Translation of an STR-based biomarker into a clinically compatible SNP-based platform for loss of heterozygosity - PubMed (original) (raw)
Translation of an STR-based biomarker into a clinically compatible SNP-based platform for loss of heterozygosity
Heather D Kissel et al. Cancer Biomark. 2009.
Abstract
Loss of heterozygosity (LOH) has been shown to be a promising biomarker of cancer risk in patients with premalignant conditions. In this study we describe analytical validation in clinical biopsy samples of a SNP-based pyrosequencing panel targeting regions of LOH on chromosomes 17p and 9p including TP53 and CDKN2A tumor suppressor genes. Assays were tested for analytic specificity, sensitivity, efficiency, and reproducibility. Accuracy was evaluated by comparing SNP-based LOH results to those obtained by previously well-studied short tandem repeat polymorphisms (STRs) in DNA derived from different tissue sources including fresh-frozen endoscopic biopsies, samples from surgical resections, and formalin-fixed paraffin-embedded sections. A 17p/9p LOH panel comprised of 43 SNPs was designed to amplify with universal assay conditions in a two-step PCR and sequence-by-synthesis reaction that can be completed in two hours and 10 minutes. The methods presented can be a model for developing a SNP-based LOH approach targeted to any chromosomal region of interest for other premalignant conditions and this panel could be incorporated as part of a biomarker for cancer risk prediction, early detection, or as entry criteria for randomized trials.
Figures
Fig. 1. Steps in SNP selection for SNP-based CDKN2A/TP53 LOH panel
SNP selection process for pyrosequencing-based LOH panel. Step 1) SNPs were selected from
(release versions 36 to 38) to be ± 10Kb from the current chromosomal location of the 12 STRs, TP53, and CDKN2A as reported in the NCBI SNP database,
http://www.ncbi.nlm.nih.gov/projects/SNP/
. SNPs with extended homopolymeric regions were excluded. Homopolymers are defined as having multiple nucleotides flanking the SNP that are the same nucleotide as the one of the bases in the SNP itself. SNPs with reported heterozygosity rates below 0.15 in Caucasian populations were excluded, and SNPs with unknown heterozygosity rates were further evaluated. Step 2. The flanking DNA sequence for each polymorphism was entered into the PSQ Assay Design Software (version 1.0.6) from Biotage (Uppsala, Sweden) to determine SNPs that make optimal pyrosequencing assays using a stringent Allele Quantification setting. The software ranks each assay on a scale of 0 to 100, with 100 being most optimal. Step 3. To limit the number of assays required for analytic testing, only the highest scoring SNP assays were used (range 82 - 100). Step 4. To determine assay specificity, the resultant DNA sequences surrounding each SNP generated by the PSQ software were entered into BLAT (Blast-like analysis tool) software to BLAST (Basic Local Alignment Search Tool) against the genome (
http://genome.ucsc.edu/cgi-bin/hgBlat?command=start
) starting with the sequence of the forward primer and ending with the reverse primer. Step 5. 163 SNPs were available for analytic testing and 70 were tested to obtain 34 SNPs. These were combined with the nine SNPs created by Biotage, resulting in the final panel of 43 SNPs.
Fig. 2. (A) Pyrograms showing all three genotypes of the SNP (yellow). The RLU ratio at positions 8 and 9 does not change for different genotypes. (B) Scatter plot of peak heights at Positions 8 and 9
(A) Pyrograms from constitutive control DNAs showing peak intensity of each nucleotide sequenced for ASY167 for homozygote T/T, homozygote C/C, and heterozygote T/C. Positions 8 and 9 of the sequence are used as non-polymorphic internal sequence controls for each sample and can be used as a measure of specificity across all samples. (B) Concordance of the peak intensity ratio of control positions 8 and 9 for 790 genotypes of ASY167.
Fig. 3. Example of genotype reproducibility between independent PCR and pyrosequencing reactions
Example of genotype reproducibility between independent PCR and pyrosequencing reaction genotypes. Allele frequency (y-axis) was determined by the pyrosequencing software. For each DNA sample, four data points are plotted one after the other, representing replicate PCR reactions which were both sequenced on two different days, shown in order of sample ID. (A) near 50% allelic ratio for heterozygotes, (B) shifted allelic ratio for homozygotes, (C) shifted allelic ratio for heterozygotes, and (D) distinct genotype clusters across homozygous and heterozygous samples.
Fig. 4. Example of pyrograms from mixing experiment
Pyrosequencing pyrograms of constitutive control and flow-purified aneuploid DNA was quantitated and mixed at 0, 10, 25, 50, 75, 90, and 100% aneuploid. The first pyrogram shows that the results are very similar to the theoretical peak heights for a normal G/C heterozygote and the flanking sequence, as depicted in the final bar graph. Mixtures were PCR amplified for pyrosequencing at an informative SNP (replicate #1 and #2). LOH in the aneuploid resulted in loss of the “G” allele.
Fig. 5. Percent allele and LOH calls for constitutive control:aneuploid mixtures in two patients
Scatter plot of the mixing experiment using a constitutive control and paired aneuploid DNA for two different patients for one assay with LOH calls based on the statistical algorithm. The squares are patient #1 and the diamonds are patient #2. The blue is LOH no, the green is LOH Intermediate, and the red is LOH yes.
References
- Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyren P, Uhlen M, Lundeberg J. Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem. 2000;280:103–110. - PubMed
- Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. Roy Stat Soc, Ser B. 1995;57:289–300.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous