PROPOSAL INSENTIF PUBLIKASI JURNAL NASIONAL TERAKREDITASI DAN JURNAL INTERNASIONAL
Allele Frequency Distributions of the Drug Metabolizer Genes CYP2C9*2, CYP2C9*3, and CYP2C19*17 in the Buginese Population of Indonesia Tim Penulis Prof. Dr. Zullies Ikawati, Apt. Theresia Desy Askitosari, M.Biotech Prof. Dr. Lukman Hakim, MSc., Apt. Dr. Joseph Tucci Dr. John Mitchell
FAKULTAS FARMASI UNIVERSITAS GADJAH MADA 2015 i
DAFTAR ISI
Hal HALAMAN JUDUL
i
LEMBAR PENGESAHAN
ii
DAFTAR ISI
iii
A. LATAR BELAKANG
1
B. TUJUAN PENULISAN
1
C. KONTRIBUSI PENELITIAN
1
D. MANFAAT YANG DIHARAPKAN
2
E. TARGET PEMBACA DAN PENERBIT
2
LAMPIRAN
3
LAMPIRAN 1. BIODATA PENELITI LAMPIRAN 2. SURAT PERNYATAAN
0
PROPOSAL
A. LATAR BELAKANG
Untuk mendukung visi Universitas Gadjah Mada menjadi universitas riset bertaraf internasional, perlu pengembangan budaya publikasi jurnal khususnya jurnal internasional bereputasi (terindeks Scopus). Disamping itu, publikasi jurnal merupakan salah
satu
diseminasi
hasil
penelitian
yang
dapat
dijadikan
indikator
dari
kualitas/keunggulan penelitian. Suatu penelitian perlu didiseminasikan hasilnya untuk dapat menjadi informasi bagi saintis lain yang menggeluti bidang yang sama, untuk menjadi sumber referensi. Pemetaan genetik masyarakat Indonesia khususnya yang berfokus pada ezim pemetabolisme obat masih terbilang masih jarang. Padahal, pengetahuan mengenai pemetaan genetik ini sangat penting mengingat peran pentingnya terhadap efektivitas dan toksisitas obat yang dikonsumsi masyarakat Indonesia. Karena itulah, sangat penting hasil penelitian ini untuk dipublikasikan dalam jurnal. Jurnal dipilih karena sesuai dengan bidang kajian penelitian dan memiliki reputasi yang baik.
B. TUJUAN PENULISAN
Tujuan penulisan adalah agar dapat menjadi sumber literatur primer bagi peneliti lain yang akan meneliti pada bidang yang sama.
C. KONTRIBUSI PENELITIAN
Penelitian yang menjadi sumber penulisan artikel adalah Proyek Hibah Kompetitif Penelitian Kerjasama Internasional dalam rangka Publikasi Internasional, tahun 2010. Selain itu penulisan artikel ini juga didukung oleh penelitian-penelitian yang tercantum dalam pustaka.
D. MANFAAT YANG DIHARAPKAN
Diharapkan hasil penelitian ini dapat menjadi tambahan referensi mengenai pemetaan genetik masyarakat Indonesia khususnya yang berfokus pada ezim pemetabolisme obat. 1
E. TARGET PEMBACA DAN PENERBIT
Target pembaca adalah peneliti, akademisi, mahasiswa di bidang ilmu farmasi dan biomedis. Jurnal dipilih karena sesuai dengan kajian penelitian dan memiliki sebaran yang luas serta bereputasi baik.
2
LAMPIRAN 1. BIODATA PENGUSUL INSENTIF PUBLIKASI JURNAL NASIONAL TERAKREDITASI DAN JURNAL INTERNASIONAL
I. IDENTITAS DIRI 1.1. Nama Lengkap (dengan gelar) 1.2. Jabatan Fungsional/golongan 1.3. NIP 1.4. Tempat dan Tanggal Lahir 1.5. Alamat Rumah 1.6. 1.7. 1.8. 1.9. 1.10.
