Molecular genetic analysis of 103 sporadic colorectal tumours in Czech patients

Abstract

The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in Europe. To evaluate whether sporadic CRCs in Czech patients have specific mutational profiles we analysed somatic genetic changes in known CRC genes (APC, KRAS, TP53, CTNNB1, MUTYH and BRAF, loss of heterozygosity (LOH) at the APC locus, microsatellite instability (MSI), and methylation of the MLH1 promoter) in 103 tumours from 102 individuals. The most frequently mutated gene was APC (68.9% of tumours), followed by KRAS (31.1%), TP53 (27.2%), BRAF (8.7%) and CTNNB1 (1.9%). Heterozygous germline MUTYH mutations in 2 patients were unlikely to contribute to the development of their CRCs. LOH at the APC locus was found in 34.3% of tumours, MSI in 24.3% and MLH1 methylation in 12.7%. Seven tumours (6.9%) were without any changes in the genes tested. The analysis yielded several findings possibly specific for the Czech cohort. Somatic APC mutations did not cluster in the mutation cluster region (MCR). Tumours with MSI but no MLH1 methylation showed earlier onset and more severe mutational profiles compared to MSI tumours with MLH1 methylation. TP53 mutations were predominantly located outside the hot spots, and transitions were underrepresented. Our analysis supports the observation that germline MUTYH mutations are rare in Czech individuals with sporadic CRCs. Our findings suggest the influence of specific ethnic genetic factors and/or lifestyle and dietary habits typical for the Czech population on the development of these cancers.

Figure 1. Distribution of mutations in selected genes.

Mutational events in the APC, TP53, KRAS and BRAF genes dispersed in 103 tumours studied. Nineteen tumours carried no mutations in these genes. ^a this group includes 1 tumour with a CTNNB1 mutation; ^b this group includes 1 tumour with a germline MUTYH mutation; ^c this group includes 1 tumour with 8 point substitutions in APC.

Figure 2. Comparison of the type of mutations in the APC gene.

Light grey, frameshift mutations; dark grey, point substitutions. Frameshift mutations were more frequent in MCR while point substitutions, especially C>T resulting in Arg>STOP, were more common outside the MCR.

Figure 3. Comparison of the distribution of mutations along the TP53 coding region.

TP53 mutations in CRCs from the UMD database (top) and in our sample (bottom) are depicted. The length of the bars reflects the number of mutations. Seven hot spot positions (representing 10 most common substitutions in UMD) are indicated by codon numbers. Numbers in frames indicates two different frequent substitutions at the same position. In our sample, two different point substitutions were observed in codon 245, and only one of them belonged to the 10 hot spot variants. Asterisks indicate FS mutations in our cohort.

Figure 4. Mutational events in the TP53 gene.

Mutations found in CRCs listed in the UMD database (left) and observed in our CRCs (right). The CRCs of Czech patients had more frameshift mutations and transversions, while transitions, especially at CpG sites, were less frequent.

Figure 5. Comparison of the stage of tumours without and with TP53 mutations.

White, stage I; light grey, stage II; dark grey, stage III; black, stage IV. Segments show the number and percentage of tumours (3 tumours lacked information). Tumours with TP53 mutations may have a tendency to skip stage II or progress through it very quickly compared to tumours without TP53 mutations (P = 0.002).