Optimized PCR with sequence specific primers (PCR-SSP) for fast and efficient determination of Interleukin-6 Promoter -597/-572/-174Haplotypes

Background Interleukin-6 (IL-6) promoter polymorphisms at positions -597(G→A), -572(G→C) and -174(G→C) were shown to have a clinical impact on different major diseases. At present PCR-SSP protocols for IL-6 -597/-572/-174haplotyping are elaborate and require large amounts of genomic DNA. Findings We describe an improved typing technique requiring a decreased number of PCR-reactions and a reduced PCR-runtime due to optimized PCR-conditions. Conclusion This enables a fast and efficient determination of IL-6 -597/-572/-174haplotypes in clinical diagnosis and further evaluation of IL-6 promoter polymorphisms in larger patient cohorts.


Findings
Interleukin-6 (IL-6) is a pleiotropic cytokine with a broad range of effects that is produced by a variety of different cells and plays a crucial role at the interface of adoptive and innate immunity. The increased knowledge about individual genetic susceptibility of the immune system led to the identification of three single biallelic nucleotide polymorphisms (SNP) within the promoter region of the IL-6 gene at positions -597(G A) (rs1800797), -572(G C) (rs1800796) and -174(G C) (rs1800795) [1,2]. The three SNPs were shown to be in linkage disequilibrium and naturally occurring IL-6 -597/-572/-174 haplotypes have been characterized [2].
At present, typing of IL-6 -597/-572/-174 haplotypes requires an elaborate and time-consuming protocol [2]. A twelvereaction PCR-SSP system with eight different allele-specific primers (AS1-AS8) is needed in order to identify both -597/-572/-174 haplotypes for the three biallelic sites in each individual tested. Since PCR-SSP is DNA-consuming, this procedure requires at least 500 ng genomic DNA; this, however, may be critical with regard to a retrospective analysis, or when valuable samples need to be analyzed. However for subsequent confirmatory investigations of Here we report a modified PCR-SSP protocol which is suitable for the genotyping of IL-6 -597/-572/-174 haplotypes in the Caucasian population. It is faster, less labour-intensive and requires less DNA, since only four instead of twelve PCR reactions are necessary in order to detect all relevant -597/-572/-174 haplotypes in the Caucasian population (Table 1).
For the isolation of genomic DNA from 200 μL whole blood, we used the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's standard protocol. The DNA concentration was estimated from the absorbance at 260 nm using a UV-spectrophotometer (GeneQuant pro; Amersham Biosciences, Freiburg, Germany). For validation, DNA samples (n = 100) from a previous study [10] genotyped according to the protocol by Terry et al. [2] were used.
According to an optimized protocol for PCR-SSP [11] reactions were carried out in a total volume of 10 μL, IV AS4-F3c AS7-R1c Additional validation of the technique could be achieved by retyping 100 healthy blood donor samples previously genotyped according to the protocol by Terry et al. [10]. Our modified technique confirmed all previous typing results unambiguously (data not shown).
PCR-SSP techniques are widely employed for the genotyping of SNPs. After PCR and agarose gel electrophoresis, the genotyping result is evaluated by the presence or absence of an allele-specific PCR product. Moreover, PCR-SSP-typing is suitable for haplotyping of neighbouring SNPs; furthermore, the presence of two alleles on one chromosome can be demonstrated when two appropriate allele-specific primers are combined in a single PCR-reaction. Consequently, complete typing of three neighbouring biallelic SNPs requires twelve PCRreactions! Since this procedure is very extensive and time-consuming it is not suitable for the typing of larger cohorts.
In our study, we describe a modified PCR-SSP protocol which is suitable for rapid IL-6 -597/-572/-174 haplotyping focusing on the four major -597/-572/-174 haplotypes (AGC, GGC, GGG, GCG) representing 99.9% of all haplotypes in the Caucasian population (Table 1). Since only four, instead of twelve, PCR-reactions are needed and a protocol with optimized sensitivity is used [11], the amount of required genomic DNA could be reduced from 500 to 100 ng per genotype. This can be extremely valuable, especially if samples of limited quantity have to be analyzed.
Notably, our protocol does not distinguish between the GGG and AGG -597/-572/-174 haplotype, which has only been detected in one Caucasian individual so far (Table 1). However, as its frequency is extremely low (0.1%), this would not affect the overall outcome of association studies. Furthermore, ACC, ACG and GCC also represent minor 597/-572/-174 haplotypes that cannot be distinguished likewise. Even though they have not been observed among Caucasians and are only of theoretical importance, it cannot be ruled out that they might occur in other populations as well.
In order to run all four PCR-reactions under identical conditions, we optimized the selection of the allele-specific primers. Thereby, the runtime of the PCR-procedure could be reduced from 110 to 68 min with an overall analysis time of approximately 2 hours including all pipetting procedures, DNA-isolation (30 min), electrophoresis (10 min) and documentation (5 min).
In summary, we optimized a previous PCR-SSP protocol for IL-6 -597/-572/174 haplo-typing of Caucasian individuals with regard to the number of PCR-reactions, the amount of genomic DNA required and overall runtime. This method represents an important prerequisite for further evaluating the clinical impact of IL-6 promoter polymorphisms in larger cohorts.