Development of Methods for Loss of Heterozygosity Studies When Normal Specimens Are Not Available
JA Lefferts, GV Belanger, CT Spyris, JB Brennick, AR Schned, GJ Tsongalis. Dartmouth-Hitchcock Medical Center, Lebanon, NH; Dartmouth Medical School/Dartmouth College, Hanover, NH; Norris Cotton Cancer Center, Lebanon, NH
Background: Loss of heterozygosity (LOH) is often performed retrospectively and is often hindered by the lack of readily available normal tissue or blood specimens from which a reference DNA sample can be extracted. Microsatellite markers having numerous alleles of varying lengths are typically amplified by PCR from both tumor and normal DNA. Complete loss of one allele peak or significant changes in relative peak heights in the tumor sample can be classified as exhibiting LOH. We have examined a novel method for determining LOH in the absence of matched, normal DNA samples.
Design: Microsatellite (STR) sequences were PCR amplified from normal de-identified human genomic DNA samples and subjected to capillary electrophoresis. Peak height ratios (longer peak height/shorter peak height) were calculated for all samples found to be heterozygous with respect to each STR. The heterozygous peak height ratios were then plotted against the differences in length between the two observed alleles to create standard curves of expected ratios. For each point on the standard curve, a statistical range of expected ratios was calculated. Results from prior LOH studies based on DNA extracted from six renal carcinomas of various sub-types, seven basal cell carcinomas and three squamous cell carcinomas were then applied to these standard curves.
Results: Linear relationships were observed for each STR evaluated with R squared values as high as 0.96 when the peak height ratios for each sample were compared to differences in allele lengths. Peak height ratios of cancer specimens from previous LOH studies were applied to these standard curves. A strong correlation was found between the LOH status of samples as determined using the standard curves and the LOH status of the same samples as determined previously.
Conclusions: 1) A linear relationship exists between peak height ratios of alleles in normal heterozygous DNA and the difference in length of the two alleles 2) This relationship can be utilized to predict the expected range of peak height ratios of two alleles in normal DNA samples that exhibit a specific difference in length. 3) Tumor samples with peak height ratios outside the expected range could be considered to exhibit LOH in the absence of normal DNA samples.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 222, Wednesday Afternoon