Germline DNMT3A mutation in familial acute myeloid leukaemia

Figures & data

Figure 1. Clinical course and mutations detected in I-2(a) and II-1(b). Germline mutation in DNMT3A P709S (black) and predominant somatic mutations in IDH2 R172K and ASXL1 P808fs (grey) that were detected within bone marrow samples at various timepoints (blue) along with the regimens and protocols given (green): CIA (induction of clofarabine, idarubicin and cytarabine), SGI-110 (DNMT inhibitor), BL-8040 (CXCR4 inhibitor), TH302 (Evofosfamide, replication inhibitor), AZD1208 (Pim-kinase inhibitor), Erlotinib (epidermal growth factor receptor (EGFR) inhibitor), PRI-724 (Wnt inhibitor), J0894 + J9100 (Decitabine + Cytarabine), AG221 x 2 cycles (IDH2 inhibitor), Trametinib (MEK inhibitor), APTO-253 (MTF-1 inhibitor) are shown

Figure 2. Position of the germline DNMT3A P709 within (a) the SAM-dependent methyltransferase (arrow) (cBioPortal) [45,46] and (b) the substrate-binding pocket of the DNA methyltransferase in the crystal structure (CN3D) [47] and its positional relationship to the R882 canonical mutation

Figure 3. The P705S mutation severely impairs the ability of Dnmt3a to methylate DNA

(a) Sequence alignment of a portion of human and mouse Dnmt3a. P709 in human Dnmt3a corresponds to P705 in mouse Dnmt3a.(b) Myc-tagged mouse Dnmt3a2 or Dnmt3a2:P705S (PS) mutant was transfected into late-passage Dnmt3a/3b double knockout (7aabb) ES cells, and stable clones obtained. The cell lysates of these clones, as well as untransfected 7aabb cells, were analysed by immunoblotting with anti-Myc and anti-β-actin antibodies. The dotted line indicates where the membrane was cut.(c) Genomic DNA from cells described in (b), as well as WT (J1) ES cells, was digested with MaeII and analysed by Southern hybridization with a probe specific for the major satellite repeats (MSRs). Densitometry was used to determine the relative methylation levels (methylation scores).

Figure 4. The Dnmt3a2:P705S mutant protein interacts with and antagonizes wild-type Dnmt3a2

(a) Flag-tagged and Myc-tagged WT Dnmt3a2 or Dnmt3a2:P705S (PS) were cotransfected in HEK 293 cells, the cell lysates were immunoprecipitated with anti-Myc antibody, and the precipitated proteins (Myc IP), as well as total cell lysates (TCL), were analysed by immunoblotting with anti-Flag and anti-Myc antibodies, as indicated.(b) Flag-tagged WT Dnmt3a2 was cotransfected with Myc-tagged Dnmt3a2:P705S or empty Myc vector (as control) in late-passage 7aabb cells, and stable clones were analysed by immunoblotting with anti-Flag, anti-Myc, and anti-β-actin antibodies, as indicated.(c) Genomic DNA from cells described in (b), as well as WT (J1) ES cells, was digested with MaeII and analysed by Southern hybridization with a probe specific for the major satellite repeats (MSRs). Densitometry was used to determine the relative methylation levels (methylation scores). Assuming Dnmt3a2 activity is 100%, 50% and 0% for WT, heterozygous KO and homozygous KO, P709S provides approximately 30% methylation activity. Note that co-expression of Dnmt3a2:PS inhibited the ability of WT Dnmt3a2 to rescue DNA methylation.

Figure 5. DNMT3A p.P709S is associated with decreased DNA methylation in germline samples

(a) LINE-1 pyrosequencing methylation analysis (PMA) as surrogate of global DNA methylation levels. I-1 (Germ) and II-1 (Germ) have significantly lower methylation than normal oral DNA from a healthy donor (CTL (Germ) and normal oral DNA from 18 AML patients (AML(Germ)) without mutations in any of the following genes: DNMT3A, IDH1, IDH2, TET1, TET2, TET3, DNMT1, DNMT3B, and EZH2 by 295-gene panel sequencing. In comparison, AML samples from I-1 and II-2 present methylation level at and above the upper limits of LINE-1 methylation in primary AML (n = 25) and CMML (n = 69) samples.(b) Genome-wide methylation analysis of germline and AML samples. Reduced Represention Bisulphite Sequencing (RRBS) was performed and there were in total 534,109 CpG sites with a minimum of 20X coverage represented in all samples, excluding sex chromosomes. Frequency of unmethylated (methylation less than 25%), partially methylated (25% to 75% methylation) or methylated (more than 75% methylation) are shown.(c). Hierarchical clustering of the top 1% most variable CpG sites analysed by RRBS. The clustering revealed that patient’s germline samples are most similar, but separated from the other samples.

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