Novel base alterations at intron 3 of 6-pyruvoyl ... - iKnow

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AIP Conference Proceedings 2331, 050028 (2021); https://doi.org/10.1063/5.0042047 2331, 050028

Novel base alterations at intron 3 of 6-

pyruvoyl-tetrahydropterin synthase gene in Indonesian population

Cite as: AIP Conference Proceedings 2331, 050028 (2021); https://doi.org/10.1063/5.0042047 Published Online: 02 April 2021

A. Pustimbara, D. C. Putri, N. M. Prakoso, et al.

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Novel Base Alterations at Intron 3 of 6-Pyruvoyl-

Tetrahydropterin Synthase Gene in Indonesian Population

A Pustimbara

⁴

, DC Putri

^1,4

, NM Prakoso

¹

, R Priambodo

¹

, Y Ariani

^1,2,3

, K Yuliarti

^1,2

, A Bowolaksono

⁴

, and DR 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 Sciences, Universitas Indonesia, Depok, Indonesia

a)Corresponding author: ukk.npm.idai@gmail.com

Abstract. 6-pyruvoyl-tetrahydropterin synthase (PTPS) or tetrahydrobiopterin (BH4) deficiency is the most common enzyme synthesis defect which was reported to cause of hyperphenylalaninemia. This deficiency is caused by pathogenic variant in exons and introns of 6-pyruvoyl-tetrahydropterin synthase (PTS) gene in chromosome 11q22.3-q23.3. This study is focused on the detection of DNA variants in intron 3 especially for insertion and base alteration. Methods that has been carried out in this study are DNA isolation, polymerase chain reaction (PCR), the visualization of PCR products through DNA electrophoresis, and Sanger sequencing. A total 29 variants have been characterized in this study, obtained from the DNA of one Indonesian PTPS patients and 33 healthy individuals as control. Those alterations were categorized into substitution and intronic insertion and located in the sequence of intron 3 and 4 of PTS gene. Further analyses are required to be performed to characterize the effect of identified variants to the splicing events of PTS mRNA.

INTRODUCTION

Tetrahydrobiopterin (BH4)-deficient hyperphenylalaninemia (HPA) is a metabolic disorder that is caused by 6- pyruvoyl-tetrahydropterin synthase enzyme deficiency involved in tetrahydrobiopterin biosynthesis. This deficiency is caused by pathogenic variants in 6-pyruvoyl-tetrahydropterin synthase (PTS) gene that encodes the 6-pyruvoyl- tetrahydropterin enzyme (1,2). Tetrahydrobiopterin has several important functions, such as to break down the phenylalanine amino acid which have important role as the building block of the cell (3). Hyperphenylalaninemia can cause damage in amine neurotransmitter development, brain cells, and affects the development of epithelial cells (3,4).

PTS gene is located on chromosome 11q22.3-q23.3 with around 8 kb in size and has 6 exons with introns flanking them (5,6). Variations in this gene are inherited as autosomal recessive trait and the risk for male and female to develop the disease are equal, although the incidence of the disease is varied between ethnic groups.

According to previous reports, the disease is not only triggered by the presence of pathogenic variants in exons, but also in introns. Variations in introns could trigger the production of defective gene products, especially when it occurs in the splice sites (8). Analyzing nucleotide variations in intronic splice sites is important to understand because they may potentially affect the splicing events. Moreover, understanding intronic variants may explore the construction of the splicing machinery (spliceosome) in the process of eliminating introns and joining exons to produce mature mRNA (9). However, the information regarding the variants of PTS gene is still limited, therefore this study is performed to discover intronic variations in intron 3 of PTS gene from Indonesian population which has not been analyzed before.

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METHOD

Sample Collection and DNA Extraction

Whole blood samples of 3 PTPS patients and 33 healthy individuals were obtained from Cipto Mangunkusumo Hospital, Jakarta. The subjects had their blood taken with sterile syringe by a health care professional following procedure from Ethical Clearance Committee, and the bloods were stored in a 4 ^oC refrigerator. The DNA was extracted using Genomic DNA Blood Mini Kit from Geneaid following the manufacturer’s extraction protocol. The extracted DNA is stored at -20 °C freezer.

Primer Design and Polymerase Chain Reaction

A forward primer (5`-GCTTGTATGTTGCTAACTTGTGCT-3`) and a reverse primer (5`- GAGATAACTGGTTGGGGAGGTAG-3`) were constructed, and their possible secondary structure were verified in silico. Two steps of PCR, the PCR gradient followed by verification at optimized temperature were carried out in this study. The PCR master mix for PCR gradient were consisted by 3.6 ʅL of nuclease-free water, 0.2 ʅL of 20 ȝM forward primer, 0.2 ʅL of 20 ȝM reverse primer, 5 ʅL of MyTaq HS Red Mix [Bioline], and 1 ʅL of DNA template.

PCR procedures were performed according to MyTaq™ Mix protocol sheet, which is started by initial denaturation at 95 °C for 1 minute, denaturation at 95 °C for 15 seconds, annealing at 50--60 °C for 15 seconds, extension at 72

°C for 30 seconds, and post-extension at 72 °C for 10 minutes. Processes from denaturation to extension were repeated up to 40 cycles. PCR products were visualized by electrophoresis using 2% of agarose gel. PCR amplification process was performed using a thermal cycler [BioRad], and documented with gel documentation system.

DNA Sequencing and Data Analysis

DNA sequencing process has been carried out using Sanger principles. The sequencing results were analyzed and verified by aligning sequences with BioEdit. Identified variants in intron 3 in patients and normal individuals were categorized as basd on their type of variation and annotated to which sample the variant was detected.

