It is widely believed that somatic as well as germline mutations play important roles in the origin and progression of colorectal cancers (Calvert and Frucht, 2002). Many genes have been investigated for mutation to elucidate mechanisms of colorectal cancer development, with these investigations demonstrating the involvement of mutations in colorectal carcinogenesis and progression. Samuels et al. reported that PIK3CA, a catalytic subunit of the class IA phosphatidylinositol 3-kinases, was somatically mutated in 32% of colorectal cancers, resulting in the attenuation of apoptosis and facilitated tumor invasion(Samuels et al. 2004). A comprehensive study entitled, “The Tyrosine Phosphatome” was accomplished by sequencing all genes involved in tyrosine phosphorylation in a large cancer cohort consisting of 175 colorectal cancer patients(Wang et al. 2004). Most mutational studies, however, have been preoccupied with the prevalence of somatic mutations in a specific single candidate gene in relatively small colorectal cancer patient cohorts. Recently, Sjoblom et al. reported the genome-wide frequencies of somatically mutated genes in human breast and colorectal cancers(Sjoblom et al. 2006). However, the methods these used were extremely expensive, time-consuming, and labor-intensive for a typical laboratory to perform. More practical strategies, amenable to smaller laboratories with more conservative budgets, would be of great value in the continuing quest to answer questions in the fields of tumor genomics and mutatomics. To this end, we present herein a circumscribed, practical mutational study employing high-throughput cDNA sequencing in colon adenocarcinoma, in which we demonstrate the eminent feasibility and results of determining sequence variation efficiently and at low cost.

In the current study, we assumed that if a mutant protein was involved in carcinogenesis or tumor progression, this mutant would be expressed and therefore detectable in tumor mRNA. i.e. we assumed that somatic mutations involved in carcinogenesis or tumor progression would be detectable by direct cDNA sequencing. By using this strategy, we avoided the need for sequencing each exon of genomic DNA, reasoning that genes which are never expressed in normal or malignant colon probably do not participate in colorectal carcinogenesis. We discovered 78 sequence variants (44 of which had been previously reported as single-nucleotide polymorphisms, but 34 of which had never been reported) among the 603 genes (304,350 bp of ORFs) studied.

Recently, Sjoblom T. et al. performed genome-wide sequencing in breast and colorectal cancers, revealing that an average of 52 mutations occurred in each colorectal cancer(Sjoblom et al. 2006). According to the article by Sjoblom et al. the somatic mutation frequency in colon cancers was 3.2 somatic mutations/Mb, on average (Table 1 of their paper). Therefore, the probability of our finding zero somatic mutations among the 603 genes (304,350 bp) that we studied was 37.76% (please see formula below), suggesting that our findings were statistically quite consistent with Sjoblom’s results: Probability=(1−3.21, 000, 000)304,350                                           =0.377599353

The Sjoblom team also defined “CAN-genes” (candidate cancer genes) as those that were frequently mutated in colorectal cancers, and found that 69 genes could be included in this category. Although the CAN-genes KRAS, GNAS and TP53 were studied by us, no somatic mutations were found in these genes. Furthermore, in addition to the genes mentioned above, NRAS, HRAS, p16, and p27 were included in the current study, but these genes also contained no somatic mutations. Finally, results of MSI assays revealed MS-stability (MSS), implying an absence of mutations in the major DNA mismatch repair genes (although these genes were not studied due to their long ORFs). It is possible that other molecular pathogenetic pathways were involved in this colorectal tumorigenesis, such as those containing APC, MCC, DCC, or the TGF-β cascade: these genes were also not examined in the current study due to ORF length.

