High-resolution Melting PCR Analysis for Genotyping Lys109Arg and Gln223Arg in Patients with Renal Cell Carcinoma

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Although several studies have documented the role of leptin receptor gene polymorphisms in cancers, the association between leptin receptor gene polymorphisms and renal cell carcinoma (RCC) remains unknown. The aim of this study was to develop a high-resolution melting (HRM) approach for genotyping single nucleotide polymorphisms of leptin receptor gene on the LightCycler 480, and to explore the relation between polymorphisms of the leptin receptor gene and RCC. The study population consisted of 83 patients with renal cell carcinoma and 161 healthy control subjects. The Lys109Arg (A/G) and Gln223Arg (A/G) polymorphisms of leptin receptor gene were examined with HRM assay. Direct DNA sequencing and PCR-restriction fragment length polymorphisms were used as a reference method for genotyping Lys109Arg and Gln223Arg, respectively. Three genotypes of Lys109Arg or Gln223Arg were clearly distinguishable from the melting curve shapes with HRM assay. The data also showed the results of the direct DNA sequencing or PCR-restriction fragment length polymorphisms analysis were in complete concordance to genotyping results obtained by HRM (kappa=1.0). In addition, the data showed the G-G haplotype frequency was higher (p<0.05), and that the A-G (p<0.001) and G-A (p<0.01) haplotypes frequencies were lower in the RCC than controls. We developed a rapid, low cost, high-throughput and reliable single-tube technology for genotyping Lys109Arg and Gln223Arg polymorphisms. In addition, our data suggest that Lys109Arg and Gln223Arg gene polymorphisms are associated with RCC in Chinese Han studied population.

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