RESULT AND DISCUSSION Primer Design and PCR Optimization

Two sets of primers have been constructed in this study, each for forward and reverse primers to amplify exon 3 and 4 of PTS gene. PCR optimization has been done in two processes for each exon, the first process used PCR gradient which then verified at the optimum temperature. The result of PCR gradient is shown in Fig. 1a where amplification product were appeared in all wells, but the single target amplicon which 645 bp in size was only observed in 60 °C. The result was verified by performing PCR at 60 °C to ensure that the primers were specific to the targeted exons. The result had been visualized and a single target amplicon had succssfully observed and used to prepare the PCR product for sequencing, as seen in Fig. 1b.

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DNA Sequencing Analysis

TABLE 1. DNA Sequencing Analysis

No. Nucleotide Location Sample Type Novelty 1 c.186+285insTGACCACACTCTC Intron 3 1 normal

individual Insertion This study

2 c.186+285insTCACCACAGTCTC Intron 3 1 normal individual

Insertion This study

3 c.187-1 G>A Intron 3 Patient Substitution This study

4 c.187-1 G>C Intron 3 2 normal

individuals Substitution This study

5 c.187-2 A>C Intron 3 Patient Substitution This study

6 c.187-2 A>G Intron 3 2 normal

7 c.187-3 T>G Intron 3 3 normal

8 c.187-4 T>C Intron 3 3 normal

individuals

Substitution This study

9 c.187-5 C>T Intron 3 3 normal

11 c.187-9 A>T Intron 3 Patient Substitution This study

12 c.187-10 T>A Intron 3 Patient Substitution dbSNP

13 c.187-11 T>A Intron 3 3 normal

14 c.187-12T>A Intron 3 4 normal

individuals

Substitution This study

15 c.187-13 G>A Intron 3 3 normal

16 c.187-14 T>G Intron 3 1 normal

individual Substitution This study

17 c.187-14 T>C Intron 3 1 normal

individual Substitution This study 18 c.187-15 T>C Intron 3 Patient Substitution This study

individuals

Substitution This study 20 c.187-17 C>A Intron 3 Patient Substitution This study 21 c.187-18 C>A Intron 3 Patient Substitution This study

individual Substitution This study

23 c.187-19 A>C Intron 3 4 normal

24 c.187-20 C>G Intron 3 3 normal

individuals

Substitution This study 26 c.243+265insGGTAGAGGAGA Intron 4 Patient Insertion This study 27 c.243+285insCACCACACACACAT Intron 4 Patient Insertion This study 28 c.243+265insATCTATGGA Intron 4 Patient Insertion This study 29 c.243+265insTGATAGCTAT Intron 4 1 normal Insertion This study

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Among these 36 Indonesian individuals which were participated in this research, three of them were previously diagnosed for PTPS disease and 33 were healthy unrelated person. Our sequncing result as can be observed in Fig.

2a contains noises which qualitatively disturbs the main reads. However, the main reads can still be distinguished from th noises and can still be interpreted. Based on the alignment result, a total of 29 variants in intron 3 and 4 have been successfully identified in a PTPS patient and normal individuals. In general, the identified variants in this study can be categorized as substitution and insertion. The location of the detected variants are variable from the closest to exon-intron junction (± 3) and deep intronic variants.

Intronic substitutions identified in our PTPS patient (See Table 1) are located on 20 bases length from the coding region, where the splicing events might occurred. Splice acceptors and donors are usually marked with AG and GT consensus splice sequences (10). A PTPS patient in this study experiences two splice site variants, c.187-1G>A and c.187-2A>C located precisely in the acceptor site, therefore they have potential to possess another alternative splice site. Alternative splicing site sometimes occurred as a phenomenon in eukaryotes, but this will have consequence on protein reading in exon (9). Based on Human Splicing Finder Genetics online tool, another intron alternative PTS gene with the best consensus value among others is in c.187-12 (Table 1). The exact position has a variation also, so another alternative splice site is needed for exon 4. Novel variations are also found in some normal samples, but with different bases alteration.

Gross insertions are also found at c.243+265 and c.243+285. About 11 bases are added at c.243+265 and 14 bases at c.243+285. These gross insertions are potentially affected in the next introns arrangement. Another four variations also appeared at the same sites, in normal samples with different bases arrangement and different length.

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(b)

FIGURE 1. PCR visualization for exon 3 and 4. (a) PCR gradient result shows that segments of DNA had been successfully amplified at 50--60 °C, with the tickes band appeared in 60 °C with the target size about 645 bp. (b) PCR product visualization for exon 3 and 4 at 60 °C shows single amplification product (M: 1 kb DNA ladder, 1: P1, 2: N1, 3: N2, 4: N3, 5: N4, 6: N5, 7:

N6, 8: N7).

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FIGURE 2. (a) An example of deoxyribonucleic acid (DNA) sequencing result from PTPS patient samples. (b) Alignment for total 34 samples of intron 3. Insertion appeared in 3 samples; the second row of sequence indicates PTPS patient.

CONCLUSION

This study has found 29 variants of intron 3 and 4 in the PTS gene. Some of the variations are occurred at the splicing site, but further analyses are needed to be conducted to ensure the effect of the identified variants on the splicing events of PTS mRNA.

ACKNOWLEDGMENT

This research was funded by Direktorat Riset dan Pengabdian Masyarakat FKUI 2017. We thanked the team of IMERI Human Genetic Research Cluster for all the cooperation and technical assistance.

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