High-Resolution Melting PCR Analysis for Genotyping Lys109arg and Gln223arg in Patients with Renal Cell Carcinoma

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) 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 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 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.

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].

Figure 1. Comparison of high resolution melting curve analysis (panel A, B), PCR-RFLP analysis (panel C) and sequence 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 cases 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. The temperature phoresis in 2% agarose gel. Results were interpreted cycling protocol consisted of an initial denaturation step based on the positive amplification band (Gln/Gln at 95°C for 2 min, before the initial annealing tempera- (A/A): 416 bp; Arg/Arg (G/G): 291 bp, 125 bp; Gln/ ture of 65°C was decreased by 0.5°C each cycle for 20 Arg (A/G): 416 bp, 291 bp, 125 bp). cycles and held at 55°C for 10 seconds for the next 30 cycles. For all cycles, denaturation was performed at Statistical analysis. Statistical analysis was carried out 95°C for 10 s and extension was carried out at 72°C for using the Statistical Package for the Social Sciences 15 s. The HRM was performed at the end of each reac- (SPSS) version 15 (SPSS Inc, USA). Allele and genotype tion and consisted of an increasing temperature from 70 frequencies, Hardy-Weinberg equilibrium (HWE), and to 99°C at intervals (ramps) of 0.02°C/s. HRM analysis linkage disequilibrium (LD) were calculated using the was carried out with the gene-scanning module (version Arlequin program, version 3.5 [26]. The frequencies of 1.5). Software programs employ a three-step analysis: 1) alleles and genotypes were compared in RCC and con- normalization by selecting linear regions before (100% trol group by Chi-square test and Fisher’s exact test. fluorescence) and after (0% fluorescence) the melting Odds ratios and 95% confidence intervals (CIs) for rela- transition, 2) temperature shifting by moving the curves tive risk of RCC were calculated. The agreement be- along the x-axis, facilitating grouping and 3) use of the tween HRM method and the PCR-RFLP or PCR-direct autogroup function. sequence analysis was assessed by kappa test. A probabil- ity value of p<0.05 was considered statistically signifi- PCR-direct sequence analysis of Lys109Arg (A/G). cant, and all reported p values are two-tailed. Direct DNA sequencing was performed to confirm the results of HRM analysis for Lys109Arg polymorphism. Results The primers used for PCR and sequencing were Lys109Arg forward (5'- CCT GCT GGA CTC TCA Comparison of HRM analysis and direct DNA AAG AA -3') and reverse (5'- TGT TAA AAT CAT sequencing for genotyping Lys109Arg. HRM AGC CAT AAG ACA TCT -3'). PCR was performed in analysis effectively distinguished the polymorphism 50 μL volume; the mixture included 2.5 mM MgCl2, 0.5 μM of both forward and reverse primer, 200 μM of of the situ Lys109Arg A/G. The Figure 1A shows 370 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016

Table 1. Allele and genotype frequencies of Lys109Arg and Gln223Arg in the RCC and control subjects.

Gene Allele or RCC (n =83) Control (n =161) χ2 value P Odds 95% CI Genotype n (%) n (%) Ratio

Lys109Arg G 142 84.52 273 84.78 0.006 0.940 0.98 0.58-1.64 A 26 15.48 49 15.22 G/A 22 26.19 41 25.47 0.015 0.902 1.04 0.57-1.90 A/A 2 2.38 4 2.48 0.002 0.957 1.05 0.17-5.34 G/G 60 71.43 116 72.05 0.011 0.918 0.97 0.54-1.74 Gln223Arg G 143 85.12 256 79.50 2.302 0.129 1.48 0.89-2.44 A 25 14.88 66 20.50 G/A 21 25.00 52 32.30 0.006 0.976 0.79 0.53-1.79 A/A 2 2.38 7 4.35 0.002 0.957 1.05 0.17-5.34 G/G 61 72.62 102 63.35 0.009 0.925 1.03 0.57-1.86

