Yongtaek Oh

Polymerase chain reaction-based fluorescent Luminex assay to detect the presence of human papillomavirus types.

Becuase 40% of human papillomavirus (HPV) infections are mixed infections, the accurate identification of high‐risk HPV genotypes in mixed infections is important for defining a womans risk for progression to cervical cancer. Thus, advanced Luminex‐based HPV genotyping has been developed to simultaneously detect the presence of multiple HPV types. Here, we describe the development of a Luminex‐based HPV genotyping that combines polymerase chain reaction amplification with hybridization to fluorescence‐labeled polystyrene bead microarrays (Luminex suspension array technology). New HPV type‐specific oligonucleotide probes and YBT L1/GP6‐1 primers were used to detect the HPV types in 132 clinical samples. We simultaneously evaluated the usefulness of this technique on clinical samples. We detected 15 specific HPV types (6, 16, 18, 31, 35, 42, 51, 52, 55, 56, 58, 59, 66, 67 and 68) examined with specificity without known cross‐reaction to other HPV types. The detection limit for the different HPV types was above 500 plasmids. We compared the performance of the Luminex‐based assay to the established HPV DNA microarray chip for polymerase chain reaction products derived from 53 clinical samples. The evaluation showed excellent agreement. The Luminex‐based HPV genotyping was a sensitive, reproducible technique for the simultaneous genotyping of all clinically relevant genital HPV types. This assay system may be used to provide critical clinical information for early detection of HPV, especially in cases where the HPV copy numbers are low and the latency period of HPV infection is prolonged. (Cancer Sci 2007; 98: 549–554)

Pharmacogenetic analysis of pediatric patients with acute lymphoblastic leukemia: a possible association between survival rate and ITPA polymorphism.

Genetic polymorphisms are important factors in the effects and toxicity of chemotherapeutics. To analyze the pharmacogenetic and ethnic differences in chemotherapeutics, major genes implicated in the treatment of acute lymphoblastic leukemia (ALL) were analyzed. Eighteen loci of 16 genes in 100 patients with ALL were analyzed. The distribution of variant alleles were CYP3A4*1B (0%), CYP3A5*3 (0%), GSTM1 (21%), GSTP1 (21%), GSTT1 (16%), MDR1 exon 21 (77%), MDR1 exon 26 (61%), MTHFR 677 (63%), MTHFR 1298 (29%), NR3C1 1088 (0%), RFC1 80 (68%), TPMT combined genotype (7%), VDR intron 8 (11%), VDR FokI (83%), TYMS enhancer repeat (22%) and ITPA 94 (30%). The frequencies of single nucleotide polymorphisms (SNPs) of 10 loci were statistically different from those in Western Caucasians. Dose percents (actual/planned dose) or toxicity of mercaptopurine and methotrexate were not related to any SNPs. Event free survival (EFS) rate was lower in ITPA variants, and ITPA 94 AC/AA variant genotypes were the only independent risk factor for lower EFS in multivariate analysis, which was a different pharmacogenetic implication from Western studies. This study is the first pharmacogenetic study in Korean pediatric ALL. Our result suggests that there are other possible pharmacogenetic factors besides TPMT or ITPA polymorphisms which influence the metabolism of mercaptopurine in Asian populations.

TotalPlex gene amplification using bulging primers for pharmacogenetic analysis of acute lymphoblastic leukemia

Genetic polymorphism among patients with acute lymphoblastic leukemia (ALL) is an important factor in the effectiveness and toxicity of anti-leukemic drugs. Genotyping of various polymorphisms that impact the outcome of anti-leukemic drug therapy (pharmacogenetics) presents an attractive approach for developing individualized therapy. We developed an easy and accurate method of analyzing multiple genes using a small amount of DNA, which we termed TotalPlex amplification. We used 16 pairs of specific bulging specific primers (SBS primers) for simultaneous amplification of 16 loci in a single PCR tube. Sixteen single nucleotide polymorphisms (SNPs) (CYP3A4*1B A>G, CYP3A5*3 G>A, GSTP1 313 A>G, GSTM1 deletion, GSTT1 deletion, MDR1 exon 21 G>T/A, MDR1 exon 26 C>T, MTHFR 677 C>T, MTHFR 1298 A>C, NR3C1 1088 A>G, RFC 80 G>A, TPMT 238 G>C, TPMT 460 G>A, TPMT 719 A>G, VDR intron 8 G>A, VDR FokI T>C) that have been implicated in the pharmacogenetics of ALL therapy were analyzed by TotalPlex amplification and SNP genotyping. We successfully amplified specific gene fragments using 16 pairs of primers in one PCR reaction tube with minimal spurious amplification products using TotalPlex amplification coupled to a multiplexed bead array detection system. The genotypes of 16 loci from 34 different genomic DNA (gDNA) samples derived using the TotalPlex system were consistent with the results of several standard genotyping methods, including automatic sequencing, PCR restriction fragment length polymorphism (RFLP) analysis, PCR, and allele-specific PCR (AS-PCR). Thus, the TotalPlex system represents a useful method of amplification that can improve the time, cost, and sample size required for high-throughput pharmacogenetic analysis of SNPs.

