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Vol. 43. Issue 3.
Pages 377-381 (July - September 2021)
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Vol. 43. Issue 3.
Pages 377-381 (July - September 2021)
Case Report
Open Access
c.9253-6T>c REV3L: A novel marker of poor prognosis in Myelodysplastic syndrome
Roberta Taiane G. de Oliveiraa,b, Ivo Gabriel. F. Françaa,b, Howard L.R. Juniora,b, Giovanna B.C. Rielloa,b, Daniela de Paula Borgesa,b, Gabrielle Melo Cavalcantea,b, Silvia M.M. Magalhãesa, Ronald F. Pinheiroa,b,
Corresponding author

Corresponding author at: Núcleo de Pesquisas e Desenvolvimento de Medicamentos (NPDM), Rua Coronel Nunes de Melo, 1000. Rodolfo Teófilo, Fortaleza, CE, CEP: 60430-275 Brazil.
a Universidade Federal do Ceará (UFC), Fortaleza, Ceará, Brazil
b Núcleo de Pesquisas e Desenvolvimento de Medicamentos (NPDM), Fortaleza, CE, Brazil
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Table 1. Variant calling of somatic variants in the case herein reported.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by dysplasias, ineffective hematopoiesis and risk of acute myeloid leukemia transformation. Approximately 90% of MDS patients present mutations in genes involved in various cell signaling pathways. Specialized DNA polymerases, such as POLN, POLI, POLK, POLQ, POLH, POLL and REV3L, insert a nucleotide opposite replication–blocking DNA lesions in an error-prone manner and, in this way, sometimes can actively promote the generation of mutation. For the best of our knowledge, has not been described the mutations of these genes in MDS. DNA target sequencing CDS regions of the REV3L gene was performed in a 58-year-old man diagnosed as High Risk Myelodysplastic Syndrome. The patient presented very low hemoglobin, increased number of blasts, karyotype:47,XY,+8[6]/47,XY,del(7)(q32),+8[7], no response to hypomethylating therapy (decitabine), all markers of poor prognosis. Target sequencing identified a mutation c.9253-6T>C REV3L (Substitution - intronic) with VAF (variant allele frequency)=16% considered pathogenic according to Functional Analysis through. Hidden Markov Models (FATHMM). This is the first evidence of REV3L mutation in MDS and, of utmost importance, associated with poor prognosis.

Myelodysplastic syndrome
Translesion synthesis
REV3L gene
Full Text

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by dysplasias, clonal and ineffective hematopoiesis, peripheral cytopenias and risk of progression to acute myeloid leukemia. Recently, studies using sequencing techniques such as next-generation sequencing (NGS) have demonstrated frequent mutations in approximately 90% of MDS patients in genes involved in various cell signaling pathways, including RNA-splicing machinery (U2AF1, SRSF2 and SF3B1), epigenetic regulators (EZH2, ASXL1), cohesin components, transcription factors (EVI1, RUNX1, GATA2), DNA damage response and signal transduction molecules (JAK2, KRAS, CBL). Mutations in TP53, NRAS, RUNX1 and IDH1/IDH2 have been reported to influence overall survival in multivariate analyses while SF3B1 is associated with favorable prognosis.1–3

DNA lesions may be spontaneous or induced by radiations or chemicals, causing genomic alterations such as nucleotide substitutions, amplifications, deletions, rearrangements or chromosomal loss. To ensure genome stability, cells use DNA damage response (DDR) to sense and repair damage.3 Despite the efficient action of repair systems that remove lesions in DNA in an error-free manner, some lesions can escape these mechanisms.3 Thus, a second response to damaged DNA, termed DNA damage tolerance (DDT), has evolved to promote replication through and beyond an altered template, leaving the damage to be repaired at a later time point, thus lowering the overall risk of replication fork collapse and genome instability4 One of the DDT mechanisms, the translesion DNA synthesis (TLS) is a DNA damage tolerance process which contributes to cell survival by bypassing the unrepaired DNA lesions.5 Specialized DNA polymerases (POLs), such as POLN, POLI, POLK, POLQ, POLH, POLL and REV3L insert a nucleotide opposite replication–blocking DNA lesions in an error-free or error-prone manner and, in this way, sometimes can actively promote the generation of mutations.4

