Evidence of genetic instability in tumors and normal nearby tissues

Abstract

Background: Comprehensive analyses have recently been performed on many human cancer tissues, leading to the identification of a number of mutated genes but providing no information on the variety of mutations present in each of them. This information is of interest to understand the possible origin of gene mutations that cause tumors.

Methodology/principal findings: We have analyzed the sequence heterogeneity of the transcripts of the human HPRT and G6PD single copy genes that are not considered tumor markers. Analyses have been performed on different colon cancers and on the nearby histologically normal tissues of two male patients. Several copies of each cDNA, which were produced by cloning the RT-PCR-amplified fragments of the specific mRNA, have been sequenced. Similar analyses have been performed on blood samples of two ostensibly healthy males as reference controls. The sequence heterogeneity of the HPRT and G6PD genes was also determined on DNA from tumor tissues. The employed analytical approach revealed the presence of low-frequency mutations not detectable by other procedures. The results show that genetic heterogeneity is detectable in HPRT and G6PD transcripts in both tumors and nearby healthy tissues of the two studied colon tumors. Similar frequencies of mutations are observed in patient genomic DNA, indicating that mutations have a somatic origin. HPRT transcripts show genetic heterogeneity also in healthy individuals, in agreement with previous results on human T-cells, while G6PD transcript heterogeneity is a characteristic of the patient tissues. Interestingly, data on TP53 show little, if any, heterogeneity in the same tissues.

Conclusions/significance: These findings show that genetic heterogeneity is a peculiarity not only of cancer cells but also of the normal tissue where a tumor arises.

Figure 1. Standard deviations of percent mutated clones of HPRT and G6PD cDNAs.

Analyses of 3 independent experiments for each gene on tumour and healthy tissues of patients A and B, on reference samples C1 and C2, and on control clones. Panel A, values of HPRT transcripts; Panel B, values of G6PD transcripts. AT and BT, values of clones of Tumour tissues; AH and BH, values of clones of Healthy tissues nearby tumours. Clones, values of control clones.

Figure 2. Percent mutated clones of HPRT and G6PD cDNA and genomic regions.

Panel A, values of HPRT transcripts; Panel B, values of G6PD transcripts. Panel C, values of HPRT genomic regions; Panel D, values of G6PD genomic regions. Percent values have been calculated on pooled data from each independent analysis. AT and BT, values of clones of Tumour tissues of patients A and B, respectively; AH and BH, values of clones of Healthy tissues near tumours. C1 and C2, values of reference samples. Clones, values of control samples.

Figure 3. Mutation frequency values of all analyzed sequences of HPRT and G6PD cDNA and genes.

Panel A, values of HPRT sequences; Panel B values of G6PD sequences. AH, AT, BH, BT: values calculated for cDNA clones of Tumour and nearby Healthy tissues of patients A and B. C1 and C2: values for cDNA clones from blood tissues of 2 healthy reference individuals. AT gene: values for clones of global genomic regions of DNA from tumour tissues of patient A. AT exons: values for the exon regions of the sequenced genomic regions. Controls: values for clone samples.