Abbreviations: n, absolute number. RCC, renal cell carcinoma. CI, confidence interval. the curve of melting profiles of normalized data of Alleles and genotypes of Lys109Arg and Lys109Arg, and distinguishable inferences between Gln223Arg in the RCC and control subjects. the three genotypes are evident. Direct DNA se- Table 1 shows the allele and genotype frequencies quencing was performed to confirm the results of of Lys109Arg and Gln223Arg gene in the RCC HRM analysis for Lys109Arg of all the samples. The subjects and control individuals, respectively. The direct DNA sequencing results of three genotypes of data showed there were no significant differences Lys109Arg are shown as Figures 1D-F. When analyz- in allelic frequencies and genotype distributions ing the specimens of 83 RCC and 161 controls, we of the examined polymorphisms of the LEPR found a 100% concordance between the HRM analy- genes between the RCC and control subjects. sis and the direct DNA sequencing assay for Lys109Arg, with a kappa test value of 1.0. Haplotypes of Lys109Arg and Gln223Arg in the RCC and control subjects. Examination of the Comparison of HRM and PCR-RFLP analysis for haplotypes of Lys109Arg and Gln223Arg re- genotyping Gln223Arg. Figure 1B shows the differ- vealed evidence for association in RCC subjects. ence among the melting curves for normalized data of We found that the G-G haplotype frequency of the situ Gln223Arg A/G in a 96-well platform. It is Lys109Arg and Gln223Arg was significantly easy to distinguish three polymorphisms of higher in the RCC subjects than in the controls Gln223Arg A/G via the melting curve shapes. For (140/166 and 243/322, respectively; p=0.024, comparison, we analyzed the same samples by PCR- OR=1.75; 95% CI=1.07-2.86). However, the RFLP; Figure 1C shows the results. When analyzing A-G haplotype frequencies of Lys109Arg and the results of 83 RCC and 161 controls, we found the Gln223Arg in the RCC subjects were lower than agreement between the HRM and PCR-RFLP analy- in the controls (1/166 and 30/322, respectively; sis for genotyping Gln223Arg to be 100%, with a p=0.0002, OR=0.06; 95% CI=0.01 to 0.44). kappa test value of 1.0. Similarly, the G-A haplotype frequencies of Lys109Arg and Gln223Arg in the RCC subjects HWE test for Lys109Arg and Gln223Arg in the were lower than in the controls (0/166 and RCC and control subjects. The HWE tested for 13/322, respectively; p=0.009). The data also Lys109Arg revealed no significant deviation (p=0.99 showed no significant differences in A-A haplo- in the patients and p=0.76 in the controls). Likewise, type frequency of Lys109Arg and Gln223Arg be- SNP of Gln223Arg was in Hardy-Weinberg equilib- tween the RCC and control subjects (25/166 and rium in the patients and controls (p=0.90 in the pa- 36/322, respectively; p=0.219, OR=1.41; 95% tients and p=1.00 in the controls). CI=0.81 to 2.44). Genotyping leptin receptor of RCC patients with HRM analysis 371

