sulfate and dermatan sulfate are degraded by iduronate-2-sulfatase (IDS) which results in the removal of the sulfate group from heparan sulfate and dermatan sulfate [1]. Failure to do so will result in the occurrence of mucopolysaccharidosis type II (MPS II) which is characterized by the accumulation of heparan sulfate and dermatan sulfate in the lysosome that leads to progressive tissue and organ damage. The alteration in the iduronate-2-sulfatase (IDS) gene can affect the synthesis of IDS enzyme which leads to IDS enzyme deficiency, thus causes the accumulation of dermatan sulfate and heparan sulfate [6].

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Mucopolysaccharidosis type II (MPS II) is characterized by certain signs and symptoms which consist of coarse facial features, stiff joints, skeletal abnormalities, hepatosplenomegaly, cardiovascular and respiratory disorders, developmental delay, and deteriorating intellectual function [1]. Mucopolysaccharidosis type II (MPS II) is classified into severe, intermediate, and mild phenotype which depends on the degree of mental retardation. The severe phenotype shows the presence of mental retardation and has life expectancy for the first to the second decade of life which the death is mostly caused by cardiopulmonary complication, meanwhile, the milder phenotypes show preserved intelligence and survival into adulthood [7].

The incidence of mucopolysaccharidosis type II (MPS II) is estimated at one in 100.000 male live births [1].

However, a special case of MPS II female patients also occurs which is caused by the inactivation of the normal X chromosome [8]. The treatment for MPS II is the enzyme replacement treatment (ERT) by giving the human recombinant IDS [9]. Although the presence of ERT reduces the burden of IDS deficiency, there is an urgent need to further understand the disease in order to provide early diagnosis properly, so the treatment can be done before the disease progress further. To understand further the MPS II, the study which correlates the genotype and phenotype of the disease should be performed.

Iduronate-2-sulfatase (IDS) gene is responsible for the synthesis of IDS enzyme. The size of IDS gene is 28 kb [10]. The IDS gene is located on the Xq28 and expresses 550 amino acids to synthesize IDS enzyme [6].

In this study, the exon 1 of IDS gene is observed because of the role of exon 1 in IDS mRNA signaling to the endoplasmic reticulum. The sequence of exon 1 is translated into 33 residues which are consisted of signal peptide and propeptide [1]. The first 25 residues are formed to be a signal peptide [11]. This signal peptide will be identified by a protein-RNA complex called signal recognition particle (SRP) in the cytosol. The SRP will guide the IDS mRNA-ribosome nascent complex which the SRP itself will be recognized by SRP receptor on the membrane of endoplasmic reticulum [12]. As part of the multiprotein translocation complex, the SRP receptor is located near the sec61, a translocon, or a translocation channel. After the SRP is recognized by SRP receptor, the signal peptide then binds to sec61. As the signal peptide binds to sec61, the translocation of IDS mRNA, which is halted momentarily as the SRP binds to signal peptide, will continue and it is followed by SRP leaving the SRP receptor and going back into the cytosol [13]. The signal peptide and propeptide will be cleaved by the signal peptide cleavage peptidase, thus does not follow into the endoplasmic reticulum lumen [11], while the rest of the translocation process is continued which the growing peptides go into the endoplasmic reticulum lumen. Therefore, it is important to ensure the sequence of exon 1 does not have any alteration which will affect the function of a signal peptide for translocation of mRNA IDS-ribosome complex to the endoplasmic reticulum.

Based on the Human Gene Mutation Database, about 60% of mutations found in IDS gene are point mutation which is comprised of 43% missense, 10% nonsense, and 9% splice site alterations. Other mutations, such as small insertion/deletions is accounted for 30% of total mutations in IDS gene. The rest of the mutations are gross insertion/deletions caused by a small chromosomal structural aberration [14]. In exon 1 itself, there are only six types of mutations registered in the public Human Gene Mutation Database (HGMD). These mutations are c.1A>T, c.22C>T, c.35G>A, c.36G>A, c.79G>T, and c.95C>A (15). The c.1A>T mutation found in attenuated MPS II patient changes ATG (methionine) into TTG (leucine) which affects the low rate of mRNA IDS translation [16].

The c.22C>T mutation found in attenuated MPS II patient changes CGA (cytosine) into TGA (stop codon) [17]. The c.35G>A mutation found in intermediate MPS II patient changes TGG (tryptophan) into TGA (stop codon) [18].

The c.36G>A mutation found in attenuated MPS II patient changes TGG (tryptophan) into TGA (stop codon) which causes premature termination (N-terminus) on IDS enzyme [19]. The c.79G>T mutation also causes premature termination as the mutation changes the GAA (glutamic acid) into TAA (stop codon) [20]. The c.95C>A mutation found in severe MPS II patient causes 31 amino acid truncated as the mutation changes the TCG (serine) into TAG (stop codon) [16].

