TaqMan Genotyping Assay Method for Single Nucleotide Polymorphisms (SNPs) detection in promoter region of N-Acetyltransferase 2 (NAT2) gene

  The N-acetyltransferase 2 (NAT2) polymorphism in coding region has been studies intensively. However, there are limited studies for promoter region of NAT2 gene. Several reported study showed that the promoter region polymorphism of NAT2 gene is genotyped by PCR-sequencing approach. In this paper, we describe TaqMan based assays for the NAT2 polymorphism genotyping in promoter region with following SNP: rs4646243 [T>C], rs4646244 [T>A], rs4646267 [A>G], rs4345600 [A>G], and rs4646246 [A>G].  Our result showed a good separation cluster, trailing cluster and some mix cluster. TaqMan genotyping assay has shown a sensitivity and specificity to detect polymorphism in NAT2 promoter region

HLA-DQB1*05:02 Allele Association with Anti-Tuberculosis Drug Induced Liver Injury: A Single-Hospital Based Study in Jakarta, Indonesian Population

Past studies have delved into the genetic factors underlying anti-tuberculosis drug-induced liver injury (AT-DILI), primarily concentrating on polymorphisms in genes responsible for drug-metabolizing enzymes. However, the immune system's potential impact on drug adverse effects, specifically through genes such as HLA, has received limited attention. Previous research has notably revealed an association between HLA-DQB1*05 and AT-DILI, specifically the prevalence of the HLA-DQB*05:02 allele in AT-DILI patients. In light of this, our study aimed to investigate a potential link between HLA-DQB1*05:02 alleles and AT-DILI. In this study, we included 51 AT-DILI cases and 59 controls belonging to the Javanese ethnic group. The HLA-DQB1*05:02 genotypes were determined using a customized PCR-based typing method, and the results were further confirmed by analyzing five samples via the Luminex assay. Our findings revealed a significant association between HLA-DQA1*05: 02 and the risk of AT-DILI (P = 0.022; OR (95% CI) = 6.11 (1.25-29.74)). Moreover, the consistent results obtained from the Luminex assay validated the reliability of the custom PCR-based genotyping approach. This preliminary study sheds light on the relationship between the HLA-DQB1*05:02 allele and AT-DILI within the Indonesian population. Furthermore, our study demonstrates the dependability of custom PCR-based genotyping in detecting HLA-DQB1*05:02 alleles. Nevertheless, further research is imperative to corroborate and expand upon our findings

The Use of High Resolution Melting (HRM) Method to Detect rs1800629 of Tumor Necrosis Factor-alpha (TNF-alpha) Gene among Tuberculosis Patients

The rs1800629 polymorphism plays a crucial role in the pathogenesis of infectious and autoimmune diseases. Meanwhile, tuberculosis (TB) remains a health primary infectious disease in Indonesia. The purpose of this study is to evaluate the HRM method in detecting the rs1800629 genotype, in the TNF-α gene’s promoter region, within TB patients. The benefit of this study is to accelerate the detection of rs1800629 with a simple, rapid, and cost-effective method for genotyping and mutation screening that does not include the use of a fluorescent probe. In this experimental study, the rs1800629 genotyped in a total of 25 tuberculosis patients using KAPA HRM kit in MyGo Mini PCR, and all amplified PCR products subsequently dispatched for direct DNA sequencing to Macrogen Inc, South Korea. Based on the results, a 100% concordance find in the genotyping of rs1800629 between HRM and sequencing. The authors provided evidence to use HRM in detecting rs1800629 within the TNF-α promotor region. This application as a genotyping assay in tuberculosis patients is a low-cost, rapid, and accurate detection. However, further studies using the HRM method in case-control samples of tuberculosis are required to evaluate the method’s effectiveness and to obtain more information regarding the genotype’s susceptibility to tuberculosis and its adverse effect treatment, including anti-tuberculosis drug, induced liver injury (AT-DILI), and multidrug-resistant TB (MDR-TB), within the Indonesian population

Differentiation of N-acetyltransferase 2 (NAT2) rapid and intermediate acetylator based on genotype and urinary assay

