Contributions
Abstract: PB1548
Type: Publication Only
Session title: Hematopoiesis, stem cells and microenvironment
Background
DNMT3A is one of the most frequently mutated genes in adult hematological neoplasms, including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Probably, the effect of mutations in this gene on disease progression is to destabilize the tetrameric form of DNMT3A and reduce the enzyme activity. Here we report the unexpected discovery of a new mutation in DNMT3A at the Fsp4HI restriction site (2:25234469 - 2:25234473), which was also located in the DNA sequence we examined during the development of a test to identify the most common R882H mutation in DNMT3A using the Fsp4HI restrictase.
Aims
This study was aimed to investigate the possibility of creating a simple restriction test system to diagnose the R882H mutation and determine the possible clinical significance of the new mutation.
Methods
From September 2019 to December 2020 we consecutively recruited 81 patients (43 male and 38 female) with the median age is 57 years (18-87) diagnosed at the Clinic of Rostov State Medical University and Сity hospital №7 (Rostov-on-Don), with the following diagnoses: MDS = 31, OML = 30, MPN = 19 (chronic myeloid leukemia (CML) = 17, primary myelofibrosis (PM) = 2). We developed a specific restriction test using Fsp4HI restrictase and original primers. The sequence after PCR contained two Fsp4HI-recognition sites - the target site for R882H and the non-target site in positions (2:25234469 - 2:25234473).
Results
Despite the expected high frequency of R882H (12 to 14% according to different sources), we did not find this mutation in our study. But we did find 5 patients with a mutation in a non-targeted restrictase recognition site, which caused a length-specific unique bend on electrophoresis. We did not find mutations in this region by searching databases (e.g., COSMIC, GenBank), so we tend to believe that the DNMT3A region (2:25234469 - 2:25234473) contains a new mutation, which we plan to confirm by sequencing in the next stage of the study.
We found this mutation in 5 (6,17%) patients from our sample (AML = 1, MDS = 1, CML = 2, PM = 1) with clonal hematopoiesis together with other mutations in JAK2 (V716F), BCR-ABL and FLT3/TKD, but not MPL (W515L, W515K).
Conclusion
During the development of a test system to detect the R882H mutation, we discovered a possible new mutation in the region (2:25234469 - 2:25234473) DNMT3A, which is a common marker for clonal hematopoiesis and is associated with other mutations and requires to determine the exact location and further research as a possible clinical marker.
Keyword(s): DNA methylation, Mutation analysis, Myeloproliferative disorder
Abstract: PB1548
Type: Publication Only
Session title: Hematopoiesis, stem cells and microenvironment
Background
DNMT3A is one of the most frequently mutated genes in adult hematological neoplasms, including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Probably, the effect of mutations in this gene on disease progression is to destabilize the tetrameric form of DNMT3A and reduce the enzyme activity. Here we report the unexpected discovery of a new mutation in DNMT3A at the Fsp4HI restriction site (2:25234469 - 2:25234473), which was also located in the DNA sequence we examined during the development of a test to identify the most common R882H mutation in DNMT3A using the Fsp4HI restrictase.
Aims
This study was aimed to investigate the possibility of creating a simple restriction test system to diagnose the R882H mutation and determine the possible clinical significance of the new mutation.
Methods
From September 2019 to December 2020 we consecutively recruited 81 patients (43 male and 38 female) with the median age is 57 years (18-87) diagnosed at the Clinic of Rostov State Medical University and Сity hospital №7 (Rostov-on-Don), with the following diagnoses: MDS = 31, OML = 30, MPN = 19 (chronic myeloid leukemia (CML) = 17, primary myelofibrosis (PM) = 2). We developed a specific restriction test using Fsp4HI restrictase and original primers. The sequence after PCR contained two Fsp4HI-recognition sites - the target site for R882H and the non-target site in positions (2:25234469 - 2:25234473).
Results
Despite the expected high frequency of R882H (12 to 14% according to different sources), we did not find this mutation in our study. But we did find 5 patients with a mutation in a non-targeted restrictase recognition site, which caused a length-specific unique bend on electrophoresis. We did not find mutations in this region by searching databases (e.g., COSMIC, GenBank), so we tend to believe that the DNMT3A region (2:25234469 - 2:25234473) contains a new mutation, which we plan to confirm by sequencing in the next stage of the study.
We found this mutation in 5 (6,17%) patients from our sample (AML = 1, MDS = 1, CML = 2, PM = 1) with clonal hematopoiesis together with other mutations in JAK2 (V716F), BCR-ABL and FLT3/TKD, but not MPL (W515L, W515K).
Conclusion
During the development of a test system to detect the R882H mutation, we discovered a possible new mutation in the region (2:25234469 - 2:25234473) DNMT3A, which is a common marker for clonal hematopoiesis and is associated with other mutations and requires to determine the exact location and further research as a possible clinical marker.
Keyword(s): DNA methylation, Mutation analysis, Myeloproliferative disorder