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Tumor Discovery Somatic mutations in POLE and POLD1 in colorectal cancer

rate of 0.3°C/s, with a starting temperature of 70°C, a final org/) and the PON-P2 computational tool (http://structure.
temperature of 95°C, and fluorescence being measured bmc.lu.se/PON-P2/) were used to predict the possible
between 80 and 95°C. The HR-1 analysis tool and custom functional effects of the POLE and POLD1 mutations. 19-21 The
software were used to analyze all the data, and both identification numbers of POLE and POLD1 were retrieved
derivative plots and difference plots were generated after from ENSEMBL (ENSP number, www.ensenbl.org), NCBI
normalizing and temperature shifting. A 4% difference in (RefSeq number, www.ncbi.nlm.nih.gov/protein), and
fluorescence between the known wild-type control and test UniProt (SwissProt protein identifier number, www.uniprot.
samples was used as the threshold for the variant call, as org) and inputted together with the protein positions and
previously described. 2 amino acid substitutions into the PROVEAN and PON-P2
software. 19,20 The PROVEAN batch protein software displays
2.6. Sequencing results of both the PROVEAN and SIFT (Sorting Intolerant
The PCR products of tumor samples with abnormal HRM From Tolerant, another protein function prediction software,
21
patterns were subjected to direct, bidirectional Sanger http://sift.jcvi.org) algorithms. Mutations that would likely
sequencing. First, the PCR products were purified using cause alterations in protein functions are called “deleterious”
the QIAquick PCR Purification Kit (Qiagen, Netherlands) (PROVEAN), “damaging” (SIFT), or “pathogenic” (PON-P2),
according to the manufacturer’s instructions. The purified whereas those unlikely to result in functional changes are
products were then sequenced directly using the appropriate displayed as “neutral” (PROVEAN, PON-P2) or “tolerant”
PCR primers. The resulting chromatograms were viewed and (SIFT). The PON-P2 computational tool also returns a
interpreted using Chromas Lite software V2.01. To analyze prediction call of “unknown” if the effect of the amino acid
the sequencing data, the Basic Local Alignment Search Tool substitution is indeterminate. 20
2 sequences program (http://www.ncbi.nlm.nih.gov/blast/
bl2seq/wblast2.cgi) was used to compare the sequences against 3. Results
the wild-type sequences for both POLE and POLD1 genes. DNA from 102 tissue samples was tested for mutations
in the endonuclease domains of POLE and POLD1
2.7. Statistical analysis with optimized QMC-PCR followed by HRM analyses.

The clinicopathological and molecular significance of The HRM analysis results were confirmed by Sanger
the POLE and POLD1 mutations were tested using SPSS sequencing. When Sanger sequencing failed to confirm
version 22. Categorical data were analyzed using the Chi- aberrant HRM analysis results, the samples were amplified
squared test (or Fisher’s exact test, when the expected with COLD-PCR and then re-sequenced for confirmation.
count in each cell was <5), whereas continuous variables
were analyzed using the independent t-test. The results 3.1. POLE and POLD1 endonuclease domain
were significant at P < 0.05. mutations
A total of 10 novel somatic variants (eight in POLE and
2.8. Prediction of the functional effects of mutations two in POLD1) were found in ten different cases, giving
The batch protein function of the protein variation effect a variant rate of 9.6% and 2.4% in POLE and POLD1,
analyzer (PROVEAN) online software (http://provean.jcvi. respectively, in the entire cohort of patients. Nine of these

Table 2. Molecular characteristics of POLE and POLD1 mutations
Subject id. Gene/Exon Nucleotide position Codon Functional amino acid change Microsatellite status Ploidy status
4 POLE/14 c. 1364 T>A L455Q Hydrophobic to polar MSS A
32 POLE/9 c. 833 C>T T278M Hydrophilic to hydrophobic MSS A
38 POLE/13 c. 1295 T>C L432P Hydrophobic to hydrophobic MSS A
40 POLE/13 c. 1240 G>A D414N Polar to polar MSS D
44 POLE/12 c. 1121 C>T A374V Hydrophobic to hydrophobic MSS A
63 POLE/13 c. 1298 G>A G433D Hydrophilic to polar MSS A
64 POLE/14 c. 1369 A>G T457A Polar to hydrophobic MSS A
83 POLE/14 c. 1382 C>A S461* Truncated protein MSI D
50 POLD1/10 c. 1211 C>T P404L Hydrophobic to hydrophobic MSS A
52 POLD1/10 c. 1229 C>T A410V Hydrophobic to hydrophobic MSS A
Abbreviations: MSI: Microsattelite instability; MSS: Microsattelite stable.

Volume 3 Issue 3 (2024) 4 doi: 10.36922/td.3659