Nomor Telepon/Fax Nomor HP Alamat Kantor Nomor Telepon/Fax Alamat e-mail
1.11. Lulusan yg telah dihasilkan 1.12 Mata Kuliah yg diampu
Prof. Dr. Zullies Ikawati, Apt. P Guru Besar (IV/c) 196812061993032001 Purwokerto, 6 Desember 1968 Jl. Kaliurang Km 6,7 Gg Sumatra E-117, Yogyakarta 0274-886728 0815 685 4012 Sekip Utara 0274-552956
[email protected],
[email protected] S1= 125 orang ; S2= 75 orang S3= 2 orang; 1. Farmakologi 2. Farmakologi Molekuler 3. Farmakoterapi Sistem Pernafasan 4. Farmakoterapi Sistem Syaraf 5. Uji Klinik 6. Farmakogenetik-farmakogenomik
II. RIWAYAT PENDIDIKAN Program S1 Nama PT Universitas Gadjah Mada
--
Bidang Ilmu Tahun masuk Tahun lulus Judul thesis
Farmasi 1987 1992 Pengaruh pemberian suksinat pada biosintesis eritromisin dari Saccharopolyspora erithrea
--
Nama pembimbing
Prof. Dr. Retno Sunarminingsih, Apt
S2
3
S3 Ehime University, Japan
The role of mast cells in tracheal contraction, study using Ws/Ws rats Prof. Kazutaka Maeyama, MD, PhD
III. PENGALAMAN PENELITIAN 5 tahun terakhir (bukan skripsi, tesis, maupun disertasi) No. Tahun Judul Penelitian Pendanaan Sumber Jml ( Rp) 1 2008 Kajian interaksi Proyek Riset Unggulan 50.000.000 farmakokinetika kombinasi Universitas Gadjah ekstrak daun legundi dan Mada rimpang temulawak dengan cetirizin dan pseudoefedrin sebagai anti alergi 2 2009Pengembangan formula Proyek Insentif Riset 475.000.000 2011 ekstrak daun legundi (Vitex Peningkatan Kapasitas trifolia L) dan rimpang Sistem Produksi temulawak (Curcuma Kementerian Negara Xanthorrhiza Roxb) sebagai Riset dan Teknologi fitofarmaka untuk anti alergi 3 2009 Pengaruh Marmin, senyawa Proyek Hibah 130.000.000 aktif dari Aegle marmelos Kolaborasi Internasional Correa, terhadap sintesis LPPM UGM dan pelepasan histamin pada sel mast 4 2009 Pemetaan polimorfisme Proyek Hibah Kompetitif 100.000.000 genetik sitokrom P450 Penelitian Kerjasama subtipe CYP2D6, CYP2C9, Internasional dalam dan CYP2C19 pada rangka Publikasi populasi etnis Jawa di Internasional, DP2M Indonesia DIKTI 5 2010Genetic Polymorphism Proyek Hibah Kompetitif 120.000.000 2011 Mapping of CYP2D6, Penelitian Kerjasama CYP2C9, and CYP2C19 on Internasional dalam Makassar Population in rangka Publikasi Indonesia Internasional, DP2M DIKTI 6 2013 – Pengembangan Nano-GVT- Proyek Hibah 282.500.000 2014 0 dengan teknik SNEDDS Kompetensi, DP2M (Self-Nano Emulsifying DIKTI Drug Delivery System) sebagai obat anti arthritis, kajian toksisitas, farmakokinetik, dan mekanisme aksi molekulernya 7 2013 Pengembangan Formula Riset Unggulan 100.000.000 Minuman Nano-herbal Anti- Komprehensif kolesterol dari Universitas Gadjah Kombinasi Ekstrak Mada Temulawak (Curcuma xanthorrhiza) dan Daun Sambung Nyawa (Gynura procumbens) 8 2013 Pengembangan Formula Hibah Penelitian Non30.000.000 Herbal Antikalkuli kompetitif Fakultas Farmasi UGM 9 2014 Scale up dan produksi Direktorat 75.000.000 minuman herbal Pengembangan Usaha 4
antikolestrol dari Kombinasi Ekstrak Temulawak (Curcuma xanthorrhiza) dan Daun Sambung Nyawa (Gynura procumbens) Pengembangan Bahan Baku Obat Tradisional Ekstrak Daun Sambungnyawa [Gynura procumbens (Lour.) Merr.]