BackgroundDetermination of the acetylator type of NAT2 generally can be predicted based on genotype data from the NAT2 database. However, in some reported studies, it does not show 100 per cent concordance with the phenotype based on urinary assay. The assay generally only differentiates the rapid and slow acetylator but does not consider the intermediate one. AimsWe conducted this study to define the phenotype of NAT2 based on both genotyping and urinary assay and to determine the concordance rate between both methods in rapid and intermediate acetylator groups. Methods NAT2 genotyping was done using the PCR-direct sequencing in a total of 30 healthy subjects. However, for the NAT2 phenotypes we only selected 19 healthy subjects that carry rapid or intermediate acetylator genotype, without involving slow acetylator phenotype. The assay was done by measuring the ratios of urinary caffeine metabolites following controlled diet exposure.Results Both data obtained from genotyping and urinary assay showed 2 samples that belonged to the rapid acetylator and 17 samples that belonged to the intermediate acetylator. The mean metabolic ratio of the rapid acetylator group showed a higher level (0.5) than the intermediate group (0.28). The predicted acetylation status of NAT2 SNPs from genotyping was matched with the phenotype which was determined by urinary analysis.ConclusionOur result showed a 100 per cent concordance of NAT2 phenotype based on the genotyping and urinary assay. Based on this study we suggest that NAT2 phenotype based on genotyping method is simpler and faster, rather than using the urinary assay that is more laborious and costly

Deteksi Mutasi Langka, Delesi 619 Bp, Pada Gen Beta-Globin Dari Etnis Melayu Mahasiswa Fakultas Kedokteran Universitas YARSI

Beta-thalassemia merupakan gangguan hematologis autosomal yang secara genetis mengakibatkan berkurangnya sintesis beta-globin di hemoglobin. Beta-talasemia sebagian besar disebabkan oleh mutasi titik, insersi atau delesi dalam gen beta-globin yang terletak pada lengan pendek kromosom 11. Organisasi Kesehatan Dunia (WHO) memperkirakan terdapat sekitar 1,5% dari populasi global (80-90 juta orang) adalah pembawa ?-thalassemia. Tidak ada studi komprehensif untuk mendeteksi pembawa beta-thalassemia di Indonesia, terutama untuk mutasi delesi 619 bp, yang mencakup ekson 3 dan memiliki prevalensi yang tinggi. Kami menggunakan metode gap-PCR yang di-kombinasikan dengan metode elektroforesis gel untuk memper-kirakan adanya mutasi delesi 619 bp pada 48 siswa Fakultas Kedokteran Universitas YARSI dengan etnis Melayu. Analisis Blast hasil sekuensing dari ketiga sampel menunjukkan bahwa terdapat similaritas 98% antara hasil amplifikasi dengan ke daerah gen beta-globin pada kromosom 11 (No. Aksesi U01317.1). Berdasarkan hasil visualisasi elektroforesis gel, semua produk PCR dari 48 sampel, menunjukkan bahwa semua sampel tidak membawa mutasi delesi 619 bp yang ditunjukkan dengan ukuran produk PCR yang sama dari semua sampel, yaitu berukuran 1.457 bp dan 2.291 bp dari PCR I dan 1.212 bp dari PCR II.Beta-thalassaemia is an autosomal haematological disorder resulting in a genetically deficient synthesis of the ?-globin chain in haemoglobin. It is mostly caused by point mutations, a small deletions or insertions within the beta-globin gene which is located as a cluster on the short arm of chromosome 11. The World Health Organization has estimated that about 1.5% of the global population (80 to 90 million people) were carriers of ?-thalassemia. There are no comprehensive study to detect carrier of ?-thalassemia in Indonesia especially for 619 bp deletion mutation, which encompasses exon 3, that has greater prevalence. We used gap-PCR combined with gel electrophoresis methods to roughly screen the presence of major indel mutation in 48 Medical Faculty, Universitas YARSI students with Malay ethnic. To validate whether the PCR product obtained is the beta-globin gene, a direct sequencing of 3 PCR products were performed. The Blast analysis of the sequence was also done using NCBI database. The result showed that the PCR products obtained in this study showed 98% identity to human beta-globin gene region on chromosome 11 (No. Acc. U01317.1). In the electrophoresis of all PCR products of 48 samples, the result showed that all the samples did not carry any major indel mutation showing by the presence of similar length of PCR products in gel electrophoresis, which has 1.457 bp and 2.291 bp product from PCR I and 1.212 bp product from PCR II