Linkage disequilibrium analysis between HRM analysis technology is a recently developed Lys109Arg and Gln223Arg alleles. The linkage gene detection technique and is rapidly being ad- disequilibrium (LD) for Lys109Arg and Gln223Arg opted for SNP analysis. HRM involves a PCR of polymorphisms was analysed. The data showed the target gene in the presence of a saturating inter- Lys109Arg A allele was in LD with the Gln223Arg calating dye and subsequent melting of the ampli- A allele in the RCC subjects (p<0.05, standardized cons by gradual increase of the temperature. The disequilibrium values r2=0.9547). However, in the melting profile depends on the base composition, control subjects, the Lys109Arg A allele was not in length, sequence, or strand complementarity of the LD with the Gln223Arg A allele (r2=0.3091). product [34]. HRM analysis is highly suitable for the detection of single-base variants, deletions, or Discussion insertions [35]. In addition, HRM method offers several attractive advantages over other convention- Specific SNPs in the human genome are tags of dif- al gene scanning methods, such as no post-PCR ferent ethnic populations and their individual dif- processing steps, complete closed-tube format, and ferences. SNPs are also markers for susceptibility to rapid turnaround time [36]. disease and drugs [27]. A number of SNP genotyping methods have emerged and vary in terms of de- Here we show that HRM is an effective method for tection system, reaction formats, and allelic dis- discriminating Lys109Arg and Gln223Arg poly- crimination. The conventional methods for morphisms. The data shows that the differences be- detecting SNPs include allele-specific oligonucle- tween the three allelic forms of Lys109Arg are dis- otide (ASO) hybridization, restriction fragment tinguishable as a result of the melting curve shape. length polymorphism (RFLP), sequence-specific Similarly, three genotypes of Gln223Arg were clear- primers PCR (PCR-SSP), single strand conforma- ly distinguishable from the melting curve shapes. tional polymorphism (SSCP), TaqMan genotyping Although HRM has a high sensitivity for detecting assay, and direct sequencing [28]. sequence variants [37], to confirm the accuracy of the HRM assays, Lys109Arg polymorphisms of all The LEPR is a single-transmembrane protein be- the samples were verified by direct DNA sequenc- longing to the cytokine receptor family. There are ing. The results of direct DNA sequencing were in several common variants of the LEPR genes, and absolute concordance with those of HRM. allelic variants at codons 109 and 223 are associated Likewise, to allow further confirmation of the spec- with amino acid changes [29]. Currently, PCR- ificity of the assays, all the samples underwent SSCP analysis and TaqMan assay are the most com- PCR-RFLP for Gln223Arg polymorphisms. The mon methods used for genotyping Lys109Arg data shows the results of the PCR-RFLP to be in polymorphism [30,31]. Similarly, the methods complete concordance with genotyping results ob- most commonly employed to genotype Gln223Arg tained by HRM. All of these suggest that the HRM polymorphism are PCR-RFLP and TaqMan assay assay is reliable single-tube technology for genotyp- [31,32]. PCR-SSCP and PCR-RFLP are time-con- ing Lys109Arg and Gln223Arg polymorphisms. suming methods for the separation of the products Further, HRM assay is a rapid and high-through- by gel electrophoresis. TaqMan assay uses two put method, which can genotype 96 samples in an TaqMan probes that carry different fluorescent dyes hour on our platform. In this study, we describe an and are specific for each SNP. The presence of an efficient and robust method for the differentiation allele is detected by the corresponding fluorescence of Lys109Arg and Gln223Arg polymorphisms. signal generated via 5’-exonuclease cleavage of the Compared to other genotyping methods, HRM- probe [33]. The main drawback for the TaqMan based genotyping offers superior resolving power SNP assay is its expensive probe and double work- and is a time, labor, and cost saving method. load. Therefore, we decided to develop an HRM analysis system to genotype Lys109Arg and Leptin, as a member of adipokine, plays a key role Gln223Arg polymorphisms, which is a time- and in the regulation of body-fat metabolism. It also labor-cost saving method. acts as an important tumor cell growth factor, 372 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 acting in cell proliferation and apoptosis [13]. limited sample size and the ethnically limited study Leptin selectively binds with the LEPR and exerts population, additional studies with larger sample its biological effects. Studies have described several sizes and in other ethnic populations are necessary common sequence variants in LEPR gene, and po- to demonstrate the relationships of these polymor- tential associations of Lys109Arg and Gln223Arg phisms with RCC risk. polymorphisms with cancer risk have been suggest- ed [22,38]. However, few studies have thus far ad- In conclusion, this report presents a rapid, low cost, dressed the association between these polymor- high throughput and reliable single-tube technolo- phisms and RCC risk. Thus, the present study was gy for genotyping Lys109Arg and Gln223Arg poly- performed to evaluate this association in a patient- morphisms. Furthermore, this analysis suggests control population. In this study, the data showed that Lys109Arg and Gln223Arg polymorphisms there were no significant differences in allelic fre- are significantly correlated with RCC risk and that quencies and genotype distributions of the exam- the G-G haplotype of Lys109Arg and Gln223Arg ined LEPR gene polymorphisms between the RCC variant is a high-risk factor for developing RCC. cases and controls. However, haplotype analysis Acknowledgements showed an association between RCC and haplo- This work was supported by the Development Fund of Wuxi types of Lys109Arg and Gln223Arg. The data Hospital Management Center [Grant no. YGZ1102]. showed the G-G haplotype frequency was higher (p<0.05), the A-G (p<0.001) and G-A (p<0.01) References haplotypes frequencies were lower in the RCC cases 1. Rocha D, Gut I, Jeffreys AJ, Kwok PY, Brookes AJ, Chanock than in controls. These suggest that he patients car- SJ. Seventh international meeting on single nucleotide poly- rying G-G haplotype were at a modestly increased morphism and complex genome analysis: 'Ever bigger scans risk of RCC, but that the patients carrying A-G or and an increasingly variable genome'. Hum Genet 2006;119:451-456. G-A haplotype were negatively associated with the 2. Edenberg HJ, Liu Y. Laboratory methods for high-throughput risk of RCC. Furthermore, our data showed the genotyping. Cold Spring Harb Protoc 2009;2009:pdb top62. 3. Hlinkova K, Babal P, Berzinec P, Majer I, Ilencikova D. 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