10
11
2014
12
2015
dan Inkubasi (PUI) UGM dan PT Phapros Tbk
Pengembangan herbal anti selulit dari kombinasi ekstrak daun pegagan dan jahe Validasi Metode Analisis Rifampisin, Atorvastatin dan Levofloxacin dalam Plasma dan Aplikasinya Dalam Rangka Meningkatkan Kemampuan Pengujian BioavailabilitasBioekuivalensi Obat di Laboratorium Terpadu Fakultas Farmasi Ugm
Pengembangan dan Peningkatan Kapasitas Produksi Bahan Baku Obat dan Bahan Baku Obat Tradisional tahun 2014, Kemenkes RI Lembaga Ilmu Pengetahuan Indonesia (LIPI)
330.000.000
Penelitian Unggulan Perguruan Tinggi
250.000.000
V. PUBLIKASI ARTIKEL ILMIAH DALAM JURNAL (5 tahun terakhir) No Judul Artikel Ilmiah Nama Jurnal 1
2
3
4
5
6
Kajian keamanan pemakaian obat antihipertensi di Poliklinik usia lanjut Instalasi Rawat Jalan RS Dr Sardjito Effects of benzylidenecyclopentanone analogues of curcumin on histamine release from mast cells Docking of Phenylsulfonamide-3trifluoromethyl-5-parabromophenyl-pyrazole to Cyclooxygenase-2 using PLANTS Effects of Marmin, isolated from Aegle marmelos Correa, on the L-histidine decarboxylase (HDC) enzyme in RBL-2H3 cells In vitro and in silico studies on Curcumin and its analogues as dual inhibitors for cyclooxigenase-1 (COX-1) and cyclooxygenase-2 (COX-2) The Importance of ARG513 as a Hydrogen Bond Anchor to Discover COX-2 Inhibitors in 5
Majalah Ilmu Kefarmasian
120.000.000
Nomer/volume /tahun 5 (3): 150169/2008
Biological Pharmaceutical Journal Indo J Chem
Vol. 32, No. 5, th 2009
Thai J Pharm Sci
35 (1-7). Th 2011
ITB Journal
Vol 44, no, 1, 51-66, th 2012
Bioinformation
6(4): 164-166, th. 2011
10(3), 348-351, th 2010
7
8
9 10 11
a Virtual Screening Campaign Effect of antihypertensive and therapy for increased intracranial pressure on Life Expectancy in Patient with intracranial Haemorrhagic stroke during Hospitalization A Safety Study of Extract Combination of Legundi (Vitex trifolia L.) Leaves and Temulawak (Curcuma xanthorrhiza R.) Rhizome as Anti-allergy in Healthy Volunteers Pharmacokinetic study of phenytoine-coffeine combination Jatropha curcas leaves for relieve of adjuvant-induced arthritis in rats The Analgesic Effect of a Curcumin Analogue 1,5-bis(4’-hydroxy-3’-methoxyphenyl)-1,4pentadien-3-on (Gamavuton-0) in acute and persistent pain
12
Acute Toxicity and Suppressive Effects of a Curcumin Analogue Gamavuton-0 (Gvt-0) On CFA-Induced Arthritis in rats
13
Therapeutic Effect of a Ribosomeinactivating protein from Mirabilis jalapa L against mouse skin cancer Induced by cocarcinogen imethylbenzantracene and UVB radiation Allele frequencies distribution of drug metabolizer genes CYP2C9*3 and CYP2C19*17 on Buginese population in Makassar Indonesia
14
Int J Pharm Teaching & Practices
2, Issue 4, 171175, th 2011
Int J Pharm Teaching & Practices
Vol.2, Issue 4, 165-170, th 2011
Int J Pharm Biomed Res Universa Medicina
Vol 4(1), 5-9
J Appl Pharm Sci
J Appl Pharm Sci
J Pharm Res
Current Pharmacogenomic s and Personalized Medicine