Deteksi Mutasi Langka, Delesi 619 bp, pada Gen Beta-Globin dari Etnis Melayu Mahasiswa Fakultas Kedokteran Universitas YARSI

Beta-thalassemia merupakan gangguan hematologis autosomal yang secara genetis mengakibatkan berkurangnya sintesis beta-globin di hemoglobin. Beta-talasemia sebagian besar disebabkan oleh mutasi titik, insersi atau delesi dalam gen beta-globin yang terletak pada lengan pendek kromosom 11. Organisasi Kesehatan Dunia (WHO) memperkirakan terdapat sekitar 1,5% dari populasi global (80-90 juta orang) adalah pembawa ?-thalassemia. Tidak ada studi komprehensif untuk mendeteksi pembawa beta-thalassemia di Indonesia, terutama untuk mutasi delesi 619 bp, yang mencakup ekson 3 dan memiliki prevalensi yang tinggi. Kami menggunakan metode gap-PCR yang di-kombinasikan dengan metode elektroforesis gel untuk memper-kirakan adanya mutasi delesi 619 bp pada 48 siswa Fakultas Kedokteran Universitas YARSI dengan etnis Melayu. Analisis Blast hasil sekuensing dari ketiga sampel menunjukkan bahwa terdapat similaritas 98% antara hasil amplifikasi dengan ke daerah gen beta-globin pada kromosom 11 (No. Aksesi U01317.1). Berdasarkan hasil visualisasi elektroforesis gel, semua produk PCR dari 48 sampel, menunjukkan bahwa semua sampel tidak membawa mutasi delesi 619 bp yang ditunjukkan dengan ukuran produk PCR yang sama dari semua sampel, yaitu berukuran 1.457 bp dan 2.291 bp dari PCR I dan 1.212 bp dari PCR II.Beta-thalassaemia is an autosomal haematological disorder resulting in a genetically deficient synthesis of the ?-globin chain in haemoglobin. It is mostly caused by point mutations, a small deletions or insertions within the beta-globin gene which is located as a cluster on the short arm of chromosome 11. The World Health Organization has estimated that about 1.5% of the global population (80 to 90 million people) were carriers of ?-thalassemia. There are no comprehensive study to detect carrier of ?-thalassemia in Indonesia especially for 619 bp deletion mutation, which encompasses exon 3, that has greater prevalence. We used gap-PCR combined with gel electrophoresis methods to roughly screen the presence of major indel mutation in 48 Medical Faculty, Universitas YARSI students with Malay ethnic. To validate whether the PCR product obtained is the beta-globin gene, a direct sequencing of 3 PCR products were performed. The Blast analysis of the sequence was also done using NCBI database. The result showed that the PCR products obtained in this study showed 98% identity to human beta-globin gene region on chromosome 11 (No. Acc. U01317.1). In the electrophoresis of all PCR products of 48 samples, the result showed that all the samples did not carry any major indel mutation showing by the presence of similar length of PCR products in gel electrophoresis, which has 1.457 bp and 2.291 bp product from PCR I and 1.212 bp product from PCR II.

