Abstract
High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.
Highlights
High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation
High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (–308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (–592 C/A, –819 T/C, –1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) single nucleotide polymorphism (SNP) were developed on the LightCycler® 480
The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0)
Summary
High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation. We can describe high, intermediate and low cytokine producer status according to the genotype. For tumor necrosis factor alpha (TNF-α), at position –308 within the promoter region, A/A, A/G and T/T genotypes correlate with a high, intermediate and low TNF-α production, respectively.[1] the substitutions in codon 10 (+869) and codon 25 (+915) of tumor growth factor beta 1 (TGF-β1) gene correlate with the protein production.[2] The codon 10 *T (Leu) and codon 25 *G (Arg) of TGF-β1 are the high responder alleles. The genotypes of ACC/ACC, ACC/ATA and ATA/ATA are classified as low, GCC/ACC and GCC/ATA as intermediate, and GCC/GCC as high IL-10 producer genotypes.[3] at position +874 within the intron 1 of interferon gamma (IFN-γ) gene, the genotypes T/T, T/A and A/A are associated with high, intermediate and low expression, respectively.[4]
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