Vol 3(91). 1017, 2014 Vol 4 (8), 4851, th 2014
Vol. 4 (11), pp. 019-023, th 2014 13 (4): 111115, 2014
Vol. 12, No. 4, 2015
VI. PEMAKALAH SEMINAR ILMIAH (Oral Presentation) dalam 5 tahun terakhir Nama Pertemuan Judul artikel Waktu dan ilmiah/Seminar tempat 1 Regional Conference on Suppresive effect of Gamavuton-0 Kelantan Molecular Medicine on Complete Freund Adjuvant(Malaysia), April induced Rheumatoid arthritis in rats 2009 2 International Congress of Acute toxicity study of extract St Petersburg Phytopharm combination of Vitex trifolia leaves (Russia), Juli and Curcuma xanthorrhiza rhizome 2010 on Wistar rats 3 FAPA Meeting Allele Frequencies of Drug Denpasar (Bali), Metabolizer Genes Cyp2c9*2, September 2012 Cyp2c9*3, And Cyp2c19*17 on Buginese Population In Makassar, 6
4
Kongres Ilmiah Ikatan Apoteker Indonesia
5
International Conference on Drug Development from Natural Resources, International Pharmacy Conference in Research and Practice
6
7
8
9
10
11
Seminar Nasional Pengembangan Terkini Sain farmasi dan Klinik Asia Federation of Pharmaceutical Sciences (AFPS) Conference Indonesian-American Kavli Frontiers of Science Symposium 2nd International Conference and Exhibition on Pharmacognosy, Phytochemistry & Natural Products Asian Conference on Clinical Pharmacy
Pengaruh Marmin, senyawa aktif dari Aegle marmelos Correa, terhadap sintesis dan pelepasan histamin pada sel mast Development of Anti Allergy from Natural Product
Makasar, Desember 2010
Pharmacogenomics and Pharmacy Practice: Impact on Drug Response and Their Applications for Disease Management Tinjauan Farmakologi Molekuler Herbal Immunomodulator
Yogyakarta, Oktober 2012
Curcumin analogue GVT-0 supress Complete Freund Adjuvant-induced arthritis in rats
Jeju City (Korea Selatan), 22 November 2013 Medan, 23-25 Juni 2014
Indonesian Herbal Medicines, from the garden to clinical setting
2 3 4 5 6
Padang, 4 Oktober 2013
Anti Allergic Effect of Extract Combination of Vitex trifolia L and Curcuma xanthorrhiza Roxb on Ovalbumin-induced Active Cutaneous Anaphylaxis Reaction
Beijing, 25-27 Agustus 2014
Distribution of alleles frequency of drug metabolizer genes CYP2C9*3 and CYP2C19*17 on Buginese population in Makassar Indonesia
Bangkok, Juni 2015
KARYA BUKU DALAM 5 TAHUN TERAKHIR No Judul Tahun Penerbit 1
Yogyakarta, Mei 2012
Jumlah halaman 300
Bahaya Alkohol dan cara Mencegah kecanduannya Cerdas mengenali obat Resep Hidup Sehat Farmakoterapi penyakit Sistem Syaraf Pusat
2009 2010 2010 2011
Elexmedia Computindo, Jakarta PT Kanisius, Yogyakarta PT Kanisius, Yogyakarta Bursa Ilmu, Yogyakarta
Penyakit Sistem Pernafasan dan Tatalaksana terapinya Farmakologi Molekuler
2011
Bursa Ilmu Yogyakarta
300
2014
Gadjah Mada University Press
175
7
50 50 300
PENGALAMAN KERJASAMA LUAR NEGERI/INDUSTRI No 1
Mitra kerjasama University Sains Malaysia
Tahun 2009
2
Universitas La Trobe, Australia
2010
3
Ehime University, Jepang
2009
4.
PT Soho Industri Pharmasi
2009
5.
PT Phapros
2013
6.