Detection of ADAM33 Gene Variants Using Sanger Sequencing

Asma adalah penyakit pernafasan yang ditandai oleh obstruksi saluran napas yang disebabkan oleh peradangan bronkus akut dan kronis. Gen disintegrin and metalloprotease 33 (ADAM33) merupakan gen terkait kerentanan terhadap asma dan diketahui memiliki lebih dari 300 polimorfisme. Studi meta-analisis melaporkan bahwa rs2280091, rs2787094, rs511898, rs2280089 dan rs2280090 memiliki asosiasi kuat dengan asma pada populasi Asia. Penelitian pendahuluan ini mengumpulkan 10 partisipan dengan penyakit asma dan 10 partisipan sehat untuk mengidentifikasi alel dan genotipe dari masing-masing polimorfisme menggunakan metode amplifikasi dan sekuensing Sanger. Hasil sekuensing menunjukkan bahwa 60% dari sampel yang diuji memiliki genotipe heterozigot untuk rs2280090 dan rs228091 baik pada sampel kasus maupun kontrol. Varian SNP rs2280096 menunjukkan bahwa frekuensi alel homozigot wildtype sebesar 60% baik pada sampel kasus maupun kontrol. Penelitian lanjutan perlu dilakukan untuk mengetahui apakah varian SNP tersebut memiliki aosisasi yang positif dengan penyakit asma

THE RISK FACTORS FOR DRUG INDUCED HEPATITIS IN PULMONARY TUBERCULOSIS PATIENTS IN DR. SOETOMO HOSPITAL

Tuberculosis (TB) is still a major public health problem in Indonesia. Anti-tuberculosis drug-induced hepatotoxicity (DIH) is common side effect leading to changes in treatment regimens, and the less effective second-line treatments. Several risk factors such as age, sex, body mass index (BMI) and acetylization status for hepatotoxicity were suggested in previous studies but in the fact, those are often not related to DIH incidence after receiving standard TB treatment regimen. The aim of this study was to asses the role of risk factors in the DIH incidence in pulmonary TB patients receiving standard TB treatment regimen in Dr. Soetomo Hospital, Surabaya. Study design was analytic observational with case control. The subjects were 30 TB DIH patients and 31 TB non-DIH patients receiving standard national TB program therapy. DIH severity was divided based on International DIH Expert Working Group. Demographic data and BMI status were taken from medical records. The age classification are ≥35 years old and <35 years old as one of the risk factors studied. DNA sequencing was used to assess single-nucleotide polymorphisms in NAT2 coding region to evaluate acetylator status from blood samples. The risk factors were evaluated using chi-square test and Mantel-Haenszel test. Significant association between low BMI and DIH in general was identified (OR=3.017; 95% CI=1.029-8.845) and more significant association between low BMI and moderate DIH (OR=15.833; 95% CI=1.792-139.922). Age, sex, and acetylization status has no significant correlation with DIH incidence in general. Significant association between slow acetylator phenotype and incidence of moderate DIH was identified (OR=7.125; 95% CI= 1.309-38.711). In conclusion, some risk factors were correlated to DIH incidence in pulmonary TB patients receiving standart TB treatment regimen

Frequencies of HLA-B alleles in Indonesian Malay Ethnic

Background: The HLA-B alleles have been used as a marker to predict drug-induced adverse reactions and as a major contributor to hypersensitivity reactions. We examined the feasibility of HLA-B alleles as pharmacogenomic markers of drug-induced hypersensitivity in an Indonesian Malay Ethnic. Methods: Fifty-eight Indonesian individuals of Malay ethnicity were enrolled in this study. HLA-B alleles were determined using reverse sequence-specific oligonucleotide probe coupled with xMAP technology. Results: HLA-B*15:02 (15.52%), HLA-B*35:05 (9.48%), and HLA-B*07:05 (7.76%) were frequent alleles in the Indonesian Malay ethnic populations. We discovered at least eight pharmacogenomics markers of drug-induced hypersensitivity: HLA-B*15:02, HLA-B*15:21, HLA-B*13:01, HLA-B*35:05, HLA-B*38:02, HLA-B*51:01, HLA-B*57:01, and HLA-B*58:01. HLA-B*15:02 was in the same serotype group with HLA-B*15:21, which is a B-75 serotype associated with genetic predisposition for carbamazepine-induced Stevens–Johnson syndrome and toxic epidermal necrolysis. The Indonesian population, represented by Malay, Javanese, and Sundanese ethnicities, was similar to South East Asian, Han Chinese, and Taiwanese populations based on HLA-B*15:02 frequency as the most common allele found in Malay ethnics. Conclusion: We provided valuable information on the frequency of drug hypersensitivity-associated HLA-B alleles in Indonesian Malay ethnic population, which can improve treatment safety