Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 367 High-resolution Melting PCR Analysis for Genotyping Lys109Arg and Gln223Arg in Patients with Renal Cell Carcinoma Hai-Ping Zhang1, Jian Zou2, Ying Yin2, Jun Ruan3, Zhuo-Qun Xu3, Shu-Dong Yang4, and Hui-Jun Mu2 1Department of Derma Science Laboratory, Wuxi No.2 People's Hospital affiliated to Nanjing Medical University, 2De- partment of Clinical Laboratory Science, Wuxi People's Hospital affiliated to Nanjing Medical University, 3Department of urinary surgery, Wuxi People's Hospital of Nanjing Medical University, and 4Department of pathology, Wuxi People's Hospital of Nanjing Medical University, Wuxi, Jiangsu Province, P. R. China Abstract. 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) re- mains 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 ex- plore 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 com- plete 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. Key words: high-resolution melting PCR analysis; leptin receptor; single nucleotide polymorphism; renal cell carcinoma. Introduction SNP analysis. A large number of distinct approach- es applying different allele differentiation strategies Single nucleotide polymorphisms (SNPs) are the and detection methods have been developed [2]. most frequently appearing genetic variation in the human genome, the total number exceeding 9 mil- High-resolution melting (HRM) analysis is an lion and accounting for over 90% of all the differ- emerging sequence variation scanning technology. ences between individuals [1]. SNPs are important This novel approach is based on the melting prop- markers that link sequence variations to phenotypic erties of double-stranded DNA. Bases variations in changes. Association studies involving the large- PCR amplicons are detected by changes in melting scale analysis of SNPs can help to advance the un- profiles as the temperature is modified in the pres- derstanding of human physiology and elucidate the ence of DNA intercalating dyes. HRM is a simple, molecular basis of diseases. Therefore, a great deal flexile, inexpensive, sensitive, and specific method. of effort has been committed to the development of Indeed, this technique has been widely employed to rapid, accurate, and cost-effective technologies for screen patients for pathogenic variants, including mutation detection [3], SNP typing [4,5], methyla- Address correspondence to Hui-Jun Mu, No. 299, Qing yang Road, Wuxi,JiangsuProvince,P. R.China,214023;phone:+86051085350356 tion analysis [6,7] and differentiation of bacterial and +8613914129397; e mail: [email protected] [8,9] or parasitic species [10]. 0091-7370/16/0400-367. © 2016 by the Association of Clinical Scientists, Inc. 368 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 Figure 1. Comparison of high resolution melting curve analysis (panel A, B), PCR-RFLP analysis (panel C) and se- quence analysis (panel D, E, F) of Lys109Arg and Gln223Arg. The panel A and B showed the melting curves of Lys109Arg and Gln223Arg in a 96-well platform, respectively. Each melting curve is derived from a single amplicon. Three genotypes were identified and are depicted by red, blue and green differential melting curves. The panel C showed the PCR-RFLP analysis for Gln223Arg. The panel D, E and F showed the three genotype of Lys109Arg by direct DNA sequencing (The SNP position is indicated by*). Leptin receptor (LEPR), a member of the cytokine the molecular basis of RCC development has not receptor superfamily, is a single transmembrane been clarified. The relationship between the poly- protein [11]. LEPR interacting with leptin plays an morphisms of LEPR and RCC also remains important role in energy expenditure [12], cell unclear. proliferation, and apoptosis [13,14]. LEPR has six alternatively spliced isoforms, which are classified The aim of this study is to develop an HRM tech- according to the length of their cytoplasmic do- nology for Lys109Arg and Gln223Arg genotyping main to four short, one long, and one soluble form and to evaluate the associations of the LEPR gene [15]. The long LEPR isoform is believed to have variations with RCC. biological activity and is essential for signal trans- duction