Myelodysplastic Neoplasm (MDS) is characterized by cytogenetic alterations in 40-60% of cases and 94% have at least one oncogenic mutation. Tolerance to non-repairable DNA damage is performed by translesion synthesis DNA polymerases (TLS), which bypass or correct the lesions in a relatively error-prone way, which can generate point mutations. TLS polymerases are likely key contributors to mutagenic signatures and their study is important in oncological diseases.

This study aimed to perform a genetic screening to identify, characterize and classify genetic variants in DNA polymerases with TLS activity (REV3L, POLQ, REV1, POLI, POLH, POLK, POLL, and POLN) in MDS.

Bone marrow samples from 30 patients (17 MDS with excess blasts - MDS-EB1 and 2, and 13 MDS with ring sideroblasts - MDS-RS-SLD/MDS-RS-MLD, according to the 2016 WHO Classification) were sequenced by NGS-Targeted Sequencing-Illumina. Variants pathogenicity analysis was performed by five in silico pathogenicity prediction tools (Ensembl VEP, Mutation Taster, Sift, Mutpred, and Provean). For the oncogenicity analysis of these variants, standards for the classification of oncogenicity of somatic variants (SOP), recommended by the Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC) was performed. This study selected loss-of-function (LoF-frameshift, inframe, stop gained, stop loss, splice donor, and splice acceptor) variants for oncogenicity classification.

Targeted sequencing of the POLQ gene detected 194 variants (85 missenses, 38 synonymous, and 17 LoF), 10 were classified as VUS and 1 as Probably Benign, according to SOP. For the REV3L, 284 variants were identified (85 missenses, 38 synonymous, and 17 LoF), 18 were classified as Potentially Oncogenic, 7 as VUS, and 3 as Benign, according to SOP. For the POLI, 58 variants were identified (22 missenses, 7 synonymous, and LoF), 3 were classified as Potentially Oncogenic, 1 as VUS, and 2 as Benign or Probably Benign, according to SOP. For the REV1, 108 variants were identified (39 missenses, 12 synonymous, and 8 LoF); 2 were classified as Potentially Oncogenic and 4 as VUS, according to SOP. For the POLH, 22 variants were identified (18 missenses and 4 synonymous).

Altered levels or polymorphism and mutations in TLS polymerases are associated with cancer progression. We identify that patients classified as MDS-EB had a higher incidence of LoF variants in the POLQ and REV3L genes, compared to patients of the MDS-RS subtype, representing a loss of the translesion synthesis mechanism in high-risk patients. Patients of the MDS-RS subtype with normal karyotype had less frequency of LoF variants and higher frequency of missense variants in REV1 and POLI genes, as well as high rates of non-LoF variants, which may represent a biomarker of genomic instability in MDS. A patient with a complex karyotype presented an accumulation of LoF mutations in POLQ and REV3L genes, which suggests that the higher genetic instability observed in patients with multiple chromosomal alterations may be present due to the absence of the TLS mechanism. The increased loss of function and oncogenic variants in REV3L and POLQ genes could contribute to genome instability during MDS progression to acute leukemia and reinforce the impact of genetic mutations on the pathophysiological process of MDS, mainly affecting genes not included in current prognostic risk scores.