PT Phapros
2014
Kegiatan Penelitian Kerjasama Luar Negeri DIKTI Penelitian Kerjasama Luar Negeri DIKTI Penelitian Hibah Kolaborasi Internasional LPPM UGM Penelitian Insentif Ristek Peningkatan Kapasitas Sistem Produksi Penelitian Hibah Unggulan Komprehensif UGM Pengembangan Produk Minuman Herbal antikolesterol
PENGALAMAN PEROLEHAN PENGHARGAAN No.
Tahun
Macam penghargaan
Pemberi
2009
Second Winner for Oral presentation in Regional Conference on Molecular Medicine di Malaysia Pemakalah terbaik di bidang Farmakologi dan Toksikologi pada Kongres Ilmiah Ikatan Apoteker Indonesia (IAI) ke XVIII di Makassar Insan Beprestasi UGM, pengembang RPKPS terbaik
Institute for Research on Molecular Medicine USM
2010
2010
Ikatan Apoteker Indonesia
Universitas Gadjah Mada
Semua data yang saya isikan dan tercantum dalam biodata ini adalah benar dan dapat dipertanggung jawabkan secara hukum. Dan apabila dikemudian hari ternyata dijumpai ketidak sesuaian dengan kenyataan, saya sanggup menerima resikonya. Yogyakarta, 18 Agustus 2015
Prof. Dr. Zullies Ikawati, Apt.
8
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Current Pharmacogenomics and Personalized Medicine, 2014, 12, 236-239
Allele Frequency Distributions of the Drug Metabolizer Genes CYP2C9*2, CYP2C9*3, and CYP2C19*17 in the Buginese Population of Indonesia Zullies Ikawati1,*, Theresia D. Askitosari2, Lukman Hakim1, Joseph Tucci3 and John Mitchell4 Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia; 2University of Surabaya, Biotechnology Faculty, Raya Kalirungkut, Tenggilis, 60293 Surabaya, Indonesia; 3School of Pharmacy and Applied Science, La Trobe University, Bendigo Campus PO Box 199 Bendigo Victoria 3552, Australia; 4Faculty of Science, Technology and Engineering, School of Molecular Sciences, Department of Genetics, La Trobe University, Australia
1
Abstract: The present study is part of the genetic mapping of Indonesia focusing on drug metabolizing enzymes, which started with the Buginese population of Makassar, South Sulawesi. The two CYP450 gene subfamilies, i.e. CYP2C9 and CYP2C19 are of interest as they exhibit wide inter-individual variation in expression, which influence the drug metabolism capacity. The CYP2C9 alleles of interest in this study were CYP2C9*2 and *3, and of CYP2C19 was CYP2C19*17. The study aimed to determine the frequencies of the CYP2C9 genotype, which contains *1, *2 and *3 alleles, and the CYP2C19 genotype, which comprises the *1 and *17 alleles in the Buginese. Ninety six Buginese subjects, comprising 48 males and 48 females were studied. CYP2C9 and CYP2C19 alleles were detected by a PCR-RFLP assay method. Results showed that there was no CYP2C9*2 allele present, while the frequencies of CYP2C9*3 and CYP2C19*17 overall were 1.56 % and 4.68 %, respectively. The frequency of the CYP2C9*3 allele in females was 2.08%, and not statistically different from that in males (1.05%). The frequency of the CYP2C19*17 allele in females (8.33%), was significantly different (P<0.05) from that in males (1.05%). No subject carried the CYP2C9*2/*2, CYP2C9*3/*3, CYP2C19*17/*17, or CYP2C9*3/CYP2C19*17 genotype. The study is the first to describe the drug metabolizing enzyme polymorphisms, CYP2C9 and CYP2C19, in the Indonesian Buginese population.