through JAK-STAT pathway [16]. There Materials and Methods are several common variants of the LEPR genes, and four allelic variants are associated with amino Patients. In this study, 83 patients who were histologi- acid changes (Lys109Arg, Gln223Arg, Ser343Ser, cally diagnosed as RCC between December 2005 and and Lys656Asn). Potential relevance of these vari- September 2012 were enrolled. The patients comprised 17 females and 66 males, aged from 17 to 85 years (me- ants with different cancers have been evaluated, in- dian 57 years). One hundred and sixty-one controls cluding cancers of breast [17,18], colorectum were selected from the Health Screening Center of [19,20], gastric [21], lung [22], and oral [23]. Wuxi People's Hospital. Controls were matched to cas- es by ethnicity, age, and sex. Subjects with underlying RCC is the second largest and the most lethal can- diseases or abnormal laboratory data were excluded cer in the urinary system, accounting for about from the controls. 2.1% of malignant tumors in adults [24]. RCC is a This study was approved by the Ethics Committee of multi-gene-related cancer, and pathogenesis is com- the institution, and patients have provided informed plex. The majority of cases contain mutations or consent. All protocols conformed to the ethical guide- epigenetic silencing of the VHL gene [25]. However, lines of the 1975 Helsinki Declaration. Genotyping leptin receptor of RCC patients with HRM analysis 369 DNA extraction. Tissue samples from 83 patients with each dNTP, 1 U Taq DNA polymerase (Promega, RCC were obtained from surgical specimens. Peripheral Madison, USA), and about 150 ng DNA. The amplifi- blood samples from 161 healthy individuals were col- cation conditions were as follows: 5 min initial denatur- lected in tubes containing EDTA-K2 as the anticoagu- ation at 94°C followed by 35 cycles of amplification, lant. Genome DNA was extracted with Wizard® genom- each including 30 s denaturation at 94°C, 45 s anneal- ic DNA purification Kit (Promega, Madison, USA) ing at 58°C, and 45 s extension at 72°C. The PCR prod- following the manufacturer’s instructions. ucts were sequenced by Sangon Biotech Co. Ltd (Shanghai, China) on the ABI Genetic Analyzer 3730 Genotyping of Lys109Arg (A/G) and Gln223Arg (A/G) XL (Applied Biosystems, Foster City, USA). SNPs with HRM analysis. Genotyping for Lys109Arg (A/G) and Gln223Arg (A/G) gene polymorphisms was PCR-RFLP analysis of Gln223Arg (A/G). Polymerase performed on a LightCycler 480 instrument (Roche chain reaction and restriction fragment length polymor- Applied Science, Basel, Switzerland) with HRM analy- phism (PCR-RFLP) was used as a reference method for sis. HRM analysis comprised three phases: a PCR reac- genotyping of the Gln223Arg. The primers used for tion, DNA melting process, and gene scanning for data PCR were Gln223Arg forward (5'- ACC CTT TAA analysis. The primers used for the HRM assay were GCT GGG TGT CCC AAA TAG -3') and reverse (5'- Lys109Arg (A/G) forward (5'-CGG AGT GAG CAA AGC TAG CAA ATA TTT TTG TAA GCA ATT -3'). GAT AGA-3') and reverse (5'- AGC TAA TGC TTA The final reaction mixtures consisted of 150 ng of DNA, CCT ATT TGT-3'), Gln223Arg (A/G) forward (5'- 1×PCR buffer, 1.5 mmol/L MgCl2, 0.5 μM of both for- GCC AAA CTC AAC GAC ACT CTC -3') and reverse ward and reverse primer, 200 μmol/L of each dNTP, and (5'- GGG CTG AAC TGA CAT TAG AGG -3'). PCR 0.5 U of Taq (Promega, Madison, USA) in a volume of was performed with 96-well closed-tube platforms 25 μL. The Gln223Arg cycling protocol was initiated by (BIOplastics, Landgraaf, Holland). The 20 μL PCR mix- one cycle at 94°C for 5 min, followed by 35 cycles of ture included 2.5 mM MgCl2, 0.5 μM of both forward denaturation at 94°C for 30 s, annealing at 55°C for 45 and reverse primer, 200 μM of each dNTP, 0.5 U Taq s, extension at 72°C for 45 s. The PCR products were DNA polymerase (Promega, Madison, USA), 1.0 μL digested with Msp I (Fermentas, Vilnius, Lithuania) re- EvaGreen (Biotium, Canifornia, USA), and about 150 striction enzyme following the manufacturer’s instruc- ng DNA. A universal touchdown PCR program was tions. The digested products then underwent electro- used for amplifying the two amplicons.