In-Cheol Baek

HLA-Cw polypmorphism and killer cell immunoglobulin-like receptor (KIR) gene analysis in Korean colorectal cancer patients

PURPOSE Natural killer cells (NK cells) play important roles in protecting the patient from the early development of cancers, and are activated or inhibited by killer cell immunoglobulin-like receptors (KIR), which bind to HLA class I. In the present study, we investigated the KIR genotype of Korean colorectal cancer patients. METHODS DNA samples were extracted from peripheral blood cell samples taken from Korean colorectal cancer patients and a control group. KIR genes were amplified using PCR-SSP methods, and HLA-Cw genes were characterized using PCR methods. The results were analyzed to assess the difference between colorectal cancer patients and the normal control group. RESULTS In the present study, the frequency of KIR2DS5 (33.2% vs. 20.8%, p-value < 0.007) was higher in the colorectal cancer group, and in the rectal cancer subgroup, the frequencies of KIR3DL1 (93.2%, vs. 98.1%, p-value < 0.05), KIR2DS2 (7.8% vs. 19.5%, p-value < 0.01), and KIR2DS4 (93.2% vs. 98.1%, p-value < 0.05) were lower significantly. The frequencies of HLA-Cw6 (9.1% vs. 15.7%, p-value < 0.05) and HLA-Cw7 (17.4% vs. 27.7%, p-value < 0.02) were lower in the colorectal cancer group. Of the patients with HLA-C1 homozygote, the frequency of KIR2DS2 was decreased significantly (5.8% vs. 14.5%, p-value < 0.004). CONCLUSIONS The frequency of KIR2DS5 is higher in Korean colorectal cancer patients, and in the rectal cancer subgroup, the frequencies of KIR3DL1, KIR2DS2 and KIR2DS4 are lower. Among the patients with HLA-C1 homozygote, the frequency of KIR2DS2 is decreased. Therefore, KIR2DS2 in presence of its ligand (HLA-C1 group) may have a protective effect against colorectal cancer.

Multiplex Genotyping of Cytokine Gene SNPs Using Fluorescence Bead Array

Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1α, IL-1R, IL-1RA, IL-1β, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-β1, and TNF-α, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.

Microarrays for high‐throughput genotyping of MICA alleles using allele‐specific primer extension

The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

The HLA-DRB1*09:29 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐DRB1*09:29 differs from HLA‐DRB1*09:01:02:01 in a codon 75 in exon 2.

The HLA-C*07:478 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐C*07:478 differs from HLA‐C*07:02:01:01 in codon 81 in exon 2.

HLA-B*40:330 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐B*40:330 differs from HLA‐B*40:06:01:01 in codons 113 and 116 in exon 3.

The HLA-A*33:110 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐A*33:110 differs from HLA‐A*33:03:01:01 in a codon 149 in exon 3.

MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8+ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations.

The HLA-DQB1*04:02:03 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐DQB1*04:02:03 differs from HLA‐DQB1*04:02:01:01 in a codon 62 in exon 2.

The HLA-C*06:66 allele identified in a volunteer donor for hematopoietic stem cell transplant

HLA‐C*06:66 differs from HLA‐C*06:02:01:01 in codon 151 in exon 3.