Keywords: Buginese, CYP2C9*2, CYP2C9*3, CYP2C19*17, Indonesia. 1. INTRODUCTION Genetic polymorphisms of drug metabolizing enzymes are one of the major determinants of inter-individual variability in drug response and are becoming a rapidly growing field in pharmacogenetics [1-3]. The cytochrome P450 (CYP450) family are the primary metabolic pathway for the metabolism of most drugs. The two CYP2C subfamilies, i.e. CYP2C9 and CYP2C19, exhibit wide interindividual variation in expression that affects drug metabolism capacity [4]. Among these functional variants, CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) have been extensively studied. The CYP2C9*2 allele encodes a moderately defective protein whereas the CYP2C9*3 allele has lower af nity and markedly lower intrinsic clearance for numerous drugs, both in vitro and in vivo [5]. These alleles have reduced catalytic activity compared to that of the wild type CYP2C9*1 [6]. The reduced activity may decrease the clearance of the parent drug and therefore increase its toxicity. In the case of CYP2C19, the variant allele of interest is CYP2C19*17 (rs12248560). This allele is associated with an increase in transcription rate and drug metabolism compared to those seen in the wild type CYP2C19*1 [7], and this
results in a lack of response to the parent drug due to its low availability. In contrast, it causes an extensive response when the parent drug is activated to the more active metabolites by this enzyme (as in prodrug). CYP2C9 and CYP2C19 variant alleles show considerable variation across different ethnic and continental populations [7-9]. The archipelago of Indonesia consists of hundreds of different ethnic groups. Sulawesi, is located near the Wallace line, which marks the transtition zone between Asia and the Australia continent. Sulawesi, therefore, has different flora, fauna, and also ethnic diversity, compared to other islands within Indonesia. One ethnic group in Sulawesi is the Buginese. Buginese is the third biggest ethnic in Indonesia, after Javanese and Sundanese which may have specific physical features and a socio-culture compared to other ethnic groups in Indonesia [10]. To date, however, there is still a lack of data on polymorphism in the CYP450 genes in Indonesians. This paper is one of the first to focus on the polymorphism of CYP450 in Indonesia, especially on the distribution and frequency of CYP2C9 and CYP2C19 variants in Buginese population. 2. MATERIALS AND METHODS 2.1. Samples
*Address correspondence to this author at the Faculty of Pharmacy Gadjah Mada University, Yogyakarta, Indonesia; Tel: 62-274-543120; Fax: 62-274-543120; E-mail:
[email protected]
1875-6913/14 $58.00+.00
Blood samples were taken from 96 healthy subjects of Buginese ancestry, consisting of 48 males and 48 females. Informed consent was obtained from all subjects and the © 2014 Bentham Science Publishers
Allele Frequency Distributions of the Drug Metabolizer Genes
Current Pharmacogenomics and Personalized Medicine, 2014, Vol. 12, No. 4 237
study protocol was approved by the Ethics Committee of Faculty of Medicine, Universitas Gadjah Mada. DNA samples were prepared from 200 l of blood using 20% Chelex (Bio-Rad) following the manufacturer’s instructions, then stored at -4oC until genotyping was performed. 2.1. Genotyping Strategy The genotyping strategies involved polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis, based on previously developed techniques for CYP2C9 and CYP2C19 genes [11, 12], with modification in the primer sequences. The primers were designed to contain a mismatch site to generate the digestion site of the respective restriction enzyme. This digestion site was disrupted if the subject DNA contained the mutation. The CYP2C9*2 allele results from a CT mutation at the 416 nucleotide position in exon 3 (11). The forward primer, 5’-TTTGGGATGGGGAAGAGGAGCATTGGGTA C(CT)-3’, and the reverse primer, 5’-GCTAACAACC AGACTCATAATGAAAGA-3’, were designed utilising two mismatches to generate the digestion site of the Kpn1 enzyme, if the sample is wild type. The digestion site is destroyed if the sample contains the CT mutation. The amplicon is 198 base pairs (bp) in size. Following PCR and digestion by the Kpn1 enzyme homozygotes for the wild type allele (*1/*1) will be seen as