Sung-Min Chun

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.