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"ABSTRAKLatar Belakang: Kanker Kolorektal termasuk ke dalam lima besar kanker dengan tingkat insidensi dan mortalitas yang tinggi di Indonesia. Sebesar 20%-25% kejadian kanker kolorektal merupakan faktor keturunan melalui pewarisan mutasi gen-gen high penetrance yang berkontribusi signifikan terhadap pembentukan kanker kolorektal, sedangkan 80%-85% merupakan kanker kolorektal sporadik. Pada kanker kolorektal sporadik mutasi terjadi pada gen-gen low penetrance yang berisiko rendah terhadap pembentukan kanker kolorektal. Identifikasi mutasi pada gen-gen lain yang berpotensi memiliki kontribusi terhadap kanker kolorektal sporadik diperlukan. Prastudi sebelumnya menggunakan metode GWAS telah mengidentifikasi CNV di kromosom 7, 8, 18, dan 20 dari pasien kanker kolorektal sporadik. Penelitian ini bertujuan untuk mengidentifikasi gen dengan CNV di kromosom tersebut dan asosiasinya pada kanker kolorektal sporadik serta hubungan variasi genetik CNV terhadap tingkat ekspresi mRNA gen DOK5.Metode: Identifikasi gen berdasarkan seleksi dari analisis data CNV dan SNP prastudi GWAS. Sebanyak 70 pasang sampel jaringan kanker kolorektal dan jaringan kolorektal sehat digunakan dalam penelitian ini dengan persetujuan komisi etik serta tujuh jenis sel lestari. Metode Real-Time PCR digunakan dalam analisis CNV gen DOK5 menggunakan DNA dari sampel jaringan maupun sampel sel lestari dan analisis ekspresi mRNA gen DOK5 dari sampel RNA sel lestari.Hasil: Jumlah Copy Number (CN) gen DOK5 yang tinggi secara signifikan (P =0,01) terdapat pada kelompok usia ≥50 tahun (CN=1,58). Variabel CN gen DOK5 berasosiasi signifikan dengan variabel kelompok usia (P=0,028) dimana lebih dari 50% sampel ≥50 tahun memilik amplifikasi gen DOK5. Pada derajat diferensiasi histopatologi dan jenis kelamin tidak ada perbedaan dan asosiasi dengan CN gen DOK5 secara signifikan (P >0,05). CNV gen DOK5 berkorelasi positif kuat (R=0,890) secara signifikan (P=0,007) dengan tingkat ekspresi mRNA gen DOK5 di beberapa sampel sel lestari dari berbagai kanker.Kesimpulan: Amplifikasi CNV gen DOK5 terkait dengan kanker kolorektal sporadik pada subyek berusia ≥50 tahun. CNV gen DOK5 yang teramplifikasi berpengaruh terhadap peningkatan ekspresi mRNA gen DOK5 di sel lestari.

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ABSTRACT

Background: Colorectal Cancer (CRC) is among top five cancers that have high level of incidence and mortality in Indonesia. About 20%-25% of CRC were familial that inherited a mutation of high penetrance genes with high predisposing risk of CRC, while the rest of 75%-80% were sporadic CRC. Most of predisposing mutation of genes in sporadic CRC were low penetrance genes with low risk of CRC. Identification of other predisposing genes that have significant and higher risk of sporadic CRC were needed. Our previous study using GWAS has identified a significant genetic variation in the form of CNV in chromosomes 7, 8, 18, dan 20 in sporadic CRC patient. This study aimed to identify the gene with CNV in those chromosomes and its association with sporadic CRC also the effect of CNV on expression level of the gene mRNA.

Method: The gene was identified by selection using analyzed data of CNV and SNP from GWAS study. Seven type of cell lines and 70 matched paired samples of cancerous and normal tissues were used in this study with approval from ethic commission. Real-Time PCR method was used to analyze both DOK5 gene CNV from DNA sample and DOK5 gene expression from RNA sample. DNA was extracted from tissues and cell lines, while RNA extracted from cell lines.

Results: Difference of DOK5 CN was significant in age group (P=0,01) with group ≥50 years old had higher CN of DOK5 gene (CN=1,58). DOK5 CN variable was associated with age group variable (P=0,028) where more than 50% samples of age ≥50 years old showed amplification of DOK5 CN. No significant difference (P >0,05) in CNV of DOK5 gene was detected in subjects if grouped based on their histopathology or gender. In cell lines, CN of DOK5 gene showed significant (P=0,007) and strong positive correlation (R=0,890) with mRNA expression level of DOK5 gene.

Conclusion: DOK5 gene was identified from GWAS CNV data. CNV amplification of DOK5 gene was associated with sporadic colorectal cancer in subject ≥50 years old. Amplified CNV of DOK5 gene affect the increased of DOK5 mRNA expression level in cell lines."

