Optimization of candidate-gene SNP-genotyping by flexible oligonucleotide microarrays; analyzing variations in immune regulator genes of hay-fever samples

Abstract

Background: Genetic variants in immune regulator genes have been associated with numerous diseases, including allergies and cancer. Increasing evidence suggests a substantially elevated disease risk in individuals who carry a combination of disease-relevant single nucleotide polymorphisms (SNPs). For the genotyping of immune regulator genes, such as cytokines, chemokines and transcription factors, an oligonucleotide microarray for the analysis of 99 relevant SNPs was established. Since the microarray design was based on a platform that permits flexible in situ oligonucleotide synthesis, a set of optimally performing probes could be defined by a selection approach that combined computational and experimental aspects.

Results: While the in silico process eliminated 9% of the initial probe set, which had been picked purely on the basis of potential association with disease, the subsequent experimental validation excluded more than twice as many. The performance of the optimized microarray was demonstrated in a pilot study. The genotypes of 19 hay-fever patients (aged 40-44) with high IgE levels against inhalant antigens were compared to the results obtained with 19 age- and sex-matched controls. For several variants, allele-frequency differences of more than 10% were identified.

Conclusion: Based on the ability to improve empirically a chip design, the application of candidate-SNP typing represents a viable approach in the context of molecular epidemiological studies.

Figure 1

Design a 23 mer oligonucleotide for SNP detection. In (a) the relevant PCR-product of 166 bp is shown. (b) exhibits the set of oligonucleotides (12 for sense and 12 for antisense strand; at n = 0 the allele is located in the middle of the oligomer, at n = -2 and n = +2 the SNP is shifted by 2 nucleotides to the left and right, respectively.

Figure 2

The dependence of signal intensity on oligonucleotide length. Hybridization was done at 45°C. I₁/I₂ labels the ratio of the signal at the full-match oligonucleotide and the signals at the mismatched oligonucleotides. 27, 25, 23, 21 and 19 indicates the length of oligomers. The SNP was located either at the center of the oligonucleotides (0) or shifted by two bases in either direction (+1, -1).

Figure 3

Differences in the performance of oligonucleotides. Set 1 to 6 label the oligonucleotides designed for detecting a SNP. Each column indicates the signal intensity at the oligomers that represent (left to right) the A, G, C or T variant of a sequence. Sets 1 to 3 are the data produced on replicate microarray positions that represent one strand, while sets 4 to 6 indicate the signal intensities produced by the complementary DNA strand. Panel (1) shows the result obtained for a heterozygous sample, panel (2) a homozygous sample. Panel (3) exhibits data obtained with an oligonucleotide that was predicted in silico to perform well but failed in the experiment. In panel (4) a result of an oligomer with a high degree of cross-hybridization is presented.

Figure 4

Gel-electrophoretic separation of the products of multiplex-PCR. Two decaplex amplifications are shown in comparison to the respective individual reactions. In both cases presented here, one product was not amplified in the multiplex reaction while the reaction worked fine in the individual amplification.

Figure 5

Image of a simultaneous hybridization of 99 PCR-products to an in situ synthesized oligonucleotide microarray. Usually, the features were scrambled across the array. For illustrative purposes, they were placed next to each other in this particular experiment.