Identification of Leukemia-Specific Fusion Gene Transcripts with a Novel Oligonucleotide Array

AbstractBackground: Identification of specific chromosomal translocations is essential for the diagnosis and prognosis of leukemia. In this study, we employ DNA microarray technology to detect chromosomal aberrations in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as well as in leukemic cell lines. Methods: Reverse transcription using a random 9-mer primer was performed with total RNA from patients and leukemic cells lines. Multiplex PCR reactions using four groups of primer sets were then performed for amplification of cDNA from reverse-transcribed total RNA samples. Normal and fusion sequences were distinguished by hybridization of the amplified cDNA to a selective oligonucleotide array (SOA) containing 20-30mer synthetic probes. A total of 23 sets of oligomers were fabricated on glass slides for the detection of normal and fusion genes, as follows: BCR/ABL, AML/EAP, AML/ETO, AML/MDS, PML/RARA, NUMA1/RARA, PLZF/RARA, and CBFB/MYH. Results: Gene translocation in leukemia was effectively identified with the SOA containing various leukemiaspecific fusion and normal control sequences. Leukemic fusion sequences from patients and cell lines hybridized specifically to their complementary probes. The probe sets differing by ≈50% at their 5′ or 3′ ends could distinguish between normal and fusion sequences. The entire process of detection was completed within 8 hours using the SOA method. Conclusions: Probe sets on SOA can effectively discriminate between leukemia-specific fusion and normal sequences with a chip hybridization procedure. The oligonucleotide array presents several advantages in identifying leukemic gene translocations, such as multiplex screening, relatively low cost, and speed.

Genotyping of CYP21A2 for Congenital Adrenal Hyperplasia Screening using Allele-Specific Primer Extension followed by Bead Array Hybridization

AbstractBackground: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease caused by mutations in the CYP21A2 gene, which codes for steroid 21-hydroxylase. More than 90% of patients with CAH have mutations in CYP21A2 or have large deletions in the RCCX module on chromosome 6p21.3, which also includes the pseudogene CYP21A1P. Genotyping of CYP21A2 is required for diagnosis of CAH, but current genotyping methods, such as direct sequencing, allele-specific PCR amplification, or PCR amplification and restriction fragment length polymorphism (PCR-RFLP) still need further improvements to reduce test time and cost. Methods: We developed a novel CAH mutation screening method based on allele-specific primer extension (ASPE), followed by bead-array hybridization, for the ten major point mutation sites and the 8 bp deletion in CYP21A2, and a long PCR assay to detect large deletions between CYP21A1P and CYP21A2. After the first long PCR amplification, a second short PCR amplification was adapted to increase the ASPE efficiency. The total genotyping procedure takes approximately 8 hours. Results: Eighteen CAH patients and two controls were tested using the bead-array method. Homozygous or heterozygous large gene deletions and three point mutation sites were detected by this method, and most of the results were consistent with sequencing or PCR-RFLP analysis. Nine of the 18 patients had a large deletion in the RCCX module, which was not easily detected using the conventional genotyping method. Conclusion: A novel CAH mutation screening method has been developed to detect ten point mutations and the 8 bp deletion in CYP21A2, as well as large deletions between CYP21A1P and CYP21A2. This novel genotyping strategy is superior to PCR-RFLP-based methods and equally as accurate as sequencing.

Semantic Search System based on Korean Medicine Ontology

We in this paper propose a semantic search system based on Korean medicine ontology. Semantic search augments search results and improves search accuracy by understanding which concept denotes terms which users is trying to find. Our semantic search system also provides these semantic search capabilities. Moreover, search scenarios which is meaningful in Korean medicine are designed and implemented by analyzing the semantics of Korean medicine ontology. Therefore, our system can help users find the useful search results with respect to Korean medicine by providing the more meaningful information as well as the connected information in ontology.

A Study of Disassembling Major Indication Terms into Minimum Meaning Units and Linking to Diseases

ABSTRACT Objectives : Ontology is a good tool to represent the knowledge and has developed for Traditional Korean Medicine(TKM) in Korea Institute of Oriental Medicine. There are a lot of TKM terms, which have a complex meaning, especially major indication terms of medicinal treatment and terms of symptom and disease. These complex meaning terms result in the low linkage between major indication terms of medicinal treatment and terms of symptom and disease in TKM ontology. We studied to enhance the percentage of the linkage among those data in TKM ontology. Methods : We disassembled major indication terms of medicinal treatment into minimum meaning units and then linked them to enhance the percentage of the linkage among medicinal material, formula and disease ontology based on Traditional Korean Medicine. To retain objectivity, several experts of Korean Medicine used a web- based tool that supports users in refining terms and disassembling them into the minimum meaning efficiently. Results : The outcome shows that the percentage of the linkage among medicinal material, formula and disease ontology increased. By linking disassembled major indication terms to symptoms and diseases, the amount of

Study on the Ontology Linking by Acupuncture Terms

Objectives : The goal of this paper is to enhance the percentage of linkages between an acupuncture point ontology and an acupuncture treatment ontology. Methods : Terms of the effect from acupuncture points and terms of the treatment method from diseases are selected in the ontologies. We have disassembled the terms into minimum meaning units and then linked them again. Results : By linking terms of the effect and terms of the treatment method, the meaninful information on the acupuncture point and treatment ontology is increased. Conclusions : The links between points for acupuncture points and treatment methods for diseases could improve the information in the ontologies. This also enables the knowledge representation for the use of acupuncture points with accompanied symptoms, causes of disease, and treatment methods.