The molecular study of MPS II in the Southeast Asian region is relatively less compared to other Asian countries such as Japan, China, Taiwan, and South Korea. In the Southeast Asian region, the mutation analysis of IDS gene which is responsible for MPS II has been conducted in Thailand [14,21], Vietnam [22], and Phillippines [7]. In Indonesia, the molecular study of MPS II has been started with the first case report from Ariani et al., which reports a novel mutation in exon 9 [23]. As part of MPS II molecular studies in Indonesia, this study aims to identify other possible mutations in exon 1 of IDS gene from MPS II Indonesian patients since there is no previous molecular data or study on exon 1 of IDS gene in Indonesia. Along with other MPS II molecular studies, this study will be used to construct the genetic profile of MPS II patients in Indonesia and to contribute further to the genetic database of MPS II patients all over the world in order to give a deep understanding of the disease and its treatment.

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METHOD

DNA Samples & Isolation

DNA samples were obtained from 10 Mucopolysaccharidosis type II (MPS II) patients and 50 normal individuals from Cipto Mangunkusumo Hospital, Jakarta. All MPS II patients in this study are male. The age of MPS II patients in this study ranges from 1-13 years old. The subjects had their blood taken with a sterile syringe by healthcare professional following procedures from Ethical Clearance Committee and then the blood was stored in a 4 ^oC refrigerator. DNA isolation was performed using the Genomic DNA Mini Kit for Blood/Cultured Cell GB004 (Geneaid). The concentration and purity of DNA samples were measured with spectrophotometer UV-Vis.

IDS Exon 1 Genetic Analysis

The exon 1 of IDS gene was amplified using forward primer (5’-GAGGGACGCAGGGAAGAG-3’) and reverse primer (5’-AAGGGACGGTAGGAAGGAGTGA-3’). These primers are used to amplify 102 bases of exon 1 sequence which also includes the 217 bases of initiator region and 120 bases of intron 1. The PCR product size based on the designed primer above is 483 bp. The following procedures of PCR (BioRad T100™ Thermal Cycler) were performed: initial denaturation at 95 °C for 60 s, 40 cycles of denaturation at 95 °C for 15 s, annealing at 56 °C for 15 s, extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 minutes. About 10 μl of PCR reaction products were then visualized by agarose gel electrophoresis and stained with GelRed. The rest of the PCR samples (about 40 μl) were sent to 1st Base Sequencing in Singapore. The sequencing result was analyzed using the BioEdit and Chromas.

RESULT AND DISCUSSION Result

DNA Isolation

The concentration of isolated DNA ranges from 175 ng/ȝl to 2.300 ng/ȝl. The DNA purity was measured with spectrophotometer UV-Vis using the 260/280 ratio. The purity of isolated DNA samples is within the range of 1,8-- 2,0 in which 1,8 shows the isolated DNA is pure. The average DNA purity of this study is 1,82. Thus, the isolated DNA is pure enough to be used as a template for polymerase chain reaction (PCR) amplification [24].

Molecular Analysis

The PCR amplification result was visualized with an agarose gel electrophoresis method to examine the size of the PCR product. Figure 1 shows the electrophoresis result of 9 MPS II samples (P1-P9) are verified in the size of 483 bp. Only one MPS II sample (P10) shows no band in the electrophoresis result.

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FIGURE 1. Visualization of PCR products of exon 1 at 56 ͼC where 483 bp bands were only observed in P1-P9 samples (1: P1, 2: P2, 3: P3, 4: P4, 5: P5, 6: P6, 7: P7, 8: P8, 9: P9, 10: P10, and 11: 1 kb DNA ladder).

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FIGURE 2. Examples of sequencing results with clear and high-quality peaks, as can be observed in the electropherogram of (a) P1 and (b) N40.

The sequencing was performed on samples which shown the electrophoresis band. The sequencing result was visualized with the electropherogram as shown in Figure 2, viewed with Chromas, and then analyzed through sequence alignment with BioEdit. Figure 3 shows the sequence alignment for exon 1 of IDS gene done for MPS II patients and normal individuals. Based on the alignment result, the mutation in exon 1 is only found in MPS II patient sample no. 9 (P9). Both mutations change the cytosine (C) to adenine (A). Based on the sequence alignment result, there is no mutation in exon 1 of IDS gene found in other MPS II patients and no variation in exon 1 of IDS gene found in normal individuals in this study.

To locate the position of the mutation in the coding sequence and to determine the nomenclature of mutation itself, the sequence alignment was performed by aligning the consensus coding sequence (CCDS) of IDS gene and the exon 1 sequence from samples. Figure 4 shows the sequence alignment of the consensus coding sequence of IDS gene and the exon 1 sequence from samples. Based on the alignment, two mutations found in P9 are in c.22 and c.54. The first mutation, c.22C>A, is a silent mutation that changes the codon CGA into AGA and both of them are translated as arginine. The second mutation, c.54C>A, is a missense which changes the codon AGC (serine) into AGA (arginine).

