Association of Polymorphisms in Toll-Like Receptors 4 and 9 with Autoimmune Thyroid Disease in Korean Pediatric Patients.

BACKGROUND: Toll-like receptors (TLRs) have been suggested to be associated with the development of AITD. METHODS: Fifteen single-nucleotide polymorphisms in 7 TLR genes were analyzed in 104 Korean children (girls = 86, boys = 18) with AITD (Hashimoto disease (HD) = 44, Graves' disease (GD) = 60, thyroid-associated ophthalmopathy (TAO) = 29, and non-TAO = 31) with 183 controls. RESULTS: GD showed higher frequencies of the TLR4 rs1927911 C allele than control. TAO showed a lower frequency of the TLR4 rs1927911 CT genotype and non-TAO showed a higher frequency of the TLR4 rs1927911 CC genotype than control. The frequency of the TLR9 rs187084 CC genotype in TAO was higher than that in non-TAO. GD females showed a higher frequency of the TLR4 rs10759932 T allele, rs1927911 CC genotype, and the rs1927911 C allele than controls. GD males showed a higher frequency of the TLR4 rs10759932 CC genotype and rs1927911 TT genotype and lower frequency of the rs1927911 CT genotype than control. The frequency of the TLR4 rs10759932 CC genotype, C allele and rs1927911 TT genotype, and T allele in a GD female were lower than in a GD male. CONCLUSIONS: Our results suggest that TLR4 and 9 polymorphisms might contribute to the pathogenesis of GD and TAO.

Comprehensive analysis of cytokine gene polymorphisms defines the association of IL-12 gene with ophthalmopthy in Korean children with autoimmune thyroid disease.

In early onset autoimmune thyroid disease (AITD) showing a strong genetic tendency, cytokines have been suggested to play a critical role in the development of AITD. To directly compare the influences of several cytokine gene polymorphisms, 25 single nucleotide polymorphisms (SNPs) in 17 cytokine genes were analyzed on 104 Korean children with AITD [Hashimoto's disease (HD) = 44, Graves' disease (GD) = 60 (thyroid-associated ophthalmopathy (TAO) = 29, non-TAO = 31)] and 192 controls. Compared with healthy controls, any significant association with polymorphisms of cytokine genes was not found in HD and GD. Among GD patients, non-TAO group only showed significant associations with IL-12 C allele (rs3212227: A > C) (76.6% vs. 51.6%, OR = 0.3 [0.15-0.71], Pc = 0.007). Particularly, the frequency of IL-12C allele was significantly lower in the non-TAO group than in the TAO group (82.8% vs. 51.6%, Pc = 0.018). Our comprehensive analysis of cytokine gene polymorphisms suggests that IL-12 gene may play impact on specific pathogenesis of ophthalmopathy in Korean children with AITD.

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 αß 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.

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-1ß, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-ß1, 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.

Association of Toll-like receptor 10 polymorphisms with autoimmune thyroid disease in Korean children.

BACKGROUND: The Toll-like receptors (TLRs) are germline-encoded receptors that play an essential role in initiating the immune response against pathogens. In this study, we assess the association of TLR polymorphism with autoimmune thyroid disease (AITD) in Korean children. METHODS: We investigated three polymorphisms in the TLR10 gene (rs4129009, rs11096956, and rs10004195) in 85 Korean AITD patients (Graves' disease, [GD]=50, Hashimoto's disease [HD]=35; thyroid-associated ophthalmopathy [TAO]=23, non-TAO=62; male=16, female=69; mean age=13.4±3.1 years) and 279 healthy control subjects. RESULTS: In patients with AITD, the frequencies of the TLR10 rs4129009 A allele (odds raio [OR]=3.9, corrected p=0.04) and rs10004195 T allele (OR=2.8, corrected p=0.02) were higher than in the healthy controls, whereas the TLR10 rs4129009 GG genotype (OR=0.3, corrected p=0.04) and rs10004195 AA genotype (OR=0.4, corrected p=0.02) showed lower frequencies. The TLR10 rs11096956 did not show any significant association. These significant associations were also found in the non-thyroid-associated ophthalmopathy (TAO) group, but not in the TAO group. The haplotype (AGT) frequency of TLR10 rs4129009, rs11096956, and rs10004195 was higher in the AITD group than in healthy controls (OR=2.1, corrected p=0.03). CONCLUSIONS: Our results suggest that TLR10 polymorphisms may contribute to the pathogenesis of AITD.

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.