MUTATIONS IN THE TP53 GENE SHOW FEATURES OF SOMATIC HYPERMUTATION PROCESS WITH PROMINENT DIFFERENCE BETWEEN IGHV MUTATED AND UNMUTATED CHRONIC LYMPHOCYTIC LEUKEMIA

Background

Introducing somatic hypermutations (SHM) into genes coding for heavy and light chains of immunoglobulins (IGHV, IGLV) is a physiological process indispensable during antibody maturation. SHM introduction is a two-step process, which in the first step involves deamination of cytosine to uracil by an enzyme Activation-Induced (Cytidine) Deaminase (AID). During the second step, uracil and potentially also flanking nucleotides are removed and error-prone DNA polymerases (mainly polymerase eta) are recruited to fill the gap. Off-targeting of AID may result in mutations in non-immunoglobulin genes, including tumor suppressor gene TP53. 

Aims
We explored the TP53 mutation patterns with regards to SHM features emphasizing the differences between mutations occurring in IGHV unmutated (U-CLL) and IGHV mutated (M-CLL) groups of patients.

Methods
In order to reduce the selection bias, we used the set of TP53 mutations detected using ultra-deep Next Generation Sequencing (NGS) with high sensitivity (0.2%) allowing to consider the minor subclonal mutations with less prominent selective advantage. For NGS analysis, Illumina Miseq platform has been used with average coverage 34788; (range 1674–177021). For variant detection, an in house bioinformatics pipeline was established. Statistical evaluation was performed by shearwater algorithm computing Bayes classifiers based on a betabinomial model. Only point substitutions were taken into account. Altogether, 464 TP53 mutations in 73 high risk CLL patients were analyzed. 121 mutations were found in M-CLL cases and 343 mutations were detected in U-CLL patients. The high number of mutations should be attributed to the high number of minor subclonal mutations detected mainly in patients in relapse after treatment. In 60% of patients, results from repeated examinations were included (2-4 examinations per patient; each mutation detected in more than one consecutive sample was considered only once). 72.6% of patients received treatment before the first or during consecutive analyses. 

Results
U-CLL showed a higher proportion of mutations in C:G pairs comparing to M-CLL (66% vs. 51% of all mutations; P=0.003). Out of these, G:C>A:T substitutions, the primary result of AID cytidine deamination, were the prevalent events observed in U-CLL but not M-CLL (59.6% vs. 40.3% of all C:G mutations; P=0.009). Focusing on the AID-targeted sequence motif RGYW/WRCY, no difference between the two subgroups was observed (2.5% vs. 3.1%; P=ns). However, we found a significant overrepresentation of mutations in GNW motif in U-CLL (14.58% of all mutations vs. 6.61% in M-CLL; P=0.025). GNW is a strand-biased motif derived from RGYW most frequently targeted by AID in IGLV genes. On the other hand, TP53mutations detected in M-CLL cases showed the features of targeting by polymerase eta: (i) frequent targeting of A:T pairs (49% vs. 34% in U-CLL; P=0.003); with prominent strand bias favoring A over T (13.8 fold vs. 3.1 fold in U-CLL; P=0.002; (ii) prevalence of mutations in WA/TW motifs (40.5% vs. 23.91% in U-CLL; P<0.0001) with strand bias favoring WA observed in both groups, but extremely prominent in M-CLL (23.5 fold in M-CLL vs. 4.37 fold in U-CLL; P= 0.014).

Summary

We documented the significantly different patterns of TP53 mutations in U-CLL vs. M-CLL. Mutations detected in U-CLL showed the features corresponding to the first step of SHM process: deamination by AID with strand bias possibly attributable to AID activity on the transcribed strand. In contrast, spectra of mutations detected in M-CLL cases suggested the more prominent involvement of polymerase eta during the second SHM step.

Supported by CZ.1.07/2.3.00/30.0009, CZ.1.05/1.1.00/02.0068, NT13519, NT13493, NT11218, MUNI/A/1180/2014, NGS-PTL/2012-2015/no.306242, MSMT-CR (2013-2015, no.7E13008)

Keyword(s): Chronic lymphocytic leukemia, Somatic hypermutation, Tumor suppressor

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