Material and methods

We performed a DNA target sequencing 108 amplicon regions of the REV3L gene from bone marrow sample of MDS patient at diagnosis. Amplicon coverage was 400 readings. The genome of interest used for alignment all of the sequencing reads was Homo sapiens (UCSC hg19) using the NextGENe software. Library preparation was performed according to Illumina Truseq Custom Amplicon (TSCA) standard procedure and run on MiSeq with the use of version 2.2 chemistry (Illumina, San Diego, CA, USA). The Q30 quality score bases was of 93.60%. For initial analysis, paired-end reads (175bp) were de-multiplexed into individual BAM files and aligned to UCSC Hg19 (GRCh37) by the on-board Illumina reporter software Miseq Reporter Software 2.4 with the use of the TruSeq Amplicon software. The single-nucleotide variations (SNVs) with ≥2% variant allele frequency (VAF) and more than 400 reads depth was considered significant. For secondary analysis, the variants candidates were loaded into a database NextGENe using BAM files for viewing of each variant. As tertiary analysis, we detected the variants in SNPs, mutations or variants of unknown significance using dbSNP, COSMIC and ESEMBL databases.

Case report

A 58-year-old man was referred for investigation of anemia. At physical examination, the patient was pale and presented petechiae. The hematology profile showed hemoglobin of 5.7g/dL, leucocytes of 1.200/mm3 and platelets of 22.000/mm3. The bone marrow smear presented dyseritropoiesis, dysgranulopoiesis and increased number of blasts (18%). Karyotype analysis of bone marrow cells showed clonal evolution: 47,XY,+8[6] and 47,XY,del(7)(q32),+8[7] (Figure 1). The patient was diagnosed as Refractory Anemia Excess Blasts II (RAEB II) according to World Health Organization (WHO)6 and IPSS-R (revised-International Prognostic Score Systems) was considered High Risk. The patient was treated with Decitabine (20mg/m2) without response and died due to sepsis after two-month follow up. DNA target sequencing CDS regions of the REV3L gene from bone marrow sample was performed at diagnosis.

Figure 1.

Next-generation sequencing showing REV3L gene mutation in MDS patient with clonal evolution. karyotype: 47, XY,+8[6]/47,XY,del(7)(q32),+8[7].


We evaluated all the polymerases with TLS activity (POLN, POLI, POLK, POLQ, POLH, POLL and REV3L) by NGS and detected a mutation in REV3L gene. Reversionless 3-like (REV3L) is the catalytic subunit of DNA polymerase zeta (POLZ) complex formed by REV3 and an accessory subunit REV7.7,8 The encode protein plays an important role in the translesion synthesis (TLS). However, the less fidelity of the REV3L due to its absence of exonuclease activity, compared with the high-fidelity replicative DNA polymerases, makes its synthesis translesion error-prone that causes spontaneous mutations. REV3L maintains genomic integrity by inserting a substitute nucleotide in the opposite DNA adducts, which increases the mutation rate and contributes to carcinogenesis.7–9 Diseases associated with REV3L include Moebius Syndrome and Poland Syndrome.10 Additionally, recent studies have presented mutations in REV3L gene in myeloid neoplasms, but not yet in MDS.11

Sequencing analysis showed 20 single-nucleotide variants of which, 15 in coding regions and 5 in intronic regions (Table 1). Variation consequences were calculated following the guidelines at ESEMBL databases and Truseq Amplicon software in missense variations (09), Synonymous (05), Stop Gained (01), intronic variants (05) and, more important, sequencing showed the presence of mutation c.9253-6T>C REV3L (Substitution - intronic) with VAF (variant allele frequency)=16% which is considered pathogenic according to Functional Analysis through Hidden Markov Models (FATHMM) (Table 1). According to Catalogue of Somatic Mutations In Cancer (COSMIC), this mutation was described in a case of Acute Myeloid Leukemia with mutation ID COSM5424379 in project entitled ICGC (LAML-KR): Acute Myeloid Leukemia – KR.

Table 1.

Variant calling of somatic variants in the case herein reported.

