Identification of Cytokine SNPs Using LightCycler Hybridization Probes and Melting Curve Analysis

Ernest Albanese, Xiao Y. Yang, and Helen Fernandes* Department of Pathology and Laboratory Medicine UMDNJ-New Jersey Medical School, Newark, U.S.A. *Corresponding author: fernande@umdnj.edu An increasing number of genetic-association studies have implicated polymorphisms of cytokine genes as host genetic factors that influence susceptibility to autoimmunity, infectious disease, and transplantation tolerance. Tumor necrosis factor (TNF-) is representative of a pro-inflammatory cytokine that can counteract the anti-inflammatory interleukin 10 (IL-10) to maintain an inflammatory milieu that favors conditions such as septic shock, allograft rejection, and other disease processes. Cytokine production is under genetic control, and certain single nucleotide polymorphisms (SNPs), which lead to allelic variants of cytokine genes are associated with higher or lower cytokine production in vitro. We have analyzed the genetic variations in the promoter region of TNF- and IL-10 with real-time amplification and melting curve analysis of the LightCycler Instrument. When compared to sequence-specific polymerase chain reaction (PCR-SSP) analysis, the results obtained on the LightCycler were rapid, reproducible, and more reliable than the conventional methodology.

Helen Fernandes

LIGHTCYCLER

Introduction
The analysis of single nucleotide polymorphisms (SNPs) in cytokine genes has begun to shed some light on the roles of cytokines in the pathophysiology of various diseases. Allelic SNPs in the promoter region of interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-) genes can affect the respective levels of cytokine produced. It has been found that the presence of a G-to-A polymorphism at position 308 of the TNF- gene can multiply transcription by six- or sevenfold. This significantly increases the level of cytokine that is produced. Also significant in cytokine expression are two polymorphisms located in the IL-10 promoter region at positions 1082 (G to A) and 819 (T to C). These allelic mutations result in lower expression levels of IL-10. Earlier studies from our laboratory analyzed TNF- and IL-10 using sequence-specific PCR (PCR-SSP) [1]. By applying such analyses we were able to correlate some of these cytokine SNPs with liver transplant rejection [2]. In this report we discuss our use of the LightCycler Instrument and specific hybridization probes to analyze these SNPs. The three cytokine SNPs (TNF- 308, IL-10 819, and IL10 1082) were differentiated based on melting peak analysis. Analysis by melting peaks using the LightCycler Instrument has proved to be specific and rapid with better discriminatory capabilities than PCR-SSP.

Materials and Methods

Sample material and DNA isolation A total of 30 healthy blood donors were screened for all three SNPs using two different methodologies. Genomic DNA of all 30 specimens was extracted using a commercial kit. a Genotyping of the TNF-a 308, IL-10 1082, and IL-10 819 SNPs All three SNPs were first genotyped using an established PCR assay with sequence-specific primers (as previously reported [1]). The same 30 specimens were then analyzed with the LightCycler SNP assay using melting peak analysis to discriminate alleles with the polymorphism from those without. The same primer pair was used for amplification of the IL-10 1082 and IL-10 819 SNPs. The sensor probes were labeled with 3-fluorescein, and the anchor probes with 5-LightCycler Red-640. The PCR mixture was prepared using LightCycler FastStart DNA Master Hybridization Probes (Roche Applied Science). The concentration of Mg2+ was 4 mM, primers 0.5 M, and probes 0.05 M. After an initial denaturation step of 95C for 8 minutes, PCR was run for 40 cycles using the following conditions: denaturation (95C, 10 seconds, ramp rate: 20C s-1), annealing (55C, 10 seconds, ramp rate: 20C s-1), and

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a
Fluorescence -d(F2/F1)/dT

0.06 0.05 0.04 0.03 0.02 0.01 0 -0.01 40 45 50 55 60 65 70 Temperature (C) 75 80 TNF G/G TNF A/G TNF A/A

b
Fluorescence -d(F2/F1)/dT

0.0030 0.0025 0.0020 0.0015 0.0010 0.0005 0 -0.0005 -0.0010 40 45 50 55 60 65 70 Temperature (C) 75 80 IL-10 T/T IL-10 C/T IL-10 C/C

0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 0 -0.005 -0.010 50

Fluorescence -d(F2/F1)/dT

IL-10 A/A IL-10 A/G IL-10 G/G

55

60 65 Temperature (C)

70

75

Figure 1: Melting peak analysis of three cytokine promotor SNPs TNF- 308 (a), IL-10 819 (b) and IL-10 1082 (c) on the LightCycler Instrument. The figures demonstrate homozygous and heterozygous peaks for each SNP.

extension (72C, 10 seconds, ramp rate: 20C s-1.). Melting curves were generated using different parameters for optimal discernment of the three SNPs.

Results
Figures 1a, 1b, and 1c correspond to the melting peak analysis, which was obtained using the LightCycler Instrument to identify the TNF- 308, IL-10 819, and IL-10 1082 genotypes, respectively. The genotypic composition of the 30 specimens tested in both assays was comparable. For TNF the incidence of the homozygous mutant allele in the normal population is less than 5% [2]. Our results from both assays picked up one TNF- mutant (A/A) in the 30 specimens tested, indicating an incidence of about 3%. Similarly, for the IL-10 819 and IL-10 1082 SNPs, the results obtained by both methodologies were alike and coincided with earlier reports. The results obtained from the melting curve analysis were unambiguous and much easier to interpret when compared to the gel identification using sequencespecific primers. The uncertainty in PCR-SSP was primarily due to the presence of faint (ghost) bands in the gels that were analyzed after amplification. These bands are present because the primers for both wild-type and mutant alleles differ by just a single nucleotide at the 3 end of the primer. The stringency of gel-based PCR sometimes does not allow for a clear discrimination of the genotype. In contrast, the LightCycler Instrument, by virtue of the probes that generate the melting curve, always gave clear and discrete genotypes.

Earlier reports have used methodologies that involve PCRSSP, PCR-RFLP, heteroduplex analysis, and invader technology. Our own recent reports have used PCR-SSP as the method of choice [2]. In this study using the LightCycler technology, we obtained rapid, reliable, and reproducible results that were very clear and easy to interpret. The LightCycler Technology has advantages over other methodologies, such as PCR-SSP, particularly because of its rapid and precise discrimination between mutant and wildtype alleles. The gel patterns obtained by PCR-SSP could result in an incorrect interpretation. In this study even though the results obtained by both methodologies were similar the confidence level was much higher with the LightCycler Technology and melting curve analysis than with PCR-SSP. In conclusion, cytokine SNP analysis on the LightCycler Instrument can be easily used in research studies. The results obtained from such investigations could offer greater insight into the understanding and monitoring of disease progression. References
1. Agarwal P et al. 2000, Diagn Mol Pathol 9: 158 164 2. Fernandes H et al. 2002, Transplantation 73: 1886 1891

Discussion
This report describes real-time PCR assays that were developed for the analysis of three different cytokine SNPs using LightCycler Technology with hybridization

Product LightCycler Fluorescein CPG LightCycler-Red-640 NHS ester

Pack Size 5 columns 1 vial

Cat. No. 3 113 906 2 015 161

LightCycler-FastStart DNA Master 1 kit (480 reactions) 2 239 272 Hybridization Probes

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probes. These immunoregulatory SNPs are present in the promoter region of the respective genes, and have been shown to affect gene transcription. SNP analysis on the LightCycler Instrument using melting curve analysis represents amplification and detection with specific probes, as well as visual discrimination, based on the melting temperatures of normal and mutant alleles in the homozygous and heterozygous configuration.