"Relaps masih menjadi masalah dalam eradikasi malaria vivaks. Primakuin adalah satu-satunya antihipnozoit yang saat ini tersedia di pasaran. Efikasi primakuin diperoleh oleh farmakokinetik dan farmakodinamik obat. Kemampuan CYP2D6 memetabolisme primakuin menjadi bentuk aktif akan memengaruhi kadar primakuin dan efikasi klinisnya. Pada penelitian ini dilakukan analisis farmakokinetik dan farmakodinamik primakuin dengan pendekatan populasi pada subjek dengan malaria vivaks; serta menganalisis hubungan variasi jumlah salinan gen CYP2D6 dengan kejadian relaps.Subjek studi adalah 174 orang Tentara Nasional Indonesia yang terinfeksi malaria vivaks dan diterapi dengan kombinasi skizontisida dan primakuin selama 14 hari. Kejadian relaps diamati selama satu tahun. Model farmakokinetik-farmakodinamik primakuin yang dikembangkan dengan metode mixed effect non linier menggunakan piranti lunak NONMEM versi 7.4.1. Kuantifikasi jumlah salinan gen CYP2D6 dilakukan pada 49 subjek. Jumlah salinan ditentukan berdasarkan nilai Cq hasil amplifikasi intron 6 dengan qPCR real-time. Jumlah salinan dihitung sesuai dengan rumus 2-ΔΔCq x jumlah salinan DNA Kalibrator, ΔΔCq = ΔCq (kalibrator)  ΔCq (sampel) dan ΔCq = Cq (CYP2D6) - Cq (RNAse P). Hubungan jumlah salinan gen CYP2D6 dan kejadian relaps malaria vivaks dianalisis dengan uji Chi-square.Hasil penelitian menunjukkan bahwa kadar primakuin plasma paling baik dideskripsikan oleh model satu kompartemen dengan penyerapan orde pertama. Berat badan diimplementasikan sebagai fungsi alometrik pada clearance (CL) dan distribusi volume (Vd). Piperakuin maupun pironaridin menurunkan CL dan Vd primakuin sebesar 3354%. Faktor genetik CYPD6 tidak memengaruhi CL primakuin. Risiko kejadian relaps malaria vivaks dideskripsikan dengan model constant hazard pada model time-to-event. Peningkatan satu poin skor aktivitas gen CYP2D6 menurunkan risiko relaps sebesar 88,3%, sehingga dapat disangkal bahwa faktor genetik CYP2D6 menjadi salah satu faktor yang dapat memengaruhi risiko kambuh vivaks malaria. Tidak didapatkan hubungan antara AUC primakuin dan kejadian relaps, sehingga hasil ini tidak dapat digunakan untuk menghitung dosis primakuin yang optimal. Kuantifikasi jumlah salinan gen CYPD6 dilakukan pada 21 subjek relaps dan 28 subjek kontrol. Mayoritas subjek memiliki jumlah salinan ≥ 2 (39 dari 49 orang). Tidak ditemukan hubungan antara jumlah salinan gen CYP2D6 dan kejadian relaps (p = 0,155).Relapse is still a problem in vivax malaria eradication. Primakuine is the only antihipnozoite currently available on the market. The efficacy of primaquine is obtained by the pharmacokinetics and pharmacodynamics of the drug. The ability of CYP2D6 to metabolize primaquine to its active form will affect primakuine levels and clinical efficacy. In this study, a pharmacokinetic and pharmacodynamic analysis of primaquine was carried out with a population approach in subjects with vivax malaria; and to analyze the relationship between variations in the number of copies of the CYP2D6 gene with the incidence of relapse.Study subjects were 174 Indonesian National Armed Forces infected with vivax malaria and treated with a combination of schizonticides and primaquine for 14 days. Relapse incidence was observed for one year. The primakuine pharmacokinetic-pharmacodynamic model was developed using a non-linear mixed effect method using NONMEM software version 7.4.1. Quantification of the number of copies of the CYP2D6 gene was performed in 49 subjects. The number of copies is determined based on the Cq value of the intron 6 amplification with real-time qPCR. The number of copies is calculated according to the formula 2-ΔΔCq x number of copies of the DNA Calibrator, ΔΔCq = ΔCq (calibrator)  ΔCq (sample) and ΔCq = Cq (CYP2D6) - Cq (RNAse P). The association between copy number of CYP2D6 gene and the incidence of vivax malaria relapse was analyzed using Chi-square test.The results showed that plasma primquine levels were best described by a one-compartment model with first-order absorption. Body weight is implemented as an allometric function on clearance (CL) and volume distribution (Vd). Piperakuine and pyronaridin reduce CL and Vd primakuin by 3354%. CYPD6 genetic factor does not affect CL primaquine. The risk of vivax malaria relapse was described using the constant hazard model in the time-to-event model. One point increase in the CYP2D6 gene activity score reduced the risk of relapse by 88.3%, so it can be denied that CYP2D6 genetic factor is one of the factors that can affect the risk of malaria vivax relapse. There was no relationship between AUC of primaquine and the incidence of relapse, so these results cannot be used to calculate the optimal primquine dose. CYPD6 gene copy count quantification was performed in 21 relapsed subjects and 28 control subjects. The majority of subjects had a number of copies ≥ 2 (39 of 49 people). No association was found between the number of copies of the CYP2D6 gene and the incidence of relapse (p = 0.155)."