a fragment of 168 bp, homozygotes for the CYP2C9*2 allele (*2/*2) will not contain the Kpn1 site, and so a fragment of 198 bp will be seen, and heterozygotes (*1/*2) will display fragment sizes of 168 bp and 198 bp. The CYP2C9*3 allele results from an AC mutation at the 1061 nucleotide position in exon 7 (11). The forward primer 5’-CCCCTGAATTGCTACAACAAATGTGCC-3’ and the reverse primer 5’-CATGGGGCAGGCTGG TGGGGAGAAGATTAA(TG)-3’ were designed utilising two mismatches to generate the digestion site of the Vsp1 enzyme, if the sample is wild type. The site is destroyed if the sample contains the AC mutation. The amplicon is 213 bp in size. Following PCR and digestion with the Vsp1 enzyme homozygotes for the wild type allele (*1/*1) will be seen as a fragment of 183 bp, homozygotes for the CYP2C9*3 allele (*3/*3) will not contain the Vsp1 site, and so will be seen as a fragment of 213 bp, and heterozygotes (*1/*3) will display fragments of 183 bp and 213 bp. The detection of CYP2C19*17 allele was carried out using a forward primer, 5’-GGTTCTATTT AATGTGAA GCC-3’ and a reverse primer, 5’- TGGCGCATTATCTCTT ACATCAGACAT-3’. This is a novel strategy whereby there is a mismatch in the reverse primer (mismatch occurs 24 bases from 5’end of primer) to introduce a Hsp9211 digestion site if DNA is wild type, resulting in a 153 bp fragment. If DNA has the CYP2C19*17 mutation, then the Hsp9211 site is destroyed and the 177 bp fragment will be seen after digestion with the Hsp9211 enzyme. 3. RESULTS The CYP2C9 and CYP2C19 alleles as well as the genotype frequencies in the Buginese population are summarized in Table 1. No individuals carried the
CYP2C9*2 allele, three individuals were heterozygous 2C9*1/*3, no subject was homozygous 2C9*3/*3, and nine were heterozygous 2C19*1/*17. The CYP2C19*17/*17 and CYP2C9*3/*17 allele was absent in the Buginese. There was no CYP2C9*2 allele in the sample, while the frequency of the CYP2C9*3 and CYP2C19*17 allele in the Buginese was 1.56 % and 4.68 %, respectively. The allele frequency of CYP2C9*3 was 1.05% in males and was not significantly different from that (2.08%) seen in females. On the other hand, the frequency of CYP2C19*17 in females was 8.33% which was significantly greater than that seen in males (1.05%), (P<0.05). Under Hardy-Weinberg equilibrium conditions one homozygotefor CYP2C9*3 is expected in every 5000 Buginese, whereas, one homozygote for CYP2C19*17 is expected in every 476 Buginese. Table 1. Allele (A) and genotype (B) frequencies of CYP2C9 and CYP2C19 in male and female Buginese populations. A Variant allele
Males (%)
Females (%)
P-value
CYP2C9*1
98.95
97.92
CYP2C9*2
0
0
-
CYP2C9*3
1.05
2.08
0.557
CYP2C19*1
98.95
91.67
CYP2C19*17
1.05
8.33
0.014
B Genotype
Male
Female
N
(%)
(N)
(%)
CYP2C9*1/*1
47
97.92
46
95.83
CYP2C9*1/*2
0
0
0
0
CYP2C9*2/*2
0
0
0
0
CYP2C9*1/*3
1
2.08
2
4.17
CYP2C9*3/*3
0
0
0
0
CYP2C19*1/*1
47
97.92
40
83.33
CYP2C19*1/*17
1
2.08
8
16.67
CYP2C19*17/*17
0
0
0
0
CYP2C19*3/*17
0
0
0
0
4. DISCUSSION Our genotyping approach was designed for the utilization of inexpensive and commercially available digestion enzymes. In order to improve the genotyping, one of each primer pairs was designed to incorporate a restriction endonuclease digestion site at its 3’ end, and each primer was designed to be around 30 bp in length, which ensured a large
238 Current Pharmacogenomics and Personalized Medicine, 2014, Vol. 12, No. 4
enough difference in band size to easily differentiate between wild type alleles and those with mutations of CYP2C9 and CYP2C19 genes when run on a 3% agarose gel. These results are novel, as ethnic groups from Indonesia have not previously been assessed for CYP450 status. The data showed a similar general trend as that seen for other Asian groups, namely, a negligible frequency of CYP2C9*2, and the presence of the CYP2C9*3 allele at a frequency between 2%-5% [9, 13, 14]. This observation is in line with the report that the CYP2C9*2 allele is primarily restricted to European, Middle Eastern and Central/South Asian populations, but is absent or found at very low frequencies in other geographic regions (Africa, East Asia, Oceania and America). The CYP2C9*3 allele has a broader geographic distribution, but the highest allele frequencies are also found in European and Central/South