Position  Variant type  Sequence context  Ref allele  Alt allele  Consequence classification  VAF  Total depth  Ref allele depth  Alt allele depth  dbSNP  Cosmic ID 
111711453  SNV  Intron  Intron_variant  13%  1421  1228  193  rs3218574   
111695887  SNV  coding  Missense_variation  100%  10055  10053  rs462779  COSM4409266 
111697881  SNV  coding  Synonymous_variant  23.4%  3762  3878  880     
111643884  SNV  Intron  Splicing_region_variant, intron_variant  15.9%  2437  2050  387     
111698946  SNV  coding  Missense_variation  6%  1154  1085  69     
111689182  SNV  coding  Missense_variation  24.9%  566  425  141     
111689082  SNV  coding  Missense_variation  37.5%  1417  885  532  rs3218606   
111696852  SNV  coding  Synonymous_variant  53.3%  6508  3039  3469  rs458486  COSM4003651/4003652 
111634667  SNV  coding  Missense_variation  16.8%  2737  2275  460     
111621365  SNV  Intron  Splicing_region_variant, intron_variant  16%  400  336  64    COSM5424379/5424380 
111695268  SNV  coding  Synonymous_variant  52%  8362  4010  4352  rs455732  COSM4406879/4406880 
111621345  SNV  coding  Missense_variation  10.4%  4779  4281  498     
111710419  SNV  Intron  Splicing_region_variant, intron_variant  7.5%  1264  1169  95     
111654447  SNV  coding  Stop_gained  15.5%  3698  3125  573     
111636564  SNV  coding  Missense_variation  24.3%  985  746  239     
111654450  SNV  coding  Missense_variation  26.6%  1581  1161  420     
111665071  SNV  Intron  Intron_variant  46.9%  3567  1893  1674  rs1623806   
111696257  SNV  coding  Synonymous_variant  100%  7182  7180  rs461646  COSM4003650 
111650837  SNV  coding  Synonymous_variant  17.5%  3116  2478  545     
111709082  SNV  coding  Missense_variation  9.3%  1154  1046  107     

POSITION: Position within reference chromosome; VARIANT TYPE: Type of variant including single nucleotide variant (SNV), insertion and deletion; SEQUENCE CONTEXT: Location of the variant based on annotations of the reference genome; REF ALLELE: The reference allele; ALT ALLELE: The altered allele; CONSEQUENCE CLASSIFICATION: Predicted transcript consequence; VAF: variant allele frequency; TOTAL DEPTH: Number of reads aligned at this position; REF ALLELE DEPTH: Number of reads containing the reference allele; ALT DEPTH: Number of reads containing the variant allele; dbSNP: Identifier in the Single Nucleotide Polymorphism Database (dbSNP), a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI); COSMIC: The numeric identifier for the variant in the Catalogue of Somatic Mutations in Cancer (COSMIC) database.


Here, we present for the first time the presence of single-nucleotide variants and mutations on REV3L gene in patient diagnosed as MDS. Of utmost importance, our report is the first study to detect mutation c.9253-6T>C REV3L in MDS. This finding suggests this mutation is an important step during acute myeloid leukemia transformation. Mutations in REV3L predispose to double strand breaks in DNA which are strongly associated with chromosomal abnormalities.12 Our case reported herein presented trisomy 8 and deletion 7q as clonal evolution, all markers of poor prognosis in MDS and genomic instability.

We suggest this mutation is associated with great genomic instability in MDS because REV3L has no exonuclease activity compared to high-fidelity replicative DNA polymerases, making its function as error-prone which causes spontaneous and DNA damage-induced mutagenesis as here presented with clonal evolution with deletion 7q. The lack of the TLS synthesis may provoke chromosome rearrangements and even chromosome loss and inactivation of the REV3L gene causes a high frequency of chromosomal breaks. Thus, REV3L is important for maintaining genome stability.

Author contribution

RTGO, GBCR, IGFF, SMMM and RFP designed the study, provided patient materials and were responsible for collection and assembly of data. HLRJ, RTGO, DPB, GMC performed the molecular procedures and analyzed the data. All drafted and edited the manuscript. All authors have approved the final version of manuscript before publication.


This study was partially supported by the National Council for Scientific and Technological Development (CNPq) (grant number # 420501/2018-5 and # 424542/2016-1).

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

All procedures were approved by the Ethics Committee of UFC (#1.292.509) and are in accordance with the 1964 Helsinki declaration and its later amendments. Informed consent was obtained from all individual participants included in the study.

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