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(a)

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FIGURE 3. (a) Alignment result of exon 1 with reference CCDS where two novel substitutions c.22C>A (red rectangle) and c.54C>A (yellow rectangle) have been detected in P9. (b) The alignment of the protein sequence of all subjects predicts c.22C>A

as a silent mutation (p.Arg8=) and c.54C>A as a missense mutation that alters the 18th amino acid from serine to arginine (p.Ser18Arg) in P9.

Discussion

In this study, two alterations in exon 1 are only found from one patient whose severe form of MPS II. The first alteration is a silent mutation, c.22C>A (p.Arg8=), which changes the codon 8^th from CGA into AGA, and both of them are translated as arginine. Based on the American Medical Genetics and Genomics (ACMG), a mutation that does not affect the gene product is classified as a benign mutation [25]. A silent mutation shown in this study by c.22C>A does not result in any structural and functional change for IDS enzyme.

The second alteration is a missense, c.54C>A (p.Ser18Arg), which changes the codon 18^th from AGC (serine) into AGA (arginine). Amino acid substitution caused by the mutation should be observed on how this amino acid alteration affects the structural and functional aspects of the protein itself. Serine is a small-sized amino acid compared to arginine. Moreover, serine is polar but neutral compared to arginine which is a positively-charged amino acid, thus makes arginine can interact with negatively-charged non-protein atoms. However, those characteristic differences do not potentially harmful for the role of exon 1 sequence as a signal peptide. Both amino acids, serine and arginine, are hydrophilic amino acids. Serine itself can be substituted with other polar amino acids or small amino acids [26]. Moreover, an amino acid substitution which does not change hydrophobic amino acid into hydrophilic amino acid and vice versa is not considered to be potentially disastrous because of the characteristic difference between hydrophobic and hydrophilic (polar) amino acid which polar amino acids tend to be on the surface of protein structure in order to bind with water or other polar amino acids [27]. In this case, the amino acid substitution that is caused by this missense does not disrupt the stability of IDS signal peptide. Therefore, the mutation is not considered to disturb the translocation of IDS mRNA-ribosome complex into the endoplasmic reticulum. Based on the American Medical Genetics and Genomics (ACMG), this mutation is classified as benign since the mutation does not cause potential harm to the structure of gene product, in this case, is the translated IDS signal peptide [25].

Both mutations, c.22C>A (p.Arg8=) and c.54C>A (p.Ser18Arg), are not located in the peptidase cleavage site where the signal peptide will be cleaved by the signal peptide peptidase on the endoplasmic reticulum membrane.

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The mutation is considered to disturb the cleavage process of signal peptide when the mutation causes the signal recognition particle (SRP) unable to identify the amino acid in the cleavage site due to amino acid substitution.

There are two putative peptidase cleavage sites in IDS signal peptide. The first putative peptidase cleavage site is located between codon 23 and codon 24, the second one is located in codon 25 and codon 26 [11]. In this study, the first mutation, c.22C>A, is located in codon 8, and the second mutation, c.54C>A, is located in codon 18. Based on these results, these mutations do not cause any potential harm to the cleavage process.

This molecular characterization of exon 1 of IDS gene in Mucopolysaccharidosis type II (MPS II) patients will be a useful contribution to build a genetic database of MPS II patients that have not been established before in Indonesia. Along with other IDS gene exon studies, the result found in exon 1 will be useful in the attempt to complete the genetic profile of MPS II patients in Indonesia. Therefore, it will provide more understanding of the molecular etiology, clinical, and biochemical nature of MPS II itself.

CONCLUSION

This study has found two novel mutations from one male MPS II patient. This finding provides data for the genetic database of MPS II patients in Indonesia which has not been recorded previously. To conclude, both mutations, c.22C>A (p.Arg8=) and c.54C>A (p.Ser18Arg), which are found in exon 1 do not cause any structural change to the IDS enzyme since the signal peptide itself will be cleaved by signal peptide peptidase in translocon on the endoplasmic reticulum membrane after the remaining sequences (exon 2--9) are continued to be translated and going into the lumen of the endoplasmic reticulum. Future experiments can be a focus on finding the variation in exon 1 from normal individuals to further enrich the molecular database of IDS gene of the Indonesian population.

ACKNOWLEDGMENT

This research is funded by PITTA grant from Direktorat Riset dan Pengembangan Masyarakat (DRPM), Universitas Indonesia. We thanked The Human Genetic Research Center IMERI FK UI, The Department of Pediatric, and The Department of Medical Biology, Universitas Indonesia for providing support and their cooperation during the research. We also thanked Dr. Anom Bowolaksono as an academic counselor from the Department of Biology, Faculty of Mathematics and Natural Science to be involved in this research.

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