Asian populations [15]. In the case of CYP2C19, we only found heterozygous 2C19*1/*17, individuals, and no homozygous 2C19*17/*17. These data are also similar to that of other Asian populations, especially Korean and Chinese [7, 8]. The prevalence of the CYP2C19*17 allele was very low [16], which therefore has no association with adverse clinical outcomes after percutaneous coronary intervention and clopidogrel. On the contrary, the CYP2C19*17 allele was found in a relatively high frequency in European population, especially Greeks, in which it was reported at 19.61% [17]. There is ongoing debate about the health consequences of such polymorphisms as those studied here. It is possible that they may be associated with adverse clinical outcomes for patients taking medications whose metabolism is controlled by the products of these genes. This is especially the case for patients taking drugs with a narrow therapeutic range (warfarin, theophylline, digoxin), and saturable kinetics (phenytoin), where small alterations in plasma levels may result in disproportionate toxicity [11, 18] or when taking other drugs which may have CYP450 inducing or inhibiting properties [19, 20]. The polymorphism also has implications for drug dose adjustment. Tentative estimates of how CYP2C9 genotyping might be applied to dose adjustments in clinical therapy were based on dose-related pharmacokinetic parameters, specially the clearance or trough drug concentrations. Mean clearances in homozygous carriers of the *3 allele were below 25% of that of the wild type for S -warfarin, tolbutamide, glipizide, celecoxib, and fluvastatin. In the more frequent heterozygote (genotype *1/*3), the clearances were between 40% and 75%. In these cases in which individual dosages are derived from clinical drug effects, such as for the oral anticoagulants, the pharmacogenetics-based dose adjustments showed a good correlation with the genotype-specific empirically derived doses [21]. In the case of polymorphism in CYP2C19, examples of clinical consequences are linked to clopidogrel, a pro drug. Carriers of the CYP2C19*2 loss-of-function allele present higher platelet reactivity and worse clinical outcomes compared to that seen in non-carriers. The hyper-function allele CYP2C19*17 increases the biotransformation of clopidogrel to its active metabolite, which in turn increase the effect to inhibit platelet aggregation. The simultaneous occurrence of the CYP2C19*17 polymorphism seems to offset the negative
Ikawati et al.
impact of the CYP2C19*2 polymorphism on platelet aggregation [22]. For other drugs, like escitalopram, the homozygous CYP2C19*17 genotype is associated with lower serum concentration of escitalopram, which might imply an increased risk of therapeutic failure in psychiatric patients [23]. Such clinical issues highlight the importance of pharmacogenomic screening. Further, wider application of such testing is being advocated in order to establish whether inter individual variation in metabolic capacity exists between participants in clinical trials [24], a factor which could influence outcomes of these studies and subsequent acceptance or rejection of new therapeutic substances. CONCLUSION The study is the first to describe the genetic polymorphism of drug metabolizing enzymes, CYP2C9 and CYP2C19, in an Indonesian population. The findings are similar to those seen in other Asian populations, especially Korean and Chinese, and contribute to genetic polymorphism mapping of drug metabolizing enzymes in Asian population. LIST OF ABBREVIATIONS CYP450 =
Cytochrome P450
PCR
=
Polymerase chain reaction
RFLP
=
Restriction fragment length polymorphism
ETHICS STATEMENT Study protocol was approved by the Ethics Committee of Faculty of Medicine, Universitas Gadjah Mada. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS The authors wish to thank Prof. Nasrum Massi from Hasanuddin University for DNA isolation of samples, The Ministry of Education for financial support and Muhammad Novrizal Abdi Sahid for assistance in writing. REFERENCES [1]
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Received: December 16, 2014
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Revised: April 8, 2015
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Accepted: April 10, 2015