Approximately 24,000,000 bp among the entire genomic DNA sequence are reported as ORFs in the UCSC database. The average density of each SNP is once per 1.9 kilobases (i.e. 1,419,190 SNPs/2.7 gigabases of human genome sequence)(Sachidanandam et al. 2001). We sequenced 304,350 bp of ORFs (viz., 1.26% of the total ORFs in the UCSC database: 304,350 bp/24,000,000 bp) and discovered 78 sequence variants, yielding a frequency of 1 alteration per 3,902 bp (78/304,350 bp). Our observed sequence variant distribution may provide a basis with which to estimate the number of SNPs in a single individual with colon cancer. That is, the SNPs reported above are one possible subset of the entire database; there is no guarantee that a given individual will always harbor all SNPs in the database.

The human ATP50 gene (X83218, NM_001697), encoding a 213-amino acid ATP synthase OSCP subunit, is a key structural component of the stalk of the mitochondrial respiratory chain F₁F₀-ATP synthase, which is a vital element in the cellular pathway of energy conversion (Senior, 1988). Although a mutant strain of yeast in which the delta subunit of F₁F₀-ATP synthase had been inactivated by insertional mutagenesis showed little or no ATPase activity(Giraud and Velours, 1994), and dysfunction of ATP synthase can cause a variety of degenerative diseases(Wallace, 1994), there have been no previous reports detailing a relationship between ATP synthase and tumorigenesis. We found restricted monoallelic (i.e. monoallelically silenced) expression of an altered allele from ATP50 in our colon cancer tissue, which would be expected to exert the same effect as would a somatic mutation of this gene. Genomic DNA sequencing of ATP50 revealed that this monoallelic expression was not due to LOH. We therefore studied the methylation status of the CpG island in the promoter region of ATP50 by MSP, but we found no methylation of this region. Other epigenetic mechanisms, such as histone deacetylation, might have contributed to monoallelic expression of ATP50. There was no monoallelic expression of ATP50 in 20 cancer cell lines that we examined. Although monoallelic expression of this altered ATP50 allele may be involved in a subset of colorectal cancers, further study is required to clarify the potential functional role of this gene in carcinogenesis.

This study poses several advantages as well as limitations. Firstly, it has been reported that some synonymous mutations may influence the stability of mRNA(Duan and Antezana, 2003; Chamary and Hurst, 2005) because they affect the thermodynamic stability of mRNA secondary structures (Fitch, 1974; Klambt, 1975). Nonsense-mediated mRNA decay (NMD) is also known as a surveillance pathway that rapidly degrades mRNAs containing premature termination codons(Culbertson and Leeds, 2003; Amrani et al. 2006). These mechanisms may cause instability of mRNA, accelerate the degradation of mRNA, and consequently result in difficulty in detecting sequence alterations by cDNA sequencing. Since we used cDNA as our starting material for sequencing, we may have ignored some key genes because of RNA degradation. Nevertheless, many sequence variants were detected reasonably well in the current study, suggesting that degradation of mRNA occurred rarely, if at all, as a consequence of sequence alterations. Instead, we considered it more important to increase our chances of finding sequence alterations by using cDNA rather than genomic DNA because of the lower cost, time, and labor involved in sequencing cDNA, as well as the increased relevance of only studying genes that are expressed in the colon.

Secondly, it is conceivable that we lost some gene sequence information due to extremely low expression levels. Therefore, we employed two-stage PCR to increase our chances of successful sequencing, thereby achieving a relatively high success rate of 862/1,038 reactions, or 83.0%. Possibly, this result still may have included genes that were not expressed in our particular colorectal cancer, even though we used the UCSC database to select genes that were purportedly expressed in colorectal cancers. Our sequencing success rate appears favorable when compared to genomic DNA sequencing, where 92% of genes were successfully analyzed (Wang et al. 2004). The total number of exons sequenced in our study was 2107, implying that at least 2107 primer pairs would have been necessary to conduct this study had it been attempted by genomic DNA sequencing; in contrast, we accomplished this task using only 1038 primer sets for cDNA sequencing. This contrast demonstrates that our method is useful to explore mutations because it is not only more cost-effective, but also less demanding in time and labor.