PMC10336536_01
Molecular characterization and 3D modeling of DNMT3A mutational profile. (A) Map of four variants identified on a linear schematization of the DNMT3A protein (lollipop plots). (B) VAF evolution of the mutations detected. (C) The Venn diagram of the reads identifiers supporting each variant was retrieved from the corresponding filtered SAM files and visually inspected. (D) Clonal configuration from diagnosis (left) to relapse (right). (E) Lateral view of the wild type DNMT3A-DNMT3L dimer in a complex with histone H3 (from 4U7T.pdb) and a DNA molecule containing 2 CpG sites (from 5YX2) is reported in the cartoon representation. Histone H3 is depicted in a green cartoon. The DNA molecule is colored orange. DNMT3A monomers are shown in white and magenta. DNMT3L monomers are shown in cyan and yellow. Residues affected by the investigated mutations are reported in yellow spheres and labeled. (F) The red cartoon representation indicates the C-terminal domain portions lost from the DNMT3A domains due to the mutations Y536* and D531Gfs*. See the previous panel legend for other color details. (G, H) Zoomed view of a DNMT3A C-terminal monomer domain containing the G707 residue [yellow stick, (G)] and the corresponding mutation G707C [white stick, (H)]. The orange cartoon indicates a groove of the DNA molecule interacting with G707 or G707C. S-adenosyl-homocysteine interacting with G707 or G707C is reported in white sticks. (I, J) Zoomed view of a DNMT3A C-terminal dimer domain containing the R899 residue [yellow sticks (I)] and the corresponding mutation R899C [white sticks, (J)]. Other residues affected by mutations are reported for comparative purposes (see previous panel legends for color details). Dashed lines and labels indicate interactions between protein residues (G-J) or between protein residues and nucleotides (G, H).
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At diagnosis, NGS analysis identified the presence of a complex DNMT3A (NM_022552.4) molecular profile consisting of four different somatic variants: c.1592_1593insG, p.D531Gfs*15 (variant allele frequency (VAF): 15.13%); c.1608C>G, p.Y536* (VAF: 47.87%); c.2119G>T, p.G707C (VAF: 9.04%) and c.2695C>T, p.R899C (VAF: 7.65%) ( Figure 1 ). In CR no DNMT3A gene variants were identified and at the DR only p.D531Gfs*15 (VAF: 32.75%) and p.Y536* (VAF: 32.12%) were detected ( Figure 1 ). Figure 1 ), suggesting that all the DNMT3A mutations were located on different alleles (in trans). Furthermore, based on the VAF value, we established the clonal configuration at diagnosis, mapping the Y536* variant (VAF~50%) on one DNMT3A allele of all leukemic cells and the other mutations (with a lower VAF) on the other allele of three different subclones. The identified subclone carrying the two novel truncating variants (Y536* and D531Gfs*15) was responsible for DR ( Figure 1 ). To rule out the occurrence of this process, the presence of transcripts carrying the above mutations was verified by direct sequencing ( Additional file  and  Figure 1 ), showing that the two truncating variants identified did not cause nonsense-mediated mRNA decay. A 3D modeling analysis ( Figures 1E ) was performed to predict the effect of the DNMT3A mutational status ( Additional file 1 ). The truncated proteins lose most of the C-terminal domain (residues 546 to 912 and 536 to 912, respectively), containing both the ADD and the catalytic domain, making the DNMT3A structure unstable, challenging every interaction with DNA molecules and causing loss of the gene activity ( Figure 1 ). The variant producing the missense mutation G707C causes an alteration of binding interactions between the DNMT3A catalytic loop (residues G707-K721), containing the G707 residue, the SAM that will provide the methyl group and the DNA double helix that should be methylated ( Figures 1G ).
DNA methylation profiling and functional analysis. (A) Global methylation status evaluation by droplet digital PCR quantification of the Alu methylation pattern. CR: complete remission, DR: disease relapse. (B) Unsupervised hierarchical clustering of the 25000 most variably methylated probes according to standard deviation, applying the Euclidean distance as a distance metric. The heatmap scale shows the range of methylation values, from 0 (unmethylated, green) to 1 (hypermethylated, red).  Probes names. (C) Distribution of differentially methylated CpGs (dmCpGs) relative to CGIs and relative distribution of dmCpGs across different genomic regions in the three comparisons. *Statistically differential enrichment (FDR<0.05) of a specific class of CpGs. (D) Heatmap of Ingenuity Pathway Analysis (IPA) comparative functional analysis of canonical pathways for the three comparisons performed. The violet intensity is proportional to the differences highlighted in each comparison.
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Nevertheless, it was possible to observe that the investigated mutations affect residues that are highly conserved in the DNMT3A C-terminal domain ( Additional   Figure 2 ). Alu methylation level to investigate the possible effect of DNMT3A loss of function ( Additional file 1 ). Although unexpected, DNMT3A's severe loss of function at diagnosis and DR, results in a global genomic hypermethylation, as compared to CR and a statistically significant difference was observed between CR and DR (81.6% vs 86.7%, respectively, p=0.04) ( Figure 2 ). Similarly, unsupervised hierarchical clustering of the most 25000 variably methylated probes ( Additional file 1 ) showed that the samples were segregated into three distinct clusters, with diagnosis and DR clustering together and showing a global genomic hypermethylation, as compared to CR ( Figure 2 ). Table 1 ). A significant enrichment (over-representation) of CpG islands (CPGis) and shores (ie., regions up to 2 kb from CpGis) among the hypermethylated CpG sites in the diagnosis and DR samples was observed as compared to the CR sample ( Figure 2 ). The overall distribution of hypermethylated CpGs at the gene mapping was suggestive of a significant enrichment of promoters and 1-5kb regions upstream of the TSS in the diagnosis and DR samples as compared to the CR sample; in details, the enrichment of hypermethylated CpGs in these genomic regions was more evident in DR sample compared to diagnosis ( Figure 2 ,  Additional Table 2 ). Ingenuity Pathway Analysis (IPA) was also performed on the first 600 genes included in the top DMRs resulting from each comparison, to identify the biological relevance of the DMRs, and then a comparative functional analysis of the three comparisons was performed ( Figure 2 ). IPA comparison analysis across the different samples revealed that the "transcriptional regulatory network in embryonic stem cells" canonical pathway was enriched in the DMRs common to the diagnosis and DR samples.