Ultradeep sequencing of a human ultraconserved region reveals somatic and constitutional genomic instability

Abstract

Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.

Figure 1. Features of eUCR41.

(A) Genomic coordinates refer to the hg18 assembly of the human genome. The two grey bars correspond to the extremely conserved sequence , and to the genomic region tested for possible enhancer activity , respectively. Black bars indicate the 11 overlapping segments used for the amplification. (B) Percentage of homopolymers and base composition of eUCR41, of the ultraconserved core, and of the flanking regions are shown.

Figure 2. Mutation spectrum of eUCR41 in sample CC.

(A) All detected substitutions are mapped on the corresponding positions of eUCR41. Two ranges of substitution frequency are shown: 40.5%–2.5% and <1.0%, since no substitution was detected in the range 2.5%–1.0%. All substitutions reported in the range 1.0%–0.1% were manually checked and excluded as sequencing errors. (B) Mutability was calculated using sliding windows of the same length as UCR41. Values corresponding to the middle point of each window are reported. Mutability increases with the decrease of sequence conservation: it is always below average for sequence identity >50%, whereas it is above average for nonconserved segments. Similar trends were observed for all samples deriving from HNPCC (unpublished data).

Figure 3. Observed and expected mutability outside and inside UCR41.

Observed values of mutability ratios (arrows) were compared to the expected distributions computed from 1,000,000 random permutations of the raw data (red) and after removing all potential errors (blue). p represents the probability of obtaining the observed mutability ratio by chance and was calculated as the fraction of the expected ratios equal or higher than the observed value.

Figure 4. Difference in mutability ratio between HNPCC and healthy samples.

The observed difference in mutability ratios (arrows) between each of the three HNPCC samples (μ_CC, μ_NC, and μ_PBL) and the healthy control (μ_H-PBL) were compared to the corresponding expected distributions. These were computed from 1,000,000 random permutations of the raw data (red) and after removing all potential errors (blue). p represents the probability of obtaining the observed difference in μ by chance and corresponds to the fraction of the expected differences equal or higher than the observed value.

Figure 5. Variation of the mutability ratio for decreasing contribution of random errors.

By progressively decreasing the number of positions with rare substitutions, the mutability ratio (μ) outside and inside UCR41 increases in all samples from HNPCC patients. In H-PBL, errors overcome true mutations inside and outside UCR41 at any value of frequency cutoff. The corresponding mutability ratio is therefore always around 1. Values on the y-axis correspond to the observed ratio for each sample.